2000 Fiscal Year Annual Research Report

卵胞の選択的死滅を決定する新規顆粒層細胞特異的アポトーシス受容体の探索とその制御

Research Project

Project/Area Number	11876064
Research Institution	Kyoto University
Principal Investigator	眞鍋昇京都大学, 農学研究科, 助教授 (80243070)
Keywords	アポトーシス / 卵胞 / 細胞死受容体 / モノクローナル抗体 / 糖蛋白糖鎖配列 / 核磁気共鳴装置 / In situ hybridization / ブタ
Research Abstract	哺乳類卵巣では99%以上の卵胞が選択的に死滅する。この選択的死滅機構に顆粒層細胞アポトーシスが支配的に関与しているが、その誘導機構は未だ不明である。本研究の目的は、新規アポトーシス受容体を探索してこの機構を解明することである。すなわち、顆粒層細胞アポトーシスを局所的に支配する新規な卵胞特異的アポトーシス受容体とそれに関わるシグナル伝達機構を解明するため、健常卵胞の顆粒層細胞に特異的にアポトーシスを誘導できるモノクローナル抗体をプローブとして、アポトーシスを誘導する膜結合性新規アポトーシス受容体の同定、これをコードするmRNAの同定、新規アポトーシス受容体の糖鎖構造の解析、顆粒層細胞に特異的なアポトーシス・リガンドの探索、アポトーシスシグナルの細胞内伝達系の解析を行った。(1)新規アポトーシス受容体の同定・局在:抗体アフィニティーカラムを作製して受容体を精製し、発育ステージ別にアポトーシス受容体の発現と局在を明らかとした。(2)アポトーシス受容体mRNAの同定と発現・局在:細胞質リボゾーム部で「受容体-リボゾーム-mRNA複合体」を形成しているmRNAを精製し、これを鋳型として相補的DNAをin vitro合成し、アポトーシス受容体蛋白mRNAの全塩基配列を決定し、次いで相補的DNAをプローブとして、Northern hybridization法とin situ hybridization法を併用して発育ステージ別に顆粒層細胞におけるmRNAの局在の推移を明らかにした。(3)新規アポトーシス受容体の糖鎖構造の解析:受容体蛋白に結合する糖鎖は立体構造や細胞表層の配位を決定する重要な役割をもつ。グリコペプチダーゼ、エンド-β-ガラクトシダーゼ等の糖鎖配列を認識する酵素にて消化後、糖鎖構造を推定し、核磁気共鳴装置を用いて構造生物学的に構造決定を行い、Vliegenthart-糖蛋白由来糖鎖NMRスペクトルデータベースを検索して糖鎖構造を構造生物学的に決定し、アポトーシス受容体に結合している糖鎖の立体配位とその生理機能を明らかとした。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "A novel in vivo 31P-nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental endocrine disrupters in mice."Japanese Journal of Clinical Medicine. 58・2. 108-111 (2000)
[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "In vivo noninvasive nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental pollutants."Environment Measurement and Chemical Analysis. 27・1. 60-66 (2000)
[Publications] Noboru Manabe,Yasuyoshi Azuma,Hajime Miyamoto et al.: "Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anemic mice."British Journal of Nutrition. 83・2. 197-204 (2000)
[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Cytotechnology. 33・2. 189-201 (2000)
[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Species specific differences in apoptotic cell localization in granulosa and theca interna eclls during follicular atresia in porcine and bovine ovaries."Journal of Reproduction and Development. 46・2. 147-156 (2000)
[Publications] Susumu Nishihara,Noboru Manabe,Hajime Miyamoto et al.: "Changes in cell adhesion molecules during follicular atresia in porcine ovaries."Journal of Reproduction and Development. 46・3. 325-334 (2000)
[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Changes in localization of type I,III and IV collagens and their mRNA expression during follicular atresia in bovine and porcine ovaries."Journal of Reproduction and Development. 47・1. 27-36 (2001)
[Publications] Kozue Uchio,Noboru Manabe,Kotaro Tamura,Makoto Miyamoto et al.: "Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain)."Nephron. 86・1. 145-141 (2000)
[Publications] Kotaro Tamura,Noboru Manabe,Kozue Uchio,Makoto Miyamoto et al.: "Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain)."The Journal of Veterinary Medical Science. 52・2. 379-390 (2000)
[Publications] Noboru Manabe,Akiko Kinoshita,,Kozue Yamada-Uchio et al.: "Changes in quantitative profile of extracellular matrix components in the kidneys of rats with adriamycin-induced nephropathy."The Journal of Veterinary Medical Science. 53・1. 125-133 (2001)
[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Future functional -morphological studies in embryology."Journal of Reproduction and Development. 46・1. 71-77 (2000)
[Publications] Noboru Manabe,Hajime Miyamoto et al.: "Immunohistochemical quantification of fast-myosin in upper hind limb muscles of the pig."Animal Science Journal. 72・2. 154-163 (2000)
[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification."Theriogenology. 53・11. 1093-1103 (2000)
[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "A novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Tohoku University Press, Sendai, Japan.. 357 (2000)

2000 Fiscal Year Annual Research Report

卵胞の選択的死滅を決定する新規顆粒層細胞特異的アポトーシス受容体の探索とその制御

Principal Investigator

眞鍋 昇 京都大学, 農学研究科, 助教授 (80243070)

Research Products

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "A novel in vivo 31P-nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental endocrine disrupters in mice."Japanese Journal of Clinical Medicine. 58・2. 108-111 (2000)

[Publications] Noboru Manabe,Miki Sugimoto,Maki Morita,Hajime Miyamoto et al.: "In vivo noninvasive nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental pollutants."Environment Measurement and Chemical Analysis. 27・1. 60-66 (2000)

[Publications] Noboru Manabe,Yasuyoshi Azuma,Hajime Miyamoto et al.: "Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anemic mice."British Journal of Nutrition. 83・2. 197-204 (2000)

[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Cytotechnology. 33・2. 189-201 (2000)

[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Species specific differences in apoptotic cell localization in granulosa and theca interna eclls during follicular atresia in porcine and bovine ovaries."Journal of Reproduction and Development. 46・2. 147-156 (2000)

[Publications] Susumu Nishihara,Noboru Manabe,Hajime Miyamoto et al.: "Changes in cell adhesion molecules during follicular atresia in porcine ovaries."Journal of Reproduction and Development. 46・3. 325-334 (2000)

[Publications] Mizuho Nakayama,Noboru Manabe,Hajime Miyamoto et al.: "Changes in localization of type I,III and IV collagens and their mRNA expression during follicular atresia in bovine and porcine ovaries."Journal of Reproduction and Development. 47・1. 27-36 (2001)

[Publications] Kozue Uchio,Noboru Manabe,Kotaro Tamura,Makoto Miyamoto et al.: "Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain)."Nephron. 86・1. 145-141 (2000)

[Publications] Kotaro Tamura,Noboru Manabe,Kozue Uchio,Makoto Miyamoto et al.: "Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain)."The Journal of Veterinary Medical Science. 52・2. 379-390 (2000)

[Publications] Noboru Manabe,Akiko Kinoshita,,Kozue Yamada-Uchio et al.: "Changes in quantitative profile of extracellular matrix components in the kidneys of rats with adriamycin-induced nephropathy."The Journal of Veterinary Medical Science. 53・1. 125-133 (2001)

[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Future functional -morphological studies in embryology."Journal of Reproduction and Development. 46・1. 71-77 (2000)

[Publications] Noboru Manabe,Hajime Miyamoto et al.: "Immunohistochemical quantification of fast-myosin in upper hind limb muscles of the pig."Animal Science Journal. 72・2. 154-163 (2000)

[Publications] Miki Sugimoto,Noboru Manabe,Hajime Miyamoto.: "Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification."Theriogenology. 53・11. 1093-1103 (2000)

[Publications] Noboru Manabe,Akira Myoumoto,Chiemi Tajima,Hajime Miyamoto et al.: "A novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles."Tohoku University Press, Sendai, Japan.. 357 (2000)

眞鍋昇京都大学, 農学研究科, 助教授 (80243070)