内分泌攪乱物質による生殖毒性評価システムの構築:構造生物学・細胞生物学的リアルタイム評価法の確立

2003 Fiscal Year Annual Research Report

• Project/Area Number: 13027241
• Research Institution: Kyoto University
• Principal Investigator: 宮本 元 京都大学, 農学研究科, 教授 (00026618)
• Co-Investigator(Kenkyū-buntansha): 宮川 恒 京都大学, 農学研究科, 教授 (10219735) ; 杉本 実紀 京都大学, 農学研究科, 助手 (20243074) ; 眞鍋 昇 京都大学, 農学研究科, 助教授 (80243070) ; 九郎丸 正道 東京大学, 農学生命科学研究科, 助教授 (00148636) ; 奥田 潔 岡山大学, 自然科学研究科, 教授 (40177168) ; 木曾 康郎 山口大学, 農学部, 教授 (10142374)
• Keywords: 外因性内分泌攪乱物質 / 核磁気共鳴イメージング / 三次元形態解析 / 哺乳類 / 胎仔 / オーファン受容体 / 受容体ノックアウト / リアルタイム解析

Research Abstract

人類が200年以上にわたって創り出してきた農薬、食品添加物等の多くの化合物が、生体の内分泌系のシグナル伝達を攪乱する内分泌攪乱物質として人類の生存を脅かすことが分かってきた。新規な原理に基づいた非侵襲的かつリアルタイムに生殖毒性を評価できる技術システムを構築し、従来法とは比較できない精度で速やかに生殖毒性を評価できる先端技術を確立することで、化合物の生殖毒性を的確に評価できる未来を開拓する基盤技術を開発することが本研究の目的である。本年度は、内分泌撹乱物質とホルモン受容体の結合様式、受容体分子の立体構造変化等を解析し、環境系に存在する様々な化合物の生殖毒性をリアルタイムに評価するシステムの確立を目指して研究を進めた。麻酔下の生きている動物をNMRシグナルを定量検出できる特殊な生体NMRプローブに保定し、胎児におけるNMRシグナルを部位特異的にリアルタイムに観測して、このデータをワークステーションにて3次元立体画像データに再構築して解析できる測定アプリケーションの作成と最適測定条件の決定を行った。毒性発現機構の生殖生理学的解明のため、遺伝子工学的にオーファン受容体に様々な構造変異を誘導し、化合物と受容体の相互的結合特性をNMRにて観測して分子構造学的に毒性を予測する技術を開発している。加えて、卵母細胞を包み込んで保育する卵胞は遺伝子に制御された細胞死によって選択されているが、これを調節している細胞死受容体のシグナルを制御している細胞内アポトーシス阻害因子(cFLIP)を新たに見いだし、これを介した細胞死シグナル伝達機構を解明し、分子レベルで化合物を評価する系を開発した。

Research Products

• [Publications] Mizuho Nakayama, Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Hajime Miyamoto: "Changes in the expression of tumor necrosis factor (TNF)a, TNFa Receptor (TNFR) 2 and TNFR-associated factor 2 in granulosa cells during atresia in pig ovaries."Biology of Reproduction. 68(2). 530-535 (2003)
• [Publications] Kohji Komatsu, Noboru Manabe, Minako Kiso, Munetake Shimabe, Hajime Miyamoto: "Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries."Journal of Experimental Zoology. 296(1). 152-159 (2003)
• [Publications] Kohji Komatsu, Noboru Manabe, Minako Kiso, Munetake Shimabe, Hajime Miyamoto: "Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries."Molecular Reproduction and Development. 65. 345-352 (2003)
• [Publications] Toshikatsu Matsui, Noboru Manabe, Yasufumi Goto, Mizuho Nakayama, Naoko Inoue, Susumu Nishihara, Hajime Miyamoto: "Changes in the expression and activity of caspase-9 and Apaf1 in granulosa cells during follicular atresia in pig ovaries."Reproduction. 126. 113-120 (2003)
• [Publications] Istvan Egerszegi, Falk Schneider, Jozsef Ratky, Ferenc Soos, Laszlo Solti, Noboru Manabe, Klaus-Peter Brussow: "Comparison of luteinizing hormone and steroid hormone secretion during the peri- and post-ovulatory periods in mangalica and landrace gilts."Journal of Reproduction and Devevelopment. 49(4). 291-296 (2003)
• [Publications] Naoko Inoue, Noboru Manabe, Toshikatsu Matsui, Akihisa Maeda, Shynsuke Nakagawa, Satoko Wada, Hajime Miyamoto: "roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries."Journal of Reproduction and Devevelopment. 49(4). 313-321 (2003)
• [Publications] Satoshi Imanishi, Noboru Manabe, Hanako Nishizawa, Maki Morita, Miki Sugimoto, Mineko Iwahori, Hajime Miyamoto: "Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray."Journal of Reproduction and Devevelopment. 49(4). 329-336 (2003)
• [Publications] Minako Kiso, Noboru Manabe, Kohji Komatsu, Munetake Shimabe, Hajime Miyamoto: "Abnormal structural luteolysis in ovaries of the senescence accelerated mouse (SAM): expression of Fas Ligand/Fas-mediated apoptosis signaling molecules in luteal cells."Journal, of Reproduction and Devevelopment. 49(6). 457-463 (2003)
• [Publications] Hanako Nishizawa, Noboru Manabe, Maki Morita, Miki Sugimoto, Satoshi Imanishi, Hajime Miyamoto: "Effects of in utero exposure of bisphenol A on expression of RARα and RXRα mRNAs in murine embryos."Journal of Reproduction and Devevelopment. 49(6). 539-545 (2003)
• [Publications] Kozue Uchio-Yamada, Noboru Manabe, Misuzu Yamaguchi-Yamada, Yasufumi Goto, Yoshie Yamamoto, Atsuo Ogura, Hajime Miyamoto: "Changes in the localization of type I, III and IV collagen mRNAs in the kidneys of hereditary nephritic (ICGN) mice with renal fibrosis."The Journal of Veterinary Medical Science. 66(2). 123-128 (2004)
• [Publications] Ysufumi Goto, Noboru Manabe, Kozue Uchio, Misuzu Yamaguchi-Yamada, Naoko Inoue, Yoshie Yamamoto, Atsuo Ogura, Nobuo Nagano, Hajime Miyamoto: "Myofibroblastic differentiation in tubulointerstitial cells is provoked by augmented cytoplasmic Smad4 protein in hereditary nephrotic ICGN mice with chronic renal fibrosis."Cell and Tissue Research. 315. 209-221 (2004)
• [Publications] Miki Sugimoto, Noboru Manabe, Hajime Miyamoto: "Cryopreservation of ovarian tissue."Igakuno Ayumi. 204(13). 921-925 (2003)
• [Publications] Noboru Manabe, Naoko Inoue, Chiemi Tajima, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Monoclonal antibodis recognize novel cell death receptors of granulosa cells in pig ovaries."Biology of Reproduction. 67. 234 (2003)
• [Publications] Naoko Inoue, Noboru Manabe, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Changes in expression of fas-dependent apoptotic signaling molecules in granulosa cells during folliclar atresia in pig ovaries."Biology of Reproduction. 67. 234 (2003)
• [Publications] Satoshi Imanishi, Noboru Manabe, Miki Sugimoto, Hajime Miyamoto: "Changes in mRNA expression of GPRC5B/RAIG2, a novel orphan receptor, in mouse placenta during late gestation."Biology of Reproduction. 67. 154 (2003)
• [Publications] Miki Sugimoto, Noboru Manabe, Maki Morita, Kei-Ichi Sezaki, Takako Tanaka, Satoshi Imanishi, Hanako Nishizawa, Mineko Iwahori, Hajime Miyamoto: "NMR spectroscopic assessment of effects of estrogenic compounds on male reproductive organs in mice."Biology of Reproduction. 67. 186 (2003)
• [Publications] Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Takashi Miyano, Kazuhiro Sakamaki, Miki Sugimoto, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Noboru Manabe, Satoko Wada, Naoko Inoue, Toshikatu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Mizuho Nakayama, Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Naoko Inoue, Noboru Manabe, Toshikatsu Matsui, Shinsuke Nakagawa, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Toshikatu Matsui, Noboru Manabe, Yasufumi Goto, Naoko Inoue, Shinsuke Nakagawa, Akihisa Maeda, Susumu Nishihara, ' Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Misuzu Yamaguchi-Yamada, Noboru Manabe, Kozue Uchio-Yamada, Naotsugu Akashi, Yasufumi Gotoh, Masaya Nagao, Yoshie Yamamoto, Atsuo Ogura, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Minako Kiso, Noboru Manabe, Kohji Komatsu, Munetake Shimabe Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Noboru Manabe, Kozue Yamada-Uchio, Misuzu Yamada-Yamaguchi, Yasufumi Gotoh, Toshiharu Mori, Chinatsu Sakata, Yoshihiro Furuya, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Maki Morita, Miki Sugimoto, Takako Tanaka, Satoshi Imanishi, Hanako Nishizawa, Kei-Ichi Sezaki, Mineko Iwahori, Noboru Manabe, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Miki Sugimoto, Noboru Manabe, Maki Morita, Satoshi Imanishi, Hanako Nishizawa, Mineko Iwahori, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Noboru Manabe, Yasuyoshi Azuma, Miki Sugimoto, Satoshi Imanishi, Hanako Nishizawa, Maki Morita, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [Publications] Noboru Manabe, Naoko Inoue, Takashi Miyano, Kazuhiro Sakamaki, Miki Sugimoto, Hajime Miyamoto: "The Ovary, 2^<nd> edition"Academic Press/Elsevier Science, Amsterdam, The Nederland. 664 (2003)
• [Publications] Noboru Manabe: "Veterinary Histology 3^<rd> edition"Gakusoshya Pub.Co, Tokyo, Japan. 315 (2003)
• [Publications] Noboru Manabe: "Biology of Reproduction 2^<nd> edition"Asakura Pub.Co, Tokyo, Japan. 315 (2003)
• [Publications] Noboru Manabe, Minako Kiso, Munetake Shimabe, Hajime Miyamoto: "The Senescence-Accelerated Mouse (SAM) : An Animal Model of Senescence"Elsevier Science Publishers B.V., Amsterdam, The Nederland. 448 (2004)
• [Publications] Munetake Shimabe, Minako Kiso, Kohji Komatsu, Noboru Manabe, Hajime Miyamoto: "The Senescence-Accelerated Mouse (SAM) : An Animal Model of Senescence"Elsevier Science Publishers B.V., Amsterdam, The Nederland. 448 (2004)
• [Publications] Minako Kiso, Munetake Shimabe, Kohji Komatsu, Noboru Manabe, Hajime Miyamoto: "The Senescence-Accelerated Mouse (SAM) : An Animal Model of Senescence"Elsevier Science Publishers B.V., Amsterdam, The Nederland. 448 (2004)
• [Publications] Morikawa Masahiro, Wakoto Matsuyama, Miki Sugimoto, Maki Morita, Aki Nakayama, Noboru Manabe, Shinsuke Morisawa: "Environmental Toxicity Evaluation and Risk Management"The Environment Risk Management Group of the JSPS-VCC Program on Environmental Science, Engineering and Ethics 2000-2009. Kyoto, Japan. 159 (2003)
• [Publications] Noboru Manabe, Minako Kiso, Munetake Shimabe, H.Miyamoto: "Proceedings of the 2^<nd> International Conference on Senescence : The SAM model"The Council for SAM Research, Sapporo, Japan. 120 (2003)
• [Publications] Munetake Shimabe, Minako Kiso, Noboru Manabe, Hajime Miyamoto: "Proceedings of the 2^<nd> International Conference on Senescence : The SAM model"The Council for SAM Research, Sapporo, Japan. 120 (2003)
• [Publications] Minako Kiso, Munetake Shimabe, Noborn Manabe, Hajime Miyamoto: "Proceedings of the 2^<nd> International Conference on Senescence : The SAM model"The Council for SAM Research, Sapporo, Japan. 120 (2003)