Updated on 2024/02/27

写真a

 
FUNAHASHI Hiroaki
 
Organization
Faculty of Environmental, Life, Natural Science and Technology Professor
Position
Professor
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Degree

  • Ph. D. ( Okayama University )

  • Ms. of Agriculture ( Okayama University )

Research Interests

  • Animal Reproductive Science

  • Animal Science

  • Gametes (Sperm and oocytes)

  • Maturation, Fertilization and early development in vitro

Research Areas

  • Life Science / Cell biology

  • Life Science / Animal production science

Education

  • Okayama University   大学院自然科学研究科   生物資源科学

    1987.4 - 1990.3

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    Country: Japan

    Notes: 博士課程

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  • Okayama University   大学院農学研究科   畜産学専攻

    1985.4 - 1987.3

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    Country: Japan

    Notes: 修士課程

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  • Okayama University   農学部   畜産学科

    1981.4 - 1985.3

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    Country: Japan

    Notes: 学士課程

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Research History

  • Okayama University   Institute of Environmental, Life, Natural Science and Technology   Professor

    2023.4

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    Country:Japan

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  • Okayama University, Institute of Environmental and Life Science   Professor

    2021.4 - 2023.3

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    Country:Japan

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  • Okayama University   Executive Director for Academic Affairs / Provost

    2021.4 - 2023.3

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    Country:Japan

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  • Okayama University   Vice President

    2020.4 - 2021.3

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    Country:Japan

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  • Okayama University   The Graduate School of Environmental and Life Science   Dean

    2017.4 - 2021.3

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  • Assisted Reproductive Technology Center, Okayama University   Professor

    2013.10

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    Country:Japan

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  • Graduate School of Environmental and Life Science, Okayama University   Professor

    2012.4 - 2023.3

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    Country:Japan

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  • Graduate School of Natural Science and Technology, Okayama University   Professor

    2011.1 - 2012.3

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    Country:Japan

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  • University of Murcia   Faculty of Veterinary Science   Visiting Professor

    2008.12

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    Country:Spain

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  • Kurashiki Sakuyo University   Faculty of Music   Visiting Lecturer

    2000.4 - 2013.3

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    Country:Japan

    Notes:教養科目(生命の科学)担当

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  • Graduate School of Natural Science and Technology, Okayama University   Associate Professor

    2000.4 - 2010.12

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    Country:Japan

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  • Swedish University of Agricultural Sciences   Faculty of Veterinary Science   Visiting Scientist

    1999.5 - 1999.7

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    Country:Sweden

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  • National Institute for Agricultural Research (INRA)   Visiting Scientist

    1998.5 - 1998.6

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    Country:France

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  • Okayama University   Faculty of Agriculture

    1996 - 2000

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    Country:Japan

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  • Department of Animal Science, University of Missouri-Columbia   Research Assistant Professor

    1994 - 1996

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    Country:United States

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  • Department of Animal Science, University of Missouri-Columbia   Postdoctral Fellow

    1991 - 1994

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    Country:United States

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  • Central Research Institute for Feed and Livestock, National Federation of Agricultural Cooperative Association (ZEN-NOH)   Researcher

    1987 - 1991

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    Country:Japan

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Professional Memberships

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Papers

  • Negative correlations of mitochondrial DNA copy number in commercial frozen bull spermatozoa with the motility parameters after thawing Reviewed International journal

    Hai Thanh Nguyen, Son Quang Do, Hiroshi Kobayashi, Takuya Wakai, Hiroaki Funahashi

    Theriogenology   210   154 - 161   2023.10

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.theriogenology.2023.07.027

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  • Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo. Reviewed International journal

    Yuka Ikawa, Takuya Wakai, Hiroaki Funahashi, Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki

    Scientific reports   13 ( 1 )   13050 - 13050   2023.8

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    Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

    DOI: 10.1038/s41598-023-40361-9

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  • Oocyte-cumulus cells crosstalk: New comparative insights Reviewed International coauthorship International journal

    Cristina A. Martinez, Dimitrios Rizos, Heriberto Rodriguez-Martinez, Hiroaki Funahashi

    Theriogenology   205   87 - 93   2023.7

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    Authorship:Last author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.theriogenology.2023.04.009

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  • Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health Reviewed International coauthorship International journal

    Hai Thanh Nguyen, Son Quang Do, Rukmali Athurupana, Takuya Wakai, Hiroaki Funahashi

    Animal Reproduction   20 ( 3 )   e20220127   2023

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:FapUNIFESP (SciELO)  

    DOI: 10.1590/1984-3143-ar2022-0127

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  • Anti-Müllerian hormone independently affect mtDNA copy number in human granulosa cells Reviewed International coauthorship International journal

    Anom Bowolaksono, Ayu Mulia Sundari, Muhammad Fauzi, Mila Maidarti, Budi Wiweko, Kresna Mutia, Pritta Ameilia Iffanolida, Ririn Rahmala Febri, Astari Dwiranti, Hiroaki Funahashi

    Journal of Ovarian Research   15 ( 1 )   2022.10

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    Authorship:Last author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    Abstract

    Background:

    Recently, as a delayed childbearing trend is emerging in modern women’s adulthood, diminished reproductive potential due to age-related changes is more prevalent. Reduction in the abundance of mitochondrial DNA (mtDNA) copies and circulating anti-Müllerian hormone (AMH) have been separately reported with aging, contributing to the decrease in successful reproduction. However, there are limited reports on the impact of age on mtDNA and AMH in the same individual and whether mtDNA copy numbers are influenced by age and AMH.

    Methods:

    In the present study, we utilized a real-time quantitative PCR (RT-qPCR) to quantify the mtDNA copy number of granulosa cells obtained from 43 women undergoing an in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) program.

    Results:

    According to our analysis, a significant correlation was observed between age and mtDNA copy number (r = −0.54, P < 0.001) and between age and AMH level (r = −0.48, P < 0.001) of the same individual. There was also a positive correlation between mtDNA copy number and AMH (r = 0.88, P < 0.001) with AMH level falling as mtDNA decreases. In our regression, age and AMH were shown to have low collinearity (VIF = 1.297) but only AMH was correlated with mtDNA quantity (P < 0.001).

    Conclusion:

    Our study suggests that both mtDNA and AMH abundance are influenced by age and that AMH levels independently affect mtDNA copy number regardless of age. Further research is required to understand the role of AMH on mitochondria bioenergetics.

    DOI: 10.1186/s13048-022-01047-4

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    Other Link: https://link.springer.com/article/10.1186/s13048-022-01047-4/fulltext.html

  • Large-scale preparation of sialyl-Tn antigen-containing peptides from mucin-like glycoproteins in boar seminal gel Reviewed International journal

    Ryota Takeuchi, Megumi Maeda, Miran Nakano, Hiroaki Funahashi, Yoshinobu Kimura

    Bioscience biotechnology and biochemistry   85 ( 9 )   2022 - 2025   2021.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford univ press  

    Sialyl-Tn antigen, a tumor antigen, is a valuable ligand for the purification of proteins that specifically bind to it. Here, we developed a new method for the preparation of large amounts of sialyl-Tn antigen-containing peptides from an unused resource, boar seminal gel. The glycopeptides were prepared from the actinase E digests by a combination of gel filtration and hydrophilic partitioning.

    DOI: 10.1093/bbb/zbab114

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  • The thickness and density of the ovarian tunica albuginea increases with age in transgender patients Reviewed International journal

    Pilar Ferre-Pujol, Junko Otsuki, Hiroaki Funahashi, Mikiya Nakatsuka

    Reproductive sciences   28 ( 5 )   1339 - 1346   2021.5

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer heidelberg  

    It is known that the extracellular matrix structure and composition changes with aging in many organs. Despite this, knowledge on how does the extracellular part of the ovary change with increasing age in women and how those changes might be related to women's loss of fertility is still lacking. For this, we propose that recurrent injury and repair events on the outermost layers of the ovary due to ovulation are partly responsible for those changes women experience with aging. The histological analysis of the ovaries from 18 female-to-male transgender patients revealed that the ovarian tunica albuginea (TA) increases its thickness and density correlatively with increasing age of the patient (r = 0.52 and r = 0.55, P < 0.05 respectively). The increase in thickness is independent of the total androgen dose received and occurs because of the appearance of defined fibrotic areas underneath the TA layer which increase the total distance of dense connective tissue from the ovarian surface. In conclusion, the ovarian TA increases in its thickness and density with aging because of the appearance of fibrotic areas underneath the layer in transgender patients. This fact might contribute to reduce oocyte quality and cause ovulation difficulties in older women.

    DOI: 10.1007/s43032-020-00390-5

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  • Relative transcript abundance in porcine cumulus cells collected from different sized follicles Reviewed International coauthorship International journal

    Carla Moros-Nicolas, Ma Jose Izquierdo-Rico, Yang Li, Leopoldo Gonzalez-Brusi, Raquel Romar, Hiroaki Funahashi

    Reproduction in domestic animals   56 ( 2 )   374 - 380   2021.2

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    Crosstalk between the oocyte and surrounding cumulus cells (CCs) is essential for the production of competent oocytes. Previous studies have analysed the relative transcript abundance in oocytes derived from small (SF: <3 mm diameter)- and medium-sized (MF: 3-6 mm diameter) follicles to determine the potential use of SF-derived oocytes in assisted reproductive technologies (ART). The aim of this study was to examine the relative transcript abundance of CCs obtained from cumulus-oocyte complexes (COCs) derived from SF and MF. Nine genes were selected according to their importance for developmental competence: AT-rich interaction domain 1B (ARID1B), bone morphogenic protein receptor 2 (BMPR2), CD44, follicle-stimulating hormone receptor (FSHR), follistatin (FST), inhibin beta-A (INHBA), luteinizing hormone receptor (LHR), nuclear receptor subfamily 2 group F member 6 (NR2F6) and vascular endothelial growth factor A (VEGFA). The expression of these genes was analysed by RT-qPCR. The results pointed to significant differences in five genes, and the relative transcript abundance of SF-derived CCs was lower in the case of INHBA, but higher in FSHR, FST, LHR and NR2F6 compared with MF-derived CCs. We provide information of gene activity in the porcine CCs from different sized follicles, thus improving our understanding of oocyte biology and providing new markers that identify viable and competent oocytes.

    DOI: 10.1111/rda.13881

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  • Animal biotechnology roles in livestock production

    Hiroaki Funahashi

    International conference: Improving tropical animal production for food security   465 ( 1 )   2020

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (international conference proceedings)   Publisher:Iop publishing ltd  

    Currently, meat and milk productions are significantly increasing especially in Asia. The supply of these products is vital to people's health and well-being, whereas the efficiency of beef production appears to be still lower than other meat productions. Improvements in the quality and functionality of their livestock products, as well as their production efficiency, are required for further production. Animal biotechnologies have contributed to genetic improvement, genetic diversity maintenance of domestic animals, etc. Basic animal biotechnologies, such as artificial insemination and embryo transfer, have been well established and applied as powerful tools for genetic improvement of livestock. In the applications of artificial insemination techniques, the use of sexed semen has been now widely spread, and also efforts are also made in the development of the technology using a small amount of sperm. For embryo transfer, several types of vitrification technologies have been applied to improve pregnancy rates and contributed to the international/domestic supply of livestock embryos. Conventional animal biotechnologies, such as in vitro fertilization and intracellular sperm injection, have been applied to not only livestock production and also human-assisted reproductive medicine. For in-vitro production of embryos in domestic animals, currently, oocytes have been collected from medium or large follicles (3-6 mm or larger in diameter) of ovaries. Although the oocytes derived from small follicles (less than 3 mm in diameter) exist more on the surface of ovaries, the developmental competence of the oocytes has been known to be significantly lower than those from medium follicles. If we could improve the competence of oocytes derived from small follicles significantly, we may be able to increase the number of female gamete resources for in vitro embryo production. Also, the development of techniques for producing transgenic and cloned animals has greatly contributed to the creation of pharmaceuticals and organs for xenotransplantation. Recently, furthermore, genome editing technologies, such as combined use of CRISPR/Cas9 and PiggyBac, have been developed and have made it possible to correct specific parts of the genome and introduce mutations by homologous recombination. In this review, I would like to discuss the application and progress of the above biotechnologies, including our recent research results.

    DOI: 10.1088/1755-1315/465/1/012001

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  • The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles Reviewed International journal

    Chiyuki Kohata-Ono, Takuya Wakai, Hiroaki Funahashi

    Journal of reproduction and development   65 ( 6 )   527 - 532   2019.12

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Society reproduction & development-srd  

    This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

    DOI: 10.1262/jrd.2019-112

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  • Removal of cumulus cells around 20 h after the start of in vitro maturation improves the meiotic competence of porcine oocytes via reduction in cAMP and cGMP levels Reviewed International coauthorship International journal

    Pilar Ferre-Pujol, Xuan Khanh Nguyen, Tomoki Nagahara, Thi Tra Mi Bui, Takuya Wakai, Hiroaki Funahashi

    Journal of reproduction and development   65 ( 2 )   177 - 182   2019.4

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Society reproduction & development-srd  

    We examined the effect of the timing of removing cumulus cells surrounding porcine oocytes from small follicles (SFs, < 3 mm in diameter) and medium follicles (MFs; 3-6 mm in diameter) on the meiotic and developmental competence of the oocytes. Cumulus-oocyte complexes (COCs) were collected from SFs and MFs, and the oocytes were denuded at 0, 20, and 44 h after the start of in vitro maturation (IVM), and the meiotic progression of the oocytes was assessed at the end of the IVM period. The incidence of mature oocytes was significantly affected by both the origin of the COCs and the time when the oocytes were denuded. Although the percentage of mature oocytes was always higher when the COCs were collected from MFs than that when the COCs were collected from SFs, the maturation rate was significantly higher when the oocytes were denuded at 20 h than when they were denuded at 44 h after the start of IVM. When the mature oocytes were activated electrically, the developmental competence of the oocytes denuded at 20 and 44 h to reach the blastocyst stage did not differ, whereas the competence of the MF-derived oocytes was significantly higher than that of SF-derived oocytes. When the intracellular cAMP and cGMP levels in SF-derived oocytes were examined at 24 h of IVM, the levels of both were significantly decreased only in the oocytes denuded at 20 h. In conclusion, denuding oocytes at 20 h of IVM caused a significant reduction in ooplasmic cAMP and cGMP levels and increased the meiotic competence of the oocytes without any reduction in blastocyst formation, even in the case of SF-derived oocytes.

    DOI: 10.1262/jrd.2018-130

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  • Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles Reviewed International journal

    Tra M. T. Bui, Khank X. Nguyen, Asako Karata, Pilar Ferre, Minh T. Tran, Takuya Wakai, Hiroaki Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   29 ( 10 )   1902 - 1909   2017.9

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:CSIRO PUBLISHING  

    The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) on the meiotic and developmental competence of porcine oocytes from small follicles (SF; 0.5-3mmdiameter). When cumulusoocyte complexes (COCs) from medium-sized follicles (MF; 3-6mm diameter) and SF were cultured for IVM, the maturation rates were significantly higher for oocytes from MF than SF. Concentrations of VEGF in the medium were significantly higher for COCs cultured from MF than SF. When COCs from SF were exposed to 200 ngmL(-1) VEGF during the first 20 h of IVM, the maturation rate improved significantly and was similar to that of oocytes derived from MF. The fertilisability of oocytes was also significantly higher than that of VEGF-free SF controls. Following parthenogenetic activation, the blastocyst formation rate improved significantly when SF COC culture was supplemented with 200 ngmL(-1) VEGF, with the rate similar to that of oocytes from MF. The results of the present study indicate that VEGF markedly improves the meiotic and developmental competence of oocytes derived from SF, especially at a concentration of 200 ngmL(-1) during the first 20 h of IVM.

    DOI: 10.1071/RD16321

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  • Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles Reviewed International journal

    Tra M. T. Bui, Khank X. Nguyen, Asako Karata, Pilar Ferre, Minh T. Tran, Takuya Wakai, Hiroaki Funahashi

    Reproduction fertility and development   29 ( 10 )   1902 - 1909   2017.9

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Csiro publishing  

    The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) on the meiotic and developmental competence of porcine oocytes from small follicles (SF; 0.5-3mmdiameter). When cumulusoocyte complexes (COCs) from medium-sized follicles (MF; 3-6mm diameter) and SF were cultured for IVM, the maturation rates were significantly higher for oocytes from MF than SF. Concentrations of VEGF in the medium were significantly higher for COCs cultured from MF than SF. When COCs from SF were exposed to 200 ngmL(-1) VEGF during the first 20 h of IVM, the maturation rate improved significantly and was similar to that of oocytes derived from MF. The fertilisability of oocytes was also significantly higher than that of VEGF-free SF controls. Following parthenogenetic activation, the blastocyst formation rate improved significantly when SF COC culture was supplemented with 200 ngmL(-1) VEGF, with the rate similar to that of oocytes from MF. The results of the present study indicate that VEGF markedly improves the meiotic and developmental competence of oocytes derived from SF, especially at a concentration of 200 ngmL(-1) during the first 20 h of IVM.

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  • Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus–oocytes complexes derived from small follicles Reviewed International journal

    R. Matsunaga, H. Funahashi

    Reproduction in domestic animals   52 ( 4 )   672 - 679   2017.8

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    The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3–6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1–2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus–oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.

    DOI: 10.1111/rda.12967

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  • Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation Reviewed

    Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   63 ( 2 )   191 - 197   2017.4

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

    DOI: 10.1262/jrd.2016-156

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  • Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes Reviewed International journal

    Pilar Ferre, Tra Mi Thi Bui, Takuya Wakai, Hiroaki Funahashi

    THERIOGENOLOGY   86 ( 7 )   1705 - 1710   2016.10

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes. (C) 2016 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2016.05.024

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  • Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos Reviewed International journal

    Jingchun Li, Sibing Zhu, Xianjing He, Rui Sun, Qianyu He, Yi Gan, Shengjun Liu, Hiroaki Funahashi, Yanbing Li

    THERIOGENOLOGY   85 ( 7 )   1211 - 1218   2016.4

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    Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 x 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 +/- 0.03) was the highest in three observation areas (P &lt; 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 x 10(6) group (10 x 10(6) sperm/mL) were higher than in the 1 x 10(6) and 5 x 10(6) groups (P &lt; 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P &lt; 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% +/- 2.61%) than the modified standard IVF technique (24.55% +/- 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation. (C) 2016 Elsevier Inc. All rights reserved.

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  • Milk supplements in a glycerol free trehalose freezing extender enhanced cryosurvival of boar spermatozoa Reviewed

    Rukmali Athurupana, Hiroaki Funahashi

    Asian pacific journal of reproduction   5 ( 1 )   58 - 62   2016.3

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    Objective: To evaluate the effect of skim milk and/or coconut milk in a glycerol-free trehalose extender to improve cryosurvival of boar spermatozoa. Methods: Sperm samples were diluted in an egg-yolk-based freezing extender containing 100 mM trehalose and 0.25% Equex STM supplemented with coconut milk or/and skim milk at 2% or 5% (w/v). Spermatozoa were cryopreserved in 0.5 mL straws and thawed by a rapid transient method (at 70 °C for 8 s) followed with a stabilizing procedure at 39 °C. Thawed samples were analyzed for motility, viability, high mitochondrial membrane potential (HMMP), and acrosome damage. Results: Even on the presence of egg yolk, motility, HMMP and viability were significantly higher in extender supplemented with 2% skim milk than controls without skim milk (. P < 0.05). Post-thaw viability significantly improved with the addition of 2% skim milk plus 2% coconut milk as well (. P < 0.05). Acrosome damage was considerably lower when the extender was supplemented with 2% coconut milk (. P < 0.05), whereas the benefit was masked in the presence of 2% skim milk. Conclusion: 2% skim milk can be used as supplements for a glycerol-free trehalose and egg yolk-based extender to improve post-thaw survival of boar spermatozoa, whereas 2% coconut milk has an effect to protect boar spermatozoa from acrosome damage.

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  • A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium Reviewed International journal

    Sumire Ioki, Qing-Shan Wu, Osamu Takayama, Hideyuki H. Motohashi, Takuya Wakai, Hiroaki Funahashi

    THERIOGENOLOGY   85 ( 3 )   428 - 433   2016.2

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    The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 mu M), higher sildenafil concentrations (25 and 250 mu M) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 mu M sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro. (c) 2016 Elsevier Inc. All rights reserved.

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  • In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years Reviewed

    Raquel Romar, Hiroaki Funahashi, Pilar Coy

    THERIOGENOLOGY   85 ( 1 )   125 - 134   2016.1

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    Assisted reproduction technology (ART) protocols are used in livestock for the improvement and preservation of their genetics and to enhance reproductive efficiency. In the case of pigs, the potential use of embryos for biomedicine is being followed with great interest by the scientific community. Owing to the physiological similarities with humans, embryos produced in vitro and many of those produced in vivo are used in research laboratories for the procurement of stem cells or the production of transgenic animals, sometimes with the purpose of using their organs for xenotransplantation. Several techniques are required for the production of an in vitro-derived embryo. These include in vitro oocyte maturation, sperm preparation, IVF, and further culture of the putative zygotes. Without doubt, among these technologies, IVF is still a critical limiting factor because of the well-known, but still unsolved, question of polyspermy. Despite the improvements made in the past decade, current IVF systems hardly reach 50% to 60% efficiency and any progression in porcine ARTs requires an unavoidable improvement in the monospermy rate. It is time, then, to learn from what happens under in vivo physiological conditions and to transfer this knowledge into ART. This review describes the latest advances in porcine IVF, from sperm preparation procedures to culture media supplements with special attention paid to molecules with a known or potential role in in vivo fertilization. Oviductal fluid is the natural medium in which fertilization takes place, and, in the near future, could become the definitive supplement for culture media, where it would help to solve many of the problems inherent in ARTs in swine and improve the quality of in vitro-derived porcine embryos. (C) 2016 Elsevier Inc. All rights reserved.

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  • Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender Reviewed

    Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

    THERIOGENOLOGY   84 ( 6 )   940 - 947   2015.10

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    Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 degrees C. Ejaculated semen samples were diluted in an egg yolk based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 degrees C, 60 degrees C, 70 degrees C, and 80 degrees C, the calculated average warming rate in the straws from -196 degrees C to 15 degrees C was much faster when thawed at 70 degrees C and 80 degrees C than at 40 degrees C (P &lt; 0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 degrees C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P &lt; 0.05), as compared with controls (at 39 degrees C). In experiment 3, frozen straws were thawed at 39 degrees C, 60 degrees C, 70 degrees C, and 80 degrees C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 degrees C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P &lt; 0.05) when frozen straws were thawed at 70 degrees C for 8 seconds and then maintained at 39 degrees C for 52 seconds as compared with the control (39 degrees C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 degrees C for 8 seconds and maintained at 39 degrees C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 degrees C for 8 seconds followed by stabilizing procedure at 39 degrees C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions. (C) 2015 Elsevier Inc. All rights reserved.

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  • Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa Reviewed

    Rukmali Athurupana, Daisen Takahashi, Sumire Ioki, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   61 ( 3 )   205 - 210   2015.6

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    Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100-250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.

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  • Development competence and relative transcript abundance of oocytes derived from small and medium follicles of prepubertal gilts Reviewed

    Chiyuki Kohata, Maria Jose Izquierdo-Rico, Raquel Romar, Hiroaki Funahashi

    THERIOGENOLOGY   80 ( 9 )   970 - 978   2013.12

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    The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, &lt;2 mm in diameter) and medium follicles (MF, 3-6 mm) of abattoir-derived prepubertal gilt ovaries. Oocytes were selected by the presence of the first polar body (1pb) after IVM in a chemically defined medium, for sperm penetration, pronuclear formation, cleavage rate, and development to the blastocyst stage. Relative transcript abundance of genes associated with regulation of oocyte maturation (AURKA, AURKB, and MOS), fertilization (ZP3 and ZP4), maternal effect (NALP9 and HSF1), and anti-apoptosis (BCL2) were also examined in oocytes at germinal vesicle (GV) and metaphase-II (MII) stages. In SF, compared with MF, the maturation rate post-IVM was lower (P &lt; 0.05), but there were no differences in sperm penetration rate (78.2% and 68.5% at 6 hours after insemination and 90.8% and 91.9% at 9 hours after insemination, P = 0.51 and P = 0.67, respectively), the percentage of oocytes that formed both female and male pronuclei (27.9% and 25.8% at 6 hours after insemination and 79.4% and 76.1% at 9 hours after insemination), or cleavage rate at 48 hours after insemination (85.9% and 89.7%, respectively, P = 0.46), whereas blastocyst formation rate was lower (P &lt; 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P &lt; 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P &lt; 0.05) in CV oocytes from SF versus MF. In conclusion, mature oocytes from SF and MF of prepubertal gilts with a visible 1pb had similar fertilizability in vitro and relative transcript abundance of nine genes. However, follicle size affected meiotic competence, early embryonic development to the blastocyst stage, and transcript abundance of the MOS gene. (C) 2013 Elsevier Inc. All rights reserved.

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  • A microfluidic device to reduce treatment time of intracytoplasmic sperm injection Reviewed International journal

    Koji Matsuura, Takuya Uozumi, Takuya Furuichi, Ikuyo Sugimoto, Mieko Kodama, Hiroaki Funahashi

    Fertility and sterility   99 ( 2 )   400 - 407   2013.2

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    Objective: To develop a microfluidic device that can reduce the intracytoplasmic sperm injection (ICSI) treatment time by increasing sperm concentration.Design: We compared the ICSI treatment time required for porcine sperm using a method employing the microfluidic device and one using the conventional microdroplet method.Settings: Academic research laboratories at Okayama University.Animal(s): Reproductive cells of porcine sperm, oocytes, and embryos.Intervention(s): Cell manipulations, ICSI, and embryo culture.Main Outcome Measure(s): Average ICSI treatment time and sperm concentration.Result(s): The average ICSI treatment time (mean +/- SEM) using the method with the microfluidic device for poor-quality semen (sperm concentration, 2.0 x 10(4) cells/mL) was significantly shorter than the treatment time using the conventional microdroplet method (265 +/- 15 seconds [n = 43] vs. 347 +/- 19 seconds [n = 50]). When diluted semen with a sperm concentration of 2.0 x 10(5) cells/mL was used, no significant difference was observed between the two methods (n = 50 and n = 48).Conclusion(s): The microfluidic device can reduce the time required for ICSI treatment that is used to increase sperm concentration in poor-quality semen samples. The results suggest that this device may be clinically useful for ICSI treatment in human assisted reproductive technology. (Fertil Steril (R) 2013;99:400-7. (C) 2013 by American Society for Reproductive Medicine.)

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  • Effects of caffeine on sperm characteristics after thawing and inflammatory response in the uterus after artificial insemination with frozen-thawed boar semen Reviewed International journal

    S. Yamaguchi, C. Suzuki, M. Noguchi, S. Kasa, M. Mori, Y. Isozaki, S. Ueda, H. Funahashi, K. Kikuchi, T. Nagai, K. Yoshioka

    Theriogenology   79 ( 1 )   87 - 93   2013.1

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    We previously reported that AI with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, and also improved fertility of gilts and sows. The objective of the present study was to determine the effects of the addition of caffeine to a thawing solution on postthaw sperm quality and uterine inflammatory response after AI with frozen-thawed boar semen. Incubation of frozen-thawed sperm in Modena solution supplemented with 10 mM caffeine for 90 minutes improved (P < 0.05) percentages of progressive motility, straightness, and linearity of sperm movement compared with no caffeine, without causing damage to plasma or acrosomal membranes. Gilts inseminated once with 2 × 109 frozen-thawed sperm suspended in Modena solution with or without caffeine, and gilts that did not receive AI, were slaughtered 4 hours later. Uteri were recovered for analysis of number of uterine PMNs and mRNA expression (quantitative reverse transcription polymerase chain reaction) of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and cyclooxygenase 2 in the endometrium. Caffeine decreased (P < 0.05) both the number of total uterine PMNs and expression of IL-8 mRNA in the endometrium after AI. The amount of IL-8 and cyclooxygenase 2 mRNA after AI in the absence of caffeine were higher than samples from gilts that did not receive AI (P < 0.05), whereas there were no significant differences between treatments in expression levels of tumor necrosis factor-α, IL-1β, IL-6, or monocyte chemoattractant protein-1 mRNA. Pregnancy rate in sows inseminated with sperm supplemented with caffeine (16 of 23; 70%) tended (P < 0.1) to exceed that without caffeine (12 of 26; 46%), but litter size was not affected. In conclusion, the addition of caffeine to the thawing solution inhibited migration of uterine PMNs, probably by downregulating IL-8 mRNA expression in the endometrium. © 2013 Elsevier Inc.

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  • What is the optimal condition for fertilization of IVM oocytes? Invited Reviewed

    Hiroaki Funahashi

    Reproductive medicine and biology   12 ( 1 )   15 - 20   2013.1

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    Application of in vitro maturation (IVM) is recently increasing for human infertility, especially to rescue patients of polycystic ovarian syndrome and ovarian hyperstimulation syndrome. To increase the application of IVM oocytes for embryo production and the efficiency of successful production of babies using IVM oocytes, quality control of oocytes and achievement of fertilization in the most suitable condition may be very important. In this paper, suitable conditions for fertilization of IVM oocytes will be discussed with recent knowledge about IVM and in vitro fertilization of oocytes in domestic animals. Currently, human oocytes are collected mainly from patients' ovaries 36 h following mild gonadotropin stimulation and used for IVM for 24-26 h. However, asynchronous progression of those oocytes to reach the metaphase-II stage may have occurred during the IVM culture. In the oocytes that have already progressed to the metaphase-II stage, sudden aging such as reduction in maturation promoting factor and MAP kinases will start to occur. Application of specific inhibitors of phosphodiesterase to control intracellular cAMP (cyclic adenosine monophosphate) level may be effective to synchronize timings of the germinal vesicle breakdown and consequently the meiotic progression of oocytes, and to improve the developmental competence. Furthermore, treatment of aging oocytes with caffeine appears to rescue them from reductions in maturation promoting factor and MAP kinases and to improve the developmental competence. Assessment methods to select oocytes with good quality may also be important to improve the successful rates.

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  • Successful delivery derived from vitrified-warmed spermatozoa from a patient with nonobstructive azoospermia Reviewed International journal

    Yuji Endo, Yoshitaka Fujii, Shozo Kurotsuchi, Hiroaki Motoyama, Hiroaki Funahashi

    Fertility and Sterility   98 ( 6 )   1423 - 1427   2012.12

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    Objective: To report the clinical outcomes following intracytoplasmic sperm injection (ICSI) with vitrified sperm from patients with severe male factor infertility. Design: Retrospective case series. Setting: IVF unit of a medical center. Patient(s): Three patients with severe oligozoospermia or nonobstructive azoospermia (NOA). Intervention(s): Cryopreservation of limited numbers of spermatozoa with the use of Cryotop and Cell Sleeper as nonbiologic containers. Main Outcome Measure(s): Four cycles underwent intracytoplasmic sperm injection (ICSI) with vitrified sperm. Result(s): A total of 148 spermatozoa in 18 containers (8.2 sperm per container) were vitrified and 36 of them (5 containers) were warmed. Thirty-three sperm (92%) were retrieved successfully and injected individually into 17 mature oocytes. Fertilization was observed in 12 oocytes (71%), and all zygotes (100%) cleaved. A couple with NOA achieved a singleton pregnancy and concluded with full-term delivery of a healthy boy (2,632 g). Conclusion(s): A successful delivery was achieved after transfer of a blastocyst derived from vitrified-warmed spermatozoa. A small number of vitrified sperm cells were used for ICSI to fertilize oocytes with predictable timing. © 2012 by American Society for Reproductive Medicine.

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  • Glycosaminoglycans Improves Early Development of Zona-free 8-cell Rat Embryos to Blastocysts in a Chemically Defined Medium, but Not the Pregnancy Rate Following Transfer of the Blastocysts Reviewed

    Masanobu Okuyama, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   58 ( 3 )   295 - 301   2012.6

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    The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined m RIECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 mu g/ml) or heparan sulfate proteoglycan (HS; 15 mu g/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.

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  • Effect of the addition of beta-mercaptoethanol to a thawing solution supplemented with caffeine on the function of frozen-thawed boar sperm and on the fertility of sows after artificial insemination Reviewed International journal

    S. Yamaguchi, H. Funahashi

    Theriogenology   77 ( 5 )   926 - 932   2012.3

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    We have reported that artificial insemination (AI) with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting the migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, thereby improving the fertility of gilts and sows. The objective of the present study was to examine the effects of the addition of the antioxidant beta-mercaptoethanol (bME) and caffeine to the thawing solution on the function of frozen-thawed sperm, on the phagocytic activity of PMNs for sperm, and on the fertility of sows after AI. When frozen-thawed sperm were cultured in the presence of 25 or 50 μm bME, sperm capacitation and spontaneous acrosome reactions were inhibited (P &lt
    0.01). There was no effect of bME on phagocytic activity of PMNs for sperm in vitro. When hormonally treated (400 IU of equine chorionic gonadotropin + 200 IU of human chorionic gonadotropin) weaned sows experienced a single intrauterine insemination with frozen-thawed sperm (25 × 10 8 sperm per 50 ml dose) 40 h after subsequent hCG administration, pregnancy and farrowing rates were unaffected by the addition of 50 μm bME (pregnancy rate, 20 vs 21% in controls
    farrowing rate, 20 vs 21%
    n = 15 and 14, respectively). However, litter size tended to be higher than in the presence of 50 μm bME compared to its absence (10.0 ± 1.0 vs 5.7 ± 1.5, respectively
    P &lt
    0.07). Thus, the addition of bME to the thawing solution containing caffeine could be of benefit for improving the function of frozen-thawed sperm without influencing the phagocytic activity of PMNs for sperm. Although there were no statistically significant effects of bME on pregnancy or farrowing rates, the litter size tended to be higher in the sows subjected to a fixed-time single AI treatment with synchronized ovulation. © 2012 Elsevier Inc..

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  • Simple vitrification for small numbers of human spermatozoa Reviewed International journal

    Yuji Endo, Yoshitaka Fujii, Kasumi Shintani, Momoyo Seo, Hiroaki Motoyama, Hiroaki Funahashi

    Reproductive BioMedicine Online   24 ( 3 )   301 - 307   2012.3

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    Conventional freezing procedures and containers are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility, and various types of container have been utilized to freeze small numbers of spermatozoa. This study tried to develop a vitrification method for small numbers of spermatozoa using the Cell Sleeper, which is a closed type of cell-cryopreservation container. The container with spermatozoa were cooled in liquid nitrogen vapour and then stored in a cryotank. Sperm motility parameters improved significantly (P &lt
    0.05) by vitrification in oil-free droplets rather than in droplets covered with oil. After vitrification of five spermatozoa per container, all spermatozoa were recovered and the viable sperm rate was significantly higher when spermatozoa were vitrified in a 3.5-μl droplet rather than in 0.5 μl (72.0% versus 38.0%
    P &lt
    0.01). Recovery, motility and viability rates of vitrified-warmed spermatozoa were similar between the Cell Sleeper and the CryoTop groups. In conclusion, the Cell Sleeper is a highly effective tool for the cryopreservation of small numbers of spermatozoa and limited cells can be vitrified quickly and simply without significant loss. © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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  • Boar seminal plasma or hen's egg yolk decrease the in-vitro chemotactic and phagocytotic activities of neutrophils when co-incubated with boar or bull sperm Reviewed International journal

    J. C. Li, S. Yamaguchi, H. Funahashi

    Theriogenology   77 ( 1 )   73 - 80   2012.1

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    The objective was to determine the effects of boar seminal plasma and hen's egg yolk on chemotaxis and phagocytosis of porcine and bovine polymorphonuclear neutrophils (PMNs) in vitro. Chemotactic activity of PMNs was determined following culture for 90 min in a blind well chamber. Phagocytosis was assayed after co-culture of PMNs with sperm for 60 min. In the presence of ≥ 5% boar seminal plasma, chemotactic activity of PMNs was reduced (P &lt
    0.05) in both pigs (from 1126.1 to 934.2-1009.1 cells/mm 2) and in cows (from 1067.1 to 768.9-800.0 cells/mm 2). Furthermore, ≥ 5% boar seminal plasma reduced (P &lt
    0.05) leukocyte phagocytosis in pigs (26.2-32.1%) and cows (27.2-30.0%) compared to controls (41.7 and 42.1%, respectively). Although 20% hen's egg yolk increased (P &lt
    0.05) chemotactic activity of PMNs in pigs (from 790.4 to 1006.1 cells/mm 2) and cows (from 789.9 to 953.5 cells/mm 2), egg yolk increased (P &lt
    0.05) phagocytotic activity of porcine PMNs (from 24.3 to 33.8%), but not the activity of bovine PMNs (15.1 vs 15.8% in controls). Boar seminal plasma and caffeine reduced (P &lt
    0.05) the egg yolk-induced increase in chemotaxis in both species (from 988.6 to 795.2 or 813.2 cells/mm 2 in pigs and from 953.5 to 779.4 or 833.8 cells/mm 2 in cows), and phagocytotic activities of PMN (from 33.8% to 15.2 or 13.3%) only in pigs (but not in cows
    11.2-15.1%). In conclusion, hen's egg yolk increased chemotactic activity of PMNs in both pigs and cows, whereas egg yolk increased only phagocytosis of PMNs in pigs, but not in cows. Even in the presence of egg yolk, boar seminal plasma and caffeine significantly reduced chemotactic activity of PMNs in pigs and cows, and phagocytotic activity of porcine PMNs. © 2012 Elsevier Inc.

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  • Caffeine, dibutyryl cyclic-AMP and heparin affect the chemotactic and phagocytotic activities of neutrophils for boar sperm in vitro Reviewed International journal

    J. C. Li, S. Yamaguchi, Y. Kondo, H. Funahashi

    Theriogenology   75 ( 7 )   1336 - 1345   2011.4

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    The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P &lt
    0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm2 vs serum, 1226.3 cells/mm2), regardless of the presence of sperm (control, 1121.1 cells/mm2 vs serum, 1245.8 cells/mm2), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm2 and with sperm, 1132.6 cells/mm2). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P &lt
    0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P &lt
    0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm2) was also reduced (P &lt
    0.05) with 2 mM caffeine (942.5 cells/mm2). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P &lt
    0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P &lt
    0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm2 to 1008.8-1026.3 cells/mm2). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities. © 2011.

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  • Single spermatozoon freezing using cryotop Reviewed

    Yuji Endo, Yoshitaka Fujii, Kasumi Shintani, Momoyo Seo, Hiroaki Motoyama, Hiroaki Funahashi

    Journal of Mammalian Ova Research   28 ( 1 )   47 - 52   2011

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    Conventional freezing procedures are not appropriate for surgically retrieved spermatozoa from the epididymis or testis because of their low numbers. Techniques for the cryopreservation of small numbers of spermatozoa have not been fully established. We tried to develop a cryopreservation method for a single spermatozoon using Cryotop, which has a simple structure and is easy to handle. Different parameters influencing the freezing procedure, types of container, sources of spermatozoa, and cryoprotectants were evaluated. The sperm recovery rate after thawing was similar between the sperm frozen using Cryotop or zona pellucida as containers (98.0% vs. 88.0%). Freezing of motile single spermatozoa obtained from ejaculates and testes were evaluated for recovery rate (90.0% vs. 95.0%) and motility rate (44.4% vs. 42.1%), which were not significantly different. The survival rate was significantly higher when sperm were treated with sucrose rather than with SpermFreeze (65.3% vs. 37.3%, P &lt
    0.01). Cryotop was a highly effective tool for the cryopreservation of a single spermatozoon, and sucrose was determined to be an efficient cryoprotectant.

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    Other Link: https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2011&ichushi_jid=J05623&link_issn=&doc_id=20110506280007&doc_link_id=10028124034&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F10028124034&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_1.gif

  • Hydrophobic silicone elastomer chamber for recording trajectories of motile porcine sperms without adsorption Reviewed

    Koji Matsuura, Yuka Kuroda, Keisuke Yamashita, Hiroaki Funahashi

    Journal of Reproduction and Development   57 ( 1 )   163 - 167   2011

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    Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P&lt
    0.05) and ALHD (P&lt
    0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation
    instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels. © 2011 by the Society for Reproduction and Development.

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  • Internalization of REIC/Dkk-3 protein by induced pluripotent stem cell-derived embryoid bodies and extra-embryonic tissues Reviewed International journal

    Ken Kataoka, Masakiyo Sakaguchi, Kun Peng Li, Chika Taketa, Ken Ichi Yamamoto, Gang Du, Hiroaki Funahashi, Hitoshi Murata, Nam Ho Huh

    International Journal of Molecular Medicine   26 ( 6 )   853 - 859   2010.12

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    REIC/Dkk-3 was first identified as a down-regulated gene in a number of human immortalized cells and human tumor-derived cell lines. Overexpression of the REIC/Dkk-3 gene using an adenovirus vector (Ad-REIC) has showed a potent selective therapeutic effect on various human cancers through induction of ER stress. Furthermore, we recently showed that Ad-REIC has an indirect host-mediated anti-tumor activity by induction of IL-7. However, the physiological function of REIC/Dkk-3 is still unclear. As a first step to study the possible receptor(s) for secreted REIC/Dkk-3, we analyzed the internalization of Cy3-labeled recombinant REIC/Dkk-3 protein. Among the cell lines screened, mouse induced pluripotent stem (iPS) cells showed a unique pattern of internalization. The internalization was observed in peripheral cells of spherical colonies formed spontaneously, but not in undifferentiated iPS cells. When we analyzed embryoid bodies (EBs) derived from iPS cells, REIC/Dkk-3 protein was internalized specifically by differentiated cells located at the periphery of EBs. Interestingly, Dkk-1 was internalized by undifferentiated cells at the center of the EBs. When developmental tissue was analyzed, internalization of REIC/Dkk-3 protein was strictly limited to extra-embryonic tissue, such as the trophectoderm layer of 4.5 days post-coitus (dpc) blastocysts and the chorionic membrane at 16.5 dpc. The mechanism of the internalization was confirmed to be endocytosis. These findings will contribute to knowledge on the interaction of REIC/Dkk-3 with a possible receptor(s).

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  • In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes Reviewed

    Takayuki Koike, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   56 ( 5 )   552 - 557   2010.10

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    Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P&lt;0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.

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  • Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows Reviewed International journal

    J. C. Li, H. Funahashi

    Theriogenology   74 ( 4 )   691 - 698   2010.9

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    Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P &lt
    0.01) when fresh serum was included in the medium (1226 cells/mm2 in serum vs. 1110 cells/mm2 in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm2) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P &lt
    0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P &lt
    0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm2) was also decreased (P &lt
    0.01) in the presence of caffeine (1090 cells/mm2). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P &lt
    0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm2). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs. © 2010 Elsevier Inc.

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  • Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration Reviewed International journal

    Hikaru Sano, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    THERIOGENOLOGY   74 ( 5 )   863 - 870   2010.9

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    The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P &lt; 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P &lt; 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P &lt; 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber. (C) 2010 Elsevier Inc. All rights reserved.

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  • Application of mechanical stimuli using a microfluidic air actuating system to cultured mammalian embryos Reviewed

    Jing Chun Li, Koji Matsuura, Yuka Kuroda, Hiroaki Funahashi, Keiji Naruse

    2010 International Symposium on Micro-NanoMechatronics and Human Science: From Micro and Nano Scale Systems to Robotics and Mechatronics Systems, MHS 2010, Micro-Nano GCOE 2010, Bio-Manipulation 2010   29 - 34   2010

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    Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression, and friction force, in the fallopian tube before nidation. We aim to develop a novel and simple system to apply mechanical stimuli (MS) similar to those generated inside the oviduct to cultured mammalian embryos. Possible MS include shear stress (SS) caused by fluid dynamics and compression of embryos due to interactions with the wall of the oviduct. A new culture system was developed to increase SS and to apply MS during in vitro embryo cultures. We developed an air actuating system with microfluidic channels to apply MS by deforming a 0.1-mm-thick poly(dimethylsiloxiane) membrane and evaluated MS applied to ICR mouse embryos inside the microfluidic channel. Using this air actuating system, we applied compression to mouse embryos inside the medium channel and estimated SS on the basis of the velocity of the embryos' motion. By changing the syringe velocity, we applied different types of MS to the em bryos. These results suggested that multiple MS such as SS and compression can be applied at the same time. MS applied using this system was similar to those generated in the physiological environment of the oviduct. ©2010 IEEE.

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  • Improved Fertility in Gilts and Sows after Artificial Insemination of Frozen-Thawed Boar Semen by Supplementation of Semen Extender with Caffeine and CaCl2 Reviewed

    Shoichiro Yamaguchi, Hiroaki Funahashi, Tetsuya Murakami

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 6 )   645 - 649   2009.12

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    Supplementation of semen extender with caffeine and CaCl2 for artificial insemination (Al) of fresh spermatozoa has been demonstrated to reduce recruitment of uterine polymorphonuclear leukocytes (PMNs) and the activity of phagocytosis. Here, we determined if addition of caffeine and CaCl2 to semen extender improves the fertility of frozen-thawed boar semen. In experiment 1, gilts were cervically inseminated twice with froxzen-thawed boar spermatozoa (25 x 10(8) cells per dose) suspended in Modena solution (n=7) or modified Beltsville Thawing Solution supplemented with caffeine and CaCl2 (BCC, n=7). The gilts were slaughtered 4 h later, and their oviducts and uterine horns plus the body of the uterus were flushed to recover PMNs and non-phagocytosed spermatozoa. There was no difference in the total number of uterine PMNs between gilts inseminated with Modena solution and those inseminated with BCC (3.8 x 10(8) vs. 1.5 x 10(8) cells, respectively); however, the total number of uterine spermatozoa was higher when gilts were inseminated with BCC (40.6 x 10(6) cells) compared with those inseminated with Modena solution (1.4 x 10(6) cells, P&lt;0.05). In experiment 2, gilts and sows were subjected to intrauterine insemination twice with frozen-thawed spermatozoa suspended (25 x 10(8) sperm per dose) in Modena (n=21) or BCC (n=21). The overall pregnancy and farrowing rates were higher in females inseminated with BCC (71.4 and 61.9%, respectively) compared with those inseminated with Modena Solution (38.1 and 28.6%, respectively, P&lt;0.05). However, no significant difference in litter size of piglets was observed between treatments (7.2 +/- 1.6 piglets for Modena solution vs. 8.2 +/- 0.9 piglets for BCC solution). In conclusion, we demonstrated that use of BCC solution for frozen-thawed boar semen produced better pregnancy and farrowing rates following Al. than Modena solution, probably by reducing the phagocytosis of spermatozoa.

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  • Successful Piglet Production in a Chemically Defined System for In-vitro Production of Porcine Embryos: Dibutyryl Cyclic AMP and Epidermal Growth Factor-family Peptides Support In-vitro Maturation of Oocytes in the Absence of Gonadotropins Reviewed

    Yuka Akaki, Koji Yoshioka, Michiko Noguchi, Hiroyoshi Hoshi, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 4 )   446 - 453   2009.8

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    To induce meiotic resumption of porcine oocytes, it is thought to be necessary to expose the cumulus-oocyte complexes (COCs) to gonadotropins during in-vitro maturation (IVM). However, the detailed mechanism of meiotic resumption by gonadotropins is still unknown, and successful piglet production has not been reported by using oocytes matured in gonadotropin-free media and fertilized in vitro. The present study was undertaken to examine the combinational effects of epidermal growth factor (EGF)-family members and dibutyryl cyclic AMP (cAMP) in a chemically defined medium on IVM of porcine oocytes and the developmental competence following in vitro fertilization (IVF). The basic IVM medium was a chemically defined medium, modified porcine oocyte medium (n-LPOM.). Supplementation of the IVM medium with 10 or 1000 ng/ml EGF, amphiregulin and betacellulin during the whole IVM period, except for 10 ng/ml amphiregulin, increased the percentage of oocytes maturing to the metaphase-II stage. When COCs were exposed to both dibutyryl cAMP and EGF-family members during the first 20-h of IVM and then culture was continued in the absence of EGF-family members and dibutyryl cAMP, the incidence of metaphase-II oocytes was significantly increased and was not different from that of oocytes cultured in a standard IVM system with gonadotropins. The developmental competence of the oocytes to the blastocyst stage following IVF was no different from that of control oocytes matured with gonadotropins. When these blastocysts were transferred into the uterine horn of three recipients, all of gilts became pregnant and delivered a total of 11 piglets. These observations indicate that supplementation of a chemically defined maturation medium with EGF-family members and dibutyryl cAMP during the first 20 h of IVM can support well the meiotic progress and developmental competence of porcine oocytes.

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  • Exogenous adenosine Reduces the mitochondrial membrane potential of murine oocytes during the latter half of in vitro maturation and pronuclear formation following chemical activation Reviewed

    Wataru Fujii, Hiroaki Funahashi

    Journal of Reproduction and Development   55 ( 2 )   187 - 193   2009.5

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    The present study was undertaken to determine the effect of nucleosides on nuclear and cytoplasmic maturation of mouse oocytes. Oocyte-cumulus complexes (OCCs) were collected from large antral follicles 4 h after eCG-hCG treatment and cultured in maturation medium with or without nucleosides (4 ribo- and 4 deoxyribonucleosides) for 12 h. A majority of the oocytes examined developed to the metaphase-II stage, and the same result was found with in-vivo matured oocytes. However, mitochondrial membrane potential (MMP) was significantly lower in the oocytes matured in the presence of nucleosides than in the nucleoside-free controls. Oocyte MMP increased in vivo between 8 to 12 h after hCG injection, whereas no increases in MMP were observed in oocytes matured in the presence of nucleosides. Oocyte MMP was significantly lower only when OCCs were exposed to nucleosides for the latter 8 h of IVM. When OCCs were exposed to adenosine during the latter 8 h of IVM, MMP was lower compared with in-vivo matured oocytes, whereas a mixture of other ribonucleosides (guanosine, cytidine and uridine) did not affect the level of MMP. The developmental competence of oocytes exposed to adenosine during the latter 8 h of IVM was lower after parthenogenetic activation due to the lower pronuclear formation of the oocytes. These observations indicate that adenosine inhibits increases in oocyte MMP during the latter half of meiotic maturation and detrimentally affects cytoplasmic maturation.

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  • Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium Reviewed International journal

    H. Funahashi, T. Koike, R. Sakai

    Theriogenology   70 ( 7 )   1041 - 1047   2008.10

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    The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6 mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55 mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P < 0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (≥10 μM 6-AN and ≥10 nM DPI) inhibited resumption of meiosis (P < 0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P < 0.05). More mature oocytes were obtained in the presence of pyruvate + glucose (P < 0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P < 0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content. © 2008 Elsevier Inc. All rights reserved.

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  • Metal mesh vitrification (MMV) method for cryopreservation of porcine embryos Reviewed

    Y. Fujino, T. Kojima, Y. Nakamura, H. Kobayashi, K. Kikuchi, H. Funahashi

    Theriogenology   70 ( 5 )   809 - 817   2008.9

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    The objective was to develop a simpler, more reliable vitrification method for porcine embryos. Prepubertal donor gilts were induced to ovulate with eCG and hCG, and then inseminated artificially. Morulae and expanding blastocysts approximately 200 μm in diameter were collected 6 or 7 d after hCG treatment. Embryos collected from donor gilts were maintained, so as to be individually recognizable, and handled in batches of four or five. The embryos together with a minimum volume (&lt
    2 μL) of vitrification solution were placed onto stainless steel metal meshes or plastic plates, and then plunged into liquid nitrogen-metal mesh vitrification (MMV) and plastic plate vitrification (PPV), respectively. The meshes or plates were stored in 1.8-mL cryotubes submerged in liquid nitrogen. Stored embryos were subsequently removed, cultured in medium for 24 h, and then assessed for viability. The survival rate (84.4%) of expanding blastocysts cooled by MMV was higher than that (53.1%) of embryos cooled by PPV (P &lt
    0.05). There was no significant difference in total cell number between MMV and PPV. The survival rate of morulae cooled by MMV was 55.0%. Transfer of 200 expanding blastocysts cooled by MMV to 10 synchronized recipient gilts resulted in 37 live piglets from 7 recipients. In conclusion, the MMV method was an effective vitrification procedure for cryopreservation of expanding porcine blastocysts. However, there was a batch effect on embryo survival after vitrification. © 2008 Elsevier Inc. All rights reserved.

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  • In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation Reviewed International journal

    Wataru Fujii, Hiroaki Funahashi

    Zygote   16 ( 2 )   117 - 125   2008.5

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    The present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocytecumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14 h after human chorionic gonadotropin (hCG) injection. Cumulus nuclei were injected into ovulated oocytes and then stimulated in the presence of 5 mM SrCl2 for 20 min at various time points (03.5 h) after injection. Some of the reconstituted eggs were cultured to observe the pronuclear formation, cleavage, and blastocyst formation. The incidences of eggs forming at least one pronucleus or containing two pronuclei were not significantly different among the periods (82.483.5% and 43.451.9%, respectively). Nor did the incidences of eggs cleaving (86.797.7%) and developing to the blastocyst stage (03.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 11.5 h after injection, 71.7% of the eggs caused premature chromatin condensation, but only 46.2% of them formed two spindles around each of maternal and somatic chromatins. However, the morphology of the somatic spindles differed from that of the spindles, which formed around the oocyte chromatins. Only 7.5% of the eggs contained the normal chromosomal number. In many reconstituted oocytes, before activation, an abnormal spindle formation was observed in the somatic chromatins. In conclusion, these results show that non-enucleated rat oocytes injected with cumulus nuclei can form pronuclei and cleave following chemical activation, whereas blastocyst formation is very limited, probably caused by abnormalities in the spindle formation and distribution of somatic chromatids. © 2008 Cambridge University Press.

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  • Up date of in vitro production of porcine embryos Invited Reviewed International journal

    Takashi Nagai, Hiroaki Funahashi, Koji Yoshioka, Kazuhiro Kikuchi

    Frontiers in bioscience-landmark   11 ( SUPPL. 2 )   2565 - U34   2006.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Bioscience research inst-bri  

    There have been intensive attempts to establish reliable in vitro maturation (IVM) and fertilization (IVF) methods in pigs. Although a great deal of progress has been made, current IVM-IVF systems still suffer from a low rate and poor quality of in vitro produced embryos. In this review, we will review the recent studies about IVM-IVF of porcine oocytes and the in vitro culture (IVC) system, especially modified in vitro production (IVP) system that produces high quality of porcine blastocysts. We then try to suggest practical ways to solve the problems mentioned above in the pigs.

    DOI: 10.2741/1991

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  • In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34(cdc2)/cyclin B kinase Reviewed International journal

    R Romar, H Funahashi

    ANIMAL REPRODUCTION SCIENCE   92 ( 3-4 )   321 - 333   2006.5

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    Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 mu M ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-1 stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1 %) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) fort-nation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current lVM system. (c) 2005 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.anireprosci.2005.04.017

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  • Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro. Reviewed International journal

    Hiroaki Funahashi

    Reproduction (cambridge, england)   130 ( 6 )   889 - 98   2005.12

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    This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.

    DOI: 10.1530/rep.1.00702

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  • Select antioxidants improve the function of extended boar semen stored at 10°C Reviewed

    H. Funahashi, T. Sano

    Theriogenology   63 ( 6 )   1605 - 1616   2005.4

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    DOI: 10.1016/j.theriogenology.2004.06.016

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  • Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine Reviewed

    Hiroaki Funahashi, Raquel Romar

    Reproduction   128 ( 6 )   789 - 800   2004.12

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    To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culturein vitrofertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 μl modified Medium 199-suspended spermatozoa at 2.5 ×105sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 ± 2.2) than 8 h (207.6 ± 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 μmol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

    DOI: 10.1530/rep.1.00295

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  • Polyspermic penetration in porcine IVM - IVF systems Reviewed

    Hiroaki Funahashi

    Reproduction, Fertility and Development   15 ( 3 )   167 - 167   2003

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    Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

    DOI: 10.1071/rd02076

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  • Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system Reviewed

    H Funahashi

    Reproduction   857 - 864   2002.12

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    The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO(2)-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N(omega)-nitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

    DOI: 10.1530/rep.0.1240857

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  • Effect of Methyl-.BETA.-Cyclodextrin and Fertilization Promoting Peptide on Capacitation of Boar Spermatozoa in a Protein-Free Medium. Reviewed

    Hiroaki FUNAHASHI

    Journal of Reproduction and Development   48 ( 1 )   57 - 63   2002

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    DOI: 10.1262/jrd.48.57

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  • Nuclear Transfer of Blastomeres Expressing EGFP-Reporter Gene May Improve the Efficiency of Transgenic Cattle Reviewed

    H. Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

    Cloning and Stem Cells   3 ( 4 )   183 - 190   2001.12

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    DOI: 10.1089/15362300152725891

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  • Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro Reviewed

    H Funahashi, H Ekwall, K Kikuchi, H Rodriguez-Martinez

    Reproduction   443 - 452   2001.9

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    The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

    DOI: 10.1530/rep.0.1220443

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  • Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine Reviewed

    Hiroaki Funahashi, Takashi Nagai

    Molecular Reproduction and Development   58 ( 4 )   424 - 431   2001.4

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    DOI: 10.1002/1098-2795(20010401)58:4<424::aid-mrd10>3.0.co;2-1

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  • Involvement of oviduct in sperm capacitation and oocyte development in pigs Invited Reviewed

    H Rodriguez-Martinez, P Tienthai, K Suzuki, H Funahashi, H Ekwall, A Johannisson

    CONTROL OF PIG REPRODUCTION VI   58 ( 58 )   129 - 145   2001

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    An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for &lt; 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

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  • Zona Reaction in Porcine Oocytes Fertilized In Vivo and In Vitro as Seen with Scanning Electron Microscopy1 Reviewed

    Hiroaki Funahashi, Hans Ekwall, Heriberto Rodriguez-Martinez

    Biology of Reproduction   63 ( 5 )   1437 - 1442   2000.11

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    DOI: 10.1095/biolreprod63.5.1437

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  • Modulation of the Function of Boar Spermatozoa via Adenosine and Fertilization Promoting Peptide Receptors Reduce the Incidence of Polyspermic Penetration into Porcine Oocytes1 Reviewed

    Hiroaki Funahashi, Toshimitsu Fujiwara, Takashi Nagai

    Biology of Reproduction   63 ( 4 )   1157 - 1163   2000.10

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    DOI: 10.1095/biolreprod63.4.1157

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  • Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro Reviewed

    Hiroaki Funahashi, Atsushi Asano, Toshimitsu Fujiwara, Takashi Nagai, Koji Niwa, Lynn R. Fraser

    Molecular Reproduction and Development   55 ( 1 )   117 - 124   2000.1

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    DOI: 10.1002/(sici)1098-2795(200001)55:1<117::aid-mrd16>3.0.co;2-7

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  • Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes. Reviewed

    Hiroaki FUNAHASHI, Takashi NAGAI

    Journal of Reproduction and Development   46 ( 5 )   319 - 324   2000

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    DOI: 10.1262/jrd.46.319

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  • Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes Reviewed

    Hiroaki Funahashi, Naoya Bandoh, Shinobu Nakahira, She-Hoon Oh, Seiji Tsuboi

    Zygote   7 ( 4 )   301 - 305   1999.11

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    The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 ± 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 ± 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 ± 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 ± 74.1 nmol/egg in unfertilised eggs, 146.0 ± 50.0 nmol/egg in DSH eggs and 39.7 ± 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p &lt; 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

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  • DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes Reviewed

    Syahruddin Said, Hiroaki Funahashi, Koji Niwa

    Zygote   7 ( 3 )   249 - 254   1999.8

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    DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 ± 0.16) and testicular spermatozoa (4.03 ± 0.34), and then decreased sharply (p &lt; 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 ± 0.03). However, the ratio was not different among corpus (0.69 ± 0.01), cauda epididymis (0.68 ± 0.11) and ejaculated spermatozoa (0.63 ± 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p &lt; 0.01) during passage of spermatozoa from testis (4.74 ± 0.16) through epididymis (caput, 2.72 ± 0.08; corpus, 1.07 ± 0.03; cauda, −0.05 ± 0.05; ejaculated, 0.08 ± 0.03). The acridine orange red/green fluorescence ratio increased (p &lt; 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

    DOI: 10.1017/s0967199499000635

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  • Production of Plasminogen Activators (PAs) in Bovine Cumulus-Oocyte Complexes during Maturation In Vitro: Effects of Epidermal Growth Factor on Production of PAs in Oocytes and Cumulus Cells1 Reviewed

    Kwang-Wook Park, Sun-Ho Choi, Xue-Xiong Song, Hiroaki Funahashi, Koji Niwa

    Biology of Reproduction   61 ( 1 )   298 - 304   1999.7

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    DOI: 10.1095/biolreprod61.1.298

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  • Co-culture of Cumulus-Enclosed Bovine Oocytes with Theca Cells Induces the Meiotic Arrest but does not Inhibit Germinal Vesicle Development. Reviewed

    Kwang-Wook Park, Hiroaki Funahashi, Koji Niwa

    Journal of Reproduction and Development   45 ( 3 )   223 - 231   1999

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    DOI: 10.1262/jrd.45.223

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  • The Presence of Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) During Meiosis Improves Porcine 'Oocyte Competence' as Determined by Early Embryonic Development After In-vitro Fertilization. Reviewed

    Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

    Journal of Reproduction and Development   45 ( 4 )   265 - 271   1999

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    DOI: 10.1262/jrd.45.265

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  • Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo Culture Medium Containing a High Sodium Chloride Concentration and Bovine Serum Albumin Maintain Developmental Ability to the Blastocyst Stage Reviewed

    She-Hoon Oh, Kazuchika Miyoshi, Hiroaki Funahashi

    Biology of Reproduction   59 ( 4 )   884 - 889   1998.10

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    DOI: 10.1095/biolreprod59.4.884

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  • Effects of Cumulus Cells on the Ability of Pig Oocytes to Utilize Cysteine or Cystine During Maturation In Vitro. Reviewed

    Ken SAWAI, Hiroaki FUNAHASHI, Koji NIWA

    Journal of Reproduction and Development   44 ( 2 )   161 - 168   1998

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    DOI: 10.1262/jrd.44.161

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  • Stage-Specific Requirement of Cysteine during in Vitro Maturation of Porcine Oocytes for Glutathione Synthesis Associated with Male Pronuclear Formation1 Reviewed

    Ken Sawai, Hiroaki Funahashi, Koji Niwa

    Biology of Reproduction   57 ( 1 )   1 - 6   1997.7

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    DOI: 10.1095/biolreprod57.1.1

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  • Synchronization of Meiosis in Porcine Oocytes by Exposure to Dibutyryl Cyclic Adenosine Monophosphate Improves Developmental Competence Following in Vitro Fertilization1 Reviewed

    Hiroaki Funahashi, Thomas C. Cantley, Billy N. Day

    Biology of Reproduction   57 ( 1 )   49 - 53   1997.7

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    DOI: 10.1095/biolreprod57.1.49

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  • Chlortetracycline fluorescence patterns andin vitrofertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations Reviewed

    Lalantha R. Abeydeera, Hiroaki Funahashi, Nam-Hyung Kim, Billy N. Day

    Zygote   5 ( 2 )   117 - 125   1997.5

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    Summary

    Porcine oocyte-cumulus complexes were cultured in bovine serum albumin(BSA)-free North Carolina State University (NCSU)23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml)and hormonal supplements (eCG and hCG: 10IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM)199 containing caffeine (5mM), fetal calf serum (FCS; 10%) and varying concentrations (26–56 mM)of NaHCO3for 9h(experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3concentrations. Experiment 3 examined the effect of FCS (1% and 10%)and NaHCO3(26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p&lt;0.05)at 36–56mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p&lt; 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56mM NaHCO3, respectively. The fertilisation medium containing 46mM NaHCO3and 1% FCS showed a higher penetration rate (84%)with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

    DOI: 10.1017/s0967199400003798

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  • Developmental Changes in the Intracellular Ca 2+ Release Mechanisms in Porcine Oocytes1 Reviewed

    Zoltán Macháty, Hiroaki Funahashi, Billy N. Day, Randall S. Prather

    Biology of Reproduction   56 ( 4 )   921 - 930   1997.4

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    DOI: 10.1095/biolreprod56.4.921

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  • Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro Reviewed International journal

    H Funahashi, TC Cantley, BN Day

    THERIOGENOLOGY   47 ( 3 )   679 - 686   1997.2

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    The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 10% porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage. (C) 1997 by Elsevier Science Inc.

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  • Effects of Cysteine in Serum-Free Maturation Medium on Male Pronuclear Formation of Maturing Pig Oocytes Penetrated In Vitro. Reviewed

    Ken SAWAI, Hiroaki FUNAHASHI, Wei-Hua WANG, Koji NIWA

    Journal of Reproduction and Development   43 ( 1 )   73 - 80   1997

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.43.73

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  • gamma-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs Reviewed International journal

    H Funahashi, Z Machaty, RS Prather, BN Day

    MOLECULAR REPRODUCTION AND DEVELOPMENT   45 ( 4 )   485 - 490   1996.12

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-LISS  

    The presence of gamma-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pi of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 +/- 1.4%)was not different(P &gt; 0.05) from HEPES-injected controls (24.9 +/- 1.3%) at 6 h after injection. Injected GTP-gamma-S, however, activated 76.0 +/- 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 +/- 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P &gt; 0.05) between GTP-gamma-S injected oocytes (4.2 +/- 0.7 pmol/oocyte) and noninjected oocytes (4.0 +/- 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P &lt; 0.01) in GGT-injected oocytes (2.1 +/- 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 +/- 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pi of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that(1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-gamma-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes. (C) 1996 Wiley-Liss, Inc.

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  • γ-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs Reviewed

    Hiroaki Funahashi, Zoltan Machaty, Randall S. Prather, Billy N. Day

    Molecular Reproduction and Development   45 ( 4 )   485 - 490   1996.12

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/(sici)1098-2795(199612)45:4<485::aid-mrd11>3.0.co;2-w

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  • Microtubule Organization in Porcine Oocytes during Fertilization and Parthenogenesis1 Reviewed

    Nam-Hyung Kim, Calvin Simerly, Hiroaki Funahashi, Gerald Schatten, Billy N. Day

    Biology of Reproduction   54 ( 6 )   1397 - 1404   1996.6

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    DOI: 10.1095/biolreprod54.6.1397

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  • Presence of Organic Osmolytes in Maturation Medium Enhances Cytoplasmic Maturation of Porcine Oocytes1 Reviewed

    Hiroaki Funahashi, Nam-Hyung Kim, Todd T. Stumpf, Thomas C. Cantley, Billy N. Day

    Biology of Reproduction   54 ( 6 )   1412 - 1419   1996.6

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    DOI: 10.1095/biolreprod54.6.1412

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  • Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro Reviewed

    N-H. Kim, H. Funahashi, L. R. Abeydeera, S. J. Moon, R. S. Prather, B. N. Day

    Reproduction   107 ( 1 )   79 - 86   1996.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Bioscientifica  

    DOI: 10.1530/jrf.0.1070079

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  • Effects of Injecting Calcium Chloride into in Vitro-Matured Porcine Oocytes1 Reviewed

    Z. Macháty, H. Funahashi, M. A. Mayes, B. N. Day, R. S. Prather

    Biology of Reproduction   54 ( 2 )   316 - 322   1996.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    DOI: 10.1095/biolreprod54.2.316

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  • Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation Reviewed

    Nam‐Hyung Kim, Hiroaki Funahashi, Randall S. Prather, Gerald Schatten, Billy N. Day

    Molecular Reproduction and Development   43 ( 2 )   248 - 255   1996.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/(sici)1098-2795(199602)43:2<248::aid-mrd14>3.0.co;2-#

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  • Current status of in vitro production of porcine embryos Invited Reviewed

    H Funahashi, BN Day

    ADVANCES IN SWINE IN BIOMEDICAL RESEARCH, VOLS 1 AND 2   491 - 502   1996

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    Authorship:Lead author   Language:English   Publishing type:Research paper (international conference proceedings)   Publisher:PLENUM PRESS DIV PLENUM PUBLISHING CORP  

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  • Factors Affecting Development In Vitro of Bovine and Rat 1-Cell Embryos. Reviewed

    Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

    Journal of Mammalian Ova Research   13 ( 2 )   71 - 80   1996

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Mammalian Ova Research  

    DOI: 10.1274/jmor.13.71

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  • Low Salt Maturation Medium Enhances the Histone H1 Kinase Activity of Porcine Oocytes at the End of In Vitro Maturation. Reviewed

    Hiroaki FUNAHASHI, Todd T. STUMPF, Nam-Hyung KIM, Billy N. DAY

    Journal of Reproduction and Development   42 ( 2 )   109 - 115   1996

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.42.109

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  • Pronuclear formation and intracellular glutathione content ofin vitro-matured porcine oocytes followingin vitrofertilisation and/or electrical activation Reviewed

    Hiroaki Funahashi, Todd T. Stumpf, Thomas C. Cantley, Nam-Hyung Kim, Billy N. Day

    Zygote   3 ( 3 )   273 - 281   1995.8

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Cambridge University Press (CUP)  

    Summary

    Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes maturedin vitrowere determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisationin vitrodiffer from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

    DOI: 10.1017/s0967199400002677

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  • Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos Reviewed

    H.-M. Kubisch, M.A. Larson, H. Funahashi, B.N. Day, R.M. Roberts

    Theriogenology   44 ( 3 )   391 - 401   1995.8

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    DOI: 10.1016/0093-691x(95)00193-c

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  • Use of Low-Salt Culture Medium for in Vitro Maturation of Porcine Oocytes is Associated with Elevated Oocyte Glutathione Levels and Enhanced Male Pronuclear Formation after in Vitro Fertilization1 Reviewed

    Hiroaki Funahashi, Thomas C. Cantley, Todd T. Stumpf, Steven L. Terlouw, Billy N. Day

    Biology of Reproduction   51 ( 4 )   633 - 639   1994.10

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    DOI: 10.1095/biolreprod51.4.633

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  • In Vitro Development of in Vitro-Matured Porcine Oocytes Following Chemical Activation or in Vitro Fertilization1 Reviewed

    Hiroaki Funahashi, Thomas C. Cantley, Todd T. Stumpf, Steven L. Terlouw, Billy N. Day

    Biology of Reproduction   50 ( 5 )   1072 - 1077   1994.5

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    DOI: 10.1095/biolreprod50.5.1072

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  • Different hormonal requirements of pig oocyte-cumulus complexes during maturation in vitro Reviewed

    H. Funahashi, T. Cantley, B. N. Day

    Reproduction   101 ( 1 )   159 - 165   1994.5

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Bioscientifica  

    DOI: 10.1530/jrf.0.1010159

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  • Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate and osmolarity Reviewed

    K. Miyoshi, H. Funahashi, K. Okuda, K. Niwa

    Reproduction   100 ( 1 )   21 - 26   1994.1

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    DOI: 10.1530/jrf.0.1000021

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  • Developmental ability of porcine oocytes matured and fertilized in vitro Reviewed

    H. Funahashi, T.T. Stumpf, S.L. Terlouw, T.C. Cantley, A. Rieke, B.N. Day

    Theriogenology   41 ( 7 )   1425 - 1433   1994.1

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    DOI: 10.1016/0093-691x(94)90193-m

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  • Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes Reviewed

    Hiroaki Funahashi, Todd T. Stumpf, Steve L. Terlouw, Billy N. Day

    Molecular Reproduction and Development   36 ( 3 )   361 - 367   1993.11

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/mrd.1080360312

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  • Effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes Reviewed

    H. Funahashi, B. N. Day

    Reproduction   99 ( 1 )   97 - 103   1993.9

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    DOI: 10.1530/jrf.0.0990097

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  • Effects of the duration of exposure to hormone supplements on cytoplasmic maturation of pig oocytes in vitro Reviewed

    H. Funahashi, B. N. Day

    Reproduction   98 ( 1 )   179 - 185   1993.5

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    DOI: 10.1530/jrf.0.0980179

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  • Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium Reviewed

    J.H. Kim, H. Funahashi, K. Niwa, K. Okuda

    Theriogenology   39 ( 4 )   875 - 886   1993.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/0093-691x(93)90425-5

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  • Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes Reviewed

    H. Funahashi, B.N. Day

    Theriogenology   39 ( 4 )   965 - 973   1993.4

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/0093-691x(93)90433-6

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  • Fertilization and early cleavage in vitro of ageing bovine oocytes after maturation in culture Reviewed

    R.C. Chian, H. Nakahara, K. Niwa, H. Funahashi

    Theriogenology   37 ( 3 )   665 - 672   1992.3

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    DOI: 10.1016/0093-691x(92)90146-i

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  • Developmental Abilities of Rat and Mouse Pronuclear Eggs Following Interspecific Nuclear or Cytoplasmic Transplantation. Reviewed

    Hiroaki FUNAHASHI, Koji NIWA

    Journal of Reproduction and Development   38 ( 3 )   179 - 183   1992

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.38.179

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  • First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6°C for various durations Reviewed

    K. Niwa, H. Funahashi, K. Ohmae, M. Kattoh

    Theriogenology   36 ( 3 )   411 - 417   1991.9

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    DOI: 10.1016/0093-691x(91)90469-t

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  • Developmental capacity of bovine oocytes collected from ovaries of individual heifers and fertilized in vitro Reviewed

    H. Funahashi, Y. Aoyagi, T. Takeda, T. Onihara

    Theriogenology   36 ( 3 )   427 - 434   1991.9

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/0093-691x(91)90471-o

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Books

  • 生殖補助医療技術学入門

    岡山大学生殖補助医療学教科書作成委員会( Role: Contributor ,  第Ⅰ章 生殖補助医療の歴史と現状)

    岡山大学出版会  2017.9  ( ISBN:9784904228555

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    Total pages:126p   Language:Japanese

    CiNii Books

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  • 生殖補助医療技術学入門

    岡山大学生殖補助医療学教科書作成委員会( Role: Contributor ,  第Ⅳ章 関係基礎技術)

    岡山大学出版会  2017.9  ( ISBN:9784904228555

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    Total pages:126p   Language:Japanese

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  • 生殖補助医療技術学入門

    岡山大学生殖補助医療学教科書作成委員会( Role: Contributor ,  第Ⅲ章 卵母細胞と精子の基礎)

    岡山大学出版会  2017.9  ( ISBN:9784904228555

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    Total pages:126p   Language:Japanese

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  • 基礎演習産業生物科学

    産業生物科学教科書編纂委員会( Role: Contributor ,  クローン動物・細胞周期・体細胞分裂と減数分裂・有性生殖の戦略)

    岡山大学出版会  2009.9  ( ISBN:9784904228050

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    Total pages:x, 314p   Language:Japanese

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  • 瞬時共培養法を用いた新たな豚体外受精系の構築

    舟橋, 弘晃

    舟橋弘晃  2006.3 

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    Total pages:58p   Language:English

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  • 基礎のためのIVM.エンブリオロジストのためのART必須ラボマニュアル(日本臨床エンブリオロジスト研究会編,監修:鈴木秋悦・福田愛作

    医歯薬出版  2005 

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  • 緑色蛍光蛋白質(GFP)を利用した形質転換動物の効率的システムの開発

    舟橋, 弘晃

    舟橋弘晃  2002.3 

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    Total pages:108p  

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  • Polyspermic penetration in porcine IVF systems;why so frequent?

    Reproductive Biotechnology:Reproductive Biotechnilogy Update and its Related Physiology, Eds.  2001 

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  • Oviduct involvement in sperm capacitation and oocyte development.

    Control of Pig Reproduction VI.(ISBN 0-906545-32-3), Eds.Foxcroft, G.R., Geisert, R.D.and Doberska, C.Journal of Reproduction and Reproduction and Fertility Ltd.,Cambridge,UK  2001 

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  • Oviduct involvement in sperm capacitation and oocyte development.

    Control of Pig Reproduction VI.(ISBN 0-906545-32-3), Eds.Foxcroft, G.R., Geisert, R.D.and Doberska, C.Journal of Reproduction and Reproduction and Fertility Ltd.,Cambridge,UK  2001 

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  • Polyspermic penetration in porcine IVF systems;why so frequent?

    Reproductive Biotechnology:Reproductive Biotechnilogy Update and its Related Physiology, Eds.  2001 

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  • 哺乳動物雄性ゲノムのプロタミン封入および雄性前核形成の調節機構の解析

    舟橋, 弘晃

    舟橋弘晃  1999.3 

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    Total pages:60p  

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  • Factors affecting the efficiency of in-vitro production of porcine embryos. "Reproductive Biology update : Novel tools for assessment of environmental toxicity"Nakanishi Publishing Co. , Kyoto,

    Japan  1997 

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  • Factors affecting the efficiency of in-vitro production of porcine embryos.

    "Reproductive Biology update : Novel tools for assessment of environmental toxicity"Nakanishi Publishing Co. , Kyoto, Japan  1997 

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  • In vitro maturation and fertilization of pig oocytes.(共著)

    'Beltsville Symposia in Agricultural Research XX. Biotechnology's Role in the Genetic Improvement of Farm Animals' The American Society of Animal Science, Savoy, IL, USA  1996 

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  • Current status of in vitro production of porcine embryos.(共著)

    'Advances in Swine Biomedical Research'Plenum Publishing Co., New York, USA  1996 

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  • スマート酪農の海外事情 2)各種機関の連携により牛群情報をリアルタイムに酪農場へ提供 最新技術導入とデータ共有進めるイスラエル その2

    舟橋弘晃

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  • 着床前胚発生におけるミトコンドリア分裂因子Drp1の機能解析

    野村瑠莉, 月向はるな, 舟橋弘晃, 若井拓哉

    Journal of Mammalian Ova Research   36 ( 1 )   S43 - S43   2019.4

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    Language:Japanese   Publisher:(一社)日本卵子学会  

    J-GLOBAL

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  • 体外成熟時のブタ卵母細胞のオートファジー能は由来する小中卵胞で異なる

    小野千由貴, 若井拓哉, 舟橋弘晃

    Journal of Mammalian Ova Research   36 ( 1 )   S56 - S56   2019.4

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  • Trim‐Away法を用いたマウス卵母細胞におけるDrp1の機能解析

    月向はるな, 野村瑠莉, 舟橋弘晃, 若井拓哉

    Journal of Mammalian Ova Research   36 ( 1 )   S44 - S44   2019.4

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  • 体外成熟時のブタ卵母細胞のオートファジー能は由来する小中卵胞で異なる

    小野 千由貴, 若井 拓哉, 舟橋 弘晃

    Journal of mammalian ova research   36 ( 1 )   S56 - S56   2019.4

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  • Trim-Away法を用いたマウス卵母細胞におけるDrp1の機能解析

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    Journal of mammalian ova research   36 ( 1 )   S44 - S44   2019.4

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  • 着床前胚発生におけるミトコンドリア分裂因子Drp1の機能解析

    野村 瑠莉, 月向 はるな, 舟橋 弘晃, 若井 拓哉

    Journal of mammalian ova research   36 ( 1 )   S43 - S43   2019.4

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  • Are changes on the extracellular environment another factor contributing to the loss of fertility in women?

    プジョルピラー フェレー, 大月純子, 舟橋弘晃, 中塚幹也

    Journal of mammalian ova research   36 ( 1 )   S51 - S51   2019

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  • 光応答的に働く卵活性化因子の開発

    大本和正, 若井拓哉, 舟橋弘晃, 渡邉和則, 大槻高史

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  • マウス卵母細胞におけるTrim-Away法を用いたリン酸化タンパク質の分解

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    Journal of reproduction and development   65 ( Suppl Japanese Issue )   j119 - j119   2019

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  • ミトコンドリア分裂因子Drp1の阻害による着床前マウス胚の発生停止

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  • 受精後のDNAメチル化リプログラミングにおける種間差異

    澤田友季乃, 舟橋弘晃, 若井拓哉

    岡山実験動物研究会報   ( 34 )   76 - 77   2018

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  • ブタ顕微授精卵での精子頭部膨化・雄性前核形成不全の解析

    林雄平, 若井拓哉, 舟橋弘晃

    日本卵子学会誌   3 ( 1 )   S45 - S45   2018

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  • 胚培養士が行う不妊予防啓発授業

    高山修, 大月純子, 舟橋弘晃, 舟橋弘晃, 中塚幹也, 中塚幹也

    日本不妊カウンセリング学会誌   17 ( 1 )   66 - 67   2018

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  • 初期胚におけるミトコンドリア分裂因子Drp1の役割

    野村瑠莉, 若井拓哉, 舟橋弘晃

    日本卵子学会誌   3 ( 1 )   S58 - S58   2018

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  • 異なる還元剤による精子前処理がICSIブタ卵での雄性前核形成に及ぼす影響

    林雄平, 若井拓哉, 舟橋弘晃

    日本畜産学会大会講演要旨   122nd   156 - 156   2017

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  • Epidermal Growth Factorはヒト初期卵胞の体外発育を促進する

    高山修, 奥平裕一, 若井拓哉, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本卵子学会誌   2 ( 1 )   S58 - S58   2017

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  • 生殖補助医療現場で使用されている手技等に関する全国調査

    高山修, 倉田裕子, 小倉初音, 坂東裕香里, 大和礼, 本橋秀之, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃, 舟橋弘晃

    日本卵子学会誌   2 ( 2 )   59 - 64   2017

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    我々は胚培養士のキャリア養成カリキュラムの開発を試みているが、生殖補助医療に関連する手技は、非常に多くの種類があり、実技指導でどの手技を選択するべきかという問題に直面している。実際に実施されている手技に関する全国規模の調査はこれまでになく、カリキュラム充実化のため本調査を実施した。日本産科婦人科学会諸登録592施設へ調査票を郵送し、271施設より回収(回収率45.8%、有効回答数268)した。常勤胚培養士1人あたりの年間採卵周期数の中央値は88.3周期であった。胚移植の時期別実施率はDay5が最も多く(98.9%)、二段階胚移植およびSEET法はそれぞれ30.2%、37.3%の施設で実施されていた。精液処理法別実施率は、遠心濃縮法、密度勾配法、swim-up法でそれぞれ31.2%、86.5%、76.3%であった。ICSIは、スパイク型ピペット法が90.2%、ピエゾ法が15.4%、レスキューICSIは15.4%、1 day old ICSIは31.9%の施設で実施されていた。これらの結果を踏まえて養成カリキュラムを作成することで、より高い教育効果が得られると考えられる。(著者抄録)

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  • Improvement of decreased the farrowing rate after artificial insemination with liquid boar semen during hot season by supplementation of semen extender with caffeine

    山口昇一郎, 金子和典, 金子和典, 川島幸子, 川島幸子, 竹村陽, 竹村陽, 増本憲考, 笠正二郎, 笠正二郎, 舟橋弘晃, 吉岡耕治

    日本養豚学会誌   54 ( 4 )   2017

  • ウシ精巣からの精原幹細胞採取の試み

    永原知樹, 若井拓哉, 舟橋弘晃

    岡山実験動物研究会報   ( 33 )   50 - 50   2017

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  • ブタ卵母細胞中のミトコンドリア細胞内分布の変化

    葛原大貴, 若井拓哉, 舟橋弘晃

    岡山実験動物研究会報   ( 33 )   50 - 51   2017

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  • クワの実は上皮細胞死を抑制することでインドメタシン誘発性胃潰瘍発症を阻止する

    定兼侑里, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃, 畑生俊光

    日本農芸化学会大会講演要旨集(Web)   2016   2016

  • 生殖補助医療現場で使用されている手技等に関する全国調査結果

    高山修, 倉田裕子, 小倉初音, 坂東裕香里, 大和礼, 本橋秀之, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃, 舟橋弘晃

    日本卵子学会誌   1 ( 1 )   S46 - S46   2016

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  • 生殖補助医療技術者の養成とスキルアップに高等教育機関が果たすべき役割

    舟橋弘晃, 高山修, 本橋秀之, 中塚幹也

    日本卵子学会誌   1 ( 1 )   S12 - S12   2016

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  • クワの実凍結乾燥粉末摂取による肥満抑制効果の検討

    畑生俊光, 渡邉智, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃

    日本農芸化学会大会講演要旨集(Web)   2016   2016

  • IVM中のブタ小中卵胞由来COC,裸化卵母細胞,卵丘細胞内のcAMPおよびcGMP量

    奥平裕一, 若井拓哉, 舟橋弘晃

    Journal of Reproduction and Development   61 ( Suppl Japanese Issue )   J91 - j91   2015.9

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    【目的】体外成熟 (IVM)中の細胞内cAMPとcGMP量の劇的な変化は,卵母細胞の減数分裂再開に大きく関与している。本研究は,卵丘細胞-卵母細胞複合体 (COC),裸化卵母細胞 (DO)および卵丘細胞塊 (CC)内のcAMPとcGMP量のIVM中の変動を,小卵胞 (SF:直径1–3 mm)および中卵胞 (MF:3–6 mm)間で比較した。【方法】SFまたはMFからCOCsを採取し,24時間IVMを行った(eCG,hCG,dibutyryl cAMP添加mPOM中で20時間培養,その後4時間はそれらを不含のmPOM中で培養)。IVM培養開始0,10,20,24時間に,COCsを回収し,COC,DO,CCそれぞれのcAMPとcGMP量をELISA法で測定した。また,IVM培養開始0, 10, 20時間に,COCあたりの卵丘細胞数を測定し,卵丘細胞あたりのcAMPとcGMP量を推定した。【結果】IVM中のSFおよびMF由来DOのcAMPとcGMP量に差は無かった。MF由来のCOCとCCのcAMP量は,IVM培養開始後20時間にSF由来のそれらより有意に高かった (P &lt; 0.05)。また,IVM培養開始後20時間のSF由来CCのcAMP量は,IVM培養開始後10時間のそれより,有意に低下した (P &lt; 0.01)。MF由来のCOCとCCのcGMP量は,IVM培養開始後0および10時間にSF由来のそれらより有意に高かった (P &lt; 0.05)。IVM中のMF由来のCOC中の卵丘細胞数は,SF由来のそれより有意に多かった (P &lt; 0.01)。IVM培養開始後10時間のSF由来卵丘細胞の推定cAMP量は,MF由来のそれより有意に高かった (P &lt; 0.01)。IVM培養開始0時間のMF由来卵丘細胞の推定cGMP量は,SF由来のそれより有意に高かった (P &lt; 0.01)。以上の結果から,IVM時のSFおよびMF由来COC中の卵丘細胞内cAMPとcGMP量の変動パターンは大きく異なっていることが明らかになった。

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  • Methods for improving in vitro and in vivo boar sperm fertility International journal

    H. Funahashi

    Reproduction in domestic animals = Zuchthygiene   50   40 - 47   2015.7

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    Fertility of boar spermatozoa is changed after ejaculation in vivo and in vitro. During processing for in vitro fertilization (IVF), although spermatozoa are induced capacitation, resulting in a high penetration rate, persistent obstacle of polyspermic penetration is still observed with a high incidence. For artificial insemination (AI), we still need a large number of spermatozoa and lose a majority of those in the female reproductive tract. Fertility of cryopreserved boar spermatozoa is still injured through freezing and thawing process. In the present brief review, factors affecting fertility of boar sperm during IVF, AI and cryopreservation are discussed in the context of discovering methodologies to improve it.

    DOI: 10.1111/rda.12568

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  • クワの実凍結乾燥粉末投与によるインドメタシン誘発性胃潰瘍形成抑制効果の検討

    畑生俊光, 定兼侑里, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃

    日本農芸化学会大会講演要旨集(Web)   2015   2015

  • ヒト初期卵胞体外培養時の調整培地の効果

    高山修, 本橋秀之, 奥平裕一, FERRE Pillar, ATHURUPANA Rukmali, MI Bui Thi Tra, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本IVF学会雑誌   18 ( 2 )   79 - 79   2015

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  • ヒト小卵胞および中卵胞由来卵丘細胞-卵母細胞複合体の体外成熟能

    奥平裕一, 高山修, BUI Thi Tra Mi, FERRE Pilar, 本橋秀之, 若井拓哉, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本IVF学会雑誌   18 ( 2 )   81 - 81   2015

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  • 段階的な精子の融解はイノシシの精子の融解後の質を改善する(Stepwise thawing enhanced post-thaw quality of boar sperms)

    Athurupana Rukmali, 舟橋 弘晃

    日本畜産学会大会講演要旨集   118回   201 - 201   2014.3

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  • ヒト中卵胞および小卵胞由来裸化卵母細胞の体外成熟能

    奥平裕一, 李楊, RUKMALI Athurupana, PILAR Ferre, 高山修, 本橋秀之, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本生殖医学会雑誌   59 ( 4 )   391 - 391   2014

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  • ヒト初期卵胞の体外培養(IVG)における培養条件の検討

    高山修, 本橋秀之, 奥平裕一, 李楊, PILAR Ferre, RUKMALI Athurupana, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本生殖医学会雑誌   59 ( 4 )   317 - 317   2014

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  • 完全合成溶液中での未成熟ブタ卵母細胞のガラス化保存加温温度の検討

    高橋大山, 舟橋弘晃

    Journal of mammalian ova research   31 ( 2 )   S43 - S43   2014

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  • 現役の胚培養士におけるリカレント教育士への意識

    本橋秀之, 高山修, 沖津摂, 沖津摂, 舟橋弘晃, 舟橋弘晃, 中塚幹也, 中塚幹也

    Journal of mammalian ova research   31 ( 2 )   S97 - S97   2014

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  • 中卵胞由来卵丘細胞塊分泌因子は小卵胞由来ブタ卵母細胞の体外成熟能を改善する

    清水舞, 舟橋弘晃, 船橋弘晃

    Journal of mammalian ova research   31 ( 2 )   S103 - S103   2014

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  • ヒト卵巣組織ガラス化保存融解後の生存性はFreshと同等か?

    本橋秀之, 高山修, RUKMALI Athurupana, 奥平裕一, PILAR Ferre, 李楊, 中塚幹也, 舟橋弘晃

    日本生殖医学会雑誌   59 ( 4 )   411 - 411   2014

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  • タンパク質合成阻害剤クロラムフェニコールおよびシクロヘキシミド存在下でのブタ精子の受精能獲得および体外受精能

    奥平裕一, 舟橋弘晃

    Journal of reproduction and development   59 ( Suppl Japanese Issue )   j136 - j136   2013

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  • 完全合成溶液による未成熟ブタ卵丘細胞-卵母細胞複合体のガラス化保存

    高橋大山, 舟橋弘晃

    Journal of reproduction and development   59 ( Suppl Japanese Issue )   j165 - j165   2013

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  • 豚の非外科的胚移植と新しい人工授精技術の実用化・産業化のための研究開発

    吉岡耕治, 舟橋弘晃, 三角浩司, 星宏良, 阿部宏之, 中根崇, 坂上信忠, 柴田貴子, 中野貞雄

    農林水産省農林水産技術会議事務局研究成果   ( 481 )   2013

  • 中小卵胞由来ブタ卵丘細胞卵母細胞複合体のヒアルロナン産生能

    中小路宗洋, 舟橋弘晃

    Journal of reproduction and development   58 ( Suppl Japanese Issue )   j97 - j97   2012

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  • グリセリンおよびトレハロースがブタ精液の凍結融解後の精子機能の正常性に及ぼす影響

    廣瀬悠人, 舟橋弘晃, 舟橋弘晃

    Journal of reproduction and development   58 ( Suppl Japanese Issue )   j205 - j205   2012

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  • 豚液状精液へのカフェイン添加が人工授精後の繁殖成績に及ぼす影響

    増本憲考, 山口昇一郎, 笠正二郎, 舟橋弘晃, 吉岡耕治

    日本養豚学会大会講演要旨   97th   2012

  • 子宮内免疫反応制御による豚人工授精技術

    山口昇一郎, 鈴木千恵, 野口倫子, 笠正二郎, 森美幸, 磯崎良寛, 舟橋弘晃, 吉岡耕治

    Journal of reproduction and development   58 ( Suppl Japanese Issue )   j77 - j77   2012

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  • ブタ受精卵の体外生産技術

    舟橋弘晃

    Journal of reproduction and development   58 ( Suppl Japanese Issue )   j76 - j76   2012

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  • 津高牧場への発情発見システム「牛歩」の導入と今後の課題

    舟橋弘晃, 舟橋弘晃, 野久保隆, 片山佳司

    岡山大学農学部センター報告   ( 33 )   2011

  • Brilliant cresyl blueで判別された中小卵胞由来ブタ卵母細胞の体外成熟能とRNA量

    THANH Lam Thi Ngoc, 舟橋弘晃

    Journal of reproduction and development   57 ( Suppl Japanese Issue )   2011

  • 豚凍結精液人工授精における精液希釈液へのカフェイン添加が融解精子の膜正常性,子宮内白血球数および炎症性サイトカインmRNA発現に及ぼす影響

    山口昇一郎, 鈴木千恵, 野口倫子, 笠正二郎, 森美幸, 磯崎良寛, 舟橋弘晃, 吉岡耕治

    Journal of reproduction and development   57 ( Suppl Japanese Issue )   j138 - j138   2011

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  • 精子内RNA量を指標としたブタ精液の評価の可能性

    奥平裕一, 佐々木真也, 舟橋弘晃

    Journal of reproduction and development   57 ( Suppl Japanese Issue )   j118 - j118   2011

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  • ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

    R. Romar, M. J. Izquierdo-Rico, H. Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   23 ( 1 )   221 - 221   2011

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  • REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

    M. J. Izquierdo-Rico, R. Romar, C. Kohata, H. Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   23 ( 1 )   229 - 230   2011

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  • 卵黄がウシ及びブタ多核白血球の貪食性と走化性に及ぼす影響

    李井春, 舟橋弘晃

    日本畜産学会大会講演要旨   112th   86 - 86   2010

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  • 小卵胞由来体外成熟ブタ卵母細胞の体外受精および前核形成能

    小畑千由貴, 舟橋弘晃

    日本畜産学会大会講演要旨   112th   85 - 85   2010

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  • マイクロ空圧駆動型人工卵管で負荷される力学的刺激

    李井春, 松浦宏治, 兒玉美恵子, 黒田ユカ, 舟橋弘晃, 成瀬恵治

    化学とマイクロ・ナノシステム研究会講演要旨集   21st   2010

  • タンパク質合成阻害剤がブタ精子内RNA量および運動性に及ぼす影響

    山下佳佑, 舟橋弘晃

    Journal of reproduction and development   56 ( Suppl Japanese Issue )   j79 - j79   2010

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  • 分化誘導したマウスES細胞及びiPS細胞におけるCy3標識REIC/Dkk-3タンパク質のエンドサートーシスによる取り込み

    片岡健, 阪口政清, LI Kun Peng, DU Gang, 武田千佳, 舟橋弘晃, 村田等, HUH Nam-Ho

    組織培養研究   29 ( 1 )   74 - 74   2010

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  • ラット胚盤胞の非外科的受精卵移植による産子の獲得

    金時宇, 柏崎直巳, 舟橋弘晃

    Journal of reproduction and development   56 ( Suppl Japanese Issue )   j70 - j70   2010

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  • 動的環境における胚培養成績上昇機構に関する考察

    松浦宏治, 黒田ユカ, 舟橋弘晃, 成瀬恵治

    日本生殖医学会雑誌   55 ( 3 )   97 - 97   2010

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  • 繁殖技術に関する最新動向 グローバルな視点での人工授精の技術開発動向

    舟橋弘晃

    養豚の友   ( 494 )   2010

  • Reduction of immunological sperm loss following artificial insemination of boar semen

    FUNAHASHI Hiroaki, LI Jing-Chun, YAMAGUCHI Shoichiro, MURAKAMI Tetsuya

    46 ( 4 )   211 - 212   2009.12

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  • 授精のベストタイミングとTop Quality Embryoの獲得 IVM成熟卵子の最適受精条件とは何か?

    舟橋 弘晃

    日本IVF学会誌   12   51 - 53   2009.9

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  • 傾斜培養によるマウス胚の培養成績とそのマイクロ空間制御

    黒田ユカ, 黒田ユカ, 松浦宏治, 松浦宏治, 舟橋弘晃, 成瀬恵治

    化学とマイクロ・ナノシステム研究会講演要旨集   20th   2009

  • PDMSの物性を利用した生殖細胞操作デバイスの作製

    松浦宏治, 松浦宏治, 黒田ユカ, 黒田ユカ, 舟橋弘晃, 成瀬恵治

    化学とマイクロ・ナノシステム研究会講演要旨集   20th   2009

  • 小卵胞由来および中卵胞由来ブタ卵母細胞に付随する卵丘細胞の生存性およびアポトーシス頻度の比較

    松永利恵, 舟橋弘晃

    日本畜産学会大会講演要旨   110th   89 - 89   2009

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  • カフェイン及びヘパリンが豚白血球の走化性と貪食性に及ぼす影響

    李井春, 舟橋弘晃

    Journal of reproduction and development   55 ( Supplement )   j158 - j158   2009

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  • シリコンエラストマー製チャンバーによるガラス吸着性精子の運動軌跡測定

    松浦宏治, 山下佳佑, 黒田ユカ, 舟橋弘晃, 舟橋弘晃

    Journal of reproduction and development   55 ( Supplement )   j75 - j75   2009

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  • 卵丘細胞の共培養が小卵胞由来ブタ卵母細胞の成熟率に与える影響

    松永利恵, 舟橋弘晃

    Journal of reproduction and development   55 ( Supplement )   j137 - j137   2009

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  • カフェイン添加希釈液への抗酸化剤の添加が豚凍結融解精子の先体反応及びAI後の繁殖成績に及ぼす影響

    山口昇一郎, 舟橋弘晃, 村上徹哉, 徳満茂

    日本養豚学会大会講演要旨   92nd   2009

  • 卵黄含有希釈液の注入がラット子宮内多核白血球数に及ぼす影響

    藪崎雅紀, 舟橋弘晃

    Journal of reproduction and development   55 ( Supplement )   j158 - j158   2009

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  • PKC活性化剤によるゴナドトロピン・フリー完全合成培地中でのブタ卵母細胞の体外成熟

    中務結貴, 舟橋弘晃, 舟橋弘晃

    Journal of reproduction and development   55 ( Supplement )   j139 - j139   2009

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  • DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

    Y. Akaki, K. Yoshioka, H. Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   21 ( 1 )   218 - 219   2009

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  • 牛多核白血球の精子および血清に対する走化性

    李井春, 薮崎雅紀, 舟橋弘晃, 舟橋弘晃

    Journal of reproduction and development   54 ( Supplement )   j137 - j137   2008

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  • ラットおよびマウス透明帯除去初期胚の体外培養とヒアルロン酸による卵割促進効果

    奥山正信, 舟橋弘晃

    Journal of reproduction and development   54 ( Supplement )   j123 - j123   2008

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  • ラット子宮内多核白血球の性周期および交配後の変動

    薮崎雅紀, 李井春, 舟橋弘晃, 舟橋弘晃

    Journal of reproduction and development   54 ( Supplement )   j137 - j137   2008

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  • 体外成熟時のシェアストレスがブタ卵母細胞の初期発生能に及ぼす影響

    小池孝幸, 成瀬恵治, 舟橋弘晃

    日本畜産学会大会講演要旨   109th   2008

  • 子宮内免疫制御による豚凍結精液人工授精方法の検討

    山口昇一郎, 舟橋弘晃, 村上徹哉

    日本畜産学会大会講演要旨   109th   2008

  • 揺動培養によるマウス胚盤胞到達率の上昇

    黒田ユカ, 松浦宏治, 片野坂友紀, 毛利聡, 舟橋弘晃, 成瀬恵治

    日本生体医工学会大会プログラム・論文集(CD-ROM)   47th   2008

  • 揺動培養器を用いたブタ卵母細胞の体外成熟と単為発生後の初期発生

    舟橋弘晃, 小池孝幸, 成瀬恵治

    日本生殖医学会雑誌   53 ( 1/2 )   44 - 44   2008

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  • 老齢種雄豚精子希釈液へのカルニチン添加が精子ミトコンドリア活性に及ぼす影響

    佐野光, 舟橋弘晃

    日本畜産学会大会講演要旨   109th   2008

  • ゴナドトロピン・フリー完全合成培地中でのブタ卵母細胞の体外成熟

    赤木友香, 舟橋弘晃

    日本畜産学会大会講演要旨   109th   2008

  • 哺乳動物卵母細胞の体外成熟の現況

    舟橋 弘晃

    Journal of clinical embryologist   9   4 - 8   2007.6

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  • 限定培地へのPyruvate添加がブタ卵母細胞の核・細胞質成熟に及ぼす影響

    小池孝幸, 坂井涼子, 舟橋弘晃

    日本畜産学会大会講演要旨   108th   67 - 67   2007

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  • グルコースおよびピルビン酸がブタ卵母細胞の核成熟およびグルタチオン濃度に及ぼす影響

    小池孝幸, 舟橋弘晃

    日本畜産学会大会講演要旨   107th   74 - 74   2007

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  • カルニチンがブタ卵母細胞の体外成熟に及ぼす影響

    赤木友香, 舟橋弘晃

    日本畜産学会大会講演要旨   108th   67 - 67   2007

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  • 小中卵胞由来ブタ卵母細胞-卵丘細胞塊におけるグルコース6リン酸脱水素酵素活性の所在

    舟橋弘晃, 池原あかり, 石田有希

    日本畜産学会大会講演要旨   107th   74 - 74   2007

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  • Nucleosideが成熟過程のマウス卵母細胞内ミトコンドリア膜電位活性に及ぼす影響

    藤井渉, 舟橋弘晃

    Journal of reproduction and development   53 ( Supplement )   j177 - j177   2007

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  • 豚凍結精液の人工授精で用いる融解液の違いが人工授精後の繁殖成績および豚凍結精子の先体反応に及ぼす影響

    山口昇一郎, 舟橋弘晃, 村上徹哉, 伊東正吾

    日本畜産学会大会講演要旨   106th   101 - 101   2006

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  • 体細胞を導入したラット卵母細胞の活性化後の発生能

    藤井渉, 舟橋弘晃

    Journal of reproduction and development   52 ( Supplement )   j77 - j77   2006

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  • 豚精液の深部注入器によるAI技術の現状 海外および国内の事例から

    舟橋弘晃

    養豚の友   ( 444 )   2006

  • Select antioxidants improve the function of extended boar semen stored at 10 degrees C International journal

    H Funahashi, T Sano

    THERIOGENOLOGY   63 ( 6 )   1605 - 1616   2005.4

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    The objective was to determine the effects of antioxidant addition to extender on viability, acrosome integrity and penetrability in vitro of boar spermatozoa preserved at 10 degrees C. Washed spennatozoa were resuspended at 1 x 10(8) cells/mL in modified Modena solution containing 20% (v/ v) boar seminal plasma and 5 mM antioxidant (glutathione, cysteine or hypotaurine). Control aliquots were the same suspension without added antioxidants. Sperm suspensions were then chilled to 10 degrees C with a computerized cooling program. Sperm viability after 7 and 14 d was higher in the presence of glutathione or cysteine, whereas hypotaurine did not improve the survival rate. Percentage of chlortetracycline (CTC) fluorescence pattern as intact live cells was higher in spermatozoa preserved with glutathione or cysteine at 7 and 14 d of preservation. When the preservation period was prolonged until 57 d, survival rate was higher with cysteine than controls. When spermatozoa were preserved with cysteine and then inseminated in an IVF system, penetration rate was not different until 15 d of preservation and higher than controls at 15-29 d, whereas no sows became pregnant after AI with spermatozoa preserved for 21-23 d. Therefore, glutathione and cysteine can improve the viability and functional status of boar spermatozoa during liquid preservation and boar spermatozoa penetrated in vitro even after preservation in the presence of cysteine at 10 degrees C for 29 d. (c) 2004 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2004.06.016

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  • 豚人工授精における技術的課題と技術開発方向 2.精液注入器の改良.

    舟橋弘晃

    畜産技術   第599号(2005年4月号) pp13-18 ( 599 )   2005

  • プロテアーゼ阻害剤による体外成熟豚卵子の前処理が体外受精系での精子侵入に及ぼす影響

    舟橋弘晃

    日本畜産学会大会講演要旨   104th   129 - 129   2005

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  • Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine International journal

    H Funahashi, R Romar

    REPRODUCTION   128 ( 6 )   789 - 800   2004.12

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    To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 mul modified Medium 199-suspended spermatozoa at 2.5 x 10(5) sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 +/- 2.2) than 8 h (207.6 +/- 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 mumol adenosine/l for 0 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

    DOI: 10.1530/rep.1.00295

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  • Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine International journal

    H Funahashi, R Romar

    REPRODUCTION   128 ( 6 )   789 - 800   2004.12

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    To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 mul modified Medium 199-suspended spermatozoa at 2.5 x 10(5) sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 +/- 2.2) than 8 h (207.6 +/- 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 mumol adenosine/l for 0 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

    DOI: 10.1530/rep.1.00295

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  • Development of an effective system to control sperm penetrability for artificial insemination and in vitro fertilization: Utilization of oxidant and anti-oxidant

    舟橋弘晃

    食肉に関する助成研究調査成果報告書   Vol.22, p55-61.   2004

  • 瞬時共培養法による豚卵子への多精子侵入頻度の軽減

    舟橋弘晃, ROMAR R

    日本畜産学会第103回大会講演要旨   p108   108 - 108   2004

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  • In vitro fertility of boar spermatozoa preserved at 10oC for 22 days.

    Reproduction Fertility and development   16(1,2):255   2004

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  • IVM-IVF of porcine oocytes treated with roscovitine、

    日本畜産学会第103回大会講演要旨   p107   2004

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  • 10℃保存された豚精子の体外受精能と人工授精の成績

    豚の繁殖衛生セミナー通信   30, 7-12   2003

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  • Polyspermic penetration in porcine IVM-IVF systems International journal

    Hiroaki Funahashi

    Reprodution, Fertility and Development   15 ( 3 )   167 - 177   2003

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    Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm-zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

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  • 豚卵母細胞の体外成熟と体外受精

    舟橋弘晃

    日本不妊学会雑誌   48, 274-275 ( 3/4 )   274 - 275   2003

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  • 体外培養システムにおけるブタ卵子の細胞質成熟

    IVF Conference 2003   68 - 71   2003

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  • 哺乳動物初期胚の対外培養および操作が着床後の成長・分化に残す課題

    舟橋 弘晃

    Journal of Chinical Embryologist   6, 15-22   15 - 22   2003

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  • Polyspermic penetration in porcine IVM-IVF systems

    H Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   15 ( 3 )   167 - 177   2003

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    Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization ( IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm - zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

    DOI: 10.1071/RD02076

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  • 同種および異種精しょうが豚凍結精子の生存性および受精能に及ぼす効果

    舟橋弘晃, 佐野通

    日本養豚学会大会講演要旨   79th   2003

  • Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system International journal

    H Funahashi

    REPRODUCTION   124 ( 6 )   857 - 864   2002.12

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    The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO2-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N-omega-vitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N-omega-vitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

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  • Effect of methyl-beta-cyclodextrin and fertilization promoting peptide on capacitation of boar spermatozoa in a protein-free medium

    H Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   48 ( 1 )   57 - 63   2002.2

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    The present study was undertaken to examine the effect of methyl-beta-cyclodextrin (MBCD) and fertilization promoting peptide (FPP; pGlu-Glu-ProNH(2)) on the capacitation and acrosome reaction of boar spermatozoa in a protein-free medium. After washing with a protein-free medium, ejaculated spermatozoa (resuspended at 1 x 10(6) cells/ml) were cultured in 2 ml of modified Medium-199 containing polyvinylalcohol (mM199-PVA) supplemented or not supplemented with MBCD and/or FPP for 2 h in an atmosphere of 5% CO2 in air at 39 C. Effects of MBCD and FPP on boar spermatozoa were determined by chlortetracycline fluorescence assessment. When spermatozoa were cultured in various MBCD concentrations, capacitation was induced in a concentration-dependent manner, but spontaneous acrosome reaction was not affected. When the effect of FPP in mM199-PVA was determined, sperm capacitation was induced and spontaneous acrosome reaction was inhibited in 100 nM FPP, regardless of the absence or presence of BSA. In the last experiment, spermatozoa were cultured in mM199-PVA supplemented with various concentrations of MBCD and/or FPP. An accelerative effect of MBCD on sperm capacitation was observed in a concentration-dependent manner even in the presence of FPP, whereas 1-2 mM of MBCD neutralized the stimulatory effect of FPP on capacitation. Furthermore, FPP inhibited spontaneous acrosome reaction even in the presence of MBCD. These results demonstrate that MBCD and PFF can induce capacitation of boar spermatozoa in a protein-free medium and that FPP alone also inhibits spontaneous acrosome reaction.

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  • Induction of capacitation and the acroxome reaction of boar spermatozoa by 1-arginine and nitric oxide synthesis associated with the anion transport system.

    Reproduction   124 ( 6 )   857 - 864   2002

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  • カフェインおよび受精促進ペプチド存在下での豚新鮮射出精子のNO産生

    第95回日本繁殖生物学会講演要旨集   p161   2002

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  • 豚凍結精液融解後のグリセリン濃度が精子の受精能に及ぼす影響

    佐野通, 荒金知宏, 舟橋弘晃

    第78回日本養豚学会大会講演要旨集   p11   2002

  • チオール類が豚液状保存精子の生存性および自発的先体反応に及ぼす影響

    舟橋弘晃, 原田護, 佐野通, 伊藤述史

    第77回日本養豚学会大会講演要旨集   p23   2002

  • 豚精子のHCO3-/C1-輸送がアルギニンによる受精態獲得・先体反応促進効果に及ぼす影響

    日本畜産学会第100回大会講演要旨集   p115   2002

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  • Effect of methyl-beta-cyclodextrin and fertilization promoting peptide on sperm capacitation of boar spermatozoa in a protein-free medium.

    Hiroaki FUNAHASHI

    Journal of Reproduction and Development   48 ( 1 )   57 - 63   2002

  • 豚精子のHCO3-/Cl-輸送がアルギニンによる受精能獲得・先体反応促進効果に及ぼす影響

    舟橋弘晃

    日本畜産学会大会講演要旨   100th   2002

  • Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro International journal

    H Funahashi, H Ekwall, K Kikuchi, H Rodriguez-Martinez

    REPRODUCTION   122 ( 3 )   443 - 452   2001.9

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    The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 mum) and in vitro (5.95 +/- 0.51 mum) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

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  • Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro

    H Funahashi, H Ekwall, K Kikuchi, H Rodriguez-Martinez

    REPRODUCTION   122 ( 3 )   443 - 452   2001.9

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    The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 mum) and in vitro (5.95 +/- 0.51 mum) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

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  • Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine International journal

    H Funahashi, T Nagai

    MOLECULAR REPRODUCTION AND DEVELOPMENT   58 ( 4 )   424 - 431   2001.4

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    Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa. (C) 2001 Wiley-Liss, Inc.

    DOI: 10.1002/1098-2795(20010401)58:4<424::AID-MRD10>3.0.CO;2-1

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  • Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine

    H Funahashi, T Nagai

    MOLECULAR REPRODUCTION AND DEVELOPMENT   58 ( 4 )   424 - 431   2001.4

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    Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa. (C) 2001 Wiley-Liss, Inc.

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  • An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

    Theriogenology   55 ( 1 )   522   2001

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  • アルギニンが豚精子の受精能獲得・先体反応に及ぼす影響

    舟橋弘晃

    日本畜産学会第98回大会講演要旨   98th   110   2001

  • 緑色蛍光蛋白質の顕微注入法と核移植技術を組み合わせた形質転換牛胚の効率的生産手法の開発

    第12回西日本胚移植研究会・第20回北海道牛受精卵移植研究会合同研究発表大会講演要旨集   36   2001

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  • Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

    Sixth International Conference on Pig Reproduction   76   2001

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  • Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

    Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

    Cloning and Stem Cells   3 ( 4 )   183 - 190   2001

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    The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

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  • Oviduct involvement in sperm capacitation and oocyte development

    Sixth International Conference on Pig Reproduction   62   2001

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  • 豚の新しい繁殖技術とその展望

    舟橋弘晃

    豚の繁殖衛生セミナー通信   27 ( 28 )   79 - 88   2001

  • Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

    Sixth International Conference on Pig Reproduction   76   2001

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  • Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

    Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

    Cloning and Stem Cells   3 ( 4 )   183 - 190   2001

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    The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

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  • An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

    Theriogenology   55 ( 1 )   522   2001

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  • Involvement of oviduct in sperm capacitation and oocyte development in pigs. International journal

    H. Rodriguez-Martinez, P. Tienthai, K. Suzuki, H. Funahashi, H. Ekwall, A. Johannisson

    Reproduction (cambridge, england) supplement   58   129 - 145   2001

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    An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for < 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

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  • Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy International journal

    H Funahashi, H Ekwall, H Rodriguez-Martinez

    BIOLOGY OF REPRODUCTION   63 ( 5 )   1437 - 1442   2000.11

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    Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 mum(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 mum(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

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  • Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy

    H Funahashi, H Ekwall, H Rodriguez-Martinez

    BIOLOGY OF REPRODUCTION   63 ( 5 )   1437 - 1442   2000.11

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    Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 mum(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 mum(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

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  • Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

    H Funahashi, T Fujiwara, T Nagai

    BIOLOGY OF REPRODUCTION   63 ( 4 )   1157 - 1163   2000.10

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    Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes, These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.

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  • Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes International journal

    H Funahashi, T Fujiwara, T Nagai

    BIOLOGY OF REPRODUCTION   63 ( 4 )   1157 - 1163   2000.10

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    Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes, These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.

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  • 越壁IVF法による精子選別は豚卵子への多精子受精頻度を減少させる

    舟橋 弘晃, 永井 卓

    The journal of reproduction and development   46 ( 5 )   319 - 324   2000.10

  • Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro International journal

    H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

    MOLECULAR REPRODUCTION AND DEVELOPMENT   55 ( 1 )   117 - 124   2000.1

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    Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome toss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these re sponses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln+FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization. (C) 2000 Wiley-Liss, Inc.

    DOI: 10.1002/(SICI)1098-2795(200001)55:1<117::AID-MRD16>3.0.CO;2-7

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  • Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

    H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

    MOLECULAR REPRODUCTION AND DEVELOPMENT   55 ( 1 )   117 - 124   2000.1

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    Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome toss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these re sponses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln+FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization. (C) 2000 Wiley-Liss, Inc.

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  • The zona reaction in porcine oocytes fertilized in vivo and vitro as seen with scanning electron microscopy.

    Theriogenology   53 ( 1 )   420   2000

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  • Sperm selection by a climbing-over-a-wall method reduces the incidence of polyspermic penetration of porcine oocytes.

    Hiroaki FUNAHASHI, Takashi NAGAI

    Journal of Reproduction and Development   46 ( 5 )   319 - 324   2000

  • Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes

    Hiroaki Funahashi

    Journal of Reproduction and Development   46 ( 5 )   319 - 324   2000

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    Effect of sperm selection according to the degree of motility after insemination on in-vitro penetration was examined by using a new in-vitro fertilization system designated as a climbing-overa-wall (COW) IVF method. When the sperm penetration rate in the COW-IVF method was compared with a standard method at the same sperm concentration (5 X 10s cells/ml), the rates (95.1 ±1.9 and 98.2 ±1.0%, respectively) were similar, but the incidence of monospermic penetration was higher in the COW-IVF (25.5 ±4.5%) than the standard method (10.4 ±2.5%). When sperm concentration was changed from 0.5 × 105 to 10 × 105 cells/ml in the COW-IVF method, sperm penetration rate was higher at a higher concentration, whereas monospermic penetration rate was increased at a lower concentration. The proportion of monospermic oocytes in matured oocytes was similar among sperm concentrations, 0.5 × 105 to 5 × 105 cells/ml, at fertilization in the COW method. These results demonstrate that the COW-IVF method, selection according to the degree of sperm motility after insemination, can increase the normal penetration of frozen-thawed boar spermatozoa into IVM oocytes without any reduction in the sperm penetration rate.

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  • Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes. (Other,)

    The 14th International Congress on Animal Reproduction   2000

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  • Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes.

    The 14th International Congress on Animal Reproduction   2000

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  • 豚精子の受精能獲得を調節するアデノシン・レセプターの役割

    舟橋弘晃, 藤原俊満

    日本畜産学会第97回大会講演要旨   97th   124   2000

  • The zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy.

    Biology of Reproduction   63 ( 5 )   1447 - 1452   2000

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  • Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

    H Funahashi, N Bandoh, S Nakahira, SH Oh, S Tsuboi

    ZYGOTE   7 ( 4 )   301 - 305   1999.11

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    The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilised eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p &lt; 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilised oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

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  • Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes International journal

    H Funahashi, N Bandoh, S Nakahira, SH Oh, S Tsuboi

    ZYGOTE   7 ( 4 )   301 - 305   1999.11

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    The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilised eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p &lt; 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilised oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

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  • Recent development in embryo technology in pigs - Review

    K Niwa, H Funahashi

    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES   12 ( 6 )   966 - 975   1999.9

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    Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage, has made it possible to utilize follicular oocytes for in, vitro production of embryos and thus stimulated research on various, embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality, Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

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  • Recent development in embryo technology in pigs - Review

    K Niwa, H Funahashi

    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES   12 ( 6 )   966 - 975   1999.9

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    Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage, has made it possible to utilize follicular oocytes for in, vitro production of embryos and thus stimulated research on various, embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality, Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

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  • DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes

    S Said, H Funahashi, K Niwa

    ZYGOTE   7 ( 3 )   249 - 254   1999.8

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    DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p &lt; 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p &lt; 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p &lt; 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

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  • DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes International journal

    S Said, H Funahashi, K Niwa

    ZYGOTE   7 ( 3 )   249 - 254   1999.8

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    DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p &lt; 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p &lt; 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p &lt; 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

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  • Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: Effects of epidermal growth factor on production of PAs in oocytes and cumulus cells

    KW Park, SH Choi, XX Song, H Funahashi, K Niwa

    BIOLOGY OF REPRODUCTION   61 ( 1 )   298 - 304   1999.7

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    We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed, Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex, In COCs before culture, however, no activity of PAs was detected. Al 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

    DOI: 10.1095/biolreprod61.1.298

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  • Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: Effects of epidermal growth factor on production of PAs in oocytes and cumulus cells International journal

    KW Park, SH Choi, XX Song, H Funahashi, K Niwa

    BIOLOGY OF REPRODUCTION   61 ( 1 )   298 - 304   1999.7

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    We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed, Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex, In COCs before culture, however, no activity of PAs was detected. Al 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

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  • 卵胞膜細胞との共培養による卵丘付着牛卵母細胞の卵核胞発育阻害を伴わない減数分裂の休止

    朴 光旭, 舟橋 弘晃, 丹羽 晧二

    The journal of reproduction and development   45 ( 3 )   223 - 231   1999.6

  • Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development

    Kwang Wook Park, Hiroaki Funahashi, Koji Niwa

    Journal of Reproduction and Development   45 ( 3 )   223 - 231   1999

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    The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.

    DOI: 10.1262/jrd.45.223

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  • The presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis improves porcine 'oocyte competence' as determined by early embryonic development after in-vitro fertilization

    Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

    Journal of Reproduction and Development   45 ( 4 )   265 - 271   1999

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    The present studies were designed to determine the effect of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis or after in vitro fertilization on early embryonic development of porcine embryos. The presence of purified ovine TIMP-1 in culture medium after in vitro fertilization did not affect the incidence of embryo cleavage or development to the blastocyst stage at 48 h or 6 days after insemination, respectively. In contrast, when various concentrations (1.25, 2.50 and 5.00 μg/ml) of purified ovine TIMP-1 were added to the culture medium during the second half (24 h) of oocyte maturation, normal cleavage rates (64 ± 3% at 1.25 μg/ml, 57 ± 5% at 2.50 μg/ml and 56 ± 1% at 5.00 μg/ml) were higher (p&lt
    0.05) as compared to controls (43 ± 2%). In addition, the incidence of embryos that developed to the blastocyst stage was increased (p&lt
    0.05) when TIMP-1 was added (34 ± 3% at 1.25 Mg/ml, 32 ± 1% at 2.50 μg/ml and 28 ± 1% at 5.00 μg/ml) as compared to controls (22 ± 3%). There was no difference in the incidence (53 ± 1%) of oocytes fertilized normally. These results indicate that the presence of TIMP-1 during the second half of in vitro maturation enhanced the competence of porcine oocytes to develop to the 2- to 4-cell stages after in vitro fertilization, consequently increasing the incidence of blastocysts.

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    Other Link: https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=1999&ichushi_jid=J04678&link_issn=&doc_id=19990903330004&doc_link_id=10.1262%2Fjrd.45.265&url=https%3A%2F%2Fdoi.org%2F10.1262%2Fjrd.45.265&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

  • 卵子への侵入過程におけるラット精子核内チオール類の酸化還元状態の観察と変化(共著)

    SAID S, 舟橋弘晃, 丹羽こう二

    第95回日本畜産学会大会講演要旨   95th   79 - 79   1999

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  • Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development

    Kwang Wook Park, Hiroaki Funahashi, Koji Niwa

    Journal of Reproduction and Development   45 ( 3 )   223 - 231   1999

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    Language:English   Publisher:The Japanese Society of Animal Reproduction (JSAR)  

    The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.

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  • The presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis improves porcine 'oocyte competence' as determined by early embryonic development after in-vitro fertilization

    Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

    Journal of Reproduction and Development   45 ( 4 )   265 - 271   1999

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    Language:English   Publisher:The Japanese Society of Animal Reproduction (JSAR)  

    The present studies were designed to determine the effect of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis or after in vitro fertilization on early embryonic development of porcine embryos. The presence of purified ovine TIMP-1 in culture medium after in vitro fertilization did not affect the incidence of embryo cleavage or development to the blastocyst stage at 48 h or 6 days after insemination, respectively. In contrast, when various concentrations (1.25, 2.50 and 5.00 μg/ml) of purified ovine TIMP-1 were added to the culture medium during the second half (24 h) of oocyte maturation, normal cleavage rates (64 ± 3% at 1.25 μg/ml, 57 ± 5% at 2.50 μg/ml and 56 ± 1% at 5.00 μg/ml) were higher (p&lt
    0.05) as compared to controls (43 ± 2%). In addition, the incidence of embryos that developed to the blastocyst stage was increased (p&lt
    0.05) when TIMP-1 was added (34 ± 3% at 1.25 Mg/ml, 32 ± 1% at 2.50 μg/ml and 28 ± 1% at 5.00 μg/ml) as compared to controls (22 ± 3%). There was no difference in the incidence (53 ± 1%) of oocytes fertilized normally. These results indicate that the presence of TIMP-1 during the second half of in vitro maturation enhanced the competence of porcine oocytes to develop to the 2- to 4-cell stages after in vitro fertilization, consequently increasing the incidence of blastocysts.

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  • Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage International journal

    SH Oh, K Miyoshi, H Funahashi

    BIOLOGY OF REPRODUCTION   59 ( 4 )   884 - 889   1998.10

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    A suitable chemically defined culture medium far 1-cell rat embryos (mR1ECM) was modified to obtain sperm penetration, and the developmental competence of oocytes fertilized in the medium was compared to that of oocytes fertilized in a traditional medium, modified Krebs-Ringer bicarbonate medium (mKRB), Sperm penetration was not observed when polyvinyl alcohol was replaced with ESA in mR1ECM (mR1ECM-BSA); the incidence was improved only when the osmolarity in mR1ECM-BSA was increased to that in mKRB (310 mOsm) by addition of NaCl, The proportion of oocytes penetrated in mR1ECM-BSA with NaCl increased (71.6 +/- 6.9%), which was not different compared to that in mKRB (76.7 +/- 13.7%). High incidences of sperm penetration (88.8 +/- 4.1% to 93.1 +/- 5.1%) were also observed when NaCl concentration in mR1ECM-BSA was increased from 76.7 mM to 100-130 mM. The incidence of embryos developing to the morula and blastocyst stages was higher when fertilized in mR1ECM-BSA containing 110-130 mM NaCl (91-94%) than in mKRB (70%), A total of 5 offspring were obtained after transfer of the morulae and blastocysts (69 embryos/ 7 females), These results demonstrate that a high developmental ability of rat embryos to the blastocyst stage is attained when the embryos have been fertilized in mR1ECM-BSA containing 110-130 mM NaCl and then cultured in mR1ECM.

    DOI: 10.1095/biolreprod59.4.884

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  • Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage

    SH Oh, K Miyoshi, H Funahashi

    BIOLOGY OF REPRODUCTION   59 ( 4 )   884 - 889   1998.10

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    A suitable chemically defined culture medium far 1-cell rat embryos (mR1ECM) was modified to obtain sperm penetration, and the developmental competence of oocytes fertilized in the medium was compared to that of oocytes fertilized in a traditional medium, modified Krebs-Ringer bicarbonate medium (mKRB), Sperm penetration was not observed when polyvinyl alcohol was replaced with ESA in mR1ECM (mR1ECM-BSA); the incidence was improved only when the osmolarity in mR1ECM-BSA was increased to that in mKRB (310 mOsm) by addition of NaCl, The proportion of oocytes penetrated in mR1ECM-BSA with NaCl increased (71.6 +/- 6.9%), which was not different compared to that in mKRB (76.7 +/- 13.7%). High incidences of sperm penetration (88.8 +/- 4.1% to 93.1 +/- 5.1%) were also observed when NaCl concentration in mR1ECM-BSA was increased from 76.7 mM to 100-130 mM. The incidence of embryos developing to the morula and blastocyst stages was higher when fertilized in mR1ECM-BSA containing 110-130 mM NaCl (91-94%) than in mKRB (70%), A total of 5 offspring were obtained after transfer of the morulae and blastocysts (69 embryos/ 7 females), These results demonstrate that a high developmental ability of rat embryos to the blastocyst stage is attained when the embryos have been fertilized in mR1ECM-BSA containing 110-130 mM NaCl and then cultured in mR1ECM.

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  • Both bovine oocytes and cumulus cells produce plasminogen activator during in vitro maturation

    SH Choi, H Funahashi, K Niwa

    THERIOGENOLOGY   49 ( 1 )   309 - 309   1998.1

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  • Both bovine oocytes and cumulus cells produce plasminogen activator during in vitro maturation

    SH Choi, H Funahashi, K Niwa

    THERIOGENOLOGY   49 ( 1 )   309 - 309   1998.1

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  • Cytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic development

    87   205 - 214   1998

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  • EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

    Journal of Reproduction and Fertility, Abstract Series   21   10   1998

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  • Co-culture of bovine cumulus-oocyte complexes with theca cells inhibits the meiotic resumption at a specific phase of germinal vesicle.

    KW Park, H Funahashi, K Niwa

    BIOLOGY OF REPRODUCTION   58 ( Suppl.1 )   219 - 219   1998

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  • Viability of boar spermatozoa frozen in 0.5ml-plastic straw by 2-step freezing method(共著)

    Proceedings of the 8th World Conference on Animal Production, Contributed Papers   2   252 - 253   1998

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  • Recent Development in Embryo Technology in Pigs. (共著)

    Proceedings of the 8th World Conference on Animal Production, Symposium Series   2   240 - 251   1998

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  • EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

    Journal of Reproduction and Fertility, Abstract Series   21   10   1998

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  • Intracellular calcium release and cortical reaction after thimerosal-treatment of porcine oocytes matured in vitro or ovulated.

    H Funahashi, Z Machaty, WH Wang, TC Cantley, RS Prather, BN Day

    BIOLOGY OF REPRODUCTION   58 ( Suppl.1 )   219 - 219   1998

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  • Inhibiting effect of theca cells on meiotic resumption of bovine oocytes may not be via cumulus cells(共著)

    Proceedings of the 8th World Conference on Animal Production, Contributed PApers   2   920 - 921   1998

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  • Effects of cumulus cells on the ability of pig oocytes to utilize cysteine or cystine during maturation in vitro

    Ken Sawai, Hiroaki Funahashi, Koji Niwa

    Journal of Reproduction and Development   44 ( 2 )   161 - 168   1998

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    The present study examined the effect of cysteine and cystine associated with cumulus cells in maturation medium on oocyte glutathione (GSH) concentration at the end of maturation culture (for 48 h) and male pronuclear (MPN) formation following in vitro fertilization of pig oocytes. When cumulus-enclosed oocytes were cultured in a serum-free medium containing 0.57 mM cysteine or cystine, their GSH concentration and incidence of MPN formation were higher than when they were cultured without cysteine and cystine. Supplementation with cysteine during the final 24 h of culture resulted in an increased GSH concentration of oocytes and a higher incidence of MPN formation, regardless of the presence of cumulus cells, than in those cultured without cysteine or cystine. The same high incidence of MPN formation was observed even when oocytes were denuded after 36 h of culture and exposed to cysteine for only 12 h. In contrast, when oocytes were denuded after 24 h of culture and exposed to cystine, neither GSH synthesis nor MPN formation was improved, but both parameters did improve when oocytes were not denuded. Exposure of cumulus-enclosed oocytes to cystine from 36 h of culture did not promote MPN formation. These results indicate that cumulus-free pig oocytes can synthesize GSH in the presence of cysteine and that the presence of cumulus cells is essential for maintaining a high concentration of GSH in oocytes in the presence of cystine.

    DOI: 10.1262/jrd.44.161

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  • Effects of cumulus cells on the ability of pig oocytes to utilize cysteine or cystine during maturation in vitro

    Ken Sawai, Hiroaki Funahashi, Koji Niwa

    Journal of Reproduction and Development   44 ( 2 )   161 - 168   1998

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    The present study examined the effect of cysteine and cystine associated with cumulus cells in maturation medium on oocyte glutathione (GSH) concentration at the end of maturation culture (for 48 h) and male pronuclear (MPN) formation following in vitro fertilization of pig oocytes. When cumulus-enclosed oocytes were cultured in a serum-free medium containing 0.57 mM cysteine or cystine, their GSH concentration and incidence of MPN formation were higher than when they were cultured without cysteine and cystine. Supplementation with cysteine during the final 24 h of culture resulted in an increased GSH concentration of oocytes and a higher incidence of MPN formation, regardless of the presence of cumulus cells, than in those cultured without cysteine or cystine. The same high incidence of MPN formation was observed even when oocytes were denuded after 36 h of culture and exposed to cysteine for only 12 h. In contrast, when oocytes were denuded after 24 h of culture and exposed to cystine, neither GSH synthesis nor MPN formation was improved, but both parameters did improve when oocytes were not denuded. Exposure of cumulus-enclosed oocytes to cystine from 36 h of culture did not promote MPN formation. These results indicate that cumulus-free pig oocytes can synthesize GSH in the presence of cysteine and that the presence of cumulus cells is essential for maintaining a high concentration of GSH in oocytes in the presence of cystine.

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  • Recent Development in Embryo Technology in Pigs. (共著)

    Proceedings of the 8th World Conference on Animal Production, Symposium Series   2   240 - 251   1998

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  • Cytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic developmentCytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic development

    87   205 - 214   1998

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  • Viability of boar spermatozoa frozen in 0.5ml-plastic straw by 2-step freezing method(共著)

    Proceedings of the 8th World Conference on Animal Production, Contributed Papers   2   252 - 253   1998

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  • In vitro production of porcine embryos: On the developmental competence.

    舟橋弘晃

    Journal of reproduction and development   44 ( 6 )   j47-j52 - j52   1998

  • 受精促進ペプチド(FPP)およびアデノシンの豚射出精液に及ぼす自発的先体反応阻止作用における競合(共著)

    浅野敦之, 舟橋弘晃, 丹羽こう二, FRASER L R

    第91回日本繁殖生物学会講演要旨集   91st ( 6 )   45 - a24   1998

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  • リン酸によるラット初期胚の発生阻害時期とマイクロフィラメント構造への影響(共著)

    呉世薫, 舟橋弘晃, 丹羽こう二

    第91回日本繁殖生物学会講演要旨集   91st ( 6 )   43 - a22   1998

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  • Co-culture of bovine cumulus-oocyte complexes with theca cells inhibits the meiotic resumption at a specific phase of germinal vesicle.

    KW Park, H Funahashi, K Niwa

    BIOLOGY OF REPRODUCTION   58 ( Suppl.1 )   219 - 219   1998

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  • Intracellular calcium release and cortical reaction after thimerosal-treatment of porcine oocytes matured in vitro or ovulated.

    H Funahashi, Z Machaty, WH Wang, TC Cantley, RS Prather, BN Day

    BIOLOGY OF REPRODUCTION   58 ( Suppl.1 )   219 - 219   1998

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  • Inhibiting effect of theca cells on meiotic resumption of bovine oocytes may not be via cumulus cells(共著)

    Proceedings of the 8th World Conference on Animal Production, Contributed PApers   2   920 - 921   1998

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  • 受精促進ペプチド(FPP)およびアデノシンが豚精子の自発的先体反応に及ぼす阻止効果(共著)

    15 ( 2 )   S26   1998

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  • Extracorporeal production and artificial operation of fertilized egg of pig.

    舟橋弘晃

    家畜人工授精   ( 185 )   14 - 22   1998

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  • 豚受精卵の体外生産技術の現状

    日本畜産学会関西支部報   ( 138 )   18 - 20   1998

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  • Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization

    H Funahashi, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   57 ( 1 )   49 - 53   1997.7

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    The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (fetal mean, 82.1 +/- 2.1%) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p &lt; 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p &lt; 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

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  • Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation International journal

    Ken Sawai, Hiroaki Funahashi, Koji Niwa

    Biology of Reproduction   57 ( 1 )   1 - 6   1997.7

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    The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-I) to metaphase II (M-II) and M-II at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-I to M-II stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

    DOI: 10.1095/biolreprod57.1.1

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  • Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation

    K Sawai, H Funahashi, K Niwa

    BIOLOGY OF REPRODUCTION   57 ( 1 )   1 - 6   1997.7

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    The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-l) to metaphase II (M-II) and M-Il at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-l to M-ll stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

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  • Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization International journal

    H Funahashi, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   57 ( 1 )   49 - 53   1997.7

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    The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (fetal mean, 82.1 +/- 2.1%) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p &lt; 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p &lt; 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

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  • Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations International journal

    LR Abeydeera, H Funahashi, NH Kim, BN Day

    ZYGOTE   5 ( 2 )   117 - 125   1997.5

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    Porcine oocyte-cumulus complexes were cultured in bovine serum albumin (BSA)-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM) 199 containing caffeine (5 mM), fetal calf serum (FCS; 10%) and varying concentrations (26-56 mM) of NaHCO3 for 9 h (experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%) and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p &lt; 0.05) at 36-56 mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46 mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p &lt; 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56 mM NaHCO3, respectively. The fertilisation medium containing 46 mM NaHCO3 and 1% FCS showed a higher penetration rate (84%) with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

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  • Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations

    LR Abeydeera, H Funahashi, NH Kim, BN Day

    ZYGOTE   5 ( 2 )   117 - 125   1997.5

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    Porcine oocyte-cumulus complexes were cultured in bovine serum albumin (BSA)-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM) 199 containing caffeine (5 mM), fetal calf serum (FCS; 10%) and varying concentrations (26-56 mM) of NaHCO3 for 9 h (experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%) and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p &lt; 0.05) at 36-56 mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46 mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p &lt; 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56 mM NaHCO3, respectively. The fertilisation medium containing 46 mM NaHCO3 and 1% FCS showed a higher penetration rate (84%) with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

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  • Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes

    Z Machaty, H Funahashi, BN Day, RS Prather

    BIOLOGY OF REPRODUCTION   56 ( 4 )   921 - 930   1997.4

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    The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP(3)), the InsP(3) receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP(3)-induced Ca2+ release was inhibited by heparin, the InsP(3) receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP(3) and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.

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  • Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes International journal

    Z Machaty, H Funahashi, BN Day, RS Prather

    BIOLOGY OF REPRODUCTION   56 ( 4 )   921 - 930   1997.4

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    The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP(3)), the InsP(3) receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP(3)-induced Ca2+ release was inhibited by heparin, the InsP(3) receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP(3) and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.

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  • Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro. International journal

    H Funahashi, T C Cantley, B N Day

    Theriogenology   47 ( 3 )   679 - 86   1997.2

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    The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

    DOI: 10.1016/S0093-691X(97)00026-5

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  • Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro

    H. Funahashi, T. C. Cantley, B. N. Day

    Theriogenology   47 ( 3 )   679 - 686   1997

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    The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 10% porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 ± 3.1%) was higher in the experimental group than in the control group (8.7 ± 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

    DOI: 10.1016/S0093-691X(97)00026-5

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  • Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

    Fifth International Conference on Pig Reproduction   142   1997

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  • Production of hyaluronic acid by porcine oocyte-cumulus cell complexes during in vitro maturation.

    Fifth International Conference on Pig Reproduction   139   1997

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  • Tris-buffered medium (TBM) is better for in vitro fertilization of porcine oocytes than mM199 which has been traditionally used.

    H Funahashi, Y Kajiwara, K Sawai, K Niwa

    BIOLOGY OF REPRODUCTION   56 ( Supplementl )   514 - 514   1997

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  • Effect of tissue inhibitor of metalloproteinase(TIMP-1)on early development of seine oocytes matured and fertilized in vitro(共著)

    Theriogenology   46 ( 1 )   277   1997

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  • Effects of pig transparent layer sugar moiety on localization and spermatozoon combination/invasion.

    宋学雄, 楊信志, 舟橋弘晃, 丹羽こう二

    日本繁殖生物学会講演要旨   ( 90th )   56   1997

  • クローン動物とコピー動物

    岡山地区高分子懇話会平成9年度第2回高分子講演会   11 - 18   1997

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  • Advances in in vitro production of pig embryos

    H Funahashi, BN Day

    JOURNAL OF REPRODUCTION AND FERTILITY   Supplement52   271 - 283   1997

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    A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

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  • Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

    Fifth International Conference on Pig Reproduction   142   1997

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  • Production of hyaluronic acid by porcine oocyte-cumulus cell complexes during in vitro maturation.

    Fifth International Conference on Pig Reproduction   139   1997

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  • Effect of tissue inhibitor of metalloproteinase (TIMP-1) on early development of swine oocytes matured and fertilized in vitro

    FUNAHASHI H

    Theriogenology   47 ( 1 )   277 - 277   1997

  • Tris-buffered medium (TBM) is better for in vitro fertilization of porcine oocytes than mM199 which has been traditionally used.

    H Funahashi, Y Kajiwara, K Sawai, K Niwa

    BIOLOGY OF REPRODUCTION   56 ( Supplementl )   514 - 514   1997

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  • Advances in in vitro production of pig embryos

    H Funahashi, BN Day

    JOURNAL OF REPRODUCTION AND FERTILITY   Supplement52   271 - 283   1997

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    Language:English   Publisher:J REPROD FERTIL INC  

    A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

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  • Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocytes penetrated in vitro

    Ken Sawai, Hiroaki Funahashi, Wei Hua Wang, Koji Niwa

    Journal of Reproduction and Development   43 ( 1 )   73 - 80   1997

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    Pig immature oocytes were cultured for 24, 36 and 48 h in serum-free maturation medium with or without 0.57 mM cysteine. The addition of cysteine to the medium was associated with increased glutathione synthesis by oocytes but did not promote nor inhibit nuclear maturation, sperm penetration in vitro, and decondensation of sperm nuclei in penetrated oocytes. The incidence of activation of penetrated oocytes 14 h after insemination in vitro was lower in those cultured both in the presence and absence of cysteine for 24 h than 36 and 48 h. The lower ability of oocytes cultured for 24 h to be activated was not improved by neither the addition of cysteine (0.57 mM) in fertilization medium nor prolonged culture time after insemination. However, male pronuclear formation in activated oocytes after sperm penetration at any stages of maturation was largely accelerated when cysteine was added to maturation medium. An increased concentration of glutathione may induce full decondensation of sperm nuclei in immature pig oocytes penetrated in vitro ensuring transformation of the decondensed sperm nuclei to male pronuclei only in synchronization with oocyte activation.

    DOI: 10.1262/jrd.43.73

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  • Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocytes penetrated in vitro

    Ken Sawai, Hiroaki Funahashi, Wei Hua Wang, Koji Niwa

    Journal of Reproduction and Development   43 ( 1 )   73 - 80   1997

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    Language:English   Publisher:The Japanese Society of Animal Reproduction (JSAR)  

    Pig immature oocytes were cultured for 24, 36 and 48 h in serum-free maturation medium with or without 0.57 mM cysteine. The addition of cysteine to the medium was associated with increased glutathione synthesis by oocytes but did not promote nor inhibit nuclear maturation, sperm penetration in vitro, and decondensation of sperm nuclei in penetrated oocytes. The incidence of activation of penetrated oocytes 14 h after insemination in vitro was lower in those cultured both in the presence and absence of cysteine for 24 h than 36 and 48 h. The lower ability of oocytes cultured for 24 h to be activated was not improved by neither the addition of cysteine (0.57 mM) in fertilization medium nor prolonged culture time after insemination. However, male pronuclear formation in activated oocytes after sperm penetration at any stages of maturation was largely accelerated when cysteine was added to maturation medium. An increased concentration of glutathione may induce full decondensation of sperm nuclei in immature pig oocytes penetrated in vitro ensuring transformation of the decondensed sperm nuclei to male pronuclei only in synchronization with oocyte activation.

    DOI: 10.1262/jrd.43.73

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  • Freezing preservation of pig spermatozoon using a 0.5ml straw for cattle semen.

    野上与志郎, 原田護, 関哲生, 舟橋弘晃, 丹羽こう二

    日本畜産学会大会講演要旨   93rd   1997

  • Effect of NaCl concentration in fertilization medium on sperm penetration and ealry development of rat eggs.

    OH S-H, 舟橋弘晃, 丹羽こう二

    Journal of mammalian ova research   14 ( 1 )   1997

  • Advances in in vitro production of pig embryos. International journal

    H. Funahashi, B. N. Day

    Journal of reproduction and fertility. Supplement   52   271 - 283   1997

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    Language:English  

    A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

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  • gamma-Glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs. International journal

    H Funahashi, Z Machaty, R S Prather, B N Day

    Molecular reproduction and development   45 ( 4 )   485 - 90   1996.12

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    The presence of gamma-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 +/- 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 +/- 1.3%) at 6 h after injection. Injected GTP-gamma-S, however, activated 76.0 +/- 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 +/- 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-gamma-S injected oocytes (4.2 +/- 0.7 pmol/oocyte) and noninjected oocytes (4.0 +/- 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 +/- 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 +/- 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-gamma-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes.

    DOI: 10.1002/(SICI)1098-2795(199612)45:4<485::AID-MRD11>3.0.CO;2-W

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  • Microtubule organization in porcine oocytes during fertilization and parthenogenesis International journal

    NH Kim, C Simerly, H Funahashi, G Schatten, BN Day

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1397 - 1404   1996.6

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    Microtubule configurations in porcine oocytes after sperm penetration or after artificial activation by electrical stimulation were imaged by immunocytochemistry and laser scanning confocal microscopy. Soon after sperm penetration, an aster was seen adjacent to the incorporated sperm head. Polyspermic penetrations led to the presence of multiple sperm asters in association with each sperm. The sperm aster enlarged and, at the time of pronuclear apposition, filled the cytoplasm. After male and female gamete union, the microtubule matrix was reduced. At the mitotic metaphase stage, microtubules were detected in the spindle, which was anastral and fusiform. At anaphase, asters assembled at each spindle pole, and at telophase, large asters filled the cytoplasm. Artificial activation by electrical stimulation induced in the cytoplasm a dense network of microtubules, which seem to be involved in proper positioning of the female pronucleus. At mitotic metaphase, microtubules were concentrated around the chromatin. The results of experiments using taxol, a microtubule stabilizing agent, suggest that maternal centrosomal material is present in the mature porcine oocyte as dispersed undetectable material that can form a microtubule network after parthenogenetic activation. However, at fertilization, the paternal centrosome collects centrosomal material to form a sperm aster. These results suggest that the functional centrosome that forms during fertilization is a result of the blending of paternal and maternal centrosomal components.

    DOI: 10.1095/biolreprod54.6.1397

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  • Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes

    H Funahashi, NH Kim, TT Stumpf, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1412 - 1419   1996.6

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    The effects of organic osmolytes on cytoplasmic maturation of porcine oocytes were examined in maturation medium (modified Whitten's medium) containing various NaCl concentrations. The presence of organic osmolytes, such as taurine and sorbitol, at 6 and 12 mM in maturation medium containing 68.49 or 92.40 mM NaCl increased oocyte glutathione content. Microfilament organization in oocytes was disrupted in maturation medium containing the higher level of NaCl (92.40 mM). However, supplementation with 12 mM sorbitol to the medium reduced the severity of the abnormality. Early embryonic development in vitro to the blastocyst stage was 8.3 +/- 0.9% for oocytes matured in modified Whitten's medium (68.49 mM NaCl) supplemented with 12 mM sorbitol, and 7.9 +/- 0.8% in modified NCSU23 medium (containing 108.73 mM NaCl, 7 mM taurine, 5 mM hypotaurine, and 1 mM glutamine), compared to 4.7 +/- 0.6% in modified Whitten's medium (68.49 mM NaCl), which did not contain organic osmolytes. These results indicate that the presence of organic osmolytes, such as sorbitol and taurine, reduces the detrimental effects of high NaCl concentration in media used for the maturation of porcine oocytes. This effect is reflected by oocyte glutathione content and microfilament organization at the end of maturation and early development following in vitro maturation and in vitro fertilization.

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  • Microtubule organization in porcine oocytes during fertilization and parthenogenesis

    NH Kim, C Simerly, H Funahashi, G Schatten, BN Day

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1397 - 1404   1996.6

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    Language:English   Publisher:SOC STUDY REPRODUCTION  

    Microtubule configurations in porcine oocytes after sperm penetration or after artificial activation by electrical stimulation were imaged by immunocytochemistry and laser scanning confocal microscopy. Soon after sperm penetration, an aster was seen adjacent to the incorporated sperm head. Polyspermic penetrations led to the presence of multiple sperm asters in association with each sperm. The sperm aster enlarged and, at the time of pronuclear apposition, filled the cytoplasm. After male and female gamete union, the microtubule matrix was reduced. At the mitotic metaphase stage, microtubules were detected in the spindle, which was anastral and fusiform. At anaphase, asters assembled at each spindle pole, and at telophase, large asters filled the cytoplasm. Artificial activation by electrical stimulation induced in the cytoplasm a dense network of microtubules, which seem to be involved in proper positioning of the female pronucleus. At mitotic metaphase, microtubules were concentrated around the chromatin. The results of experiments using taxol, a microtubule stabilizing agent, suggest that maternal centrosomal material is present in the mature porcine oocyte as dispersed undetectable material that can form a microtubule network after parthenogenetic activation. However, at fertilization, the paternal centrosome collects centrosomal material to form a sperm aster. These results suggest that the functional centrosome that forms during fertilization is a result of the blending of paternal and maternal centrosomal components.

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  • Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes International journal

    H Funahashi, NH Kim, TT Stumpf, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1412 - 1419   1996.6

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    Language:English   Publisher:SOC STUDY REPRODUCTION  

    The effects of organic osmolytes on cytoplasmic maturation of porcine oocytes were examined in maturation medium (modified Whitten's medium) containing various NaCl concentrations. The presence of organic osmolytes, such as taurine and sorbitol, at 6 and 12 mM in maturation medium containing 68.49 or 92.40 mM NaCl increased oocyte glutathione content. Microfilament organization in oocytes was disrupted in maturation medium containing the higher level of NaCl (92.40 mM). However, supplementation with 12 mM sorbitol to the medium reduced the severity of the abnormality. Early embryonic development in vitro to the blastocyst stage was 8.3 +/- 0.9% for oocytes matured in modified Whitten's medium (68.49 mM NaCl) supplemented with 12 mM sorbitol, and 7.9 +/- 0.8% in modified NCSU23 medium (containing 108.73 mM NaCl, 7 mM taurine, 5 mM hypotaurine, and 1 mM glutamine), compared to 4.7 +/- 0.6% in modified Whitten's medium (68.49 mM NaCl), which did not contain organic osmolytes. These results indicate that the presence of organic osmolytes, such as sorbitol and taurine, reduces the detrimental effects of high NaCl concentration in media used for the maturation of porcine oocytes. This effect is reflected by oocyte glutathione content and microfilament organization at the end of maturation and early development following in vitro maturation and in vitro fertilization.

    DOI: 10.1095/biolreprod54.6.1412

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  • Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro International journal

    NH Kim, H Funahashi, LR Abeydeera, SJ Moon, RS Prather, BN Day

    JOURNAL OF REPRODUCTION AND FERTILITY   107 ( 1 )   79 - 86   1996.5

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    The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

    DOI: 10.1530/jrf.0.1070079

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  • Effects of injecting calcium chloride into in vitro-matured porcine oocytes International journal

    Z Machaty, H Funahashi, MA Mayes, BN Day, RS Prather

    BIOLOGY OF REPRODUCTION   54 ( 2 )   316 - 322   1996.2

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    In vitro-matured porcine oocytes were given injections of 0.1 M CaCl2 and after 6 h evaluated for signs of early and late activation events. CaCl2 injection caused cortical granule exocytosis in 75% (3 of 4) of the oocytes tested. It also induced cell cycle resumption as monitored by the histone H1 kinase assay: the phosphorylation rate of histone H1 decreased to 36.7% of the original value. Treated oocytes completed meiosis, extruded the second polar body, and progressed to first interphase: 79.4% of them formed one or more pronuclei. The elevated intracellular Ca2+ level resulted in activation-related changes in the protein synthetic profile in 90% (9 of 10) of the oocytes. Furthermore, 14.7% (9 of 61) of the treated oocytes developed to the compact morula/early blastocyst stage after a 7-day culture in ligated porcine oviduct, and one blastocyst hatched from the zona pellucida. Control oocytes given injections of 0.1 M MgCl2 or carrier medium (10 mM Hepes) did not show the changes mentioned. The results strengthen the idea that Ca2+ is a cell messenger that plays a central part in oocyte activation; it is concluded that elevated intracellular Ca2+ level caused by a single injection of CaCl2 leads to both early and late events of porcine oocyte activation.

    DOI: 10.1095/biolreprod54.2.316

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  • Effects of injecting calcium chloride into in vitro-matured porcine oocytes

    Z Machaty, H Funahashi, MA Mayes, BN Day, RS Prather

    BIOLOGY OF REPRODUCTION   54 ( 2 )   316 - 322   1996.2

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    In vitro-matured porcine oocytes were given injections of 0.1 M CaCl2 and after 6 h evaluated for signs of early and late activation events. CaCl2 injection caused cortical granule exocytosis in 75% (3 of 4) of the oocytes tested. It also induced cell cycle resumption as monitored by the histone H1 kinase assay: the phosphorylation rate of histone H1 decreased to 36.7% of the original value. Treated oocytes completed meiosis, extruded the second polar body, and progressed to first interphase: 79.4% of them formed one or more pronuclei. The elevated intracellular Ca2+ level resulted in activation-related changes in the protein synthetic profile in 90% (9 of 10) of the oocytes. Furthermore, 14.7% (9 of 61) of the treated oocytes developed to the compact morula/early blastocyst stage after a 7-day culture in ligated porcine oviduct, and one blastocyst hatched from the zona pellucida. Control oocytes given injections of 0.1 M MgCl2 or carrier medium (10 mM Hepes) did not show the changes mentioned. The results strengthen the idea that Ca2+ is a cell messenger that plays a central part in oocyte activation; it is concluded that elevated intracellular Ca2+ level caused by a single injection of CaCl2 leads to both early and late events of porcine oocyte activation.

    DOI: 10.1095/biolreprod54.2.316

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  • Co-culture with follicular shell pieces (FSP) increases the developmental competence of pig oocytes matured in vitro.

    LR Abeydeera, H Funahashi, TC Cantley, A Rieke, BN Day

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   100 - 100   1996

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  • In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

    Theriogenology   45 ( 1 )   265   1996

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  • Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

    Journal of Reproduction and Development   42 ( 2 )   109 - 115   1996

  • Factors affecting nuclear and cytoplasmic maturation in pig oocytes

    P Coy, S Ruiz, N Ouhibi, H Funahashi, BN Day, RM Moor

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   412 - 412   1996

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  • Co-culture with follicular shell pieces (FSP) increases the developmental competence of pig oocytes matured in vitro.

    LR Abeydeera, H Funahashi, TC Cantley, A Rieke, BN Day

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   100 - 100   1996

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  • Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.

    H Funahashi, H Tatemoto, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   408 - 408   1996

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  • Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

    Congress Programme of the 13th International Congress on Animal Reproduction   2   10 - 4   1996

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  • In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

    Theriogenology   45 ( 1 )   265   1996

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  • Gamma-glutamyltranspeptidase of spermatozoa may reduce oocyte glutathione content at sperm penetration(共著)

    Hiroaki Funahashi, Zoltan Machaty, Randall S. Prather, Billy N. Day

    Molecular Reproduction and Development   45 ( 4 )   485 - 490   1996

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    The presence of γ-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine 5'-O-(3'-thiotriphosphate) (GTP-γ-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 ± 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 ± 1.3%) at 6 h after injection. Injected GTP-γ-S, however, activated 76.0 ± 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 ± 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-γ-S injected oocytes (4.2 ± 0.7 pmol/oocyte) and noninjected oocytes (4.0 ± 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 ± 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 ± 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP- γ-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes.

    DOI: 10.1002/(SICI)1098-2795(199612)45:4<485::AID-MRD11>3.0.CO;2-W

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  • Factors Affecting Development in Vitro of Bovine and Rat 1-Cell Embryos

    Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

    Journal of Mammalian Ova Research   13 ( 2 )   71 - 80   1996

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  • Factors Affecting Development in Vitro of Bovine and Rat 1-Cell Embryos

    Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

    Journal of Mammalian Ova Research   13 ( 2 )   71 - 80   1996

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  • Factors affecting nuclear and cytoplasmic maturation in pig oocytes

    P Coy, S Ruiz, N Ouhibi, H Funahashi, BN Day, RM Moor

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   412 - 412   1996

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  • Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

    Congress Programme of the 13th International Congress on Animal Reproduction   2   10 - 4   1996

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  • Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

    Hiroaki FUNAHASHI, Todd T. STUMPF, Nam-Hyung KIM, Billy N. DAY

    Journal of Reproduction and Development   42 ( 2 )   109 - 115   1996

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    The effect of NaCl in maturation media on histone H1 kinase activity of porcine oocytes was examined at the end of culture for maturation. Oocyte-cumulus complexes were cultured in modified Whitten's medium containing different concentrations of NaCl (44.50, 68.49, 92.40, 116.40 or 140.35 mM). The media were supplemented with 10% porcine follicular fluid and hormones for 20 h and then without hormonal supplements for an additional 20 h. At the end of culture, oocytes were sampled for morphological observation, for glutathione assay and for histone H1 kinase assay. At the end of culture, the incidence of oocytes at each meiotic stage was not different among groups. However, histone H1 kinase activity and glutathione content were higher (P < 0.01) in oocytes cultured in media with lower NaCl concentrations. Histone H1 kinase activity in the oocytes at the end of maturation culture was correlated with intracellular glutathione content (r = 0.899, P < 0.01). These data indicate that histone H1 kinase activity and glutathione content of porcine oocytes at the end of culture for maturation are reduced when a high salt medium is used for maturation.

    DOI: 10.1262/jrd.42.109

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  • Effect of saccharides on the semination into a pig ovum.

    宋学雄, 沢井健, 許東けい, 舟橋弘晃, 丹羽こう二

    日本繁殖生物学会講演要旨   89th   57   1996

  • Effect of cysteine and cystine on the male pronucleus forming ability of pig’s follicular ovum.

    沢井健, 舟橋弘晃, 丹羽こう二

    日本繁殖生物学会講演要旨   89th   44   1996

  • Effect of somatotropin on the progress rate of the maturation division of cattle’s follicular ovum.

    伊賀浩輔, 舟橋弘晃, 丹羽こう二

    日本繁殖生物学会講演要旨   89th   41   1996

  • Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.

    H Funahashi, H Tatemoto, TC Cantley, BN Day

    BIOLOGY OF REPRODUCTION   54 ( Supplement 1 )   408 - 408   1996

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  • PRONUCLEAR FORMATION AND INTRACELLULAR GLUTATHIONE CONTENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING IN-VITRO FERTILIZATION AND/OR ELECTRICAL ACTIVATION

    H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

    ZYGOTE   3 ( 3 )   273 - 281   1995.8

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    Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

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  • PRONUCLEAR VISIBILITY, DEVELOPMENT AND TRANSGENE EXPRESSION IN IVM/IVF-DERIVED PORCINE EMBRYOS

    HM KUBISCH, MA LARSON, H FUNAHASHI, BN DAY, RM ROBERTS

    THERIOGENOLOGY   44 ( 3 )   391 - 401   1995.8

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    A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). Alter 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

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  • PRONUCLEAR FORMATION AND INTRACELLULAR GLUTATHIONE CONTENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING IN-VITRO FERTILIZATION AND/OR ELECTRICAL ACTIVATION International journal

    H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

    ZYGOTE   3 ( 3 )   273 - 281   1995.8

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    Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

    DOI: 10.1017/S0967199400002677

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  • PRONUCLEAR VISIBILITY, DEVELOPMENT AND TRANSGENE EXPRESSION IN IVM/IVF-DERIVED PORCINE EMBRYOS

    HM KUBISCH, MA LARSON, H FUNAHASHI, BN DAY, RM ROBERTS

    THERIOGENOLOGY   44 ( 3 )   391 - 401   1995.8

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    A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). Alter 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

    DOI: 10.1016/0093-691X(95)00193-C

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  • EFFECT OF BICARBONATE ON IN-VITRO PENETRATION OF PIG OOCYTES BY COMMERCIALLY PREPARED FROZEN-EJACULATED BOAR SEMEN

    LR ABEYDEERA, H FUNAHASHI, NH KIM, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement 1 )   122 - 122   1995

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  • EFFECT OF TAXOL ON MICROTUBULE ORGANIZATION IN PORCINE OOCYTES AFTER FERTILIZATION AND PARTHENOGENETIC ACTIVATION

    NH KIM, LR ABEYDEERA, RE EVERTS, H FUNAHASHI, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement 1 )   139 - 139   1995

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  • Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

    Theriogenology   43 ( 1 )   213   1995

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  • GAMMA-GLUTAMYL-TRANSPEPTIDASE (GGT) OF SPERMATOZOA MAY REDUCE OOCYTE GLUTATHIONE (GSH) CONTENT AT SPERM PENETRATION

    H FUNAHASHI, Z MACHATY, RS PRATHER, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement1 )   140 - 140   1995

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  • Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著) Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's

    Role in the Genetic Improvement of Farm Animals   19   1995

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  • Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation International journal

    Nam Hyung Kim, Hiroaki Funahashi, Randall S. Prather, Gerald Schatten, Billy N. Day

    Molecular reproduction and development   43 ( 2 )   248 - 255   1995

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    Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic loci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

    DOI: 10.1002/(SICI)1098-2795(199602)43:2<248::AID-MRD14>3.0.CO;2-#

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  • Microtuble and microfilament dynamics in porcine oocytes during meiotic maturation.(共著)

    Molecular Reproduction and Development   43 ( 2 )   248 - 255   1995

  • EFFECT OF TAXOL ON MICROTUBULE ORGANIZATION IN PORCINE OOCYTES AFTER FERTILIZATION AND PARTHENOGENETIC ACTIVATION

    NH KIM, LR ABEYDEERA, RE EVERTS, H FUNAHASHI, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement 1 )   139 - 139   1995

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  • EFFECT OF BICARBONATE ON IN-VITRO PENETRATION OF PIG OOCYTES BY COMMERCIALLY PREPARED FROZEN-EJACULATED BOAR SEMEN

    LR ABEYDEERA, H FUNAHASHI, NH KIM, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement 1 )   122 - 122   1995

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  • Effects of cumulus cells on glutathione content of porcine oocyte during in vitro maturation

    FUNAHASHI H

    J Anim Sci   73 ( 1 )   90 - 90   1995

  • Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

    Journal of Animal Science   73 ( Supplement 1 )   90   1995

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  • Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

    Theriogenology   43 ( 1 )   262   1995

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  • Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

    Theriogenology   43 ( 1 )   213   1995

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  • GAMMA-GLUTAMYL-TRANSPEPTIDASE (GGT) OF SPERMATOZOA MAY REDUCE OOCYTE GLUTATHIONE (GSH) CONTENT AT SPERM PENETRATION

    H FUNAHASHI, Z MACHATY, RS PRATHER, BN DAY

    BIOLOGY OF REPRODUCTION   52 ( Supplement1 )   140 - 140   1995

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  • Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著)

    Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's Role in the Genetic Improvement of Farm Animals   19   1995

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  • Effects of cumulus cells on glutathione content of porcine oocytes during in vitro maturation.(共著)

    Journal of Animal Science   73 ( Supplement 1 )   90   1995

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  • Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

    Journal of Animal Science   73 ( Supplement 1 )   90   1995

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  • Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

    Theriogenology   43 ( 1 )   262   1995

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  • USE OF LOW-SALT CULTURE-MEDIUM FOR IN-VITRO MATURATION OF PORCINE OOCYTES IS ASSOCIATED WITH ELEVATED OOCYTE GLUTATHIONE LEVELS AND ENHANCED MALE PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   51 ( 4 )   633 - 639   1994.10

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    The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supple ments; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl(59.3 +/- 8.1 and 70.9 +/- 2.0%,respectively) than in medium containing 68.49 mM NaCl (33.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

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  • USE OF LOW-SALT CULTURE-MEDIUM FOR IN-VITRO MATURATION OF PORCINE OOCYTES IS ASSOCIATED WITH ELEVATED OOCYTE GLUTATHIONE LEVELS AND ENHANCED MALE PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION International journal

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   51 ( 4 )   633 - 639   1994.10

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    The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supple ments; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl(59.3 +/- 8.1 and 70.9 +/- 2.0%,respectively) than in medium containing 68.49 mM NaCl (33.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

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  • DEVELOPMENTAL ABILITY OF PORCINE OOCYTES MATURED AND FERTILIZED IN-VITRO

    H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

    THERIOGENOLOGY   41 ( 7 )   1425 - 1433   1994.5

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    The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17 beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42/71) and with monospermic penetration and male and female pronuclei (32%, 23/71) were higher (P &lt; 0.01) in the PFF-treated group than in controls (25%, 18/71 and 8%, 6/71, respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28/288 and 13%, 41/318, respectively) were also higher (P &lt; 0.05) than in controls (2%, 6/284 and 6%, 16/248, respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9/288 in PFF-treated group and 2%, 6/284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3/100) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

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  • IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( 5 )   1072 - 1077   1994.5

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    Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p &lt; 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p &lt; 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (P &lt; 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p &lt; 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p &lt; 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (P &lt; 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes) as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

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  • DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO International journal

    H FUNAHASHI, T CANTLEY, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   101 ( 1 )   159 - 165   1994.5

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    Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml(-1), hCG (10 iu ml(-1)) and oestradiol (1 mu g ml(-1)) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 +/- 72.6 ng ml(-1)) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 +/- 13.3 ng ml(-1)), during the first 20 h of culture (42.8 +/- 19.4 ng ml(-1)) and also during the second 20 h period of culture without hormonal supplements (0.925 +/- 0.544 ng ml(-1)). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 +/- 0.2 ng ml(-1) at 0 h to 6.9 +/- 1.5 ng ml(-1) after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.

    DOI: 10.1530/jrf.0.1010159

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  • DEVELOPMENTAL ABILITY OF PORCINE OOCYTES MATURED AND FERTILIZED IN-VITRO International journal

    H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

    THERIOGENOLOGY   41 ( 7 )   1425 - 1433   1994.5

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    The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17 beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42/71) and with monospermic penetration and male and female pronuclei (32%, 23/71) were higher (P &lt; 0.01) in the PFF-treated group than in controls (25%, 18/71 and 8%, 6/71, respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28/288 and 13%, 41/318, respectively) were also higher (P &lt; 0.05) than in controls (2%, 6/284 and 6%, 16/248, respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9/288 in PFF-treated group and 2%, 6/284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3/100) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

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  • IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION International journal

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( 5 )   1072 - 1077   1994.5

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    Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p &lt; 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p &lt; 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (P &lt; 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p &lt; 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p &lt; 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (P &lt; 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes) as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

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  • DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO

    H FUNAHASHI, T CANTLEY, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   101 ( 1 )   159 - 165   1994.5

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    Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml(-1), hCG (10 iu ml(-1)) and oestradiol (1 mu g ml(-1)) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 +/- 72.6 ng ml(-1)) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 +/- 13.3 ng ml(-1)), during the first 20 h of culture (42.8 +/- 19.4 ng ml(-1)) and also during the second 20 h period of culture without hormonal supplements (0.925 +/- 0.544 ng ml(-1)). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 +/- 0.2 ng ml(-1) at 0 h to 6.9 +/- 1.5 ng ml(-1) after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.

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  • DEVELOPMENT OF RAT ONE-CELL EMBRYOS IN A CHEMICALLY-DEFINED MEDIUM - EFFECTS OF GLUCOSE, PHOSPHATE AND OSMOLARITY

    K MIYOSHI, H FUNAHASHI, K OKUDA, K NIWA

    JOURNAL OF REPRODUCTION AND FERTILITY   100 ( 1 )   21 - 26   1994.1

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    Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l(-1) (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-(1). This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l(-1) and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mu mol phosphate l(-1), but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mu mol phosphate l(-1). In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l(-1) (67%) and 10.0 mmol glucose l(-1) (60%) were not statistically different from the percentage at 5.0 mmol glucose l(-1) (46%), but was significantly greater than the percentage at 2.5 mmol l(-1) (33%). When osmolarity of the medium with 5.0 mmol glucose l(-1) was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the few-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

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  • DEVELOPMENT OF RAT ONE-CELL EMBRYOS IN A CHEMICALLY-DEFINED MEDIUM - EFFECTS OF GLUCOSE, PHOSPHATE AND OSMOLARITY

    K MIYOSHI, H FUNAHASHI, K OKUDA, K NIWA

    JOURNAL OF REPRODUCTION AND FERTILITY   100 ( 1 )   21 - 26   1994.1

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    Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l(-1) (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-(1). This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l(-1) and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mu mol phosphate l(-1), but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mu mol phosphate l(-1). In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l(-1) (67%) and 10.0 mmol glucose l(-1) (60%) were not statistically different from the percentage at 5.0 mmol glucose l(-1) (46%), but was significantly greater than the percentage at 2.5 mmol l(-1) (33%). When osmolarity of the medium with 5.0 mmol glucose l(-1) was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the few-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

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  • Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   71   1994

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  • HISTONE-H1 KINASE (H1K) ACTIVITY AND GLUTATHIONE (GSH) CONTENT OF PORCINE OOCYTES DURING PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

    H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   128 - 128   1994

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  • Histone H1 kinase activity after electrical activation of in vitro matured porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   74   1994

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  • Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   71   1994

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  • Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   75   1994

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  • MICROTUBULE ORGANIZATION OF PORCINE OOCYTES DURING IN-VITRO FERTILIZATION

    NH KIM, H FUNAHASHI, TT STUMPF, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   146 - 146   1994

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  • 受精卵あるいは卵子輸送装置.(共著)

    特許公報   特公平6-94401   1994

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  • MICROTUBULE ORGANIZATION OF PORCINE OOCYTES DURING IN-VITRO FERTILIZATION

    NH KIM, H FUNAHASHI, TT STUMPF, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   146 - 146   1994

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  • Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   75   1994

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  • In vitro maturation/in vitro fertilization of porcine oocytes.(共著)

    Proceeding for Annual Meeting of the Society for Theriogenology   206 - 214   1994

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  • HISTONE-H1 KINASE (H1K) ACTIVITY AND GLUTATHIONE (GSH) CONTENT OF PORCINE OOCYTES DURING PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

    H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( Supplement 1 )   128 - 128   1994

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  • Effects of sodium chloride concentration in maturation media on histone H1 kinase(H1K)activity and cytoplasmic maturation of porcine oocytes.(共著)

    Journal of Reproduction and Fertility   72 ( Abstract Series 13 )   38   1994

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  • Histone H1 kinase activity after electrical activation of in vitro matured porcine oocytes.(共著)

    Journal of Animal Science   72 ( Supplement 1 )   74   1994

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  • EFFECTS OF ELECTRICAL-STIMULATION BEFORE OR AFTER IN-VITRO FERTILIZATION ON SPERM PENETRATION AND PRONUCLEAR FORMATION OF PIG OOCYTES

    H FUNAHASHI, TT STUMPF, SL TERLOUW, BN DAY

    MOLECULAR REPRODUCTION AND DEVELOPMENT   36 ( 3 )   361 - 367   1993.11

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    The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm-1 AC followed by a 30 musec pulse at 120 V mm-1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88-100%) and polyspermic rates (79-100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 +/- 0.5 vs. 4.6 +/- 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. (C) 1993 Wiley-Liss, Inc.

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  • EFFECTS OF ELECTRICAL-STIMULATION BEFORE OR AFTER IN-VITRO FERTILIZATION ON SPERM PENETRATION AND PRONUCLEAR FORMATION OF PIG OOCYTES International journal

    H FUNAHASHI, TT STUMPF, SL TERLOUW, BN DAY

    MOLECULAR REPRODUCTION AND DEVELOPMENT   36 ( 3 )   361 - 367   1993.11

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    The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm-1 AC followed by a 30 musec pulse at 120 V mm-1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88-100%) and polyspermic rates (79-100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 +/- 0.5 vs. 4.6 +/- 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. (C) 1993 Wiley-Liss, Inc.

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  • EFFECTS OF FOLLICULAR-FLUID AT FERTILIZATION IN-VITRO ON SPERM PENETRATION IN PIG OOCYTES International journal

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   99 ( 1 )   97 - 103   1993.9

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    The effects of porcine follicular fluid (PFF) on sperm penetration of pig oocytes and on prevention of polyspermy were examined and characteristics of spermatozoa exposed to PFF were determined. The addition of PFF at the level of 1 and 10% to the prefertilization and fertilization media decreased penetration rates and the mean number of spermatozoa in penetrated eggs regardless of the origin of PFF. In the presence of BSA, supplementation of 0.1% PFF to prefertilization and fertilization media and 1% PFF to prefertilization media did not decrease the penetration rates but did increase monospermic penetration to 54 and 68%, respectively. When PFF was added to prefertilization media, the number of spermatozoa binding to the zona and the percentage of acrosome-intact spermatozoa decreased with increased PFF concentration (from 43.1 +/- 2.8 and 73.1 +/- 4.9% to 7.2 +/- 1.3 and 15.7 +/- 15.4%, respectively). At the end of prefertilization incubation, sperm agglutination was observed and the degree depended on PFF concentration. Supplementation of fetal calf serum to prefertilization and fertilization media blocked the effects of PFF on sperm penetration and binding of spermatozoa to the zona. These results indicate that the prefertilization incubation of porcine spermatozoa in suitable concentrations of porcine follicular fluid will effectively reduce both the number of spermatozoa that attach to the surface of pig eggs and the incidence of polyspermy.

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  • EFFECTS OF FOLLICULAR-FLUID AT FERTILIZATION IN-VITRO ON SPERM PENETRATION IN PIG OOCYTES

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   99 ( 1 )   97 - 103   1993.9

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    The effects of porcine follicular fluid (PFF) on sperm penetration of pig oocytes and on prevention of polyspermy were examined and characteristics of spermatozoa exposed to PFF were determined. The addition of PFF at the level of 1 and 10% to the prefertilization and fertilization media decreased penetration rates and the mean number of spermatozoa in penetrated eggs regardless of the origin of PFF. In the presence of BSA, supplementation of 0.1% PFF to prefertilization and fertilization media and 1% PFF to prefertilization media did not decrease the penetration rates but did increase monospermic penetration to 54 and 68%, respectively. When PFF was added to prefertilization media, the number of spermatozoa binding to the zona and the percentage of acrosome-intact spermatozoa decreased with increased PFF concentration (from 43.1 +/- 2.8 and 73.1 +/- 4.9% to 7.2 +/- 1.3 and 15.7 +/- 15.4%, respectively). At the end of prefertilization incubation, sperm agglutination was observed and the degree depended on PFF concentration. Supplementation of fetal calf serum to prefertilization and fertilization media blocked the effects of PFF on sperm penetration and binding of spermatozoa to the zona. These results indicate that the prefertilization incubation of porcine spermatozoa in suitable concentrations of porcine follicular fluid will effectively reduce both the number of spermatozoa that attach to the surface of pig eggs and the incidence of polyspermy.

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  • EFFECTS OF THE DURATION OF EXPOSURE TO HORMONE SUPPLEMENTS ON CYTOPLASMIC MATURATION OF PIG OOCYTES INVITRO International journal

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   98 ( 1 )   179 - 185   1993.5

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    The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte-cumulus complexes in a medium with PMSG (10 iu ml-1), hCG (10 iu ml-1) and oestradiol (1 mug ml-1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte-cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact, and oocytectomized oocyte-cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte-cumulus complexes were improved after the removal of oocyte-cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte-cumulus complexes to hormone were similar to those of intact oocyte-cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

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  • EFFECTS OF THE DURATION OF EXPOSURE TO HORMONE SUPPLEMENTS ON CYTOPLASMIC MATURATION OF PIG OOCYTES INVITRO

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   98 ( 1 )   179 - 185   1993.5

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    The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte-cumulus complexes in a medium with PMSG (10 iu ml-1), hCG (10 iu ml-1) and oestradiol (1 mug ml-1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte-cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact, and oocytectomized oocyte-cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte-cumulus complexes were improved after the removal of oocyte-cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte-cumulus complexes to hormone were similar to those of intact oocyte-cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

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  • EFFECTS OF DIFFERENT SERUM SUPPLEMENTS IN MATURATION MEDIUM ON MEIOTIC CYTOPLASMIC MATURATION OF PIG OOCYTES International journal

    H FUNAHASHI, BN DAY

    THERIOGENOLOGY   39 ( 4 )   965 - 973   1993.4

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    The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P &lt; 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.

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  • EFFECTS OF DIFFERENT SERUM SUPPLEMENTS IN MATURATION MEDIUM ON MEIOTIC CYTOPLASMIC MATURATION OF PIG OOCYTES

    H FUNAHASHI, BN DAY

    THERIOGENOLOGY   39 ( 4 )   965 - 973   1993.4

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    The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P &lt; 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.

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  • GLUCOSE REQUIREMENT AT DIFFERENT DEVELOPMENTAL STAGES OF INVITRO FERTILIZED BOVINE EMBRYOS CULTURED IN SEMI-DEFINED MEDIUM

    JH KIM, H FUNAHASHI, K NIWA, K OKUDA

    THERIOGENOLOGY   39 ( 4 )   875 - 886   1993.4

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    Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).

    DOI: 10.1016/0093-691X(93)90425-5

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  • GLUCOSE REQUIREMENT AT DIFFERENT DEVELOPMENTAL STAGES OF INVITRO FERTILIZED BOVINE EMBRYOS CULTURED IN SEMI-DEFINED MEDIUM International journal

    JH KIM, H FUNAHASHI, K NIWA, K OKUDA

    THERIOGENOLOGY   39 ( 4 )   875 - 886   1993.4

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    Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).

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  • Assessment of porcine oocytes using brilliant cresyl blue.(共著)

    Theriogenology   39 ( 1 )   214   1993

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  • Cytoplasmic maturation and in vitro development of porcine oocytes matured in Whitten's medium and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   36   1993

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  • Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   23   1993

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  • Effects of hormonal conditions during last 20 h of in vitro maturation on cytoplasmic maturation and vitro fertilization of pig oocytes.(共著)

    Journal of Animal Science   71 ( Supplement 1 )   70   1993

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  • Pig oocyte activation and processing of transplanted nuclei.(共著)

    39 ( 1 )   329   1993

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  • Electrical stimulation of pig pig oocytes in the presence or absence of calcium and magnesium.(共著)

    Fourth International Conference on Pig Reproduction   42   1993

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  • Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Theriogenology   39 ( 1 )   322   1993

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  • Effects of artificial activation before in vitro fertilization on sperm penetration and pronuclear formation in pig oocytes.(共著)

    Theriogenology   39 ( 1 )   222   1993

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  • ラット前核期胚の体外発生におよぼすリン酸およびグルコ-スの影響.(共著)

    三好和睦, 丹羽こう二, 奥田潔, 舟橋弘晃

    家畜繁殖学会講演要旨   39 ( 5 )   a13   1993

  • Electrical stimulation of pig pig oocytes in the presence or absence of calcium and magnesium.(共著)

    Fourth International Conference on Pig Reproduction   42   1993

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  • Cytoplasmic maturation and in vitro development of porcine oocytes matured in Whitten's medium and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   36   1993

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  • Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   23   1993

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  • Premature chromosomal condensation of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Biology of Reproduction   48 ( Supplement 1 )   175   1993

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  • Pronuclear formation and in vitro development folowing artificial activation of pig oocytes matured different culture media.(共著)

    Biology of Reproduction   48 ( Supplement 1 )   171   1993

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  • Effects of hormonal conditions during last 20 h of in vitro maturation on cytoplasmic maturation and vitro fertilization of pig oocytes.(共著)

    Journal of Animal Science   71 ( Supplement 1 )   70   1993

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  • Pig oocyte activation and processing of transplanted nuclei.(共著)

    39 ( 1 )   329   1993

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  • Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Theriogenology   39 ( 1 )   322   1993

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  • Effects of artificial activation before in vitro fertilization on sperm penetration and pronuclear formation in pig oocytes.(共著)

    Theriogenology   39 ( 1 )   222   1993

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  • Premature chromosomal condensation of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Biology of Reproduction   48 ( Supplement 1 )   175   1993

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  • Pronuclear formation and in vitro development folowing artificial activation of pig oocytes matured different culture media.(共著)

    Biology of Reproduction   48 ( Supplement 1 )   171   1993

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  • Assessment of porcine oocytes using brilliant cresyl blue

    ERICSSON S

    Theriogenology   39 ( 1 )   214 - 214   1993

  • FERTILIZATION AND EARLY CLEAVAGE INVITRO OF AGING BOVINE OOCYTES AFTER MATURATION IN CULTURE International journal

    RC CHIAN, H NAKAHARA, K NIWA, H FUNAHASHI

    THERIOGENOLOGY   37 ( 3 )   665 - 672   1992.3

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    Bovine follicular oocytes cultured for 20 to 48 hours were inseminated with frozen thawed spermatozoa. Significantly higher penetration rates (94 to 100% vs 86 to 94%: P &lt; 0.05) and proportions of polyspermy (35 to 78% vs 22 to 45%: P &lt; 0.01) were obtained for oocytes cultured continuously for 24 hours with spermatozoa than for those separated from spermatozoa 8 hours after insemination. The most prominent effect of ageing of oocytes on early events of penetration was observed in the incidence of polyspermy rather than in the penetration rate and the proportion of pronuclear plus cleaved oocytes: the proportion of polyspermic oocytes significantly increased (P &lt; 0.05) in oocytes inseminated after 28 to 48 hours of culture (36 to 78%) compared with those cultured for 20 to 24 hours (22 to 35%) for maturation. Culture experiments for early development of penetrated oocytes indicated that no significant differences were observed in the proportions of oocytes cleaved to the two- to four- cell stage 48 hours after insemination among those cultured for 20 to 40 hours for maturation. However, further cleavage to the four- to sixteen-cell stage 72 to 96 hours after insemination was greatly inhibited as ageing of oocytes proceeded from 28 hours in culture for maturation.

    DOI: 10.1016/0093-691X(92)90146-I

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  • FERTILIZATION AND EARLY CLEAVAGE INVITRO OF AGING BOVINE OOCYTES AFTER MATURATION IN CULTURE

    RC CHIAN, H NAKAHARA, K NIWA, H FUNAHASHI

    THERIOGENOLOGY   37 ( 3 )   665 - 672   1992.3

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    Bovine follicular oocytes cultured for 20 to 48 hours were inseminated with frozen thawed spermatozoa. Significantly higher penetration rates (94 to 100% vs 86 to 94%: P &lt; 0.05) and proportions of polyspermy (35 to 78% vs 22 to 45%: P &lt; 0.01) were obtained for oocytes cultured continuously for 24 hours with spermatozoa than for those separated from spermatozoa 8 hours after insemination. The most prominent effect of ageing of oocytes on early events of penetration was observed in the incidence of polyspermy rather than in the penetration rate and the proportion of pronuclear plus cleaved oocytes: the proportion of polyspermic oocytes significantly increased (P &lt; 0.05) in oocytes inseminated after 28 to 48 hours of culture (36 to 78%) compared with those cultured for 20 to 24 hours (22 to 35%) for maturation. Culture experiments for early development of penetrated oocytes indicated that no significant differences were observed in the proportions of oocytes cleaved to the two- to four- cell stage 48 hours after insemination among those cultured for 20 to 40 hours for maturation. However, further cleavage to the four- to sixteen-cell stage 72 to 96 hours after insemination was greatly inhibited as ageing of oocytes proceeded from 28 hours in culture for maturation.

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  • 体外受精由来和牛胚の凍結融解後の生存性ならびに移植・分娩成績について.(共著)

    北海道牛受精卵移植研究会会報   ( 11 )   19 - 21   1992

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  • Developmental abilities of mouse and rat pronuclear eggs following interspecific pronuclear and cytoplasmic transplantation.(共著)

    Journal of Reproduction and Development   38 ( 3 )   179 - 183   1992

  • Developmental Abilities of Rat and Mouse Pronuclear Eggs Following Interspecific Nuclear or Cytoplasmic Transplantation

    Hiroaki Funahashp, Koji Niwa

    Journal of Reproduction and Development   38 ( 3 )   179 - 183   1992

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    Homologous and heterologous replacement of a small amount of cytoplasm or both pronuclei were performed in rat and mouse pronuclear eggs. Rat eggs after receiving rat or mouse cytoplast never developed beyond the 2-cell stage in vitro. On the other hand, the in-vitro development of mouse eggs that received rat cytoplasm was possible, but it was greatly inhibited compared with those that received mouse cytoplasm. None of enucleated rat eggs that received rat or mouse pronuclei also developed beyond the 2-cell stage. Although few enucleated mouse eggs after receiving rat pronuclei developed beyond the 2-cell stage, high proportion (64%) of those that received mouse pronuclei could develop to blastocysts in vitro. When 2-cell embryos obtained 18 h after fusing with donor karyoplast were transferred to pseudopregnant rats, 2 (9%) of 23 enucleated rat embryos and 1 (7%) of 15 enucleated mouse embryos each fused with heterologous karyoplast developed to early blastocyst 48-55 h after transfer. These results may indicate that the embryonic genome can be activated in the heterospecific cytoplasm. © 1992, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.

    DOI: 10.1262/jrd.38.179

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  • 牛体外受精卵の初期発生におよぼすグルコースの影響

    金宗興, 舟橋弘晃, 伊賀浩輔, 丹羽こう二

    日本畜産学会大会講演要旨   85th   1992

  • Study on repeated superovulation treatment of a cow using a corpus luteum hormone preparation.

    青柳敬人, 武富敏郎, 板倉はつえ, 亀山賢次, 舟橋弘晃, 武田哲男, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   ( 20(1991) )   1992

  • Experiment on transplantation of frozen in vitro fertilized bovine ova obtained under a complete in vitro culture system.

    青柳敬人, 武富敏郎, 舟橋弘晃

    JA全農飼料畜産中央研究所試験研究報告   ( 20(1991) )   1992

  • Experiment on production of in vitro fertilized bovine ova with donor identification.

    青柳敬人, 舟橋弘晃, 武田哲男

    JA全農飼料畜産中央研究所試験研究報告   ( 20(1991) )   1992

  • 1ST CLEAVAGE OF ENUCLEATED RAT EGGS FOLLOWING TRANSPLANTATION OF KARYOPLAST REMOVED FROM PRONUCLEAR EGGS STORED AT 2-DEGREES-C TO 6-DEGREES-C FOR VARIOUS DURATIONS

    K NIWA, H FUNAHASHI, K OHMAE, M KATTOH

    THERIOGENOLOGY   36 ( 3 )   411 - 417   1991.9

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    Pronuclear rat eggs were cultured for 24 to 48 hours at 37-degrees-C after storage at 2 to 6-degrees-C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6-degrees-C).

    DOI: 10.1016/0093-691X(91)90469-T

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  • 1ST CLEAVAGE OF ENUCLEATED RAT EGGS FOLLOWING TRANSPLANTATION OF KARYOPLAST REMOVED FROM PRONUCLEAR EGGS STORED AT 2-DEGREES-C TO 6-DEGREES-C FOR VARIOUS DURATIONS

    K NIWA, H FUNAHASHI, K OHMAE, M KATTOH

    THERIOGENOLOGY   36 ( 3 )   411 - 417   1991.9

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    Pronuclear rat eggs were cultured for 24 to 48 hours at 37-degrees-C after storage at 2 to 6-degrees-C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6-degrees-C).

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  • DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES COLLECTED FROM OVARIES OF INDIVIDUAL HEIFERS AND FERTILIZED INVITRO

    H FUNAHASHI, Y AOYAGI, T TAKEDA, T ONIHARA

    THERIOGENOLOGY   36 ( 3 )   427 - 434   1991.9

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    Bovine follicular oocytes from individual heifers (n = 49) were separately matured, fertilized with frozen-thawed spermatozoa and cultured with cumulus cells. Although there were great variations in the number (mean +/- SD = 19.1 +/- 11.9) of oocytes collected from individual heifers and the percentages of the oocytes cleaved 48 hours after insemination (mean +/- SD = 69.5 +/- 18.4) and developed to the morula stage 7 days after insemination (mean +/- SD = 10.9 +/- 10.9), there were significant correlations between the numbers of oocytes collected and cleaved (the correlation coefficient: r = 0.9336) or developed to morula stage (r = 0.6633), indicating that oocytes from different heifers have the same developmental ability after in vitro fertilization. Ten morulae and 12 blastocysts which were obtained 7 and 8 days after insemination were transferred, one by one, to each uterine horn of 11 recipients. At Day 60 of pregnancy, 8 (80%) fetuses were identified in 4 (80%) of 5 recipients into which 10 embryos were transferred at Day -1 of synchrony. However, only 3 (25%) fetuses were identified in 2 (40%) of 6 recipients into which 12 embryos were transferred at Day 0 or +1 of synchrony.

    DOI: 10.1016/0093-691X(91)90471-O

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  • DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES COLLECTED FROM OVARIES OF INDIVIDUAL HEIFERS AND FERTILIZED INVITRO International journal

    H FUNAHASHI, Y AOYAGI, T TAKEDA, T ONIHARA

    THERIOGENOLOGY   36 ( 3 )   427 - 434   1991.9

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    Bovine follicular oocytes from individual heifers (n = 49) were separately matured, fertilized with frozen-thawed spermatozoa and cultured with cumulus cells. Although there were great variations in the number (mean +/- SD = 19.1 +/- 11.9) of oocytes collected from individual heifers and the percentages of the oocytes cleaved 48 hours after insemination (mean +/- SD = 69.5 +/- 18.4) and developed to the morula stage 7 days after insemination (mean +/- SD = 10.9 +/- 10.9), there were significant correlations between the numbers of oocytes collected and cleaved (the correlation coefficient: r = 0.9336) or developed to morula stage (r = 0.6633), indicating that oocytes from different heifers have the same developmental ability after in vitro fertilization. Ten morulae and 12 blastocysts which were obtained 7 and 8 days after insemination were transferred, one by one, to each uterine horn of 11 recipients. At Day 60 of pregnancy, 8 (80%) fetuses were identified in 4 (80%) of 5 recipients into which 10 embryos were transferred at Day -1 of synchrony. However, only 3 (25%) fetuses were identified in 2 (40%) of 6 recipients into which 12 embryos were transferred at Day 0 or +1 of synchrony.

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  • 黄体ホルモン製剤を用いた黒毛和種牛の反復過剰排卵処理に関する検討.(共著)

    北海道牛受精卵移植研究会会報   ( 10 )   19 - 21   1991

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  • ラット前核期胚および桑実期胚の低温保存におよぼすショ糖の影響.(共著)

    家畜繁殖学雑誌   37 ( 4 )   a21   1991

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  • Experiment on intergeneric muclear transplantation between early-stage embryos of rats and mice.

    舟橋弘晃, 武田哲男, 丹羽こう二

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • Experiment on trasplantaion of bovine embryos produced by in vitro fertilization those were developed under a complete in vitro culture system.

    舟橋弘晃, 武富敏郎

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • Study on nuclear transplantation in cattle.

    亀山賢次, 青柳敬人, 舟橋弘晃, 武田哲男, 武富敏郎, 板倉はつえ

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • Study n a low-cost narcotic method for pig.

    武富敏郎, 亀山賢次, 舟橋弘晃, 板倉はつえ

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • Experiment on developemtn in vivo of rat and mouse embryos produced by intergeneric nuclear transplantation.

    舟橋弘晃, 武田哲男, 丹羽こう二

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • Experiment on transplantation of frozen bovine embryo splits filled in artificial zona pellucida.

    武田哲男, 青柳敬人, 亀山賢次, 武富敏郎, 舟橋弘晃, 板倉はつえ, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   ( 19(1990) )   1991

  • ラット2-細胞期胚における電気的割球融合

    舟橋 弘晃, 丹羽 皓二

    家畜繁殖学雑誌   36 ( 2 )   114 - 119   1990.6

  • Electric fusion of 2-cell rat embryo balastomeres.

    FUNAHASHI Hiroaki, NIWA Koji

    Journal of Reproduction and Development   36 ( 2 )   114 - 119   1990

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    Two-cell rat embryos were exposed to one or a train of two electric pulses with different field strengths and durations. It was difficult to induce blastomere fusion with a field strength of 1.0 kV/cm of 50μs duration: only 1 (7%) of 15 treated embryos fused after either one or a train of two pulses. The rate of fusion reached a maximum (80-100%) with 1.5-2.0 kV/cm and with 100-200μs duration. While the fusion rates were improved when the embryos were treated with a train of two pulses of 1.0 kV/cm for 100-200 μs, little effect was observed with 1.5 and 2.0 kV/cm. With 1.0 and 1.5 kV/cm, the fusion times were shortened when the embryos were exposed to a train of two pulses of 50 to 200 μs duration; no marked shortening of the time by the increased number of pulses was observed with 2.0 kV/cm. When fused embryos were transferred into pseudopregnant females and recovered 3 days later, 15 (83%), 12 (80%) and 9 (64%) of recovered embryos, which were originally submitted to 1.0, 1.5 and 2.0 kV/cm of field strengths each for 150 μs, respectively, had developed to the morula-blastocyst stage. There were no significant differences in the rates of development among these different field strengths.

    DOI: 10.1262/jrd1977.36.114

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  • 牛体外受精卵の凍結保存におよぼす凍結保護物質の影響.(共著)

    舟橋弘晃, 板倉はつえ, 武田哲男, 鬼原辰夫

    第83回日本畜産学会大会講演要旨   83rd   15   1990

  • 平成元年度野外実証試験について.

    第15回農協畜産技術研究会講演要旨   103 - 107   1990

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  • Electric fusion of 2-cell rat embryo balastomeres.

    FUNAHASHI Hiroaki, NIWA Koji

    Journal of Reproduction and Development   36 ( 2 )   114 - 119   1990

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    Two-cell rat embryos were exposed to one or a train of two electric pulses with different field strengths and durations. It was difficult to induce blastomere fusion with a field strength of 1.0 kV/cm of 50μs duration: only 1 (7%) of 15 treated embryos fused after either one or a train of two pulses. The rate of fusion reached a maximum (80-100%) with 1.5-2.0 kV/cm and with 100-200μs duration. While the fusion rates were improved when the embryos were treated with a train of two pulses of 1.0 kV/cm for 100-200 μs, little effect was observed with 1.5 and 2.0 kV/cm. With 1.0 and 1.5 kV/cm, the fusion times were shortened when the embryos were exposed to a train of two pulses of 50 to 200 μs duration; no marked shortening of the time by the increased number of pulses was observed with 2.0 kV/cm. When fused embryos were transferred into pseudopregnant females and recovered 3 days later, 15 (83%), 12 (80%) and 9 (64%) of recovered embryos, which were originally submitted to 1.0, 1.5 and 2.0 kV/cm of field strengths each for 150 μs, respectively, had developed to the morula-blastocyst stage. There were no significant differences in the rates of development among these different field strengths.

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  • Study of on cryoprotectant materials for split embryos of cattle and mouse.

    武田哲男, 亀山賢次, 武富敏郎, 舟橋弘晃, 板倉はつえ

    JA全農飼料畜産中央研究所試験研究報告   ( 18(1989) )   1990

  • Study on cryoprotectant materials for split bovine embryos.

    武田哲男, 武富敏郎, 舟橋弘晃, 板倉はつえ

    JA全農飼料畜産中央研究所試験研究報告   ( 18(1989) )   1990

  • Experiment on transfer of porcine embryos after short-term preservation.

    亀山賢次, 武富敏郎, 板倉はつえ, 舟橋弘晃

    JA全農飼料畜産中央研究所試験研究報告   ( 18(1989) )   1990

  • Surgical techniques for recovering and transplanting porcine embryos.

    武富敏郎, 立花文夫, 舟橋弘晃, 武田哲男, 鈴木正, 兼子よう子

    JA全農飼料畜産中央研究所試験研究報告   16 ( 1 )   493 - 501   1989

  • ラットおよびマウス1細胞期胚の属間核置換と置換卵の発生能.(共著)

    舟橋 弘晃

    第82回日本畜産学会大会講演要旨   82回   20 - 20   1989

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  • Method of in vitro culture of an in vitro fertilized bovine ova.

    舟橋弘晃, 武富敏郎

    JA全農飼料畜産中央研究所試験研究報告   17 ( 1 )   396 - 406   1989

  • Method of culturing a porcine embryo for 24 hours or longer.

    亀山賢次, 武富敏郎, 舟橋弘晃, 武田哲男, 板倉はつえ

    JA全農飼料畜産中央研究所試験研究報告   17 ( 1 )   390 - 395   1989

  • Cryoprotectants used in frozen preservation of in vitro fertilized bovine ova.

    舟橋弘晃, 武富敏郎, 亀山賢次, 板倉はつえ, 武田哲男, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   17 ( 1 )   381 - 389   1989

  • Transplantation of bovine embryos produced by in vitro fertilization of ova and cultured under a variety of condition.

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   17 ( 1 )   371 - 380   1989

  • Non-surgical techniques for transplanting frozen-thawed bovine embryos.

    武富敏郎, 鈴木正, 舟橋弘晃, 武田哲男, 立花文夫, 兼子よう子

    JA全農飼料畜産中央研究所試験研究報告   16 ( 1 )   486 - 492   1989

  • 体外受精技術を利用した牛受精卵移植の現状と問題点.

    第14回農協畜産技術研究会講演要旨   64 - 69   1989

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  • Transplantation of porcine embryos after a longdistance transport.

    武富敏郎, 武田哲男, 舟橋弘晃, 板倉はつえ, 中谷正史, 普川一雄

    JA全農飼料畜産中央研究所試験研究報告   17 ( 1 )   365 - 370   1989

  • Influences of heparin and caffeine on in vitro fertilization of ova and subsequent development of embryos in cattle.

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   16 ( 1 )   507 - 513   1989

  • Influences of FSH (folicle stimulating hormone) and HCG (human chorionic gonadotrophin) on in vitro maturation of follicular oocytes.

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    JA全農飼料畜産中央研究所試験研究報告   16 ( 1 )   502 - 506   1989

  • Development of rat eggs with pronuclei transplanted by electrofusion.

    舟橋弘晃, 丹羽こう二

    家畜繁殖学雑誌   34 ( 3 )   133 - 137   1988

  • 凍結・融解した牛体外受精卵の非手術的移植後の受胎能.(共著)

    家畜繁殖学雑誌   34 ( 4 )   末   1988

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  • カフェインとヘパリンの添加培地で得られた牛体外受精卵の発生能.(共著)

    家畜繁殖学雑誌   34 ( 4 )   末   1988

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  • ラットにおける前核置換卵の移植による産子の生産.(共著)

    家畜繁殖学雑誌   33 ( 4 )   末   1987

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  • ラット2細胞期胚割球の電気的融合条件.(共著)

    第79回日本畜産学会大会講演要旨   11   1987

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  • ラット前核期卵における核移植に関する基礎的研究.(共著)

    哺乳動物卵子研究会誌   2 ( 1 )   75 - 78   1985

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  • マウス排卵卵子への精巣精子、精巣上体精子の顕微注入.(共著)

    日本不妊学会雑誌   31 ( 1 )   160   1985

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  • ラット前核期卵における雄性前核の紫外線スポット照射による不活化と作出された雌性二倍体胚の発育性について.(共著)

    日本畜産学会関西支部会報   101,23   1985

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  • The basic study on unclear transplantation in rat zygote by HVJ-mediated cell fusion karyoplast.

    Journal of Mammalian Ova Research   2 ( 1 )   75 - 78   1985

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  • The basic study on uuclear transplantation in rat zygote by HVJ-mediated cell fusion karyoplast.

    Journal of Mammalian Ova Research   2 ( 1 )   75 - 78   1985

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  • Nuclear transplantation in pronuclear stage rat eggs : Inactivation of nucleus by micro-spot UV-irradiation.(共著)

    International Symposium on Mammalian Reproduction and Early Develelopment, Abstracts   48   1984

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  • Nuclear transplantation into pronuclear-stage rat eggs : Enucleation of eggs by UV-irradiation.

    Journal of Mammalian Ova Research   1 ( 1 )   85 - 89   1984

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  • ラット前核期卵の核移植:特に紫外線照射による非外科的核除去について.(共著)

    哺乳動物卵子研究会誌   1 ( 1 )   85 - 89   1984

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  • Nuclear transplantation in pronuclear stage rat eggs : Inactivation of nucleus by micro-spot UV-irradiation.(共著) International Symposium on Mammalian Reproduction and Early

    Develelopment, Abstracts   48   1984

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  • ラット前核期卵におよぼす前核への紫外線照射の影響.(共著)

    日本畜産学会関西支部会報   ( 98 )   30   1984

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  • ラット受精卵の体外培養について -血清を中心とした培養液要素についての検討.(共著)

    日本不妊学会雑誌   28 ( 2 )   148   1982

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Industrial property rights

  • 運動性良好な精子採取装置

    松浦 宏治, 舟橋 弘晃, 古市 卓也

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    Applicant:国立大学法人 岡山大学

    Application no:特願2012-081396  Date applied:2012.3.30

    Announcement no:特開2012-213395  Date announced:2012.11.8

    Patent/Registration no:特許第5877512号  Date registered:2016.2.5 

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  • 光学的観察用チャンバー及び試料の光学的観察方法、並びに下側透明板の製造方法

    松浦 宏治, 舟橋 弘晃

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    Applicant:国立大学法人 岡山大学

    Application no:特願2011-523592  Date applied:2010.6.24

    Publication no:WO2011-010525  Date published:2011127

    Patent/Registration no:特許第5610313号  Date registered:2014.9.12 

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  • 卵細胞の培養装置

    成瀬 恵治, 舟橋 弘晃, 石田 敬雄, 松浦 宏治, 原 鐵晃

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    Applicant:国立大学法人 岡山大学

    Application no:特願2007-194968  Date applied:2007.7.26

    Announcement no:特開2009-027973  Date announced:2009.2.12

    Patent/Registration no:特許第5083952号  Date registered:2012.9.14 

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  • 再構築受精卵の作製方法及びそれを用いたトランスジェニック胚の作製方法

    小西 正人, 青柳 敬人, 舟橋 弘晃, 岡部 勝

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    Applicant:全国農業協同組合連合会

    Application no:特願2000-232041  Date applied:2000.7.31

    Announcement no:特開2001-103867  Date announced:2001.4.17

    Patent/Registration no:特許第4845073号  Date registered:2011.10.21 

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  • アデノシンおよび受精促進ペプチドを利用した効率的な体外受精卵生産方法

    舟橋 弘晃

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    Applicant:岡山大学長

    Application no:特願2000-070752  Date applied:2000.3.14

    Announcement no:特開2001-251990  Date announced:2001.9.18

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  • 受精卵あるいは卵子輸送装置

    舟橋 弘晃, 井田 脩三

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    Applicant:全国農業協同組合連合会

    Application no:特願平2-404910  Date applied:1990.12.21

    Announcement no:特開平5-310501  Date announced:1993.11.22

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Awards

  • 日本繁殖生物学会賞(島村賞)

    1998  

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    Country:Japan

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Research Projects

  • Development of functional photosensitizing peptide for photocontrol of biofunctions

    Grant number:18H02090  2018.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Ohtsuki Takashi

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    Grant amount:\17160000 ( Direct expense: \13200000 、 Indirect expense:\3960000 )

    In this study, we developed a molecular tool combining a photosensitizer and a peptide/protein (functional photosensitizing peptide; FPP) to establish a technology for the spatiotemporal control and observation of cellular functions by light. First, we elucidated the effects of the amino acid sequence near the photosensitizers to support FPP design. Furthermore, by using this technology, we established a method for light-directed cytosolic delivery of two different RNAs, clarified the cell cycle dependence of apoptosis photo-triggered using a FPP, and succeeded in photoinduced RNA delivery into a single cell in mouse embryos.

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  • Creation of mammalian oocytes with high fertility by spatiotemporal regulation of the function of mitochondria

    Grant number:18H02328  2018.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    FUNAHASHI Hiroaki

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    The aim of this study was to generate oocytes with high developmental competence by regulating the mitochondrial (mt) DNA copy number in the ooplasm. Porcine ooplasmic mtDNA copy number did not change during in vitro maturation (IVM) and was lower in oocytes from small than medium follicles. Drp1 (mt mitogen) was reduced during early development, and its inhibition impaired the mt distribution in oocytes and early developmental competence. Overexpression of PGC1alpha (mt synthesis-related genes) resulted in increased reactive oxygen species and mtDNA copy number reduction in oocytes, and reduced IVM rate and early developmental competence. The autophagic potential of oocytes varied greatly during IVM and early development, and differed significantly by follicle diameter of origin. Attempts to introduce photosensitizers and peptide/protein molecular tools into oocytes have not resulted in significant improvement.

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  • Cryopreservation of ovarian tissue

    Grant number:16K11089  2016.04 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Motohashi Hideyuki

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    The cancer treatments in young women such as anti-cancer drugs and radiation can affect fertility by several biologic systems. The purpose of this study was to develop a novel cryopreservation method for ovarian tissue. In this study, an improved vitrification for ovarian tissue resulted in survival and subsequent development in vivo and oocyte maturation in vitro after cryopreservation.

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  • Characteristic analyses of boar seminal gel and the application

    Grant number:24658236  2012.04 - 2014.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    FUNAHASHI Hiroaki

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    Grant amount:\2990000 ( Direct expense: \2300000 、 Indirect expense:\690000 )

    Characteristic analyses of seminal gel in ejaculated boar semen were analysed. The gel could be dissolved in both conditions of severe acidic and alkaline conditions, whereas it is stable around neutral conditions. Chemotaxis of polymorphonuclear leukocytes for seminal gel was not observed. Preparation of sperm capsule by mixing frozen-dried seminal gel with boar semen or washed sperm allowed survival and release of sperm from the capsule. Chemical analyses of seminal gel demonstrated that the gel is o-glycan-rich. Analyses of sugar chains and peptides showed that seminal gel contains specific sugar chains and protein. These information may have possibilities to contribute in wide fields including biomedical area.

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  • Investigation for effects of blood coagulation factor XI deficiency on productivity to the genetic improvement of Japanese beef cattle

    Grant number:23380166  2011.04 - 2014.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    KUNIEDA Tetsuo, TSUJI Takehito, IBI Takayuki, FUNAHASHI Hiroaki, ACOSTA Tomas

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    Grant amount:\18200000 ( Direct expense: \14000000 、 Indirect expense:\4200000 )

    Blood coagulation factor XI deficiency is a hereditary disorder observed in Japanese Black cattle. While the clinical condition of this disorder is mild, the economical impact of this disorder is apprehended to be serious because of the remarkably high allelic frequency of the mutant allele in the population of Japanese Black cattle. We, therefore, investigated the effect of the factor XI deficiency on defects in pregnancy and delivery and carcass traits using large samples of Japanese Black cattle. As a result, we found that affected animals of factor XI deficiency have a tendencies for increased frequencies of abortion and stillborn, but no effect on the condition of calf and carcass traits. These findings will be useful to future breeding of Japanese Black cattle.

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  • Immunological regulation of the female reproductive tract to establish an efficient new artificial insemination system in mammals

    Grant number:20380154  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    FUNAHASHI Hiroaki, KONDOU Yasuhiro

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    Grant amount:\9360000 ( Direct expense: \7200000 、 Indirect expense:\2160000 )

    In the female reproductive tracts, immunological system exists to remove sperm from the tract rapidly after mating or artificial insemination. In the present study, we could make clear that both phagocytotic and chemotactic activities of bovine and porcine polymorphonuclear leukocytes (PMNs), which mainly perform in the system, are stimulated in the presence of fresh serum and decreased in the presence of caffeine, heparin, and seminal plasma. Furthermore, PMNs phagocytize equally both active motile and dead sperm.

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  • Development of a transient co-incubation IVF system in the pig

    Grant number:16580230  2004 - 2005

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    FUNAHASHI Hiroaki

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    Grant amount:\3600000 ( Direct expense: \3600000 )

    This research project was undertaken to develop a transient-co-incubation IVF system in the pig, in order to obtain an efficient normal monospermic sperm penetration. Obtained sights are as follows :
    (A)Effect of pretreatment of spermatozoa with adenosine and a transient gamete co-incubation with caffeine
    A new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermic penetration.
    (B)Effective regulation of sperm capacitation during transient IVF period
    The presence of beta-mercaptoethanol (bME) prevents induction of sperm capacitation even in the presence of caffeine, whereas H_2O_2 induces sperm capacitation and the spontaneous acrosome reaction.
    (C)Regulation of oxidative stress of gametes during transient co-incubation
    Supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.
    (D)Selection of antioxidants to improve the function of extended boar semen stored at a low temperature
    Glutathione and cysteine can improve the viability and functional status of boar spermatozoa during liquid preservation and boar spermatozoa penetrated in vitro even after preservation in the presence of cysteine at 10℃ for 29 days.
    (E)Development of an IVM system for the transient IVF system
    Although cytoplasmic maturation in porcine oocytes pretreated with roscovitine for 48 h did not equal that of control oocytes in the current IVM system, those stored oocytes can matured, fertilized and developed in vitro to the blastocyst stage.

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  • Development of an efficient transgenic method bu using GFP-reporter gene and nuclear transfer

    Grant number:10556065  1998 - 2001

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    FUNAHASHI Hiroaki, AOYAGI Yoshito, NIWA Koji, OKABE Masaru

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    Grant amount:\12300000 ( Direct expense: \12300000 )

    The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12-24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to difficulty in visualizing the pronucleus, the incidence of successful injection of linear DNA into pronucleus was higher when injected from 20 to 24 h, as compared with an early period from 12 to 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP-expression rate were not different among the variously-timed microinjections. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5 % of morulae that developed from the NT-eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of non-integrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.
    Efficient methods to produce porcine zygotes in vitro for the microinjection of DNA constructs were also developed. Replacement of caffeine with adenosine or fertilization promoting peptide, which induced capacitation but prevented acrosome reaction of boar spermatozoa, reduced the incidence of polyspermic penetration of porcine oocytes without any reduction in penetration.

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  • Packaging of Mammalian Paternal Genome and Male Pronuclear Formation

    Grant number:09660303  1997 - 1998

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    FUNAHASHI Hiroaki

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    Grant amount:\3800000 ( Direct expense: \3800000 )

    DNA-stability and thiol-disulfide status of rat sperm nuclei was observed during maturation in the epididymis and during penetration to the oocytes in vivo. When spermatids and spermatozoa were stained with acridine orange (AO) after fixation with acetic alcohol, the ratio of red/green fluorescence under a confocal microscope decreased until caput epididymis. On the other hand, when spermatozoa were labeled with rnBBr the fluorescence intensities gradually decreased during transportation of spermatozoa from testis through epididymis. The ratio of red/green AO fluorescence increased during penetration of zona pellucida and sperm decondensation. When spermatozoa in the perivitelline space were labeled with mBBr and examined fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidization of protamine during sperm maturation in the epididymis and that an initial event of the unpackaging process in rat genome begins during or just after zona-penetration but before penetration into the ooplasm.
    The contents of glutathione and other thiols in rat eggs were examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilized oocytes and penetrated eggs with a decondensed sperm nucleus than eggs at the pronuclear stage. The content of oxidized glutathione (GSSG) was not different among experimental groups. The ratio of GSSG/GSH was not different during fertilization. Although reduced cysteinylglycine content did not changed among experimental groups, oxidized form of cysteinylglycine increased between sperm decondensation and pronuclear formation. Low contents of cystine were detected during fertilization but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

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  • Study on in vitro production and manipulation of mammalian gametes.

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    Grant type:Competitive

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  • 哺乳動物精子の受精能獲得機構に関する研究

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    Grant type:Competitive

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  • 哺乳動物受精卵における雄性前核形成のメカニズムに関する研究

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    Grant type:Competitive

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  • 哺乳動物生殖細胞の体外生産及び操作に関する研究

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  • Study on the capacitation of mammalian spermatogoa

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  • Study on the mechanisn of male pronuclear formation in mammalian zygotes.

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Academic Activities

  • International Embryo Transfer Society Foundation, trustee

    Role(s):Planning, management, etc.

    International Embryo Transfer Society  2010.1 - 2015.1

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    Type:Academic society, research group, etc. 

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