Faculty Profiles - FUNAHASHI Hiroaki

MISC - FUNAHASHI Hiroaki

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

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.

DOI： 10.1017/S0967199400002677

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.

DOI： 10.1016/0093-691X(95)00193-C

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EFFECT OF BICARBONATE ON IN-VITRO PENETRATION OF PIG OOCYTES BY COMMERCIALLY PREPARED FROZEN-EJACULATED BOAR SEMEN

LR ABEYDEERA, H FUNAHASHI, NH KIM, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 122 - 122 1995

GAMMA-GLUTAMYL-TRANSPEPTIDASE (GGT) OF SPERMATOZOA MAY REDUCE OOCYTE GLUTATHIONE (GSH) CONTENT AT SPERM PENETRATION

H FUNAHASHI, Z MACHATY, RS PRATHER, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement1 ) 140 - 140 1995

Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著) Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's

Role in the Genetic Improvement of Farm Animals 19 1995

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation International journal

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

Molecular reproduction and development 43 ( 2 ) 248 - 255 1995

Language：English

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic loci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

DOI： 10.1002/(SICI)1098-2795(199602)43:2<248::AID-MRD14>3.0.CO;2-#

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Microtuble and microfilament dynamics in porcine oocytes during meiotic maturation.(共著)

Molecular Reproduction and Development 43 ( 2 ) 248 - 255 1995

EFFECT OF TAXOL ON MICROTUBULE ORGANIZATION IN PORCINE OOCYTES AFTER FERTILIZATION AND PARTHENOGENETIC ACTIVATION

NH KIM, LR ABEYDEERA, RE EVERTS, H FUNAHASHI, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 139 - 139 1995

EFFECT OF BICARBONATE ON IN-VITRO PENETRATION OF PIG OOCYTES BY COMMERCIALLY PREPARED FROZEN-EJACULATED BOAR SEMEN

LR ABEYDEERA, H FUNAHASHI, NH KIM, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 122 - 122 1995

Effects of cumulus cells on glutathione content of porcine oocyte during in vitro maturation

FUNAHASHI H

J Anim Sci 73 ( 1 ) 90 - 90 1995

Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995

Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

Theriogenology 43 ( 1 ) 262 1995

Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

Theriogenology 43 ( 1 ) 213 1995

GAMMA-GLUTAMYL-TRANSPEPTIDASE (GGT) OF SPERMATOZOA MAY REDUCE OOCYTE GLUTATHIONE (GSH) CONTENT AT SPERM PENETRATION

H FUNAHASHI, Z MACHATY, RS PRATHER, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement1 ) 140 - 140 1995

Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著)

Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's Role in the Genetic Improvement of Farm Animals 19 1995

Effects of cumulus cells on glutathione content of porcine oocytes during in vitro maturation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995

Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995

Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

Theriogenology 43 ( 1 ) 262 1995

Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

Theriogenology 43 ( 1 ) 213 1995

EFFECT OF TAXOL ON MICROTUBULE ORGANIZATION IN PORCINE OOCYTES AFTER FERTILIZATION AND PARTHENOGENETIC ACTIVATION

NH KIM, LR ABEYDEERA, RE EVERTS, H FUNAHASHI, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 139 - 139 1995

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