ETV5介导的小鼠精原干细胞自我更新转录调控机制研究

Spermatogonial stem cells (SSCs) serve as the foundation of spermatogenesis. In vitro growth of SSCs is dependent on the growth factor GDNF. We have identified a critical transcriptional factor, ETV5 not only as a downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes, including Bcl6b and LIM homeobox 1 (Lhx1), but also as a regulator of Brachyury (T) and CXC chemokine receptor type 4 (Cxcr4), which is of biological importance and functions to promote self-renewal or differentiation of mouse SSCs cultured in vitro. Our previous data indicated that remarkable conservation of gene expression, especially for self-renewal genes, in these prepubertal germline cells between mouse and human that diverged phylogenetically ~75 million years ago. Notably, Etv5 was also found greatly elevated in the early stage of male germline cells in both species, which indicates the regulation of Etv5 could be essential in SSCs. In current study, we would combine chromatin immunoprecipitation (CHIP) and next-generation high throughput sequencing to explore ETV5 mediated regulation in SSCs, together with stem cell culture, small RNA interference, lentivirus transfection and in vivo transplantion. We will also analyze the SSC growth dynamics, SSC migration and SSC differentiation after transplantation, as well as the number of colonies from transplanted SSCs. Base on those data, we expect to disclose the details of ETV5 mediated regulation in the self-renewal of SSCs and contribute valuable information to the stability of male's genetic information passed to the next generation.

精原干细胞(SSCs)是雄性生精基础。SSCs体内自我更新、体外培养和增殖依赖于GDNF。我们前期的研究提示转录因子蛋白ETV5，不仅自身表达受GDNF影响，同时还在转录水平上调控GDNF下游基因，如Bcl6b、Lhx1、Brachyury(T)、Cxcr4等，在SSCs自我更新的过程中起关键作用。此外，我们发现，从小鼠到人类，物种间有七千多万年进化差距，但其SSCs的自我更新机制具有保守性，ETV5在这两个雄性物种早期生殖细胞中都高表达，提示它在SSCs中的作用可能也具有保守性。本项目通过染色体免疫沉淀方法，结合新一代DNA深度测序技术，探讨SSCs中受到ETV5调控的下游基因；并通过RNA干扰，慢病毒转染等方法，研究这些下游基因表达的改变是否会影响到SSCs增殖、迁移、分化以及再生精等生物学功能，从而在分子水平阐明雄性生殖干细胞的调控机制，为个体遗传信息稳定传递提供理论依据。

生长因子依赖的干细胞自我更新是组织稳定和再生的基础。精原干细胞支持雄性一生的精子发生及育性，但目前尚无文章来全景式揭示磷酸化生物学事件在SSCs自我更新和增殖过程中的重要作用。我们分析揭示了干细胞中受生长因子GDNF调控的定量蛋白质组学和磷酸化蛋白质组，发现3382个磷酸化蛋白和12141个位点，其中325个蛋白和570个位点受到调控，ERK1/2, GSK3, CDK1,和 CDK5是主要的底物富集激酶。进一步我们发现GDNF/ERK1/2通路调控干细胞G2/M期细胞阻滞，同时mTORC1复合物的Raptor Ser863位点磷酸化是细胞增殖所必须的。而这些信号通路调控转录因子ETV5的表达。组织特异敲除Raptor小鼠精子发生严重损伤，精原细胞进行性丢失，而体外过表达Raptor，干细胞增殖显著加快。这些研究发现了以前未揭示的重要信号通路，而这些通路改变可能影响干细胞调控，精子发生维持，是潜在雄性生育的病理损伤结果成因。.此外，我们发现了一个新的精原干细胞特异表达的lncRNA（lncRNA033862），它是小鼠精原干细胞维持所必需的。LncRNA033862主要表达于包括精原干细胞在内的早期精原细胞，并且是受到GDNF调控的805个lncRNA中的一个，而GDNF是维持精原干细胞自我更新最重要的细胞因子。LncRNA033862是Gfrα1反义链上起源且不能编码蛋白的转录本，通过与Gfrα1的染色体相结合从而调节Gfrα1的表达。更为重要的是，当lncRNA033862被敲减之后，精原干细胞的生存能力明显受到损害，ETV5表达显著下调，并且通过移植实验检测其再生精能力也受到影响。我们的研究发现了第一个调节精原干细胞命运的lncRNA。.综上，这些结果会提高我们对调控精子发生过程的认识，并且有助于解决男性不育的问题。