Faculty Profiles - FUNAHASHI Hiroaki

MISC - FUNAHASHI Hiroaki

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An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

Theriogenology 55 ( 1 ) 522 2001

Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

Sixth International Conference on Pig Reproduction 76 2001

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

Cloning and Stem Cells 3 ( 4 ) 183 - 190 2001

Language：English Publisher：Mary Ann Liebert Inc.

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

DOI： 10.1089/15362300152725891

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An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

Theriogenology 55 ( 1 ) 522 2001

豚の新しい繁殖技術とその展望

舟橋弘晃

豚の繁殖衛生セミナー通信 27 ( 28 ) 79 - 88 2001

アルギニンが豚精子の受精能獲得・先体反応に及ぼす影響

舟橋弘晃

日本畜産学会第98回大会講演要旨 98th 110 2001

緑色蛍光蛋白質の顕微注入法と核移植技術を組み合わせた形質転換牛胚の効率的生産手法の開発

第12回西日本胚移植研究会・第20回北海道牛受精卵移植研究会合同研究発表大会講演要旨集 36 2001

Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

Sixth International Conference on Pig Reproduction 76 2001

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

Cloning and Stem Cells 3 ( 4 ) 183 - 190 2001

Language：English Publisher：Mary Ann Liebert Inc.

DOI： 10.1089/15362300152725891

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Oviduct involvement in sperm capacitation and oocyte development

Sixth International Conference on Pig Reproduction 62 2001

Involvement of oviduct in sperm capacitation and oocyte development in pigs. International journal

H. Rodriguez-Martinez, P. Tienthai, K. Suzuki, H. Funahashi, H. Ekwall, A. Johannisson

Reproduction (cambridge, england) supplement 58 129 - 145 2001

Language：English

An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for < 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

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Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy International journal

H Funahashi, H Ekwall, H Rodriguez-Martinez

BIOLOGY OF REPRODUCTION 63 ( 5 ) 1437 - 1442 2000.11

Language：English Publisher：SOC STUDY REPRODUCTION

Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 mum(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 mum(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

DOI： 10.1095/biolreprod63.5.1437

Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy

H Funahashi, H Ekwall, H Rodriguez-Martinez

BIOLOGY OF REPRODUCTION 63 ( 5 ) 1437 - 1442 2000.11

Language：English Publisher：SOC STUDY REPRODUCTION

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Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

H Funahashi, T Fujiwara, T Nagai

BIOLOGY OF REPRODUCTION 63 ( 4 ) 1157 - 1163 2000.10

Language：English Publisher：SOC STUDY REPRODUCTION

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes, These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.

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Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes International journal

H Funahashi, T Fujiwara, T Nagai

BIOLOGY OF REPRODUCTION 63 ( 4 ) 1157 - 1163 2000.10

Language：English Publisher：SOC STUDY REPRODUCTION

DOI： 10.1095/biolreprod63.4.1157

越壁IVF法による精子選別は豚卵子への多精子受精頻度を減少させる

舟橋弘晃, 永井卓

The journal of reproduction and development 46 ( 5 ) 319 - 324 2000.10

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro International journal

H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

MOLECULAR REPRODUCTION AND DEVELOPMENT 55 ( 1 ) 117 - 124 2000.1

Language：English Publisher：WILEY-LISS

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome toss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these re sponses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln+FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1098-2795(200001)55:1<117::AID-MRD16>3.0.CO;2-7

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

MOLECULAR REPRODUCTION AND DEVELOPMENT 55 ( 1 ) 117 - 124 2000.1

Language：English Publisher：WILEY-LISS

DOI： 10.1002/(SICI)1098-2795(200001)55:1<117::AID-MRD16>3.0.CO;2-7

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The zona reaction in porcine oocytes fertilized in vivo and vitro as seen with scanning electron microscopy.

Theriogenology 53 ( 1 ) 420 2000

The zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy.

Biology of Reproduction 63 ( 5 ) 1447 - 1452 2000

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