Faculty Profiles - FUNAHASHI Hiroaki

Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes. (Other,)

The 14th International Congress on Animal Reproduction 2000

Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes.

The 14th International Congress on Animal Reproduction 2000

豚精子の受精能獲得を調節するアデノシン・レセプターの役割

舟橋弘晃, 藤原俊満

日本畜産学会第97回大会講演要旨 97th 124 2000

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

H Funahashi, N Bandoh, S Nakahira, SH Oh, S Tsuboi

ZYGOTE 7 ( 4 ) 301 - 305 1999.11

Language：English Publisher：CAMBRIDGE UNIV PRESS

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilised eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilised oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes International journal

H Funahashi, N Bandoh, S Nakahira, SH Oh, S Tsuboi

ZYGOTE 7 ( 4 ) 301 - 305 1999.11

Language：English Publisher：CAMBRIDGE UNIV PRESS

DOI： 10.1017/S0967199499000702

Recent development in embryo technology in pigs - Review

K Niwa, H Funahashi

ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 12 ( 6 ) 966 - 975 1999.9

Language：English Publisher：ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOCIETIES

Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage, has made it possible to utilize follicular oocytes for in, vitro production of embryos and thus stimulated research on various, embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality, Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

Recent development in embryo technology in pigs - Review

K Niwa, H Funahashi

ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 12 ( 6 ) 966 - 975 1999.9

Language：English Publishing type：Book review, literature introduction, etc. Publisher：ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOCIETIES

DOI： 10.5713/ajas.1999.966

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes

S Said, H Funahashi, K Niwa

ZYGOTE 7 ( 3 ) 249 - 254 1999.8

Language：English Publisher：CAMBRIDGE UNIV PRESS

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes International journal

S Said, H Funahashi, K Niwa

ZYGOTE 7 ( 3 ) 249 - 254 1999.8

Language：English Publisher：CAMBRIDGE UNIV PRESS

DOI： 10.1017/S0967199499000635

Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: Effects of epidermal growth factor on production of PAs in oocytes and cumulus cells

KW Park, SH Choi, XX Song, H Funahashi, K Niwa

BIOLOGY OF REPRODUCTION 61 ( 1 ) 298 - 304 1999.7

Language：English Publisher：SOC STUDY REPRODUCTION

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed, Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex, In COCs before culture, however, no activity of PAs was detected. Al 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

DOI： 10.1095/biolreprod61.1.298

卵胞膜細胞との共培養による卵丘付着牛卵母細胞の卵核胞発育阻害を伴わない減数分裂の休止

Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: Effects of epidermal growth factor on production of PAs in oocytes and cumulus cells International journal

KW Park, SH Choi, XX Song, H Funahashi, K Niwa

BIOLOGY OF REPRODUCTION 61 ( 1 ) 298 - 304 1999.7

Language：English Publisher：SOC STUDY REPRODUCTION

DOI： 10.1095/biolreprod61.1.298

朴光旭, 舟橋弘晃, 丹羽晧二

The journal of reproduction and development 45 ( 3 ) 223 - 231 1999.6

Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development

Kwang Wook Park, Hiroaki Funahashi, Koji Niwa

Journal of Reproduction and Development 45 ( 3 ) 223 - 231 1999

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

The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.

DOI： 10.1262/jrd.45.223

The presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis improves porcine 'oocyte competence' as determined by early embryonic development after in-vitro fertilization

Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

Journal of Reproduction and Development 45 ( 4 ) 265 - 271 1999

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

The present studies were designed to determine the effect of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis or after in vitro fertilization on early embryonic development of porcine embryos. The presence of purified ovine TIMP-1 in culture medium after in vitro fertilization did not affect the incidence of embryo cleavage or development to the blastocyst stage at 48 h or 6 days after insemination, respectively. In contrast, when various concentrations (1.25, 2.50 and 5.00 μg/ml) of purified ovine TIMP-1 were added to the culture medium during the second half (24 h) of oocyte maturation, normal cleavage rates (64 ± 3% at 1.25 μg/ml, 57 ± 5% at 2.50 μg/ml and 56 ± 1% at 5.00 μg/ml) were higher (p&lt
0.05) as compared to controls (43 ± 2%). In addition, the incidence of embryos that developed to the blastocyst stage was increased (p&lt
0.05) when TIMP-1 was added (34 ± 3% at 1.25 Mg/ml, 32 ± 1% at 2.50 μg/ml and 28 ± 1% at 5.00 μg/ml) as compared to controls (22 ± 3%). There was no difference in the incidence (53 ± 1%) of oocytes fertilized normally. These results indicate that the presence of TIMP-1 during the second half of in vitro maturation enhanced the competence of porcine oocytes to develop to the 2- to 4-cell stages after in vitro fertilization, consequently increasing the incidence of blastocysts.

DOI： 10.1262/jrd.45.265

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=1999&ichushi_jid=J04678&link_issn=&doc_id=19990903330004&doc_link_id=10.1262%2Fjrd.45.265&url=https%3A%2F%2Fdoi.org%2F10.1262%2Fjrd.45.265&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

卵子への侵入過程におけるラット精子核内チオール類の酸化還元状態の観察と変化(共著)

SAID S, 舟橋弘晃, 丹羽こう二

第95回日本畜産学会大会講演要旨 95th 79 - 79 1999

Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development

Kwang Wook Park, Hiroaki Funahashi, Koji Niwa

Journal of Reproduction and Development 45 ( 3 ) 223 - 231 1999

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

DOI： 10.1262/jrd.45.223

The presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis improves porcine 'oocyte competence' as determined by early embryonic development after in-vitro fertilization

Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

Journal of Reproduction and Development 45 ( 4 ) 265 - 271 1999

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

DOI： 10.1262/jrd.45.265