Lee Jiyoung, Kanatsu-Shinohara Mito, Inoue Kimiko, Ogonuki Narumi, Miki Hiromi, Toyokuni Shinya, Kimura Tohru, Nakano Toru, Ogura Atsuo, Shinohara Takashi
Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Development. 2007 May;134(10):1853-9. doi: 10.1242/dev.003004. Epub 2007 Apr 11.
Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.
精原干细胞具有独特的自我更新特性，并在其整个生命周期中支持精子发生。尽管胶质细胞系源性神经营养因子（GDNF）最近已被确定为精原干细胞的自我更新因子，但精原干细胞自我更新的分子机制仍不清楚。在本研究中，我们使用一种生殖系干细胞（GS）培养系统评估了磷酸肌醇-3激酶（PI3K）-Akt信号通路的作用，该系统可在体外扩增精原干细胞。当将GDNF添加到GS细胞培养物中时，Akt会迅速磷酸化，而添加PI3K的化学抑制剂会阻止GS细胞的自我更新。此外，4-羟基他莫昔芬对肉豆蔻酰化形式的Akt-Mer（myr-Akt-Mer）的条件性激活在无GDNF的情况下诱导GS细胞对数增殖至少5个月。myr-Akt-Mer GS细胞表达精原细胞标志物并保留孤雄印记模式。此外，将它们进行精原细胞移植到不育受体小鼠的睾丸后，它们支持精子发生并产生后代，表明它们功能正常。这些结果表明，PI3K-Akt信号通路的激活在精原干细胞的自我更新分裂中起核心作用。
