精子形成の試験管内誘導

• Project/Area Number: 20659027
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: General anatomy (including Histology/Embryology)
• Research Institution: Kyoto University
• Principal Investigator: 篠原 隆司 京都大学, 医学研究科, 教授 (30322770)
• Project Period (FY): 2008 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 2010: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2009: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2008: ¥1,100,000 (Direct Cost: ¥1,100,000)
• Keywords: 遺伝学 / 発生・分化 / 幹細胞 / 精子形成 / 自己複製 / 移植 / 顕微授精 / セルトリ細胞

Research Abstract

本研究の目的は精子幹細胞の分化糸を確立することである。本年度、我々は器官培養されたマウス精巣内に精子幹細胞を移植し、その分化能力の検定を行なった。半数体のマーカー分子としてacrosin Promoter下にenhanced green fluorescence Protein(EGFP)を発現するマウスから樹立したGermline stem(GS)細胞を先天的に不妊であるWBB6F1-W/Wvマウスの精巣内に移植を行なった。移植後数時間以内に精巣をピンセットで断片化し、alpha MEM培地を用いてアガロースゲル上で34度で器官培養を行なった。移植後にacrosinの発現をEGFPでUVランプ照射下においてモニターしたところ、移植後3-4週間でEGFP陽性細胞の存在が確認された。この細胞が精子形成細胞として正常に子孫を作製する能力があるか否かを調べるために、この断片から生殖細胞を回収し、その細胞を用いて顕微授精を行なった。二度にわたり異なる精巣を用いて実験をおこなったが、いずれの場合も作製された胚は子宮に着床後に死亡しており、正常な子孫を得ることが出来なかった。しかしながら、この結果は、精子幹細胞が試験管内で半数体細胞へと分化することを示すものであり、この培養系を改善することにより、より効率よく半数体細胞を得ることができると予想される。現在、我々は異なる培養系を用いてGS細胞の半数体細胞への分化能の検討を行っている。

Research Products

• [Journal Article] Homologous recombination in rat germline stem cells. (2011)
  • Author(s): Kanatsu-Shinohara M., Kato-Itoh M., Ikawa M., Takehashi M., Sanbo M., Morioka Y., Tanaka T., Morimoto H., Hirabayashi M., Shinohara T.
  • Journal Title: Biol.Reprod. Volume: (In press)
  • Peer Reviewed
• [Journal Article] Unstable side population phenotype of mouse spermatogonial stem cells in vitro. (2011)
  • Author(s): Shinohara T., Ishii K., Kanatsu-Shinohara M.
  • Journal Title: J.Reprod.Dev. Volume: (In press)
  • NAID: 10028275625
  • Peer Reviewed
• [Journal Article] Serum-and feeder-free culture of mouse germline stem cells. (2011)
  • Author(s): Kanatsu-Shinohara M., Inoue K., Ogonuki N., Morimoto H., Ogura A., Shinohara T.
  • Journal Title: Biol.Reprod. Volume: 84 Pages: 97-105
  • Peer Reviewed
• [Journal Article] Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model. (2010)
  • Author(s): Iwasa T., BabaS., Doi H., Kaichi S., Yokoo N., Miwa T., Kanatsu-Shinohara M., Shinohara T., Nakahata T., Heike T.
  • Journal Title: Biochem.Biophys.Res.Commun. Volume: 400 Pages: 27-33
  • Peer Reviewed
• [Journal Article] Germline modification using mouse spermatogonial stem cells.. (2010)
  • Author(s): Kanatsu-Shinohara M., Shinohara T.
  • Journal Title: Methods Enzymol. Volume: 477 Pages: 17-36
• [Journal Article] Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects (2009)
  • Author(s): Takashima S., et al.
  • Journal Title: Biology of Reproduction 81 Pages: 155-164
  • Peer Reviewed
• [Journal Article] Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation (2009)
  • Author(s): Lee J., et al.
  • Journal Title: Cell Stem Cell 5 Pages: 76-86
  • Peer Reviewed
• [Journal Article] Phenotypic plasticity of mouse spermatogonial stem cells (2009)
  • Author(s): Morimoto H., et al.
  • Journal Title: Plos One 4
  • Peer Reviewed
• [Journal Article] Genetic influences in mouse spermatogonial stem cell self-renewal (2009)
  • Author(s): Kanatsu-Shinohara M., et al.
  • Journal Title: Journal of Reproduction and Development 56 Pages: 145-153
  • NAID: 10026323942
  • Peer Reviewed
• [Journal Article] Heritable imprinting defect caused by epigenetic abnormalities in mouse spermatogonial stem cells. (2009)
  • Author(s): Lee J., et al.
  • Journal Title: Biol. Reprod. 80 Pages: 518-27
  • Peer Reviewed
• [Journal Article] Production of transgenic rats following lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells. (2008)
  • Author(s): Kanatsu-Shinohara M., et al.
  • Journal Title: Biol. Reprod. 79 Pages: 1121-8
  • Peer Reviewed
• [Journal Article] Homing of mouse spermatogonial stem cells to germline niche depends on β1-integrin. (2008)
  • Author(s): Kanatsu-Shinohara M., et al.
  • Journal Title: Cell Stem Cell 3 Pages: 533-42
  • Peer Reviewed
• [Presentation] Positive and negative regulators of mouse spermatogonial stem cell self-renewal (2010)
  • Author(s): Takashi Shinohara, Mito Kanatsu-Shinohara
  • Organizer: 11^<th> International Symposium on Spematology
  • Place of Presentation: 沖縄コンベンションセンター
  • Year and Date: 2010-06-26
• [Presentation] Positive and negative regulators of mouse spermatogonial stem cell self-renewal (2010)
  • Author(s): Takashi Shinohara
  • Organizer: Keystone meeting
  • Place of Presentation: Keystone, Colorado, 米国
  • Year and Date: 2010-02-17
• [Presentation] CDKインヒビターp21とp27による精子幹細胞の制御 (2010)
  • Author(s): 篠原隆司
  • Organizer: CREST 「iPS細胞」領域会議
  • Place of Presentation: 千里ライフサイエンスセンター
• [Presentation] Phenotypic plasticity of mouse spermatogonial stem cells (2010)
  • Author(s): 篠原隆司
  • Organizer: 特定領域研究「生殖細胞」班会議
  • Place of Presentation: 九州大学
• [Presentation] Derivation of embryonic germline stem cells (2009)
  • Author(s): Takashi Shinohara
  • Organizer: 36^<th> International Congress of Physiological Sciences
  • Place of Presentation: 京都
  • Year and Date: 2009-08-01