2021/10/17 更新

写真a

フナハシ ヒロアキ
舟橋 弘晃
FUNAHASHI Hiroaki
所属
本部 理事
職名
理事
外部リンク

学位

  • 農学修士 ( 岡山大学 )

  • 学術博士 ( 岡山大学 )

研究キーワード

  • 動物生殖科学

  • 応用動物科学

  • 生殖細胞

  • 体外成熟・受精・初期発生

研究分野

  • ライフサイエンス / 動物生産科学

  • ライフサイエンス / 動物生産科学

  • ライフサイエンス / 細胞生物学

学歴

  • 岡山大学   The Graduate School of Natural Science and Technology   Major of Bio-Resource Science

    1987年4月 - 1990年3月

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    国名: 日本国

    備考: Doctoral Course

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  • 岡山大学   Graduate School of Agriculture   Department of Animal Science

    1985年4月 - 1987年3月

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    国名: 日本国

    備考: Master Course

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  • 岡山大学   Faculty of Agriculture   Department of Animal science

    1981年4月 - 1985年3月

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    国名: 日本国

    備考: Bachelor's degree Course

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経歴

  • 岡山大学学術研究院環境生命科学学域   教授

    2021年4月 - 現在

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    国名:日本国

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  • 岡山大学生殖補助医療技術教育研究センター   教授

    2013年10月 - 現在

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    国名:日本国

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  • 岡山大学大学院環境生命科学研究科   教授

    2012年4月 - 現在

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    国名:日本国

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  • 岡山大学大学院自然科学研究科   教授

    2011年1月 - 2012年3月

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    国名:日本国

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  • ムルシア大学   獣医学部   客員教授

    2008年12月 - 現在

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    国名:スペイン

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  • くらしき作陽大学   音楽学部   非常勤講師

    2000年4月 - 2013年3月

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    国名:日本国

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  • 岡山大学大学院自然科学研究科   准教授

    2000年4月 - 2010年12月

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    国名:日本国

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  • スウェーデン農科大学   獣医学部   客員研究員

    1999年5月 - 1999年7月

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    国名:スウェーデン王国

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  • フランス国立農業研究所   客員研究員

    1998年5月 - 1998年6月

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    国名:フランス共和国

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  • 岡山大学   Faculty of Agriculture

    1996年 - 2000年

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    国名:日本国

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  • ミズーリ大学コロンビア校   研究専任助教授

    1994年 - 1996年

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    国名:アメリカ合衆国

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  • ミズーリ大学コロンビア校   博士研究員

    1991年 - 1994年

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    国名:アメリカ合衆国

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  • 全国農業協同組合連合会飼料畜産中央研究所   研究員

    1987年 - 1991年

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    国名:日本国

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所属学協会

  • 米国繁殖学会(The Society for the Study of Reproduction)

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  • 日本繁殖生物学会

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  • 日本畜産学会

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  • 日本哺乳動物卵子学会

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  • 国際胚移植学会(International Embryo Transfer Society)

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  • 日本胚移植学会

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  • 日本養豚学会

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  • 米国発生生物学会(The Society for Developmental Biology)

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  • 英国生殖学会(The Society for the Study of Fertility)

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論文

  • Large-scale preparation of sialyl-Tn antigen-containing peptides from mucin-like glycoproteins in boar seminal gel 国際誌

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

    Bioscience biotechnology and biochemistry   85 ( 9 )   2022 - 2025   2021年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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.

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  • The thickness and density of the ovarian tunica albuginea increases with age in transgender patients 国際誌

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

    Reproductive sciences   28 ( 5 )   1339 - 1346   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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 国際誌

    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年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元: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

    Chiyuki Kohata-Ono, Takuya Wakai, Hiroaki Funahashi

    Journal of reproduction and development   65 ( 6 )   527 - 532   2019年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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

    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年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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 国際誌

    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年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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 査読 国際誌

    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年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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 国際誌

    R. Matsunaga, H. Funahashi

    Reproduction in domestic animals   52 ( 4 )   672 - 679   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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

    Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

    Journal of reproduction and development   63 ( 2 )   191 - 197   2017年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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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  • 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 査読

    Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   63 ( 2 )   191 - 197   2017年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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.

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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

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

    Theriogenology   86 ( 7 )   1705 - 1710   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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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  • 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 査読 国際誌

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

    THERIOGENOLOGY   86 ( 7 )   1705 - 1710   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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.

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  • Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos 査読 国際誌

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

    THERIOGENOLOGY   85 ( 7 )   1211 - 1218   2016年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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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  • Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos 国際誌

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

    Theriogenology   85 ( 7 )   1211 - 1218   2016年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    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 < 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 < 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 < 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

    Rukmali Athurupana, Hiroaki Funahashi

    Asian pacific journal of reproduction   5 ( 1 )   58 - 62   2016年3月

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    掲載種別:研究論文(学術雑誌)  

    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

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

    Theriogenology   85 ( 3 )   428 - 433   2016年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    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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  • A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium 査読 国際誌

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

    THERIOGENOLOGY   85 ( 3 )   428 - 433   2016年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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 国際誌

    Raquel Romar, Hiroaki Funahashi, Pilar Coy

    Theriogenology   85 ( 1 )   125 - 134   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

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

    Raquel Romar, Hiroaki Funahashi, Pilar Coy

    THERIOGENOLOGY   85 ( 1 )   125 - 134   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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 国際誌

    Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

    Theriogenology   84 ( 6 )   940 - 947   2015年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

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

    Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

    THERIOGENOLOGY   84 ( 6 )   940 - 947   2015年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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 査読

    Rukmali Athurupana, Daisen Takahashi, Sumire Ioki, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   61 ( 3 )   205 - 210   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

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

    Rukmali Athurupana, Daisen Takahashi, Sumire Ioki, Hiroaki Funahashi

    Journal of reproduction and development   61 ( 3 )   205 - 210   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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 国際誌

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

    Theriogenology   80 ( 9 )   970 - 978   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, <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 < 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 < 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P < 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P < 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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  • Development competence and relative transcript abundance of oocytes derived from small and medium follicles of prepubertal gilts 査読

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

    THERIOGENOLOGY   80 ( 9 )   970 - 978   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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 国際誌

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

    Fertility and sterility   99 ( 2 )   400 - 407   2013年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    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.)

    DOI: 10.1016/j.fertnstert.2012.10.022

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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 国際誌

    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年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1016/j.theriogenology.2012.09.012

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

    Hiroaki Funahashi

    Reproductive medicine and biology   12 ( 1 )   15 - 20   2013年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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 査読 国際誌

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

    Fertility and Sterility   98 ( 6 )   1423 - 1427   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Fertility and sterility   98 ( 6 )   1423 - 1427   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    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. (Fertil Steril (R) 2012; 98: 1423-7. (C) 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 査読

    Masanobu Okuyama, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   58 ( 3 )   295 - 301   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    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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  • 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

    Masanobu Okuyama, Hiroaki Funahashi

    Journal of reproduction and development   58 ( 3 )   295 - 301   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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 査読 国際誌

    S. Yamaguchi, H. Funahashi

    Theriogenology   77 ( 5 )   926 - 932   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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

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

    Reproductive biomedicine online   24 ( 3 )   301 - 307   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier sci ltd  

    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 < 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-mu l droplet rather than in 0.5 mu l (72.0% versus 38.0%; P < 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. (C) 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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  • Simple vitrification for small numbers of human spermatozoa 査読 国際誌

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

    Reproductive BioMedicine Online   24 ( 3 )   301 - 307   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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 査読 国際誌

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

    Theriogenology   77 ( 1 )   73 - 80   2012年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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 査読 国際誌

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

    Theriogenology   75 ( 7 )   1336 - 1345   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    Koji Matsuura, Yuka Kuroda, Keisuke Yamashita, Hiroaki Funahashi

    Journal of reproduction and development   57 ( 1 )   163 - 167   2011年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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<0.05) and ALHD (P<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.

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  • Single spermatozoon freezing using cryotop 査読

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

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

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    記述言語:英語   出版者・発行元:(一社)日本卵子学会  

    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.

    DOI: 10.1274/jmor.28.47

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    その他リンク: 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 査読

    Koji Matsuura, Yuka Kuroda, Keisuke Yamashita, Hiroaki Funahashi

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

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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 査読 国際誌

    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年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    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年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Spandidos publ ltd  

    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

    Takayuki Koike, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    Journal of reproduction and development   56 ( 5 )   552 - 557   2010年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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<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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  • 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 査読

    Takayuki Koike, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   56 ( 5 )   552 - 557   2010年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    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 査読 国際誌

    J. C. Li, H. Funahashi

    Theriogenology   74 ( 4 )   691 - 698   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1016/j.theriogenology.2010.03.019

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  • Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration 査読 国際誌

    Hikaru Sano, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    THERIOGENOLOGY   74 ( 5 )   863 - 870   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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 a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

    Hikaru Sano, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

    Theriogenology   74 ( 5 )   863 - 870   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier science inc  

    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 < 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 < 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 < 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 査読

    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年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

    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

    Shoichiro Yamaguchi, Hiroaki Funahashi, Tetsuya Murakami

    Journal of reproduction and development   55 ( 6 )   645 - 649   2009年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

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

    Shoichiro Yamaguchi, Hiroaki Funahashi, Tetsuya Murakami

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 6 )   645 - 649   2009年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    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

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

    Journal of reproduction and development   55 ( 4 )   446 - 453   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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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  • 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 査読

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

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 4 )   446 - 453   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    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 査読

    Wataru Fujii, Hiroaki Funahashi

    Journal of Reproduction and Development   55 ( 2 )   187 - 193   2009年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1262/jrd.20122

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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

    Wataru Fujii, Hiroaki Funahashi

    Journal of reproduction and development   55 ( 2 )   187 - 193   2009年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Society reproduction & development-srd  

    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.

    DOI: 10.1262/jrd.20122

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  • Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium 国際誌

    H. Funahashi, T. Koike, R. Sakai

    Theriogenology   70 ( 7 )   1041 - 1047   2008年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1016/j.theriogenology.2008.06.025

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

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

    Theriogenology   70 ( 5 )   809 - 817   2008年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1016/j.theriogenology.2008.05.045

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  • In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation 査読 国際誌

    Wataru Fujii, Hiroaki Funahashi

    Zygote   16 ( 2 )   117 - 125   2008年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1017/S0967199408004632

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  • In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation 国際誌

    Wataru Fujii, Hiroaki Funahashi

    Zygote   16 ( 2 )   117 - 125   2008年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Cambridge univ press  

    The present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocyte-cumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14h 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 (0-3.5 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 (82.4-83.5% and 43.4-51.9%, respectively). Nor did the incidences of eggs cleaving (86.7-97.7%) and developing to the blastocyst stage (0-3.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 1-1.5h 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.

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  • Up date of in vitro production of porcine embryos 国際誌

    Takashi Nagai, Hiroaki Funahashi, Koji Yoshioka, Kazuhiro Kikuchi

    Frontiers in bioscience-landmark   11 ( SUPPL. 2 )   2565 - U34   2006年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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 査読 国際誌

    R Romar, H Funahashi

    ANIMAL REPRODUCTION SCIENCE   92 ( 3-4 )   321 - 333   2006年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    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. 国際誌

    Hiroaki Funahashi

    Reproduction (cambridge, england)   130 ( 6 )   889 - 98   2005年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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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  • Involvement of oviduct in sperm capacitation and oocyte development in pigs 査読

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

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

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:SOC REPRODUCTION FERTILITY  

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

    H Funahashi, TC Cantley, BN Day

    THERIOGENOLOGY   47 ( 3 )   679 - 686   1997年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    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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  • gamma-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs 査読 国際誌

    H Funahashi, Z Machaty, RS Prather, BN Day

    MOLECULAR REPRODUCTION AND DEVELOPMENT   45 ( 4 )   485 - 490   1996年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元: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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  • Current status of in vitro production of porcine embryos 査読

    H Funahashi, BN Day

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

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:PLENUM PRESS DIV PLENUM PUBLISHING CORP  

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  • Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B48. 国際誌

    F Giannoni, D K Bonen, T Funahashi, C Hadjiagapiou, C F Burant, N O Davidson

    The Journal of biological chemistry   269 ( 8 )   5932 - 6   1994年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Mammalian small intestine secretes a truncated apolipoprotein B (apoB48) species as a result of tissue-specific post-transcriptional RNA editing. The human liver, by contrast, contains only unedited apoB mRNA and secretes only apoB100. We have recently isolated a cDNA clone from rat small intestine which encodes an apoB mRNA editing protein, REPR (Teng, B., Burant, C.F., and Davidson, N.O. (1993) Science 260, 1816-1819). The current study demonstrates that homogenates of Xenopus oocytes expressing REPR confer editing ability upon S100 extracts prepared from human liver when tested on a synthetic apoB RNA template in vitro. Transfection of REPR into HepG2 cells resulted in editing of endogenous apoB mRNA and the appearance of an apoB48-like protein in the media. Extracts prepared from these transfected cells edit mammalian apoB RNA templates when incubated alone and with enhanced efficiency in the presence of chicken intestinal S100 extracts. The results suggest that human liver expresses factor(s) which are critical to apoB mRNA editing and which allow functional complementation of REPR in vivo.

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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年3月 

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    総ページ数:58p   記述言語:英語

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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年 

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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年3月 

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    総ページ数: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年 

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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年 

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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年 

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

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

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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年3月 

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    総ページ数: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年 

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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年 

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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年 

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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年 

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MISC

  • 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

    Dairyman   71 ( 8 )   2021年

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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  • 細胞外環境の変化は女性における妊孕性喪失に寄与するもう一つの因子だろうか

    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年

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    記述言語:英語  

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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

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019年

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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

    月向はるな, 野村瑠莉, 舟橋弘晃, 若井拓哉

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

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    記述言語:日本語   出版者・発行元:(公社)日本繁殖生物学会  

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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

    野村瑠莉, 月向はるな, 舟橋弘晃, 若井拓哉

    Journal of reproduction and development   65 ( Suppl Japanese Issue )   j120 - j120   2019年

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    記述言語:日本語   出版者・発行元:(公社)日本繁殖生物学会  

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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年

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    記述言語:日本語   出版者・発行元:岡山実験動物研究会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(NPO)日本不妊カウンセリング学会  

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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年

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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  • 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年

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    記述言語:日本語   出版者・発行元:岡山実験動物研究会  

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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年

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    記述言語:日本語   出版者・発行元:岡山実験動物研究会  

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  • 豚液状精液人工授精におけるカフェイン添加による暑熱期の分娩率低下の改善

    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年

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

    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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  • 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年

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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  • 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年9月

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    記述言語:日本語   出版者・発行元:(一社)日本繁殖生物学会  

    【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 国際誌

    H. Funahashi

    Reproduction in domestic animals = Zuchthygiene   50   40 - 47   2015年7月

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    記述言語:英語  

    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年

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  • 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年

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    記述言語:日本語   出版者・発行元:(一社)日本IVF学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本IVF学会  

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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年3月

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    記述言語:英語   出版者・発行元:(公社)日本畜産学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本生殖医学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本生殖医学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本生殖医学会  

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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年

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    記述言語:日本語   出版者・発行元:(公社)日本繁殖生物学会  

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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年

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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年

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  • 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年

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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年

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  • 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年

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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年

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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年

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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年

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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年

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  • 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年

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  • 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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:CSIRO PUBLISHING  

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:CSIRO PUBLISHING  

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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年

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  • 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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年9月

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  • 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年

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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年

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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年

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  • 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年

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  • 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年

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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年

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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年

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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年

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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年

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  • 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年

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  • 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年

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  • 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年

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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年

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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年

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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年

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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年

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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年

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  • 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年

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  • 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年6月

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    記述言語:日本語   出版者・発行元:(一社)日本臨床エンブリオロジスト学会  

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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  • Select antioxidants improve the function of extended boar semen stored at 10 degrees C 国際誌

    H Funahashi, T Sano

    THERIOGENOLOGY   63 ( 6 )   1605 - 1616   2005年4月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE INC  

    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年

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  • 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年

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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 国際誌

    H Funahashi, R Romar

    REPRODUCTION   128 ( 6 )   789 - 800   2004年12月

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    記述言語:英語   出版者・発行元:BIO SCIENTIFICA LTD  

    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.

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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 国際誌

    H Funahashi, R Romar

    REPRODUCTION   128 ( 6 )   789 - 800   2004年12月

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    記述言語:英語   出版者・発行元:BIO SCIENTIFICA LTD  

    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.

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  • 人工授精や体外受精に用いる豚精子受精能の人為制御システムの開発.

    舟橋弘晃

    平成15年度食肉に関する助成研究調査成果報告書(財団法人伊藤記念財団)   Vol.22, p55-61.   2004年

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  • 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年

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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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年

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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年

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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年

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  • Polyspermic penetration in porcine IVM-IVF systems

    H Funahashi

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

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:C S I R O PUBLISHING  

    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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  • Polyspermic penetration in porcine IVM-IVF systems 国際誌

    Hiroaki Funahashi

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

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    記述言語:英語  

    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年

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    記述言語:日本語   出版者・発行元:(一社)日本生殖医学会  

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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年

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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年

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    記述言語:日本語   出版者・発行元:(一社)日本臨床エンブリオロジスト学会  

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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年

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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 国際誌

    H Funahashi

    REPRODUCTION   124 ( 6 )   857 - 864   2002年12月

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    記述言語:英語   出版者・発行元:SOC REPRODUCTION FERTILITY  

    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年2月

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    記述言語:英語   出版者・発行元:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    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.

    DOI: 10.1262/jrd.48.57

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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年

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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年

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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年

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  • 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年

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  • 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年

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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年

  • 豚精子のHCO<sub>3</sub><sup>-</sup>/Cl<sup>-</sup>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年

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

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

    REPRODUCTION   122 ( 3 )   443 - 452   2001年9月

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    記述言語:英語   出版者・発行元:SOC REPRODUCTION FERTILITY  

    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年9月

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    記述言語:英語   出版者・発行元:SOC REPRODUCTION FERTILITY  

    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 国際誌

    H Funahashi, T Nagai

    MOLECULAR REPRODUCTION AND DEVELOPMENT   58 ( 4 )   424 - 431   2001年4月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

    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年4月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

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

    Theriogenology   55 ( 1 )   522   2001年

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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年

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  • 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年

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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年

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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年

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    記述言語:英語   出版者・発行元:Mary Ann Liebert Inc.  

    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.

    DOI: 10.1089/15362300152725891

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  • Oviduct involvement in sperm capacitation and oocyte development

    Sixth International Conference on Pig Reproduction   62   2001年

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  • 豚の新しい繁殖技術とその展望

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

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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年

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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年

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    記述言語:英語   出版者・発行元:Mary Ann Liebert Inc.  

    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.

    DOI: 10.1089/15362300152725891

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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年

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  • Oviduct involvement in sperm capacitation and oocyte development

    Sixth International Conference on Pig Reproduction   62   2001年

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  • Involvement of oviduct in sperm capacitation and oocyte development in pigs. 国際誌

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

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

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    記述言語:英語  

    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 国際誌

    H Funahashi, H Ekwall, H Rodriguez-Martinez

    BIOLOGY OF REPRODUCTION   63 ( 5 )   1437 - 1442   2000年11月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.

    DOI: 10.1095/biolreprod63.5.1437

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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年11月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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年10月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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 国際誌

    H Funahashi, T Fujiwara, T Nagai

    BIOLOGY OF REPRODUCTION   63 ( 4 )   1157 - 1163   2000年10月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.

    DOI: 10.1095/biolreprod63.4.1157

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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年10月

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

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

    MOLECULAR REPRODUCTION AND DEVELOPMENT   55 ( 1 )   117 - 124   2000年1月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

    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年1月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

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

    Theriogenology   53 ( 1 )   420   2000年

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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年

  • 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年

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    記述言語:英語   出版者・発行元:The Japanese Society of Animal Reproduction (JSAR)  

    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.

    DOI: 10.1262/jrd.46.319

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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年

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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年

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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年

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  • 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年

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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年11月

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    記述言語:英語   出版者・発行元:CAMBRIDGE UNIV PRESS  

    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 γ-glutamyl cycle during sperm penetration and pronuclear formation of rat oocytes. 国際誌

    Zygote   7 ( 4 )   301 - 305   1999年11月

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

    Asian-Australian Journal of Animal Science   12 ( 6 )   966 - 975   1999年9月

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    記述言語:英語  

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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年9月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOCIETIES  

    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.

    DOI: 10.5713/ajas.1999.966

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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年8月

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    記述言語:英語   出版者・発行元:CAMBRIDGE UNIV PRESS  

    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 国際誌

    S Said, H Funahashi, K Niwa

    ZYGOTE   7 ( 3 )   249 - 254   1999年8月

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    記述言語:英語   出版者・発行元:CAMBRIDGE UNIV PRESS  

    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 cummulus-oocyte complexes during maturation in vitro : effects of epidermal growth factor on production of Pas in oocytes and cumulus cells. (共著)

    Biology of Reproduction   61 ( 1 )   298 - 304   1999年7月

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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 国際誌

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

    BIOLOGY OF REPRODUCTION   61 ( 1 )   298 - 304   1999年7月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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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  • 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年6月

  • 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年

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    記述言語:英語   出版者・発行元: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.

    DOI: 10.1262/jrd.45.223

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

    FUNAHASHI H, MCINTUSH E W, SMITH M F, DAY B N

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

  • 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年

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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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. (共著)

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

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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年

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    記述言語:英語   出版者・発行元: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.

    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 国際誌

    SH Oh, K Miyoshi, H Funahashi

    BIOLOGY OF REPRODUCTION   59 ( 4 )   884 - 889   1998年10月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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 Nacl concentration and bovine serum albumin maintain developmental ability to the blastocyst stage(共著)

    Biology of Reproduction   59 ( 4 )   884 - 889   1998年10月

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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年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ELSEVIER SCIENCE INC  

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

    Theriogenology   49 ( 1 )   309 - 309   1998年1月

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

    87   205 - 214   1998年

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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年

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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年

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  • Co-culture of bovine cumulus-oocyte complexes with thece cells inhibits the meiotic resumption at a specific phase of germinal vesicle(共著)

    Biology of Reproduction   58 ( Suppl.1 )   219 - 219   1998年

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

    Biology of Reproduction   58 ( Suppl.1 )   219 - 219   1998年

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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年

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  • Effect of cumulus cells on the ability of pig oocytes to utilize cysteine or cystine during maturation in vitro(共著)

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

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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年

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    記述言語:英語   出版者・発行元:The Japanese Society of Animal Reproduction (JSAR)  

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

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

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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年

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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年

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  • 豚卵胞卵子の体外成熟・受精:とくに初期発生能獲得に関する研究(共著)

    舟橋弘晃

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

  • 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年

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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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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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年

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  • 豚受精卵の体外生産と人為操作

    舟橋弘晃

    家畜人工授精   ( 185 )   14 - 22   1998年

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    記述言語:日本語   出版者・発行元:(一社)日本家畜人工授精師協会  

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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年

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

    Journal of Reproduction and Fertility, Abstract Series   21   10   1998年

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

    Journal of Reproduction and Fertility, Abstract Series   21   10   1998年

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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年7月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.

    DOI: 10.1095/biolreprod57.1.49

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

    Ken Sawai, Hiroaki Funahashi, Koji Niwa

    Biology of Reproduction   57 ( 1 )   1 - 6   1997年7月

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    記述言語:英語  

    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 oysteine during in-vitro maturation of porcine oocytes for glutathione synthesis ossociated with male pronuclear formation(共著)

    Biology of Reproduction   57 ( 1 )   1 - 6   1997年7月

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  • Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic AMP improves developmental competence following in vitro fertilization(共著) 国際誌

    Biology of Reproduction   57 ( 1 )   49 - 53   1997年7月

  • Chlortetracycline fluorescence patterns and in vitro fertilization of frozen-thawed boar spermatozoa incuboted under various bicarbonate concentrations(共著) 国際誌

    Zygote   5 ( 2 )   117 - 125   1997年5月

  • 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年5月

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    記述言語:英語   出版者・発行元:CAMBRIDGE UNIV PRESS  

    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(共著)

    56 ( 4 )   921 - 930   1997年4月

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

    Z Machaty, H Funahashi, BN Day, RS Prather

    BIOLOGY OF REPRODUCTION   56 ( 4 )   921 - 930   1997年4月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.

    DOI: 10.1095/biolreprod56.4.921

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  • Preincubation of oocyte-cumulus complexes before exposing to gonadotropins enhanced the developmental ability of porcine embryos maturad and fertilized in vitro(共著)

    Theriogenology   47 ( 3 )   679 - 686   1997年

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

    Fifth International Conference on Pig Reproduction   142   1997年

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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年

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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年

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

    Biology of Reproduction   56 ( Supplementl )   514 - 514   1997年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)  

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  • Advances in in-vitro production of porcine embryos(共著)

    Journal of Reproduction and Fertility   Supplement52   271 - 283   1997年

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  • Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocyte penetrated in vitro(共著)Journal of Reproduction and Development

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

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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年

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    記述言語:英語   出版者・発行元: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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  • Preincubation of oocyte-cumulus complexes before exposing to gonadotropins enhanced the developmental ability of porcine embryos maturad and fertilized in vitro(共著)

    Theriogenology   47 ( 3 )   679 - 686   1997年

  • Advances in in vitro production of pig embryos

    H Funahashi, BN Day

    JOURNAL OF REPRODUCTION AND FERTILITY   Supplement52   271 - 283   1997年

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    記述言語:英語   出版者・発行元: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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  • Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

    Fifth International Conference on Pig Reproduction   142   1997年

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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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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  • 豚透明帯糖残基の局在性および精子結合・侵入に及ぼす影響(共著)

    宋学雄, 楊信志, 舟橋弘晃, 丹羽こう二

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

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  • 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年

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  • 受精培地中のNaCl濃度がラット卵子への精子侵入およびその後の初期発生におよぼす影響

    OH S-H, 舟橋弘晃, 丹羽こう二

    Journal of mammalian ova research   14 ( 1 )   1997年

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  • 牛精液用0.5mlストローを用いた豚精子の凍結保存

    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年

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  • Advances in in vitro production of pig embryos. 国際誌

    H. Funahashi, B. N. Day

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

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    記述言語:英語  

    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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  • Microtubule organization in porcine oocytes during fertilization and parthenogenesis 国際誌

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

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1397 - 1404   1996年6月

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    記述言語:英語   出版者・発行元: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.

    DOI: 10.1095/biolreprod54.6.1397

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

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

    BIOLOGY OF REPRODUCTION   54 ( 6 )   1412 - 1419   1996年6月

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    記述言語:英語   出版者・発行元: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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  • Microtuble organization in porcine oocytes during fertilization and parthenogenesis.(共著)

    Biology of Reproduction   54 ( 6 )   1397 - 1404   1996年6月

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  • Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes.(共著)

    Biology of Reproduction   54 ( 6 )   1412 - 1419   1996年6月

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

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

    JOURNAL OF REPRODUCTION AND FERTILITY   107 ( 1 )   79 - 86   1996年5月

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    記述言語:英語   出版者・発行元:J REPROD FERTIL INC  

    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 vitromatured porcine oocytes.(共著) 国際誌

    Biology of Reproduction   54 ( 2 )   316 - 322   1996年2月

  • 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年2月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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 development al competence of pig oocytes matured in vitro.(共著)

    Biology of Reproduction   54 ( Supplement 1 )   100 - 100   1996年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)  

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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年

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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 borine and rat 1-cell embryos(共著)

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

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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年

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  • Factors affecting nuclear and cytoplasmic maturation in pig oocytes.(共著)

    Biology of Reproduction   54 ( Supplement 1 )   412 - 412   1996年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)  

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

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

  • 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年

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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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  • 糖類が豚卵子への精子侵入におよぼす影響(共著)

    宋学雄, 沢井健, 許東けい, 舟橋弘晃, 丹羽こう二

    第89回日本繁殖生物学会講演要旨   89th   57   1996年

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  • システインおよびシスチンが豚卵胞卵子の応性前核形成能に及ぼす影響(共著)

    沢井健, 舟橋弘晃, 丹羽こう二

    第89回日本繁殖生物学会講演要旨   89th   44   1996年

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  • 成長ホルモンが牛卵胞卵子の成熟分裂の進行速度におよぼす影響(共著)

    伊賀浩輔, 舟橋弘晃, 丹羽こう二

    第89回日本繁殖生物学会講演要旨   89th   41   1996年

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

    Biology of Reproduction   54 ( Supplement 1 )   408 - 408   1996年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)  

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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年

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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年

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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年

  • 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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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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年

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  • Pronuclear formation and intracellular glutathione content of in vitro matured porcine oocytes following in vitro fertilization and/or electrical activation.(共著)

    Zygote   3 ( 3 )   273 - 281   1995年8月

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  • Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos.(共著)

    Theriogenology   44 ( 3 )   391 - 401   1995年8月

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

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

    ZYGOTE   3 ( 3 )   273 - 281   1995年8月

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    記述言語:英語   出版者・発行元:CAMBRIDGE UNIV PRESS  

    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年8月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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.(共著)

    Biology of Reproduction   52 ( Supplement 1 )   122 - 122   1995年

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  • γ-glutamyl-transpeptidase(GGT)of spermatozoa may reduce oocyte glutathione(GSH)contest at sperm penetration.(共著)

    Biology of Reproduction   52 ( Supplement1 )   140 - 140   1995年

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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年

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

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

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

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

  • 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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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

    Journal of Animal Science   73 ( Supplement 1 )   90   1995年

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

    Theriogenology   43 ( 1 )   262   1995年

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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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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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年

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

    Journal of Animal Science   73 ( Supplement 1 )   90   1995年

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

    Theriogenology   43 ( 1 )   262   1995年

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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年

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  • Effect of taxol on microtuble organization in porcine oocytes after fertilization and parthenogenetic activation.(共著)

    Biology of Reproduction   52 ( Supplement 1 )   139 - 139   1995年

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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.(共著)

    Biology of Reproduction   51 ( 4 )   633 - 639   1994年10月

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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 国際誌

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   51 ( 4 )   633 - 639   1994年10月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.(共著)

    Theriogenology   41 ( 7 )   1425 - 1433   1994年5月

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  • In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization.(共著)

    Biology of Reproduction   50 ( 5 )   1072 - 1077   1994年5月

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  • DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO 国際誌

    H FUNAHASHI, T CANTLEY, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   101 ( 1 )   159 - 165   1994年5月

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    記述言語:英語   出版者・発行元:J REPROD FERTIL INC  

    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 国際誌

    H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

    THERIOGENOLOGY   41 ( 7 )   1425 - 1433   1994年5月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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.

    DOI: 10.1016/0093-691X(94)90193-M

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  • IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION 国際誌

    H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

    BIOLOGY OF REPRODUCTION   50 ( 5 )   1072 - 1077   1994年5月

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    記述言語:英語   出版者・発行元:SOC STUDY REPRODUCTION  

    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.

    DOI: 10.1095/biolreprod50.5.1072

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  • Different hormonal requirements of pig oocyte-cumulus complexes during maturation in vitro.(共著)

    Journal of Reproduction and Fertility   101 ( 1 )   159 - 165   1994年5月

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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年1月

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    記述言語:英語   出版者・発行元:J REPROD FERTIL INC  

    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.(共著)

    Journal of Reproduction and Fertility   100 ( 1 )   21 - 26   1994年1月

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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年

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  • Histone H1 kinase(H1K)activity and glutathione(GSH)content of porcine oocytes during pronuclear formation after in vitro fertilization.(共著)

    Biology of Reproduction   50 ( Supplement 1 )   128 - 128   1994年

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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年

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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年

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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年

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  • Microtubule organization of porcine oocytes during in vitro fertilization.(共著)

    Biology of Reproduction   50 ( Supplement 1 )   146 - 146   1994年

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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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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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年

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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年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SOC STUDY REPRODUCTION  

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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年

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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年

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  • Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes.(共著)

    Molecular Reproduction and Development   36 ( 3 )   361 - 367   1993年11月

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  • EFFECTS OF ELECTRICAL-STIMULATION BEFORE OR AFTER IN-VITRO FERTILIZATION ON SPERM PENETRATION AND PRONUCLEAR FORMATION OF PIG OOCYTES 国際誌

    H FUNAHASHI, TT STUMPF, SL TERLOUW, BN DAY

    MOLECULAR REPRODUCTION AND DEVELOPMENT   36 ( 3 )   361 - 367   1993年11月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

    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.

    DOI: 10.1002/mrd.1080360312

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  • EFFECTS OF FOLLICULAR-FLUID AT FERTILIZATION IN-VITRO ON SPERM PENETRATION IN PIG OOCYTES 国際誌

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   99 ( 1 )   97 - 103   1993年9月

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    記述言語:英語   出版者・発行元:J REPROD FERTIL INC  

    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.

    DOI: 10.1530/jrf.0.0990097

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  • Effects of follicular fluid at fertilization in vitro on sperm penetration of pig oocytes.(共著)

    Journal of Reproduction and Fertility   99 ( 1 )   97 - 103   1993年9月

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  • EFFECTS OF THE DURATION OF EXPOSURE TO HORMONE SUPPLEMENTS ON CYTOPLASMIC MATURATION OF PIG OOCYTES INVITRO 国際誌

    H FUNAHASHI, BN DAY

    JOURNAL OF REPRODUCTION AND FERTILITY   98 ( 1 )   179 - 185   1993年5月

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    記述言語:英語   出版者・発行元:J REPROD FERTIL INC  

    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.

    DOI: 10.1530/jrf.0.0980179

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  • Effects of the duration of exposure to hormone supplements on cytoplasmic maturation of pig oocytes in vitro.(共著)

    Journal of Reproduction and Fertility   98 ( 1 )   179 - 185   1993年5月

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  • EFFECTS OF DIFFERENT SERUM SUPPLEMENTS IN MATURATION MEDIUM ON MEIOTIC CYTOPLASMIC MATURATION OF PIG OOCYTES 国際誌

    H FUNAHASHI, BN DAY

    THERIOGENOLOGY   39 ( 4 )   965 - 973   1993年4月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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.

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

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  • Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes.(共著)

    Theriogenology   39 ( 4 )   965 - 973   1993年4月

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  • Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium.(共著)

    Theriogenology   39 ( 4 )   875 - 886   1993年4月

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  • GLUCOSE REQUIREMENT AT DIFFERENT DEVELOPMENTAL STAGES OF INVITRO FERTILIZED BOVINE EMBRYOS CULTURED IN SEMI-DEFINED MEDIUM 国際誌

    JH KIM, H FUNAHASHI, K NIWA, K OKUDA

    THERIOGENOLOGY   39 ( 4 )   875 - 886   1993年4月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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年

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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年

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  • Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   23   1993年

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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年

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  • Pig oocyte activation and processing of transplanted nuclei.(共著)

    39 ( 1 )   329   1993年

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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年

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  • Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Theriogenology   39 ( 1 )   322   1993年

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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年

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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年

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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年

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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年

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  • Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

    Fourth International Conference on Pig Reproduction   23   1993年

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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年

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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年

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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年

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  • Pig oocyte activation and processing of transplanted nuclei.(共著)

    39 ( 1 )   329   1993年

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  • Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

    Theriogenology   39 ( 1 )   322   1993年

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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年

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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年

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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年

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  • Assessment of porcine oocytes using brilliant cresyl blue.(共著)

    Theriogenology   39 ( 1 )   214   1993年

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  • FERTILIZATION AND EARLY CLEAVAGE INVITRO OF AGING BOVINE OOCYTES AFTER MATURATION IN CULTURE 国際誌

    RC CHIAN, H NAKAHARA, K NIWA, H FUNAHASHI

    THERIOGENOLOGY   37 ( 3 )   665 - 672   1992年3月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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 in vitro of ageing bovine oocytes after maturation in culture.(共著)

    Theriogenology   37 ( 3 )   665 - 672   1992年3月

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    記述言語:英語  

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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年

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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年

  • 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年

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    記述言語:英語  

    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年

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  • 完全体外培養系で得た牛体外受精凍結卵の移植試験

    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年

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  • 牛における個体別体外受精卵の作出試験

    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年

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  • 黄体ホルモン製剤利用による牛の反復過排卵処置に関する検討

    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年

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  • First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6℃ for various durations.(共著)

    Theriogenology   36 ( 3 )   411 - 417   1991年9月

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  • Developmental capacity of bovine oocytes collected from ovaries of individual heifers and fertilized in vitro.(共著)

    Theriogenology   36 ( 3 )   427 - 434   1991年9月

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  • DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES COLLECTED FROM OVARIES OF INDIVIDUAL HEIFERS AND FERTILIZED INVITRO 国際誌

    H FUNAHASHI, Y AOYAGI, T TAKEDA, T ONIHARA

    THERIOGENOLOGY   36 ( 3 )   427 - 434   1991年9月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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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  • 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年9月

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    記述言語:英語   出版者・発行元:BUTTERWORTH-HEINEMANN  

    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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  • 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年

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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年

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  • ラットおよびマウスの初期はいにおける属間核移植試験

    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年

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  • 牛における核移植に関する検討

    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年

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  • 豚における低コスト麻酔法の検討

    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年

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  • ラットおよびマウスの属間核移植卵の体内発育試験

    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年

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  • 人工透明帯封入牛分割凍結卵の移植試験

    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年

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  • 完全体外培養系で得た牛体外受精卵の移植試験

    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年

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  • 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年6月

  • Electric fusion of 2-cell rat embryo blastomeres.(共著)

    Hiroaki FUNAHASHI, Koji NIWA

    Japanese Journal of Animal Reproduction   36 ( 2 )   114 - 119   1990年

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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年

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  • 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年

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  • Electric fusion of 2-cell rat embryo blastomeres.(共著)

    舟橋 弘晃, 丹羽 晧二

    Japanese Journal of Animal Reproduction   36 ( 2 )   114 - 119   1990年

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  • 牛およびマウス卵子を用いた分割卵凍結用保護物質の検討

    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年

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  • 牛受精卵を用いた分割卵凍結用保護物質の検討

    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年

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  • 豚受精卵の短期保存後の移植試験

    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年

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  • 豚の手術的採卵・移植技術の検討.(共著)

    武富敏郎, 立花文夫, 舟橋弘晃, 武田哲男, 鈴木正, 兼子よう子

    全農飼料畜産中央研究所試験研究報告   16 ( 1 )   493 - 501   1989年

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  • 豚受精卵の長距離輸送による移植試験.(共著)

    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

    全農飼料畜産中央研究所試験研究報告   17 ( 1 )   365 - 370   1989年

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  • 牛卵子の体外受精においてヘパリンとカフェインの受精と発生に与える影響.(共著)

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    全農飼料畜産中央研究所試験研究報告   16 ( 1 )   507 - 513   1989年

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  • 牛卵胞卵子の体外成熟に及ぼすFSHおよびhCGの影響.(共著)

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    全農飼料畜産中央研究所試験研究報告   16 ( 1 )   502 - 506   1989年

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  • 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年

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  • 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年

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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  • 牛体外受精卵の体外培養法の検討.(共著)

    舟橋弘晃, 武富敏郎

    全農飼料畜産中央研究所試験研究報告   17 ( 1 )   396 - 406   1989年

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  • 豚受精卵における24時間以上の培養法の検討.(共著)

    亀山賢次, 武富敏郎, 舟橋弘晃, 武田哲男, 板倉はつえ

    全農飼料畜産中央研究所試験研究報告   17 ( 1 )   390 - 395   1989年

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  • 牛体外受精卵の凍結保存における凍結保護物質の検討.(共著)

    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

    全農飼料畜産中央研究所試験研究報告   17 ( 1 )   381 - 389   1989年

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  • 牛体外受精の諸要件による受精卵の移植.(共著)

    舟橋弘晃, 武富敏郎, 武田哲男, 鬼原辰夫

    全農飼料畜産中央研究所試験研究報告   17 ( 1 )   371 - 380   1989年

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  • 牛における凍結受精卵の非手術的移植技術の検討.(共著)

    武富敏郎, 鈴木正, 舟橋弘晃, 武田哲男, 立花文夫, 兼子よう子

    全農飼料畜産中央研究所試験研究報告   16 ( 1 )   486 - 492   1989年

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  • 電気的融合法によるラット1細胞期卵の前核置換と置換卵の発生能.(共著)

    舟橋 弘晃, 丹羽 晧二

    家畜繁殖学雑誌   34 ( 3 )   133 - 137   1988年

  • 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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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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年

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▼全件表示

産業財産権

  • 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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    出願人:国立大学法人 岡山大学

    出願番号:特願2012-081396  出願日:2012年3月30日

    公開番号:特開2012-213395  公開日:2012年11月8日

    特許番号/登録番号:特許第5877512号  登録日:2016年2月5日 

    J-GLOBAL

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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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    出願人:国立大学法人 岡山大学

    出願番号:特願2011-523592  出願日:2010年6月24日

    公表番号:WO2011-010525  公表日:2011年1月27日

    特許番号/登録番号:特許第5610313号  登録日:2014年9月12日 

    J-GLOBAL

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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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    出願人:国立大学法人 岡山大学

    出願番号:特願2007-194968  出願日:2007年7月26日

    公開番号:特開2009-027973  公開日:2009年2月12日

    特許番号/登録番号:特許第5083952号  登録日:2012年9月14日 

    J-GLOBAL

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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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    出願人:全国農業協同組合連合会

    出願番号:特願2000-232041  出願日:2000年7月31日

    公開番号:特開2001-103867  公開日:2001年4月17日

    特許番号/登録番号:特許第4845073号  登録日:2011年10月21日 

    J-GLOBAL

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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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    出願人:岡山大学長

    出願番号:特願2000-070752  出願日:2000年3月14日

    公開番号:特開2001-251990  公開日:2001年9月18日

    J-GLOBAL

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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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    出願人:全国農業協同組合連合会

    出願番号:特願平2-404910  出願日:1990年12月21日

    公開番号:特開平5-310501  公開日:1993年11月22日

    J-GLOBAL

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▼全件表示

受賞

  • 日本繁殖生物学会賞(島村賞)

    1998年  

     詳細を見る

    受賞国:日本国

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共同研究・競争的資金等の研究

  • 生命機能の光制御のための機能性光増感ペプチドの開発

    研究課題/領域番号:18H02090  2018年04月 - 2022年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    大槻 高史, 舟橋 弘晃

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    配分額:17160000円 ( 直接経費:13200000円 、 間接経費:3960000円 )

    本研究では、光増感剤とペプチド/タンパク質を組み合わせた機能性光増感ペプチド(FPP)を開発し、細胞機能を光で時空間的制御して観察する技術を確立する。 FPPは光依存的に細胞質内に入っていく分子である。 本研究ではFPP開発とともに、FPP技術の生殖・発生・細胞分化への応用を進めている。本年度は以下に取り組んだ。
    1)FPP一過的導入に基づくFPP機能の細胞周期依存性の解明:HeLa細胞では各細胞周期phaseは1時間弱から数時間で次の周期に切り替わる。このような細胞周期と外来ペプチド/タンパク質の細胞内機能との関係性を調べるためには、その素早い細胞内導入法が必要である。本年度はアポトーシス誘導性のBimペプチドをFPPの形状にして、光で瞬時に細胞質内に導入し、アポトーシス誘導機能の細胞周期依存性を調べた。その結果、G1/S移行期にBimによるアポトーシスが起こりやすくなることを明らかにした。
    2)FPPを用いた人為的卵活性化に向けたTatPLCζの遺伝子工学的作製:哺乳動物では受精時、精子が卵子に侵入すると卵活性化因子PLCζが導入され、反復性の細胞内Ca2+濃度の上昇の誘導を経て、卵活性化して発生が始まる。この卵活性化機構が不全なケースは不妊の要因の1つである。この解決策としてPLCζの人為的導入が考えられるため、FPPの仕組みで働くTatPLCζの作製を行った。その遺伝子を作製し、発現精製したところ、少ないながらも目的産物が得られた。
    3)初期発生の時空間制御:shRNA導入による初期発生の時空間制御を目指し、内部細胞塊と栄養外胚葉の2つの細胞系譜への分化に関わる遺伝子の時空間特異的ノックダウンを試みた。狙いをつけた細胞内へのshRNA導入はできるようになり、僅かながら標的のaPKCノックダウンが見られた。

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  • 卵細胞質の時空間的ミトコンドリア機能制御による高妊孕性卵母細胞の創出

    研究課題/領域番号:18H02328  2018年04月 - 2022年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    舟橋 弘晃, 中塚 幹也, 若井 拓哉, 大槻 高史

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    配分額:17290000円 ( 直接経費:13300000円 、 間接経費:3990000円 )

    本研究は、卵細胞質を量的・質的に改変(ミトコンドリアDNA(mtDNA)コピー数制御を含む)することで、卵母細胞の時空間的制御が発生能に関与する機構を明らかにし、発生・妊孕能の高い卵母細胞の作出系を構築することを目的としている。
    令和元年度は以下について取り組んだ。
    (1)卵細胞質mtDNAコピー数の成熟時の動態解析:IVM培養前後の卵母細胞中ミトコンドリアDNAコピー数は体外成熟前後で有意差が認められなかったが、MF由来卵母細胞中のDNAコピー数と比較してSF由来卵母細胞中のそれは有意に低いことが明らかになったので、ミトコンドリア合成に係る遺伝子を強制発現させることでどのような影響が出るのかを調べるために種々の手法を試した。
    (2)ミトコンドリア融合・分裂能の解析と卵細胞質オートファジー活性の成熟・発生時の変動解析: 中卵胞(MF:直径3-6 mm)由来卵母細胞と小卵胞(SF:直径3 mm未満)由来卵母細胞でオートファジー能について調べた。その結果、体外成熟および初期発生能を指標としたオートファジー阻害剤や同促進剤に対する反応がMF由来卵母細胞はそれらに反応する能力があるものの、SF由来卵にはそれらがないことを明らかにした。また、オートファジー能にも影響することが知られているトレハロースがSF由来卵母細胞の体外成熟および初期発生能を改善することを明らかにした。一方、卵母細胞からの精子ミトコンドリア除去にオートファジーが関わる可能性を示した。
    (3)ケージドアミノアシルtRNAのミトコンドリア制御への適用: 卵母細胞への光増感剤とペプチド/タンパク質を組み合わせた分子ツール(機能性光増感ペ
    プチド:Functional Photosensitizing Peptide/Protein; FPP)の製作と卵への導入を引き続き検討した。

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  • 妊孕性温存のためのヒト卵巣凍結保存と移植後の血管新生促進による成熟卵子の作出

    研究課題/領域番号:16K11089  2016年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    本橋 秀之, 中塚 幹也, 舟橋 弘晃, 若井 拓哉, 高山 修

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    若年女性ががんに罹患すると,抗がん剤や放射線治療によって不妊になるリスクが増大する。そこで生存性に優れた卵巣組織凍結保存法を開発することを目的とした。本研究では、まず最初にガラス化保存新規デバイスを開発し、それらに最適化したガラス化保存プロトコールの確立を行った。次に動物モデルにより、開発したガラス化保存デバイスおよび最適化プロトコールにもとづいて卵巣組織を凍結融解し、移植によってその後の体内発生能や卵子の体外成熟能を検証した結果、組織の生着、卵胞発育および卵母細胞成熟が確認され、本卵巣組織ガラス化保存法の有効性が示された。

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  • ブタ精液中に存在する膠様物の機能解析と応用

    研究課題/領域番号:24658236  2012年04月 - 2014年03月

    日本学術振興会  科学研究費助成事業 挑戦的萌芽研究  挑戦的萌芽研究

    舟橋 弘晃

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    配分額:2990000円 ( 直接経費:2300000円 、 間接経費:690000円 )

    ブタ精液中の膠様物の諸特性を解析評価した。膠様物は、強酸性および強アルカリ性で溶解できたが、中性周辺では比較的安定していた。また、子宮内に多く存在する多核白血球の膠様物への走化性はなく、また、極めて高い保水性が認められた。粉末膠様物を精液と混和することで、精子カプセルを作成することが出来、そのカプセル中で一定時間精子が生存でき、また徐々に精子が放出されることを確認できた。化学的な解析により、膠様物はo-グリカンを多く含むことが明らかとなり、糖鎖解析とペプチド解析から特異的な糖鎖とタンパク質を含むことが明らかとなった。これらの知見は、医療分野を含む広い領域で有効活用できる可能性を含む。

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  • 和牛の遺伝的改良を目的とした血液凝固第XI因子欠乏症の生産性に与える影響の解明

    研究課題/領域番号:23380166  2011年04月 - 2014年03月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    国枝 哲夫, 辻 岳人, 揖斐 隆之, 舟橋 弘晃, アコスタ トマス

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    配分額:18200000円 ( 直接経費:14000000円 、 間接経費:4200000円 )

    血液凝固第XI因子欠乏症は黒毛和種牛に見られる出血性の遺伝性疾患である。本疾患の出血傾向自体は軽微であるが、その遺伝子頻度はきわめて高く、もし本疾患が肉牛の生産性に影響を与えるなら、経済的損失は多大となる。そこで本研究では、第XI因子欠乏症と異常産や肉の形質との関連について大規模な調査を行った。その結果、第XI因子欠乏症では流産等の異常産の発生頻度が高い傾向があるが、新生仔の異常やの瑕疵等の枝肉形質には顕著な影響は与えないことが明らかとなった。

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  • 哺乳動物の新たな人工授精システム構築に向けた雌性生殖道内の免疫制御技術の開発

    研究課題/領域番号:20380154  2008年 - 2010年

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    舟橋 弘晃, 近藤 康博

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    配分額:9360000円 ( 直接経費:7200000円 、 間接経費:2160000円 )

    哺乳動物の雌子宮内に交配後に子が入ると、免疫機構が働き、精子を除去する仕組みが存在する。本研究では、この機構が多核白血球という免疫細胞によって行われ、それによる精子の貪食は、血清の存在下で活発に行われるが、運動精子と死滅精子の両方を同様に貪食することを明らかにした。さらに、カフェインやへパリン、精漿などの存在下で、牛および豚多核白血球の同種精子への走化性と貪食能を抑制することを明らかにした。

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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

    研究課題/領域番号:16580230  2004年 - 2005年

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    舟橋 弘晃

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    配分額:3600000円 ( 直接経費:3600000円 )

    本研究プロジェクトで得られた研究成果は以下のとおりである。
    A.瞬時共培養体外受精システムにおける共培養条件の詳細な検討
    瞬時共培養時における種々の影響を調べた結果、共培養後にカフェインが存在すると多精子受精卵率が増加し、アデノシン存在下で精子を前培養すると、共培養時のカフェインの存否に関係なく多精子受精卵率が改善されることを明らかにした。
    B.瞬時共培養後の効率的な精子受精能獲得誘起手法の確立
    精子受精能獲得誘起手法の検討を行い、beta-mercaptoethanol(BME)の存在は精子の受精能獲得および先体反応を阻害するが、過酸化水素の添加は逆にそれらの反応を促進することを明らかにした。また、プロテアーゼ・インヒビターの存在下で精子は受精能獲得や先体反応に何等影響を受けないが、卵子透明帯への接合に結合する精子の数が減少することを明らかにした。
    C.瞬時共培養体外受精システムにおける共培養条件の詳細な検討
    共培養時およびその後の受精培養時の酸化ストレスが受精に及ぼす影響について調べるために、それぞれの培地にBMEを添加してその影響を調べた。その結果、共培養中にBMEが存在すると精子の受精能獲得・先体反応を阻害し、精子侵入率が低下されるが、その後の受精培地中での存在は卵子の表層粒反応の程度を改善し、単精子受精卵率を増大した。さらに瞬時共培養後の受精培地および初期発生培地にBMEを添加することで、卵子自体の表層顆粒反応や体外受精後に発生した胚盤胞の細胞数を改善することを明らかにした。
    D.酸化ストレス軽減による精子受精能獲得誘起阻止現象を利用した精子の液状保存法の確立
    還元剤を豚精子の液状保存に利用できないかと試みた結果、保存液中にシステインを添加することで豚精子の10℃での液状保存中の生存率や受精能未獲得状態の維持に有効であることを見出した。
    E.瞬時共培養システムに供する卵子の体外成熟システムの開発
    瞬時共培養に用いる卵子の体外成熟システムの構築を目指して、ロスコビチンによる処理で採取した卵母細胞を48時間培養保存後に体外成熟・瞬時共培養を行っても、保存培養していない対照区と大差なく成熟し、受精することが可能であることを見出した。

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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

    研究課題/領域番号:10556065  1998年 - 2001年

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    舟橋 弘晃, 青柳 敬人, 丹羽 晧二, 岡部 勝

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    配分額:12300000円 ( 直接経費:12300000円 )

    先ず、緑色蛍光蛋白質(GFPまたはEGFP)遺伝子および他の目的遺伝子としてネオマイシン耐性遺伝子を含む遺伝子断片を授精後様々な時間に牛体外受精卵の前核に注入し、注入時期による外来遺伝子の発現およびその後の初期発生を調べたところ、注入の時期に関わらず牛胚では注入遺伝子がモザイクに発現する頻度の高いこと、また前核可視化のための遠心分離が授精後20時間以降の受精卵の初期発生を著しく阻害することが明らかになった。次に、EGFP遺伝子を前核注入後、EGFPを発現している(体外受精卵由来)牛桑実期胚割球を(染色体を除いた)体外成熟卵子に核移植したところ、胚盤胞期胚を得ることができたが、中にはEGFPが発現していない胚も存在し、桑実期胚の段階でさえゲノムに組み込まれずに発現している遺伝子が存在することが明らかになった。しかし、EGFPを発現している胚では、すべての細胞でEGFPが発現しており、これらの細胞を継代培養したところ、すべての細胞でEGFPと同時に組み込んだネオマイシン耐性遺伝子の発現も確認できた。以上の研究から、緑色蛍光蛋白質遺伝子をマーカーとして利用し、かつ核移植技術を巧みに組み合わせることによって、目的遺伝子をすべての細胞で発現するトランスジェニック動物(細胞)を生産するシステムを構築することができた。
    また、豚の体外受精系を用いて、遺伝子を導入する材料である正常な前核期受精卵を体外で効率的に作製するための基礎研究にも取り組んだ。豚精子の授精能獲得および先体反応を促進する試薬に注目し、従来のカフェインを含む体外受精系では、先体反応初期まで誘起されることが多精子受精を引き起こす要因の一つであることを示唆し、アデノシンや受精促進ペプチドのような受精能獲得を促進し、自発的先体反応を阻止する試薬の存在が単精子侵入頻度を増すことを明らかにした。

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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

    研究課題/領域番号:09660303  1997年 - 1998年

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    舟橋 弘晃

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    配分額:3800000円 ( 直接経費:3800000円 )

    ラット精子のDNAの酸に対する安定性とチオールの酸化還元状態が精巣上体での成熟および卵子への精子侵入の間にどのように変化するのかを調べた。DNAの酸に対する安定性を調べるために、円形精子細胞および精子を酢酸アルコールによる固定後アクリジンオレンジで処理したところ、共焦点レーザー顕微鏡下での赤色/緑色蛍光比は、精子が精巣上体頭部に移行するまでに低下した。還元型チオールに結合して蛍光を発するmonobromobimane(mBBr)をラベルした場合、蛍光量は精子の精巣から精巣上体尾部への移行に伴い徐々に低下し、精巣上体尾部および射出精子では蛍光が観察されなかった。また、アクリジンオレンジの赤色/緑色蛍光比は、透明帯通過および膨化精子で高かった。さらに、囲卵腔の精子をmBBrでラベルしたところ蛍光が観察された。これらのことから、精子の精巣上体における成熟でDNAの酸に対する安定化はプロタミンの酸化に先行し、プロタミンの還元およびDNAの不安定化は精子が卵子透明帯を通過後にすでに始まっていることを示した。
    精子侵入および前核形成に伴うラット卵子中のグルタチオンおよび関連するチオール類量の変化を高速液体クロマトグラフィーを用いて解析した。還元型グルタチオンは、未受精卵および膨化精子を有する受精卵で前核期受精卵と比較して高かった。酸化型グルタチオン量は、精子侵入および前核形成を通じて変化しなかった。還元型システニルグリシン量に精子侵入および前核形成による変動は認められなかったが、酸化型システニルグリシン量は精子頭部の膨化後、前核形成までに増加した。また、低量のシスチンは受精過程を通じて認められたが、システインおよびγ-グルタミルシステインは、検出されなかった。これらの結果から、ラット卵子でのグルタチオン量は、精子頭部の膨化と前核形成の間に減少することが明らかとなった。この減少はγ-グルタミルトランスペプチダーゼ活性の上昇によるものと推察される。

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  • Study on in vitro production and manipulation of mammalian gametes.

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    資金種別:競争的資金

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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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    資金種別:競争的資金

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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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    資金種別:競争的資金

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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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    資金種別:競争的資金

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  • Study on the capacitation of mammalian spermatogoa

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    資金種別:競争的資金

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  • Study on the mechanisn of male pronuclear formation in mammalian zygotes.

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    資金種別:競争的資金

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学術貢献活動

  • 国際受精卵移植学会財団理事

    役割:企画立案・運営等

    国際受精卵移植学会  2010年1月 - 2015年1月

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    種別:学会・研究会等 

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