Faculty Profiles - FUNAHASHI Hiroaki

MISC - FUNAHASHI Hiroaki

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10℃保存された豚精子の体外受精能と人工授精の成績

豚の繁殖衛生セミナー通信 30, 7-12 2003

Polyspermic penetration in porcine IVM-IVF systems

H Funahashi

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

Language：English Publishing type：Book review, literature introduction, etc. Publisher：C S I R O PUBLISHING

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

DOI： 10.1071/RD02076

Polyspermic penetration in porcine IVM-IVF systems International journal

Hiroaki Funahashi

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

Language：English

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

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豚卵母細胞の体外成熟と体外受精

舟橋弘晃

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

体外培養システムにおけるブタ卵子の細胞質成熟

IVF Conference 2003 68 - 71 2003

哺乳動物初期胚の対外培養および操作が着床後の成長・分化に残す課題

舟橋弘晃

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

同種および異種精しょうが豚凍結精子の生存性および受精能に及ぼす効果

舟橋弘晃, 佐野通

日本養豚学会大会講演要旨 79th 2003

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

H Funahashi

REPRODUCTION 124 ( 6 ) 857 - 864 2002.12

Language：English Publisher：SOC REPRODUCTION FERTILITY

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

DOI： 10.1530/rep.0.1240857

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

H Funahashi

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

Language：English Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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

DOI： 10.1262/jrd.48.57

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

Reproduction 124 ( 6 ) 857 - 864 2002

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

Hiroaki FUNAHASHI

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

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

日本畜産学会第100回大会講演要旨集 p115 2002

カフェインおよび受精促進ペプチド存在下での豚新鮮射出精子のNO産生

第95回日本繁殖生物学会講演要旨集 p161 2002

豚凍結精液融解後のグリセリン濃度が精子の受精能に及ぼす影響

佐野通, 荒金知宏, 舟橋弘晃

第78回日本養豚学会大会講演要旨集 p11 2002

チオール類が豚液状保存精子の生存性および自発的先体反応に及ぼす影響

舟橋弘晃, 原田護, 佐野通, 伊藤述史

第77回日本養豚学会大会講演要旨集 p23 2002

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

舟橋弘晃

日本畜産学会大会講演要旨 100th 2002

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

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

REPRODUCTION 122 ( 3 ) 443 - 452 2001.9

Language：English Publisher：SOC REPRODUCTION FERTILITY

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

DOI： 10.1530/rep.0.1220443

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

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

REPRODUCTION 122 ( 3 ) 443 - 452 2001.9

Language：English Publisher：SOC REPRODUCTION FERTILITY

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

H Funahashi, T Nagai

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

Language：English Publisher：WILEY-LISS

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

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

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

H Funahashi, T Nagai

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

Language：English Publisher：WILEY-LISS

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

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