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

2002 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るために、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)をほとんど得られなかった。次いで、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、これでも満足な成績が得られなかった。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や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。本研究では、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るが、IおよびIII型がクローニングされている)と申請者らが見いだしたアポトーシス阻止遺伝子を卵巣の細胞に導入して、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。今年度は、卵巣に耐凍性タンパク遺伝子、申請者らが発見した新規アポトーシス阻害因子遺伝子および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。遺伝子導入に際して、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はとらず、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を進めた。すなわち、IおよびIII型耐凍タンパク、新規アポトーシス阻害因子およびp53のセンスmRNAを調製してNIH-3T3細胞に導入してタンパク産生能をスクリーニングし、最も産生能の高いクローンの選抜を進めた。

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] Uchio K, Tuchweber B, Manabe Noboru, Gabbiani G, Rosenbaum J, Desmouliere A.: "Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts"Laboratory Investigation. 82・4. 619-628 (2002)
• [文献書誌] Chiemi Tajima, Noboru Manabe, Mizuho Nakayama, Naoko Inoue, Toshikatsu Matsui, Eiichi Hondo, Hajime Miyamoto: "Monoclonal antibody recognizes follicular granulosa cell antigens in porcine ovaries"Journal of Reproduction and Development. 48・4. 567-572 (2002)
• [文献書誌] Satoshi Imanishi, Noboru Manabe, Takako Tanaka, Reika Okamoto, Maki Morita, Miki Sugimoto, Hajime Miyamoto: "mRNA of GPCR5B/Raig2, a novel orphan receptor, expresses in murine placentae"Journal of Reproduction and Development. 48・4. 627-632 (2002)
• [文献書誌] Noboru Manabe: "Molecular mechanism of follicle selection in mammalian ovaries"Journal of Laboratory Animals (Kansai). 23・1. 71-78 (2002)
• [文献書誌] Noboru Manabe, Miki Sugimoto, Hajime Miyamoto: "In vivo noninvasive nuclear magnetic resonance (NMR) technique for assessment of teratogenicity induced by chemicals"Chemical Engineering. 47・2. 110-114 (2002)
• [文献書誌] Satoko Wada, Noboru Manabe, Naoko Inoue, Mizuho Nakayama, Toshikatsu Matsui, Hajime Miyamoto: "TRAIL-decoy receptor-1 disappears in granulosa cells of atretic follicles in porcine ovaries"Journal of Reproduction and Development. 48・1. 167-173 (2002)
• [文献書誌] Satoko Wada, Noboru Manabe, Naoko Inoue, Mizuho Nakayama, Toshikatsu Matsui, Hajime Miyamoto: "TRADD is involved in apoptosis induction in granulosa cells during atresia in pig ovaries"Journal of Reproduction and Development. 48・1. 175-181 (2002)
• [文献書誌] Hideki Tonomura H, Miyake T, Taniguchi Y, Manabe Noboru, Kose H, Matsumoto K, Yamada T, Sasaki Y.: "Detection of quantitative trait locus for rat model useful for genetic analysis of intramascular fat accumulation using the OLETF rat"The Journal of Veterinary Medical Science. 64・1. 45-50 (2002)
• [文献書誌] Satoko Wada, Noboru Manabe, Mizuho Nakayama, Naoko Inoue, Tohikatsu Matsui, Hajime Miyamoto: "TRAIL-decoy receptor-1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicules"The Journal of Veterinary Medical Science. 64・5. 435-439 (2002)
• [文献書誌] Miki Sugimoto, Noboru Manabe, Maki Morita, Takako Tanaka, Reika Okamoto, Satoshi Imanishi, Hajime Miyamoto: "NMR microscopic observation of mouse embryo malformations induced by maternal administration of retinoic acid"The Journal of Veterinary Medical Science. 64・5. 427-433 (2002)
• [文献書誌] Miki Sugimoto, Noboru Manabe, Hajime Miyamoto: "Cryopreservation techniques for ovarian tissues"Hormone Frontier in Gynecology. 9・1. 135-140 (2002)
• [文献書誌] Mizuho Nakayama, Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto Sakamaki, Miki Sugimoto, Hajime Miyamoto: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Tumor necrosis factor-α acts as a survival factor in granulosa cells during follicular selection in porcine ovaries. PP. 143-149"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Naoko Inoue, Noboru Manabe, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Hajime Miyamoto: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Roles of Fas-ligand and TRAIL systems in granulosa cell apoptosis during follicular atresia in porcine ovaries. PP. 151-156"Hokuto Publication Company, Kyoto, Japan. 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 (Sato E, Miyamoto H, Manabe Noboru eds.), Roles of the mitochondria-dependent pathway in granulosa cell apoptosis during follicular atresia in pig ovaries. PP. 157-163"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Yamaguchi-Yamada M, Noboru Manabe, Uchio-Yamada K, Akashi N, Gotoh Y, Nagao M, Yamamoto Y, Ogura A, Miyamoto H.: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Anemia associated with renal failure in the ICR-derived glomerulonephritis (ICGN) mice. PP.281-285"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Minako Kiso, Noboru Manabe, Kohji Komatsu, Munetake Shimabe, Hajime Miyamoto: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Abnormal structural luteolysis in ovaries of the senescence accelerated mouse (SAM) : expression of Fas mediated apoptosis signal in luteal bodies. PP. 287-294"Hokuto Publication Company, Kyoto, Japan. 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 (Sato E, Miyamoto H, Manabe Noboru eds.), Histochemical technique quantitatively estimates the physiological and pathological properties. PP.303-309"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Noboru Manabe, Naoko Inoue, Toshikatsu Matsui, Shinsuke Nakagawa, Akihisa Maeda, Takashi Miyano, Kazuhiro Sakamaki, Miki Sugimoto Hajime: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Regulatory mechanisms of follicle selection in mammalian ovaries. PP. 117-132"Hokuto Publication Company, Kyoto, Japan. 363 (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 (Sato E, Miyamoto H, Manabe Noboru eds.), Tumor necrosis factor-related apoptosis-inducing ligand and its receptors play crucial roles in apoptosis of granulosa cells during follicular atresia in pig ovaries. PP. 133-142"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Maki Morita, Miki Sugimoto, Noboru Manabe, Hajime Miyamoto: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Morphological alterations in mammalian reproductive organ induced by exposure to diethylstilbestrol (DES). PP. 319-323"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Miki SUGIMOTO, Noboru MANABE, Maki MORITA, Hajime MIYAMOTO: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), Utilization of magnetic resonance microscope for studies in reproductive toxicology. PP. 325-330"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Noboru Manabe, Yasuyoshi Azuma, Miki Sugimoto, Kei-Ichi Sezaki, Mineko Iwahori, Satoshi Imanishi, Hanako Nishizawa, Takako Tanaka, Maki Morita, Hajime Miyamoto: "Animal Frontier Science (Sato E, Miyamoto H, Manabe Noboru eds.), In vivo and real time estimation of energy metabolism by ^<31>p-nuclear magnetic resonance. PP. 339-347"Hokuto Publication Company, Kyoto, Japan. 363 (2003)
• [文献書誌] Morikawa Masahiro, Wakoto Matsuyama, Yuko Kawamoto, Miki Sugimoto, Maki Morita, Aki Nakayama, Noboru Manabe, Minoru Yoneda, Shinsuke Morisawa: "Environmental Toxicity Evaluation and Risk Management Vol.1(Morisawa S eds.), Dynamic performance of bisphenol-A (BPA) in pregnant mouse and physiologically-based pharmacokinetic model development. PP. 60-74"The Environment Risk Management Group of The JSPS-VCC Program on Environmental Science, Engineering and Ethics 2000-2009. Kyoto, Japan. 159 (2002)