精原干细胞（SSC）在鱼类种质资源活体保存、品种改良等方面具有巨大应用潜力，但其体内数量有限，体外难以持续增殖并维持干性，极大阻碍了其应用。SSC自我更新及其调控机制，是突破其体外培养困难技术瓶颈的重要科学问题。我们前期工作证实，胶质细胞源神经营养因子Gdnf通过与其受体Gfrα1结合，可介导青鳉SG3（迄今鱼类唯一精原干细胞系）增殖及干性维持，提示其在SSC自我更新中具有重要作用，但在体内是否具有同样作用，作用机制如何，尚不清楚。本项目拟用CRISPR/Cas9、转基因过表达构建青鳉gdnf、gfrα1的功能缺失与获得模型，通过组织学观察、EdU掺入法、免疫组化等，研究其在体内SSC中的作用；结合SG3体外培养体系，通过基因敲降、抑制剂处理、免疫印迹、转录组分析等，鉴定其介导的信号分子及靶基因，揭示其作用机制。本研究将为养殖及珍稀鱼类SSC体外培养体系的建立提供理论依据与重要参考。

Spermatogonial stem cells (SSC) have great potential in the fish living preservation and breed improvement. However, the applications have been greatly impeded due to the limited number of SSC in vivo as well as the difficulty of in vitro culture of SSC. The mechanisms in the self-renewal of SSC is the foremost issue to overcome the difficulty of in vitro culture of SSC. Our previous study indicated that the glial cell line-derived neurotrophic factor (Gdnf) could mediate the self-renewal of SG3 (the only fish spermatogonial stem cell line up to date) through binding of the glial cell line-derived neurotrophic factor family receptor α1 (Gfrα1). These data suggest that Gdnf/Gfrα1 have a crucial role in the self-renewal of SSC. However, whether they have similar role in vivo and what are their regulatory mechanisms in the self-renewal of SSC still remain unknown. In the present project, using medaka as an animal model, the gdnf and gfrα1 will be depleted and overexpressed using CRISPR/Cas9 and transgenic overexpressed, respectively. The role of Gdnf/Gfrα1-mediated self-renewal of SSC in these individuals will be investigated using H&E staining, EdU incorporation assay, immunohistochemistry assay and so on. Under the well-defined culture conditions, the signaling molecules and target genes mediated by Gdnf/Gfrα1 in the SG3 will be identified through the gene knockdown, chemical inhibitor treatment, Western blotting, transcriptome analyses and so on. Our study would reveal the role and molecular mechanisms of Gdnf/Gfrα1-mediated self-renewal of SSC, which will provide theoretical evidence and reference for the in vitro culture of SSC from farmed fishes as well as rare fishes.