MISC - FUNAHASHI Hiroaki

クローン動物とコピー動物

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

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Advances in in vitro production of pig embryos

H Funahashi, BN Day

JOURNAL OF REPRODUCTION AND FERTILITY   Supplement52   271 - 283   1997

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Language：English   Publisher：J REPROD FERTIL INC  

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

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

Fifth International Conference on Pig Reproduction   142   1997

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

Fifth International Conference on Pig Reproduction   139   1997

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

FUNAHASHI H

Theriogenology   47 ( 1 )   277 - 277   1997

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CiNii Article

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

H Funahashi, Y Kajiwara, K Sawai, K Niwa

BIOLOGY OF REPRODUCTION   56 ( Supplementl )   514 - 514   1997

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

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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

　More details

Language：English   Publisher：J REPROD FERTIL INC  

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

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

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

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

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

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

DOI： 10.1262/jrd.43.73

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

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

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

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

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

DOI： 10.1262/jrd.43.73

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

OH S-H, 舟橋弘晃, 丹羽こう二

Journal of mammalian ova research   14 ( 1 )   1997

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

野上与志郎, 原田護, 関哲生, 舟橋弘晃, 丹羽こう二

日本畜産学会大会講演要旨   93rd   1997

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J-GLOBAL

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Advances in in vitro production of pig embryos. International journal

H. Funahashi, B. N. Day

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

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Language：English  

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

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

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

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

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

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

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Microtubule organization in porcine oocytes during fertilization and parthenogenesis International journal

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

BIOLOGY OF REPRODUCTION   54 ( 6 )   1397 - 1404   1996.6

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

DOI： 10.1095/biolreprod54.6.1397

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

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

BIOLOGY OF REPRODUCTION   54 ( 6 )   1412 - 1419   1996.6

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

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Microtubule organization in porcine oocytes during fertilization and parthenogenesis

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

BIOLOGY OF REPRODUCTION   54 ( 6 )   1397 - 1404   1996.6

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Language：English   Publisher：SOC STUDY REPRODUCTION  

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

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

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

BIOLOGY OF REPRODUCTION   54 ( 6 )   1412 - 1419   1996.6

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Language：English   Publisher：SOC STUDY REPRODUCTION  

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

DOI： 10.1095/biolreprod54.6.1412

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

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

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

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

DOI： 10.1530/jrf.0.1070079

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

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

BIOLOGY OF REPRODUCTION   54 ( 2 )   316 - 322   1996.2

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

DOI： 10.1095/biolreprod54.2.316

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

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

BIOLOGY OF REPRODUCTION   54 ( 2 )   316 - 322   1996.2

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Language：English   Publisher：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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