Molecular Analysis of Spermatogonial Stem Cell Aging

• Project/Area Number: 18H05281
• Research Category: Grant-in-Aid for Scientific Research (S)
• Allocation Type: Single-year Grants
• Review Section: Broad Section I
• 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): 2018-06-11 – 2023-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥193,440,000 (Direct Cost: ¥148,800,000、Indirect Cost: ¥44,640,000); Fiscal Year 2022: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000); Fiscal Year 2021: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000); Fiscal Year 2020: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000); Fiscal Year 2019: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000); Fiscal Year 2018: ¥41,600,000 (Direct Cost: ¥32,000,000、Indirect Cost: ¥9,600,000)
• Keywords: 精子形成 / 老化 / 幹細胞 / 精子幹細胞

Outline of Annual Research Achievements

生殖細胞変異は、遺伝的多様性と種の進化の根底をなすものである。これまでの研究では、主に集団と家系の遺伝子マーカーとレポーター遺伝子を分析することにより、生殖細胞の理論的な突然変異率とスペクトルが評価されていた。そこで今回我々は、マウスの培養精子幹細胞、すなわち生殖細胞系列幹（GS）細胞と、GS細胞から自発的に脱分化した多能性GS（mGS）細胞に長期培養が及ぼす影響を全ゲノム配列決定により直接測定を行なった。 GS細胞は、精細管への移植によって子孫を生成できる機能的な精子を生成するが、mGS細胞は、胚性幹細胞と同様の方法で胚盤胞へのマイクロインジェクションによって生殖細胞系列キメラに寄与する。 GS細胞とmGS細胞の推定変異率は、細胞集団が2倍になるごとに塩基あたりそれぞれ約0.22×10-9と1.0×10-9であり、GS細胞はmGS細胞と比較して変異率が低いことが明らかになった。 GS細胞とmGS細胞も明確な変異パターンを示し、GS細胞ではCからTへの移行が最も頻繁に見られ、mGS細胞ではCからAへの移行が最も優勢出会った。核型分析によると、GS細胞は染色体15および16の再発トリソミーを示しましたが、mGS細胞は染色体1、6、8、および11の増幅を頻繁に示し、異なる染色体異常が試験管内で各細胞タイプに選択的な増殖の利点を与えることを示唆する。これらのデータは、生殖細胞変異を研究するための基礎と、生殖技術および臨床応用のためのGS細胞の将来の利用のための基礎を提供する。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

GS細胞の5年間に及ぶ長期培養の遺伝子配列レベルでの異常の特徴について明らかにすることが出来た。これによりES細胞とは異なる特徴を持ち、かつ遺伝子突然変異の頻度もES細胞よりも低いことが分かった。

Strategy for Future Research Activity

本年度はマウスの個体老化に伴う精子形成細胞への影響を明らかにする。ほぼ２年間の間に渡り維持したマウスの血液中のホルモンレベルの測定や子孫の作成を行うことにより、老化が個体の妊孕性や精子幹細胞活性に及ぼす影響についての解析を行う。子孫を作製した場合には、子孫に見られる異常についても検討を行う。

Assessment Rating

Interim Assessment Comments (Rating)

A: In light of the aim of introducing the research area into the research categories, the expected progress has been made in research.

Research Products

• [Int'l Joint Research] ピッツバーグ大学(米国)
• [Journal Article] Spermatogonial stem cell transplantation into nonablated mouse recipient testes (2022)
  • Author(s): Hiroko Morimoto, Narumi Ogonuki, Mito Kanatsu-Shinohara , Shogo Matoba , Atsuo Ogura , Takashi Shinohara
  • Journal Title: Stem Cell Reports . Volume: 16 Pages: 1832-1844
  • DOI: 10.1016/j.stemcr.2021.05.013
  • Peer Reviewed / Open Access
• [Journal Article] Genomic stability of mouse spermatogonial stem cells in vitro. (2021)
  • Author(s): Chuma S, Kanatsu-Shinohara M, Katanaya A, Hosokawa M, Shinohara T.
  • Journal Title: Sci Rep Volume: 11 Pages: 24199-24199
  • DOI: 10.1038/s41598-021-03658-1
  • Peer Reviewed / Open Access
• [Journal Article] Cdc42 is required for male germline niche development in mice. (2021)
  • Author(s): Mori Y, Takashima S, Kanatsu-Shinohara M, Yi Z, Shinohara T.
  • Journal Title: Cell Reports Volume: 36 Pages: 109550-109550
  • DOI: 10.1016/j.celrep.2021.109550
  • NAID: 120007141735
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] OGG1 protects mouse spermatogonial stem cells from reactive oxygen species in culture (2021)
  • Author(s): Mori Y, Ogonuki N, Hasegawa A, Kanatsu-Shinohara M, Ogura A, Wang Y, McCarrey JR, Shinohara T.
  • Journal Title: Biol Reprod. Volume: 104 Pages: 706-716
  • DOI: 10.1093/biolre/ioaa216
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia. (2021)
  • Author(s): Hiroko Morimoto, Takuya Yamamoto, Takehiro Miyazaki, Harumi Ogonuki, Atsuo Ogura, Takashi Tanaka, Mito Kanatsu-Shinohara , Chihiro Yabe-Nishimura, Hongliang Zhang, Yves Pommier, Andreas Trumpp, Takashi Shinohara
  • Journal Title: Genes Dev Volume: 35(3-4) Pages: 250-260
  • DOI: 10.1101/gad.339903.120
  • NAID: 120006951673
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CD2 is a surface marker for mouse and rat spermatogonial stem cells. (2020)
  • Author(s): Kanatsu-Shinohara M, Chen G, Morimoto H, Shinohara T.
  • Journal Title: J. Reprod. Dev. Volume: Mar 26. Pages: 1-32
  • DOI: 10.1262/jrd.2020-019
  • NAID: 130007888738
  • Peer Reviewed / Open Access
• [Journal Article] Expression and functional analyses of EPHA2 in mouse spermatogonial stem cells. (2020)
  • Author(s): Morimoto, H., Kanatsu-Shinohara, M., Orwig, K. E. and Shinohara, T.
  • Journal Title: Biol Reprod. Volume: 102 Pages: 220-232
  • DOI: 10.1093/biolre/ioz156
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Transgenesis and genome editing of mouse spermatogonial stem cells by lentivirus pseudotyped with Sendai virus F protein. (2020)
  • Author(s): Shinohara, T. and Kanatsu-Shinohara, M.
  • Journal Title: Stem Cell Reports Volume: 14 Pages: 447-461
  • DOI: 10.1016/j.stemcr.2020.02.001
  • NAID: 120006936408
  • Peer Reviewed / Open Access
• [Journal Article] ROS amplification drives mouse spermatogonial stem cell self-renewal. (2019)
  • Author(s): Morimoto H, Kanatsu-Shinohara M, Ogonuki N, Kamimura S, Ogura A, Yabe-Nishimura C, Mori Y, Morimoto T, Watanabe S, Otsu K, Yamamoto T, *Shinohara T.
  • Journal Title: Life Science Alliance Volume: 2 Pages: 2-2
  • DOI: 10.26508/lsa.201900374
  • Peer Reviewed / Open Access
• [Journal Article] Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation. (2019)
  • Author(s): Kanatsu-Shinohara, M., Yamamoto, T., Toh, H., Kazuki, Y., Kazuki, K., Imoto, J., Ikeo, K., Oshima, M., Shirahige, K., Iwama, A., Nabeshima, Y., Sasaki, H. and Shinohara, T.
  • Journal Title: Proc. Natl. Acad. Sci. USA Volume: 116 Pages: 16404-16409
  • DOI: 10.1073/pnas.1904980116
  • Peer Reviewed
• [Journal Article] Sendai virus-mediated transduction of mammalian spermatogonial stem cells. (2019)
  • Author(s): Watanabe S., Kanatsu-Shinohara, M., Shinohara T.
  • Journal Title: Biology of Reproduction Volume: 100 Pages: 523-534
  • DOI: 10.1093/biolre/ioy192
  • Peer Reviewed
• [Journal Article] Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes (2018)
  • Author(s): Kanatsu-Shinohara M, Morimoto H, Watanabe S, Shinohara T.
  • Journal Title: J. Reprod. Dev. Volume: 64(6) Issue: 6 Pages: 1551-1564
  • DOI: 10.1262/jrd.2018-093
  • NAID: 130007530501
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed
• [Journal Article] In vivo genetic manipulateon of spermatogonial stem cells and their microenvironment by adeno-associated viruses. (2018)
  • Author(s): Watanabe S., Kanatsu-Shinohara, M., Ogonuki N., Matoba S., Ogura A., Shinohara T.
  • Journal Title: Stem Cell Reports. Volume: S2213-6711 (18) Pages: 1551-1564
  • DOI: 10.1016/j.stemcr.2018.03.005
  • Peer Reviewed / Open Access
• [Presentation] 不妊症の遺伝子治療 (2022)
  • Author(s): 篠原隆司
  • Organizer: 生殖細胞と減数分裂研究の過去、現在、未来"およびサテライトシンポジウム
  • Invited
• [Presentation] 不妊症の遺伝子治療 (2021)
  • Author(s): 篠原隆司
  • Organizer: 理研バイオリソースセンター設立20周年記念シンポジウム
  • Invited
• [Presentation] Effect of spermatogonial manipulation on offspring (2021)
  • Author(s): Takashi Shinohara
  • Organizer: Molecular and cellular mechanisms of brain systems generating individuality
  • Int'l Joint Research
• [Presentation] 宇宙環境が精子幹細胞に及ぼす影響についての解析 (2019)
  • Author(s): 篠原隆司
  • Organizer: 日本宇宙生物科学会
  • Invited
• [Presentation] 精子幹細胞の老化メカニズム (2019)
  • Author(s): 篠原隆司
  • Organizer: 若手支援技術講習会
  • Invited
• [Presentation] Aging of spermatogonial stem cells. Long Lie spermatogonial stem cells! (2019)
  • Author(s): Takashi Shinohara
  • Organizer: Brinster Symposium
  • Int'l Joint Research / Invited
• [Presentation] 精子幹細胞の移植による妊孕性の回復 (2019)
  • Author(s): 篠原隆司
  • Organizer: 日本アンドロロジー学会
  • Invited
• [Presentation] Tight junction and the fate of transplanted spermatogonial stem cells (2018)
  • Author(s): Takashi Shinohara
  • Organizer: Gordon research conference on Mammalian Reproduction
  • Int'l Joint Research / Invited
• [Patent(Industrial Property Rights)] 1.Method of introducing polynucleotide into cell in testis (2019)
  • Inventor(s): 篠原隆司、渡邉哲史
  • Industrial Property Rights Holder: 篠原隆司、渡邉哲史
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2020-004529
  • Filing Date: 2019
  • Overseas
• [Patent(Industrial Property Rights)] 雄性生殖細胞またはセルトリ細胞にポリヌクレオチドを導入する方法 (2019)
  • Inventor(s): 篠原隆司、渡邉哲史
  • Industrial Property Rights Holder: 京都大学
  • Industrial Property Rights Type: 特許
  • Filing Date: 2019
  • Overseas