Faculty Profiles - FUNAHASHI Hiroaki

Papers - FUNAHASHI Hiroaki

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Exogenous expression of PGC-1α during in vitro maturation impairs the developmental competence of porcine oocytes Reviewed

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

Theriogenology 228 30 - 36 2024.10

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes Reviewed

Wanniarachchige Tharindu Lakshitha Fonseka, Son Quang Do, Phong Ngoc Van, Hai Thanh Nguyen, Takuya Wakai, Hiroaki Funahashi

Theriogenology 226 158 - 166 2024.9

Effect of oral administration of dehydroepiandrosterone on PCOS-Like phenotype of female C57BL/6 mice Reviewed

Ayu Mulia Sundari, Nurul Muhammad Prakoso, Astari Dwiranti, Abinawanto Abinawanto, Arief Boediono, Hiroaki Funahashi, Anom Bowolaksono

Jurnal sain peternakan indonesia 19 ( 2 ) 111 - 116 2024.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Badan penerbitan fakultas pertanian (BPFP) universitas bengkulu

We aim to evaluate the efficacy and optimal dose of orally administered DHEA in the PCOS mice model by assessing their ovarian morphology and serum FSH, LH, and testosterone levels. Female C57BL/6 mice were divided into a control group (n=5, received daily injections of 0.2 ml sesame oil) and an experimental group, which was further classified into 1) D-45 group (n=5), 2) D-60 (n=5), and 3) D-90 group (n=5) that were treated with 45, 60, and 90 mg/Kg body weight of oral DHEA. After modelling, mice in the control group had a regular estrous cycle, while mice in all experimental groups had a disturbed estrous cycle. Ovarian histology showed several growing follicles and some corpora lutea (CL) in the control, D-60, and D-90 groups. However, some large antral follicles and decreased CL were observed in the D-45 group. Serum FSH was significantly lower in the D-45 group than in the control group (3.73 ± 0.12 vs. 5.28 ± 0.31 mIU/mL, P<0.01), but D-60 and D-90 groups had a similar FSH level to the control group (P>0.05). The serum level of LH and testosterone were significantly elevated in the D-45 group than in the control group (2.52 ± 0.43 vs. 1.30 ± 0.33 mIU/mL, P<0.01 and 1.80 ± 0.32 vs. 1.24 ± 0.23 ng/mL, P<0.01, respectively). Still, the level of LH and testosterone in the D-60 and the D-90 groups was comparable to the control group (P>0.05). Our study suggests that oral administration of DHEA is efficacious in establishing PCOS-like phenotype in mice with the suggested optimal dosage of 45 mg/Kg body weight.

DOI： 10.31186/jspi.id.19.2.111-116

Hormonal and Laboratory Profile of Polycystic Ovarian Syndrome (Pcos) Women Visiting Fertility Center in Indonesia Reviewed

Ayu Mulia Sundari, Tri Aprilliana Wulandari, Nurul Muhammad Prakoso, Astari Dwiranti, Abinawanto, Arief Boediono, Hiroaki Funahashi, Anom Bowolaksono

Jurnal Profesi Medika : Jurnal Kedokteran dan Kesehatan 18 ( 1 ) 67 - 75 2024.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Fakultas Kedokteran UPN Veteran Jakarta

Our study aims to scrutinize the hormonal and laboratory profile of women with PCOS visiting a fertility center in Indonesia. A computerized database of 120 PCOS women and 120 controls was retrospectively reviewed and analyzed. According to analysis, the concentration of AMH and basal LH were significantly more elevated in the PCOS group than in the control group (6.77 vs. 2.05, P<0.001; and 6.8 vs. 5.3, P<0.001, respectively). The level of basal FSH was significantly lower in the PCOS group (5.83 vs. 7.86; P<0.001). After an IVF cycle, PCOS women had significantly higher retrieved oocytes and mature oocytes compared to controls (18 vs. 8, P<0.001; 12 vs. 6, P<0.001). The number of fertilizations, cleavages, and top-quality cleavages were also higher in the PCOS group than in the control group (10 vs. 4, P<0.001; 10 vs. 4, P<0.001; 4 vs. 2, P<0.001). At the blastocyst stage, patients with PCOS had a higher number of blastocysts and top-quality blastocysts than control (9 vs. 4, P<0.001); 4 vs. 1, P<0.01). However, the clinical pregnancy was significantly higher in the control group than in the PCOS group (52.6% vs. 16.7%; P = 0.045).

DOI： 10.33533/jpm.v18i1.7546

Redistribution of fragmented mitochondria ensure symmetric organelle partitioning and faithful chromosome segregation in mitotic mouse zygotes Reviewed

Haruna Gekko, Ruri Nomura, Daiki Kuzuhara, Masato Kaneyasu, Genpei Koseki, Deepak Adhikari, Yasuyuki Mio, John Carroll, Tomohiro Kono, Hiroaki Funahashi, Takuya Wakai

2024.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Cold Spring Harbor Laboratory

Abstract

In cleavage-stage embryos, preexisting organelles partition evenly into daughter blastomeres without significant cell growth after symmetric cell division. The presence of mitochondrial DNA within mitochondria and its restricted replication during preimplantation development makes their inheritance particularly important. While chromosomes are precisely segregated by the mitotic spindle, the mechanisms controlling mitochondrial partitioning remain poorly understood. In this study, we investigate the mechanism by which Dynamin-related protein 1 (Drp1) controls the mitochondrial redistribution and partitioning during embryonic cleavage. Deletion of Drp1 in mouse zygotes causes marked mitochondrial aggregation, and the majority of embryos arrest at the 2-cell stage. Clumped mitochondria are located in the center of mitotic Drp1-depleted zygotes with less uniform distribution, thereby preventing their symmetric partitioning. Asymmetric mitochondrial inheritance is accompanied by functionally inequivalent blastomeres with biased ATP and endoplasmic reticulum Ca²⁺levels. We also find that marked mitochondrial centration in Drp1-depleted zygotes prevents the assembly of parental chromosomes, resulting in chromosome segregation defects and binucleation. Thus, mitochondrial fragmentation mediated by Drp1 ensure proper organelle positioning and partitioning into functional daughters during the first embryonic cleavage.

Impact statement

Depletion of Dynamin-related protein 1, a key regulator of mitochondrial fission, in mouse zygotes impair symmetric organelle partitioning and chromosome segregation leading to early developmental arrest.

DOI： 10.1101/2024.06.13.598818

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

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

Theriogenology 210 154 - 161 2023.10

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

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

Scientific reports 13 ( 1 ) 13050 - 13050 2023.8

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

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

DOI： 10.1038/s41598-023-40361-9

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

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

Theriogenology 205 87 - 93 2023.7

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

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

Animal Reproduction 20 ( 3 ) e20220127 2023

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

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

Journal of Ovarian Research 15 ( 1 ) 2022.10

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

Abstract

Background:

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

Methods:

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

Results:

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

Conclusion:

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

DOI： 10.1186/s13048-022-01047-4

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

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

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

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

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

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

DOI： 10.1093/bbb/zbab114

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

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

Reproductive sciences 28 ( 5 ) 1339 - 1346 2021.5

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

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

DOI： 10.1007/s43032-020-00390-5

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

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

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

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

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

DOI： 10.1111/rda.13881

Animal biotechnology roles in livestock production

Hiroaki Funahashi

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

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

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

DOI： 10.1088/1755-1315/465/1/012001

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

Chiyuki Kohata-Ono, Takuya Wakai, Hiroaki Funahashi

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

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

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

DOI： 10.1262/jrd.2019-112

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

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

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

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

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

DOI： 10.1262/jrd.2018-130

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

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

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

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

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

DOI： 10.1071/RD16321

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

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

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

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

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

DOI： 10.1071/RD16321

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

R. Matsunaga, H. Funahashi

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

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

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

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

Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

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

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

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

DOI： 10.1262/jrd.2016-156

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

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

THERIOGENOLOGY 86 ( 7 ) 1705 - 1710 2016.10

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

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

DOI： 10.1016/j.theriogenology.2016.05.024

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

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

THERIOGENOLOGY 85 ( 7 ) 1211 - 1218 2016.4

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

DOI： 10.1016/j.theriogenology.2015.12.001

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

Rukmali Athurupana, Hiroaki Funahashi

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

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

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.

DOI： 10.1016/j.apjr.2015.12.010

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

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

THERIOGENOLOGY 85 ( 3 ) 428 - 433 2016.2

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

DOI： 10.1016/j.theriogenology.2015.09.013

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

Raquel Romar, Hiroaki Funahashi, Pilar Coy

THERIOGENOLOGY 85 ( 1 ) 125 - 134 2016.1

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

DOI： 10.1016/j.theriogenology.2015.07.017

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

Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

THERIOGENOLOGY 84 ( 6 ) 940 - 947 2015.10

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

DOI： 10.1016/j.theriogenology.2015.05.033

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

Rukmali Athurupana, Daisen Takahashi, Sumire Ioki, Hiroaki Funahashi

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

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

DOI： 10.1262/jrd.2014-152

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

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

THERIOGENOLOGY 80 ( 9 ) 970 - 978 2013.12

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

DOI： 10.1016/j.theriogenology.2013.07.023

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

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

Fertility and sterility 99 ( 2 ) 400 - 407 2013.2

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

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

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

Theriogenology 79 ( 1 ) 87 - 93 2013.1

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

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

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

Hiroaki Funahashi

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

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

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.

DOI： 10.1007/s12522-012-0134-9

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

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

Fertility and Sterility 98 ( 6 ) 1423 - 1427 2012.12

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

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.

DOI： 10.1016/j.fertnstert.2012.07.1128

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

Masanobu Okuyama, Hiroaki Funahashi

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

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

DOI： 10.1262/jrd.11-092H

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

S. Yamaguchi, H. Funahashi

Theriogenology 77 ( 5 ) 926 - 932 2012.3

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

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..

DOI： 10.1016/j.theriogenology.2011.09.023

Simple vitrification for small numbers of human spermatozoa Reviewed International journal

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

Reproductive BioMedicine Online 24 ( 3 ) 301 - 307 2012.3

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

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.

DOI： 10.1016/j.rbmo.2011.11.016

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

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

Theriogenology 77 ( 1 ) 73 - 80 2012.1

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

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.

DOI： 10.1016/j.theriogenology.2011.07.018

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

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

Theriogenology 75 ( 7 ) 1336 - 1345 2011.4

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

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.

DOI： 10.1016/j.theriogenology.2010.12.005

Single spermatozoon freezing using cryotop Reviewed

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

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

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

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

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

Koji Matsuura, Yuka Kuroda, Keisuke Yamashita, Hiroaki Funahashi

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

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

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.

DOI： 10.1262/jrd.10-075N

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

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

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

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

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).

DOI： 10.3892/ijmm-00000534

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

Takayuki Koike, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

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

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

DOI： 10.1262/jrd.10-041H

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

J. C. Li, H. Funahashi

Theriogenology 74 ( 4 ) 691 - 698 2010.9

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

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

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

Hikaru Sano, Koji Matsuura, Keiji Naruse, Hiroaki Funahashi

THERIOGENOLOGY 74 ( 5 ) 863 - 870 2010.9

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

DOI： 10.1016/j.theriogenology.2010.04.011

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

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

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

Language：English Publishing type：Research paper (international conference proceedings)

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.

DOI： 10.1109/MHS.2010.5669581

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

Shoichiro Yamaguchi, Hiroaki Funahashi, Tetsuya Murakami

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

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

DOI： 10.1262/jrd.20238

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

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

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

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

DOI： 10.1262/jrd.20219

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

Wataru Fujii, Hiroaki Funahashi

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

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

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

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

H. Funahashi, T. Koike, R. Sakai

Theriogenology 70 ( 7 ) 1041 - 1047 2008.10

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

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

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

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

Theriogenology 70 ( 5 ) 809 - 817 2008.9

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

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

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

Wataru Fujii, Hiroaki Funahashi

Zygote 16 ( 2 ) 117 - 125 2008.5

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

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

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

Takashi Nagai, Hiroaki Funahashi, Koji Yoshioka, Kazuhiro Kikuchi

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

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

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

DOI： 10.2741/1991

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

R Romar, H Funahashi

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

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

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

Hiroaki Funahashi

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

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

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

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

H. Funahashi, T. Sano

Theriogenology 63 ( 6 ) 1605 - 1616 2005.4

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

Hiroaki Funahashi, Raquel Romar

Reproduction 128 ( 6 ) 789 - 800 2004.12

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

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

DOI： 10.1530/rep.1.00295

Other Link： https://rep.bioscientifica.com/downloadpdf/journals/rep/128/6/1280789.xml

Polyspermic penetration in porcine IVM - IVF systems Reviewed

Hiroaki Funahashi

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

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

DOI： 10.1071/rd02076

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

H Funahashi

Reproduction 857 - 864 2002.12

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

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

DOI： 10.1530/rep.0.1240857

Other Link： https://rep.bioscientifica.com/downloadpdf/journals/rep/124/6/857.xml

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

Hiroaki FUNAHASHI

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

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

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

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

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

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

Reproduction 443 - 452 2001.9

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

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

DOI： 10.1530/rep.0.1220443

Other Link： https://rep.bioscientifica.com/downloadpdf/journals/rep/122/3/443.xml

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

Hiroaki Funahashi, Takashi Nagai

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

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

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

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

Language：English Publisher：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 < 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.

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

Hiroaki Funahashi, Hans Ekwall, Heriberto Rodriguez-Martinez

Biology of Reproduction 63 ( 5 ) 1437 - 1442 2000.11

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

Hiroaki Funahashi, Toshimitsu Fujiwara, Takashi Nagai

Biology of Reproduction 63 ( 4 ) 1157 - 1163 2000.10

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

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

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

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

Hiroaki FUNAHASHI, Takashi NAGAI

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

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

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

Zygote 7 ( 4 ) 301 - 305 1999.11

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

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 < 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

DOI： 10.1017/s0967199499000702

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

Syahruddin Said, Hiroaki Funahashi, Koji Niwa

Zygote 7 ( 3 ) 249 - 254 1999.8

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

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 < 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 < 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 < 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

DOI： 10.1017/s0967199499000635

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

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

Biology of Reproduction 61 ( 1 ) 298 - 304 1999.7

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

Kwang-Wook Park, Hiroaki Funahashi, Koji Niwa

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

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

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

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

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

She-Hoon Oh, Kazuchika Miyoshi, Hiroaki Funahashi

Biology of Reproduction 59 ( 4 ) 884 - 889 1998.10

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

Ken SAWAI, Hiroaki FUNAHASHI, Koji NIWA

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

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

Ken Sawai, Hiroaki Funahashi, Koji Niwa

Biology of Reproduction 57 ( 1 ) 1 - 6 1997.7

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

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

Biology of Reproduction 57 ( 1 ) 49 - 53 1997.7

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

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

Zygote 5 ( 2 ) 117 - 125 1997.5

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

Summary

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

DOI： 10.1017/s0967199400003798

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

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

Biology of Reproduction 56 ( 4 ) 921 - 930 1997.4

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

H Funahashi, TC Cantley, BN Day

THERIOGENOLOGY 47 ( 3 ) 679 - 686 1997.2

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

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

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

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

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

H Funahashi, Z Machaty, RS Prather, BN Day

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

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

The presence of gamma-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pi of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5'-O-(3'-thiotriphosphate) (GTP-gamma-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 +/- 1.4%)was not different(P > 0.05) from HEPES-injected controls (24.9 +/- 1.3%) at 6 h after injection. Injected GTP-gamma-S, however, activated 76.0 +/- 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 +/- 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-gamma-S injected oocytes (4.2 +/- 0.7 pmol/oocyte) and noninjected oocytes (4.0 +/- 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 +/- 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 +/- 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 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.

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

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

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

Microtubule Organization in Porcine Oocytes during Fertilization and Parthenogenesis1 Reviewed

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

Biology of Reproduction 54 ( 6 ) 1397 - 1404 1996.6

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

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

Biology of Reproduction 54 ( 6 ) 1412 - 1419 1996.6

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

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

Reproduction 107 ( 1 ) 79 - 86 1996.5

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

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

Biology of Reproduction 54 ( 2 ) 316 - 322 1996.2

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation Reviewed

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

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

Current status of in vitro production of porcine embryos Invited Reviewed

H Funahashi, BN Day

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

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

Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

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

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

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

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

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

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

Zygote 3 ( 3 ) 273 - 281 1995.8

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

Summary

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

DOI： 10.1017/s0967199400002677

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