Faculty Profiles - FUNAHASHI Hiroaki

MISC - FUNAHASHI Hiroaki

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Co-culture with follicular shell pieces (FSP) increases the developmental competence of pig oocytes matured in vitro.

LR Abeydeera, H Funahashi, TC Cantley, A Rieke, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 100 - 100 1996

Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

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

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

The effect of NaCl in maturation media on histone H1 kinase activity of porcine oocytes was examined at the end of culture for maturation. Oocyte-cumulus complexes were cultured in modified Whitten's medium containing different concentrations of NaCl (44.50, 68.49, 92.40, 116.40 or 140.35 mM). The media were supplemented with 10% porcine follicular fluid and hormones for 20 h and then without hormonal supplements for an additional 20 h. At the end of culture, oocytes were sampled for morphological observation, for glutathione assay and for histone H1 kinase assay. At the end of culture, the incidence of oocytes at each meiotic stage was not different among groups. However, histone H1 kinase activity and glutathione content were higher (P < 0.01) in oocytes cultured in media with lower NaCl concentrations. Histone H1 kinase activity in the oocytes at the end of maturation culture was correlated with intracellular glutathione content (r = 0.899, P < 0.01). These data indicate that histone H1 kinase activity and glutathione content of porcine oocytes at the end of culture for maturation are reduced when a high salt medium is used for maturation.

DOI： 10.1262/jrd.42.109

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Effect of saccharides on the semination into a pig ovum.

宋学雄, 沢井健, 許東けい, 舟橋弘晃, 丹羽こう二

日本繁殖生物学会講演要旨 89th 57 1996

Effect of cysteine and cystine on the male pronucleus forming ability of pig’s follicular ovum.

沢井健, 舟橋弘晃, 丹羽こう二

日本繁殖生物学会講演要旨 89th 44 1996

Effect of somatotropin on the progress rate of the maturation division of cattle’s follicular ovum.

伊賀浩輔, 舟橋弘晃, 丹羽こう二

日本繁殖生物学会講演要旨 89th 41 1996

Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.

H Funahashi, H Tatemoto, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 408 - 408 1996

Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

Congress Programme of the 13th International Congress on Animal Reproduction 2 10 - 4 1996

In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

Theriogenology 45 ( 1 ) 265 1996

Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

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

Factors affecting nuclear and cytoplasmic maturation in pig oocytes

P Coy, S Ruiz, N Ouhibi, H Funahashi, BN Day, RM Moor

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 412 - 412 1996

Co-culture with follicular shell pieces (FSP) increases the developmental competence of pig oocytes matured in vitro.

LR Abeydeera, H Funahashi, TC Cantley, A Rieke, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 100 - 100 1996

Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.

H Funahashi, H Tatemoto, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 408 - 408 1996

Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

Congress Programme of the 13th International Congress on Animal Reproduction 2 10 - 4 1996

In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

Theriogenology 45 ( 1 ) 265 1996

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

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

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

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

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

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Factors Affecting Development in Vitro of Bovine and Rat 1-Cell Embryos

Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

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

Factors Affecting Development in Vitro of Bovine and Rat 1-Cell Embryos

Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

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

Factors affecting nuclear and cytoplasmic maturation in pig oocytes

P Coy, S Ruiz, N Ouhibi, H Funahashi, BN Day, RM Moor

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 412 - 412 1996

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

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

ZYGOTE 3 ( 3 ) 273 - 281 1995.8

Language：English Publisher：CAMBRIDGE UNIV PRESS

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

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PRONUCLEAR VISIBILITY, DEVELOPMENT AND TRANSGENE EXPRESSION IN IVM/IVF-DERIVED PORCINE EMBRYOS

HM KUBISCH, MA LARSON, H FUNAHASHI, BN DAY, RM ROBERTS

THERIOGENOLOGY 44 ( 3 ) 391 - 401 1995.8

Language：English Publisher：BUTTERWORTH-HEINEMANN

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). Alter 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

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