研究者詳細 - 舟橋　弘晃

MISC - 舟橋　弘晃

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Advances in in vitro production of pig embryos

H Funahashi, BN Day

JOURNAL OF REPRODUCTION AND FERTILITY Supplement52 271 - 283 1997年

記述言語：英語出版者・発行元：J REPROD FERTIL INC

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

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

Fifth International Conference on Pig Reproduction 142 1997年

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

Fifth International Conference on Pig Reproduction 139 1997年

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

FUNAHASHI H

Theriogenology 47 ( 1 ) 277 - 277 1997年

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

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

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

Journal of Reproduction and Fertility Supplement52 271 - 283 1997年

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

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

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

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

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

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

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

DOI： 10.1262/jrd.43.73

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

Theriogenology 47 ( 3 ) 679 - 686 1997年

牛精液用0.5mlストローを用いた豚精子の凍結保存

野上与志郎, 原田護, 関哲生, 舟橋弘晃, 丹羽こう二

日本畜産学会大会講演要旨 93rd 1997年

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

OH S-H, 舟橋弘晃, 丹羽こう二

Journal of mammalian ova research 14 ( 1 ) 1997年

Advances in in vitro production of pig embryos. 国際誌

H. Funahashi, B. N. Day

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

記述言語：英語

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

Molecular Reproduction and Development 45 ( 4 ) 485 - 90 1996年12月

Microtubule organization in porcine oocytes during fertilization and parthenogenesis 国際誌

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

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

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

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

DOI： 10.1095/biolreprod54.6.1397

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

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

Microtuble organization in porcine oocytes during fertilization and parthenogenesis.(共著)

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

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

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

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

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

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

DOI： 10.1095/biolreprod54.6.1412

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

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

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

記述言語：英語出版者・発行元：J REPROD FERTIL INC

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

DOI： 10.1530/jrf.0.1070079

Effects of injecting calcium chloride into in vitromatured porcine oocytes.(共著) 国際誌

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

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

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

BIOLOGY OF REPRODUCTION 54 ( 2 ) 316 - 322 1996年2月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

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

DOI： 10.1095/biolreprod54.2.316

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