哺乳類卵胞の選択的死滅制御機構とその人為支配による潜在的卵巣卵の利用

2005 年度 実績報告書

• 研究課題/領域番号: 13GS0008
• 研究機関: 東京大学
• 研究代表者: 眞鍋 昇 東京大学, 大学院農学生命科学研究科, 教授 (80243070)
• 研究分担者: 宮野 隆 神戸大学, 農学部, 教授 (80200195) ; 酒巻 和弘 京都大学, 生命科学研究科, 助教授 (20271017) ; 若山 照彦 理化学研究所, 神戸研究所・発生再生科学総合研究センター, チームリーダー(研究職) (40360672)
• キーワード: 哺乳類 / 卵巣 / 卵胞 / 選択的死滅 / アポトーシス / 細胞死受容体 / 細胞内シグナル伝達阻害因子 / 異種移植

研究概要

脊椎動物は多様な生殖戦略をもつ。魚類の多くは数万〜数億のなかで幸運で優れた仔だけが生き残る。逆に、哺乳類は性周期毎に1〜10個の卵母細胞を排卵し、極わずかの仔を産み、それを大切に育てる。成熟した哺乳類卵巣には、胎児期に減数分裂の途中・ディプロテン期で休止した5〜100万個の卵母細胞が含まれ、これが性周期毎に発育・成熟して排卵に至る。この過程で、最終的に排卵にいたるものの100倍以上の卵胞が発達を開始し、99%以上が選択的に死滅する。この卵胞の選択的死滅は優秀で強靭な子孫を残す戦略として重要であるが、これを調節している分子制御機構は未解明である。本研究は、哺乳類卵巣において繰返される卵胞の選択的死滅を支配している分子機構を解明し、これをもとに細胞死を制御している遺伝子の発現をin vivo制御することで卵胞発育を人為的に支配しようとするものである。この新技術で、食肉処理場で捨てられる卵巣内の卵母細胞の有効利用を実現しようとしている。今年度は実証的研究に注力した。卵胞顆粒層細胞に特異的に発現している新規細胞死受容体を認識するユニークなモノクロナル抗体を用いてデコイ受容体を同定し、その発現動態を明らかとした。さらに細胞死受容体を介する細胞内アポトーシスシグナル伝達系を担うカスパーゼ系を阻害するcFLIPを見いだして遺伝子とアミノ酸配列を決定した。これの発現を腫瘍壊死因子α調節し、その制御に多面的機能をもつinterleukin-6が重要に関わっていることを示唆した。併行して、大型家畜の未成熟な卵胞を含む卵巣片を免疫不全マウス腎漿膜嚢に異種移植し、in vivo遺伝子導入とRNA発現阻止(iRNA)にて人為的に制御できるシステムを開発した。

研究成果

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• [雑誌論文] Propagation of an infertile hermaphrodite mouse lacking germ cells, using nuclear transfer and embryonic stem cell technology. (2005)
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• [雑誌論文] Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia. (2005)
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• [雑誌論文] Generation of progeny from embryonic stem cells by microinsemination of male germ cells from chimeric mice. (2005)
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• [雑誌論文] Changes in expression of anti-apoptotic protein, cFLIP,in granulosa cells during follicular atresia in porcine ovaries. (2005)
  • 著者名/発表者名: Fukuo Matsuda-Minehata, Yasufumi Goto, Naoko Inoue, Noboru Manabe
  • 雑誌名: Molecular Reproduction and development 72 ページ: 145-151
• [雑誌論文] Birth of Offispring after Transfer of Mongolian Gerbil(Meriones unguiculatus)Embryos Cryopreserved by Vitrification. (2005)
  • 著者名/発表者名: Mochida K, Wakayama T, Takano K, Noguchi Y, Yamamoto Y, Suzuki O, Matsuda J, Ogura A
  • 雑誌名: Molecular Reproduction and Development 70 ページ: 464-470
• [雑誌論文] Effects of long-term grafting on follicular growth in porcine ovarian cortical grafts xenoplanted to severe combined immunodeficient(SCID)mice. (2005)
  • 著者名/発表者名: Noriko Kagawa, Yohichiro Sakurai, Takashi Miyano, Noboru Manabe
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• [雑誌論文] Ovarian activity and oocyte development during follicular development in pigs at different reproductive phases estimated by the repeated endoscopic method. (2005)
  • 著者名/発表者名: Jozsef Ratky, Helmut Torner, Istvan Egerszegi, Falk Schneider, Peter SarlOs, Noboru Manabe, Klaus-Peter Brussow
  • 雑誌名: Journal of Reproduction and Devevelopment 51 ページ: 253-263
• [雑誌論文] Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D_<9K+> (2005)
  • 著者名/発表者名: Eui-Ju Hong, Youn-Kyu Ji, Kyung-Chul Choi, Noboro Manabe, Eui-Bae Jeung
  • 雑誌名: Journal of Reproduction and Devevelopment 51 ページ: 253-263
• [雑誌論文] Effects of in utero exposure to bisphenol A on mRNA expression of arylhydrocarbon and retinoid receptors in murine embryos. (2005)
  • 著者名/発表者名: Hanako Nishizawa, Maki Morita, Miki Sugimoto, Stoshi Imanishi, Noboru Manabe
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 315-324
• [雑誌論文] Comparison of follicular and oocyte development and reproductive hormone secretion during the ovulatory period in hungarian native breed,Mangalica,and Landrace gilts. (2005)
  • 著者名/発表者名: Jozsef Ratky, Klaus-Peter Brussow, Istvan Egerszegi, Hwlmut Torner, Falk Schneider, Laszlo Solti, Noboru Manabe
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 427-432
• [雑誌論文] Effects of exposure in utero to bisphenol A on the expression of arylhydrocarbon receptor,related factors and xenobiotic metabolizing enzymes in murine embryos. (2005)
  • 著者名/発表者名: Hanako Nishizawa, Satoshi Imanishi, Noboru Manabe
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 593-605
• [雑誌論文] Chenges in expression and localization of connexin 43 mRNA and protein in porcine ovary granulosa cells during follicular atresia. (2005)
  • 著者名/発表者名: Yuan Cheng, Naoko Inoue, Fuko Matsuda-Minehata, Yasufumi Goto, Akihisa Maeda, Noboru Manabe
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 627-637
• [雑誌論文] Growth and maturation of porcine follicles and oocytes following xenotransplantation of ovarian tissues and in vitro maturation culture. (2005)
  • 著者名/発表者名: Noriko Kagawa, Takashi Miyano, Noboru Manabe
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 741-748
• [雑誌論文] In vitro growth of mammalian oocytes. (2005)
  • 著者名/発表者名: Miyano T.
  • 雑誌名: Journal of Reproduction and devevelopement 51 ページ: 169-176
• [雑誌論文] Transfroming growth factor-β1 mediated up-regulation of lysyl oxidase in the kidneys of hereditary nephrotic mouse with chronic renal fibrosis. (2005)
  • 著者名/発表者名: Yasufumi Goto, kozue Uchio-Yamada, Anan Sayuri, Yoshie Yamamoto, Atsuo Ogura, Noboru Manabe
  • 雑誌名: Virchowx Archiv 447 ページ: 859-868
• [雑誌論文] Decreased expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the kidneys of hereditary nephrotic(ICGN)mice. (2005)
  • 著者名/発表者名: Kozue Uchio-Yamada, Noboru Manabe, Yasufumi Goto, Sayuri Anann, Yoshie Yamamoto, Kaoru Takano, Atsuo Ogura, Junichiro Matsuda
  • 雑誌名: The Journal of Veterinary Medical Science 67 ページ: 35-41
• [雑誌論文] Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN)mice. (2005)
  • 著者名/発表者名: Misuzu Yamaguchi-Yamada, Noboru Manabe, Minako Kiso, Yasufumi Goto, Toshiharu Mori, Chinatsu Sakata, Sayuri Anan, Masaya Nagao, Yoshie Yamamoto, Atsuo Ogura
  • 雑誌名: The Journal of Veterinary Medical Science 67 ページ: 891-899
• [雑誌論文] Effect of human erythropoietin (hEPO)treatment on anemia in ICR-derived glomerulaonephrosis(ICGN)mice. (2005)
  • 著者名/発表者名: Yohei Miyamoto, Keiko Kuramitsu-Miyamoto, Ema Iwanaga, Kozue Uchio-Yamada, Misuzu Yamaguchi-Yamada, Atsuo Ogura, Noboru Manabe
  • 雑誌名: Experimental Animal 54 ページ: 181-184
• [雑誌論文] Disposition of disphenol A in pregnant mice and fetuses after a single and repeated oral administration. (2005)
  • 著者名/発表者名: Yuko Kawamoto, Wakoto Matsuyama, Masahiro Morikawa, Maki Morita, Miki Sugimoto, Moboru Manabe, Shinsuke Morisawa
  • 雑誌名: Toxicological and Environmental Chemistry 87 ページ: 199-213
• [雑誌論文] Effects of dietary supplementation with branched-chain amino acids(BCAAs)during nursing on plasma BCAA levels and subsequent growth in cattle. (2005)
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  • 雑誌名: Asian Australian Journal of Animal Science 18 ページ: 1440-1444
• [雑誌論文] New preservation method for mouse spermatozoa without freezing. (2005)
  • 著者名/発表者名: Van Thuan N, Wakayama S, Kishigami S, Wakayama T
  • 雑誌名: Biology of Reproduction 72 ページ: 444-450
• [雑誌論文] Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues. (2005)
  • 著者名/発表者名: Wakayama S, Ohta H, Kishigami S, Van Thuan N, Hikichi T, Mizutani E, Miyake M, Wakayama T
  • 雑誌名: Biology of Reprodution 72 ページ: 932-936
• [雑誌論文] Changes in expression of anti-apoptotic protein, cFLIP, in granulose cells during follicular atresia in porcine ovaries.(review) (2005)
  • 著者名/発表者名: Noboru Manabe, Fuko Matsuda-Minehata, Yasufumi Goto, Akihisa Maeda, Yuan Cheng, Takafumi Sai, Miyuki Kohori, Maiko Endo, Junyou Li
  • 雑誌名: European Journal of Cellular and Molecular Physiopathology 44 ページ: 120-124
• [雑誌論文] Anti-apoptotic activity of porcine cFLIP in ovarian grabulosa cell lines.(review) (2005)
  • 著者名/発表者名: Noboru Manabe, Fuko Matsuda-Minehata, Yasufumi Goto, Akihisa Maeda, Yuan Cheng, Takafumi Sai, Miyuki Kohori, Maiko Endo, Junyou Li
  • 雑誌名: European Journal of Cellular and Molecular Physiopathology 44 ページ: 125-129
• [雑誌論文] Molecular cloning of porcine; cellular flice-like inhibitory protein.(review) (2005)
  • 著者名/発表者名: Noboru Manabe, Fuko Matsuda-Minehata, Yasufumi Goto, Akihisa Maeda, Yuan Cheng, Takafumi Sai, Miyuki Kohori, Maiko Endo, Junyou Li
  • 雑誌名: European Journal of Biology and Chemistry of Proteins 18 ページ: 201-204
• [雑誌論文] Changes in the expression of bid in granulosa cells during follicular atresia in pig ovaries.(review) (2005)
  • 著者名/発表者名: Noboru Manabe, Fuko Matsuda-Minehata, Yasufumi Goto, Akihisa Maeda, Yuan Cheng, Takafumi sai, Miyuki Kohori, Maiko Endo, Junyou Li
  • 雑誌名: European Journal of Biology and Chemistry of Proteins 18 ページ: 205-208
• [雑誌論文] Characteristics of initiation and early events for muscle development in the Xenopus limb bud. (2005)
  • 著者名/発表者名: Satoh A., Sakamaki K., Ide H., Tamura K.
  • 雑誌名: Developmenta Dynamics 234 ページ: 846-857
• [雑誌論文] Expression and functional analysis of genes deregulated in mouse placental over growth models : Car2 and Ncaml. (2005)
  • 著者名/発表者名: Singh U, Sun T, Shi W, Schulz R, Nuber UA, Varanou A, Hemberger MC, Elliott RW, Ohta H, Wakayama T, Fundele R
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• [雑誌論文] Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDKI and MAP kinase during maturation and fertilization of pig oocytes (2005)
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• [雑誌論文] Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally. (2005)
  • 著者名/発表者名: Ohta H, Wakayama T
  • 雑誌名: Biology of Reproduction 73 ページ: 390-395
• [雑誌論文] Interleukin-6 regulates the expression of cellular FLICE-like inhibitory protein in granulose cells of ovaries.(abstrqact) (2005)
  • 著者名/発表者名: Akihisa Maeda, Yasufumi goto, Fuko Matsuda-Minehata, Yuan Cheng, Takafumi Sai, Noboru Manabe
  • 雑誌名: Biology of Reproduction 70 ページ: 115-116
• [雑誌論文] Granulosa cell is a typr II apoptotic cell that regulates follicular atresia in pig ovaries.(abstrqact) (2005)
  • 著者名/発表者名: Takafumi Sai, Toshikatsu Matsui, Yasufumi Goto, Fuko Matsuda-Minehata, Naoko Inoue, Akihisa Maeda, Yuan Cheng, Noboru Manabe
  • 雑誌名: Biology of Reproduction 70 ページ: 116-117
• [雑誌論文] CFLIP regulates granulose cell apoptosis during follicular atresia in porcine ovaries.(abstrqact) (2005)
  • 著者名/発表者名: Fuko Matsuda-Minehata, Yasufumi Goto, Naoko Inoue, Noboru Manabe
  • 雑誌名: Biology of Reproduction 70 ページ: 118-119
• [雑誌論文] Apoptosis is regulated by soluble Fas during luteolysis in Iuteal cells of mouse ovaries.(abstrqact) (2005)
  • 著者名/発表者名: Noboru Manabe, Kohji Komatsu, Minako Kiso, Yasufumi Goto, Sayuri Anan, Junyou Li
  • 雑誌名: Biology of Reproduction 70 ページ: 122-123
• [雑誌論文] Connexin-43 has relation to regulation of granulosa cell apoptosis during atresia in pig ovarian follicules. (abstrqact) (2005)
  • 著者名/発表者名: Yuan Cheng, Naoko Inoue, Fuko Matsuda-Minegishi, Yasufumi Goto, Akihisa Maeda, Junyou Li, Noboru Manabe
  • 雑誌名: Biology of Reproduction 70 ページ: 124-125
• [雑誌論文] Changes in the expression of Bid in granulosa cells during follicular atresia in pig ovaries.(abstrqact) (2005)
  • 著者名/発表者名: Naoko Inoue, Ryosuke Yoshioka Yasufumi Goto, Fuko Matsuda-Minehata, Akihisa Maeda, Yuan Cheng, Noboru Manabe
  • 雑誌名: Biology of Reproduction 70 ページ: 223-224
• [雑誌論文] Round spermatids stained with mitotracker can be used to produce offspring more simply (2005)
  • 著者名/発表者名: Hikichi T, Kishigami S, Thuan NV, Ohta H, Mizutani E, Wakayama S, Wakayama T.
  • 雑誌名: Zygote 13 ページ: 55-61
• [図書] Gamete Biology-Emerging Frontiers in Fertility and Contraceptive Development : Oocyte growth and acquisition of meiotic competence. (2006)
  • 著者名/発表者名: Miyano T, Manabe N.
  • 総ページ数: 312
  • 出版者: Nottinghm University Press
• [図書] Endocrine Disrupting Chemicals : Efect of various phtalates on the induction of Calbindin-D9k as an estrogen-like chemical in in vitro ans in vivo models. (2005)
  • 著者名/発表者名: Eui-Ju Hong, Youn-Kyu Ji, Kyung-Chul Choi, Noboru Manabe, Eui-Bae Jeung
  • 総ページ数: 482
  • 出版者: The Endocrine Society
• [図書] ウシの解剖・消化器系 (2005)
  • 著者名/発表者名: 眞鍋 昇
  • 総ページ数: 138
  • 出版者: チクサン出版株式会社
• [図書] 犬の繁殖(Dog Breeding) (2005)
  • 著者名/発表者名: 眞鍋 昇, 佐籐英明, 永村武美
  • 総ページ数: 338
  • 出版者: 株式会社インターズー
• [図書] 卵子形成・成熟および排卵の機能 : 生命の誕生に向けて (2005)
  • 著者名/発表者名: 宮野 隆
  • 総ページ数: 482
  • 出版者: 近代出版株式会社・日本哺乳動物卵子学会