マウス生殖細胞の発生分化制御に関わる遺伝子機構の解析

• Project/Area Number: 15080204
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: 中辻 憲夫 Kyoto University, 再生医科学研究所, 教授 (80237312)
• Co-Investigator(Kenkyū-buntansha): 中馬 新一郎 京都大学, 再生医科学研究所, 助教 (20378889) ; 野瀬 俊明 株式会社三菱化学生命科学研究所, 研究部門, 主任研究員 (70183902)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥164,800,000 (Direct Cost: ¥164,800,000); Fiscal Year 2007: ¥24,800,000 (Direct Cost: ¥24,800,000); Fiscal Year 2006: ¥25,100,000 (Direct Cost: ¥25,100,000); Fiscal Year 2005: ¥38,300,000 (Direct Cost: ¥38,300,000); Fiscal Year 2004: ¥38,300,000 (Direct Cost: ¥38,300,000); Fiscal Year 2003: ¥38,300,000 (Direct Cost: ¥38,300,000)
• Keywords: 発生・分化 / 遺伝学 / 遺伝子 / 動物 / バイオテクノロジー / 生殖細胞 / 生殖顆粒 / vasa / tudor / ES細胞 / 分化誘導 / 再プログラム化 / 核移植

Research Abstract

(1)生殖顆粒構成分子の包括的解析:マウス成体精巣から半数体精子細胞をエルトリエーション法により分離し、細胞破砕のバッファー組成、分画遠心強度、密度勾配遠心等について細胞内構造染色とウェスタンブロット解析を指標に生殖顆粒の濃縮条件を設定した。抗TDRD抗体による免疫沈降、溶出条件を詳細に検討し、RNAはマイクロアレイ解析、蛋白質はショットガンLC-MS/MS解析を行った。その結果、既知の生殖顆粒構成分子の他にRNA代謝関連蛋白質が多数同定され、また多くのmRNAが生殖顆粒に存在する事が明らかとなった。蛍光及び電子顕微鏡免疫、RNA in situハイブリザイゼーション法により細胞内局在の確認を進めている。
(2)哺乳類tudor関連遺伝子群の遺伝学的解析:Tdrd1、6、7に続いてTdrd9/spindle-Eの解析を進めた。Tdrd9遺伝子はTudorドメイン及びヘリケースドメインをコードし生殖細胞に特異的に発現するが、TDRD1、6、7とは異なり細胞質と核内にdiffuseに存在する。Tdrd9遺伝子改変マウスは雄生殖細胞の減数分裂期に異常を示し、レトロトランスポゾンのmRNA発現とゲノムDNAメチル化に異常が見られる。TDRD9蛋白質の生化学活性を探索した結果、RNA/DNAを基質とした3'-exonuclease活性を持つ事が分かった(投稿準備中)。この事からTDRD9は生殖細胞のRNA/DNA代謝を通じてレトロトランスポゾン制御とエピジェネティックスに関わる興味深い分子で有る事が明らかとなった。

Research Products

• [Journal Article] Long-Term Culture of Male Germline Stem Cells from Hamster Testes (2008)
  • Author(s): Kanatsu-Shinohara, M., et. al.
  • Journal Title: Biol. Reprod. 78 Pages: 611-617
  • Peer Reviewed
• [Journal Article] 哺乳類生殖細胞と生殖顆粒構造 (2007)
  • Author(s): 中馬新一郎、中辻憲夫
  • Journal Title: 蛋白質核酸酵素 52 Pages: 2102-2108
• [Journal Article] Tudor-related Proteins TDRD1/MTR-1,TDRD6 and TDRD7/TRAP : domain composition, intracellular localization,and function in male germ cells in mice (2007)
  • Author(s): Hosokawa, M
  • Journal Title: Dev.Biol. 301(1) Pages: 38-52
• [Journal Article] Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice (2006)
  • Author(s): Chuma, S
  • Journal Title: Proc.Natl.Acad.Sci.USA 103(43) Pages: 15894-15899
• [Journal Article] Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination (2006)
  • Author(s): Shinohara, T
  • Journal Title: Proc.Natl.Acad.Sci.USA 103(37) Pages: 13624-13628
• [Journal Article] Testatin transgenic and knockout mice exhibit normal sex-differentiation. (2006)
  • Author(s): Hasegawa, K.
  • Journal Title: Biochem.Biophys.Res.Commun. 341 Pages: 369-375
• [Journal Article] Genes associated with the formation of germ cells from embryonic stem cells in cultures containing different glucose concentrations. (2006)
  • Author(s): Mizuno, K.
  • Journal Title: Mol.Repro.Dev. 74 Pages: 437-445
• [Journal Article] RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis. (2005)
  • Author(s): Pan, J.
  • Journal Title: Development 132 Pages: 4029-4039
• [Journal Article] RNA interference during spermatogenesis in mice. (2005)
  • Author(s): Shoji, M.
  • Journal Title: Dev.Biol. 282 Pages: 524-534
• [Journal Article] Nanog expression in mouse germ cell development. (2005)
  • Author(s): Yamaguchi, S.
  • Journal Title: Gene Expr.Patterns. 5 Pages: 639-646
• [Journal Article] 培養系における生殖細胞分化 (2005)
  • Author(s): 野瀬
  • Journal Title: Hormone Frontier in Gynecology 12 Pages: 321-326
• [Journal Article] Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX/XY chimeric testis. (2005)
  • Author(s): Isotani, A.
  • Journal Title: Proc.Natl.Acad.Sci. U S A 102 Pages: 4039-4044
• [Journal Article] Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. (2005)
  • Author(s): Chuma, S.
  • Journal Title: Development 132 Pages: 117-122
• [Journal Article] 生殖細胞研究の研究動向 (2005)
  • Author(s): 野瀬 俊明
  • Journal Title: 実験医学 23 Pages: 40-45
• [Journal Article] Oligo-astheno-teratozoospermia in mice lacking Cnot7,a regulator of retinoid X receptor beta. (2004)
  • Author(s): Nakamura, T.
  • Journal Title: Nature Genetics 36 Pages: 528-533
• [Journal Article] A novel human beta1,3N-acethygalactosaminy-ltransferase that synthesizes a unique carbohydrate structure, GalNAc beta1-3GlcNAc. (2004)
  • Author(s): Hiruma, T.
  • Journal Title: J Bio.Chem. 279 Pages: 14087-114095
• [Journal Article] Cleavage-like cell division and explosive increase in cell number of neonatal gonocytes. (2004)
  • Author(s): Sakai, Y.
  • Journal Title: Develop.Growth.Differ. 46 Pages: 15-21
• [Journal Article] 培養下における生殖細胞分化 (2004)
  • Author(s): 野瀬 俊明
  • Journal Title: 蛋白質核酸酵素 49 Pages: 616-624
  • NAID: 40006137289
• [Journal Article] RNA interference during spermatogenesis in mice.
  • Author(s): Shoji, M.
  • Journal Title: Dev.Biol. (印刷中)
• [Presentation] REGULATION OF RETROELEMENT EXPRESSION AND GENOMIC DNA METHYLATION BY TDRD9/SPN-E IN THE GERMLINE (2007)
  • Author(s): Shoji, M., et. al.
  • Organizer: International Symposium on Regenerative Medical Therapy
  • Place of Presentation: Kyoto
• [Patent(Industrial Property Rights)] 哺乳動物由来のVasaホモログ遺伝子のプロモーター配列を含む発現ベクター及びその利用 (2005)
  • Inventor(s): 野瀬, 阿部, 三瀬
  • Industrial Property Rights Holder: 株式会社三菱化学生命科学研究所, 理化学研究所
  • Industrial Property Number: 2005-161279
  • Filing Date: 2005-06-01
• [Publications] Kasai, S., Chuma, S., Motoyama, N., Nakatsuji, N.: "Haploinsufficiency of Bcl-x leads to male specific defects in fetal germ cells : differential regulation of germ cell apoptosis between the sexes."Dev.Biol.. 264. 202-216 (2003)
• [Publications] Chuma, S., Hiyoshi, M., Yamamoto, A., Hosokawa, M., Nakatsuji, N.: "Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs."Mech.Dev.. 120. 979-990 (2003)
• [Publications] Y.Toyooka, N.Tsunekawa, R.Akasu, T.Noce.: "ES cell can form germ cell in culture."Proc.Natl.Acad.Sci.,USA. 100. 11457-11462 (2003)
• [Publications] S.Ina, N.Tsunekawa, A.Nakamura, T.Noce.: "Expression of the mouse Aven gene during spermatogenesis, analyzed by subtraction screening using Mvh-knock-out mice."Gene Expr.Patterns.. 3. 635-638 (2003)
• [Publications] T.Noce: "Germ cell differentiation in culture."J Mamm.Ova.Res.. 20. 69-73 (2003)
• [Publications] 野瀬俊明: "卵形成の細胞遺伝学"産婦人科治療. 87. 561-566 (2003)
• [Publications] 野瀬俊明, 恒川直樹: "Vasa遺伝子を通してわかってきた生殖細胞形成機構"細胞工学. 22. 1077-1080 (2003)
• [Publications] 野瀬俊明: "ES細胞と生殖医療"医学の歩み. 204. 927-932 (2003)
• [Publications] N.Shibata, N.Tsunekawa, S.Okamoto-Ito, R.Akasu, A.Tokumasu, T.Noce.: "Mouse Ran-BPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein."Mol.Reprod.Dev.. 67. 1-7 (2004)
• [Publications] 中辻憲夫, 中馬新一郎: "多能性幹細胞と胚形成・生殖系列 「再生医学の基礎 幹細胞と臓器形成」(中辻憲夫編)"名古屋大学出版会. 29 (2003)