耐凍タンパク等の遺伝子導入による卵巣凍結保存法の創出

2003 年度 実績報告書

• 研究課題/領域番号: 14656104
• 研究機関: 京都大学
• 研究代表者: 宮本 元 京都大学, 農学研究科, 教授 (00026618)
• 研究分担者: 眞鍋 昇 京都大学, 農学研究科, 助教授 (80243070)
• キーワード: 卵巣 / 凍結保存 / 遺伝子導入 / 哺乳類 / 耐凍タンパク質 / 凍結傷害 / 卵母細胞 / 授精能

研究概要

哺乳類の卵巣の凍結保存を実現すべく、凍結前に卵巣組織を薄切して急速に冷却・凍結しようとする手法が試みられたが、受精能をもつ卵母細胞(厳密には、哺乳類では二次卵母細胞に精子が侵入して授精が成立するので、卵子は存在しない)をほとんど得られていない。あらかじめ卵巣を摘出する前に動脈から耐凍剤を灌流した後摘出し、組織を薄切して凍結する方法が考案されたが、満足な成績が得られていない。凍結保存法が確立している精子や受精卵などの遊離細胞より格段に巨大で血管系、神経系、結合組織などに富む卵巣の凍結保存を実現するためにはブレークスルー的技術革新が必要である。極地に生息する動物の体液氷点を低下させたり氷晶表面に結合して結晶の成長を抑えて細胞破砕を阻止する耐凍タンパクの遺伝子(耐凍タンパクには化学構造の違うI、II、III型とN型糖鎖をもつタンパクがあり、IおよびIII型がクローニング済)と申請者らが見いだした細胞内アポトーシス阻止因子(cFLIP)の遺伝子クローニングベクターに組み込んで、卵巣細胞に導入し、凍害保護や細胞死阻害を実現し、雌性生殖器官の凍結保存を実現しようとしている。卵巣に耐凍性タンパク、新規アポトーシス阻害因子cFLIPと既知アポトーシス阻害因子p53を特異的に発現させる遺伝子導入法の最適化を培養細胞レベルで進めた。導入効率は高いのだがゲノムDNAに入り込む可能性の高いウイルス遺伝子を含むウイルスベクター法と併行して、ゲノムDNAに導入遺伝子が組み込まれてしまう心配のないex vivo遺伝子導入のためのリポソーム作製法の確立も進めた。

研究成果

• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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 gifts."Journal of Reproduction and Devevelopment. 49(4). 291-296 (2003)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Yasufumi 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)
• [文献書誌] Miki Sugimoto, Noboru Manabe, Hajime Miyamoto: "Cryopreservation of ovarian tissue."Igakuno Ayumi. 204(13). 921-925 (2003)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Noboru Manabe, Satoko Wada, Naoko Inoue, Toshikatu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] Mizuho Nakayama, Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] Naoko Inoue, Noboru Manabe, Toshikatsu Matsui, Shinsuke Nakagawa, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Minako Kiso, Noboru Manabe, Kohji Komatsu, Munetake Shimabe Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Miki Sugimoto, Noboru Manabe, Maki Morita, Satoshi Imanishi, Hanako Nishizawa, Mineko Iwahori, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] Noboru Manabe, Yasuyoshi Azuma, Miki Sugimoto, Satoshi Imanishi, Hanako Nishizawa, Maki Morita, Hajime Miyamoto: "Animal Frontier Science"Hokuto Pub.Co, Kyoto. 363 (2003)
• [文献書誌] 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)
• [文献書誌] Noboru Manabe: "Veterinary Histology 3^<rd> edition"Gakusoshya Pub.Co, Tokyo, Japan. 315 (2003)
• [文献書誌] Noboru Manabe: "Biology of Reproduction 2^<nd> edition"Asakura Pub.Co, Tokyo, Japan. 315 (2003)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] 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)
• [文献書誌] Minako Kiso, Munetake Shimabe, 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)