Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice

Spermatogonial stem cells (SSCs) provide a continuous spermatogenesis and male fertility. However, the underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Specific deletion of Srsf1 in mouse germ cells impairs self-renewal and homing of SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult cKO mice, which indicates severe non-obstructive azoospermia (NOA) in cKO mice. Expression of SSC-related genes (Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of conditional knockout (cKO) mice. CLIP-seq data found that SSC-related genes (Plzf, Id4, Setdb1, Stra8, Tial1, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Moreover, multi-omics analysis suggests that SRSF1 directly binds and regulates the expression of Tial1 via AS to implement SSCs self-renewal and differentiation. Collectively, our data reveal the critical role of an SRSF1-mediated AS in SSCs self-renewal and differentiation, which may provide a framework to elucidate the molecular mechanisms of the post-transcriptional network underlying SSCs. Testes were isolated and prepared from 5 dpp mouse. Total RNA was extracted from 5 dpp mouse testes精原干细胞（Spermatogonial stem cells, SSCs）可维持持续的精子发生过程与雄性生育能力。然而，小鼠精原干细胞中可变剪接（alternative splicing, AS）的潜在调控机制迄今仍未完全阐明。本研究证实，丝氨酸/精氨酸剪接因子1（SRSF1）对小鼠精原干细胞的基因表达与剪接调控至关重要。在小鼠生殖细胞中特异性敲除Srsf1基因，会损害精原干细胞的自我更新与归巢能力，进而导致雄性不育。整体免疫染色（whole-mount staining）结果显示，成年条件性敲除（conditional knockout, cKO）小鼠的睾丸内已无生殖细胞存在，表明该小鼠罹患严重的非梗阻性无精子症（non-obstructive azoospermia, NOA）。条件性敲除小鼠的睾丸中，精原干细胞相关基因（Gfra1、Pou5f1、Plzf、Dnd1、Stra8及Taf4b）的表达水平显著下调。紫外交联免疫沉淀测序（CLIP-seq）数据显示，在小鼠睾丸组织中，SRSF1可结合以下精原干细胞相关基因的转录本：Plzf、Id4、Setdb1、Stra8、Tial1、Bcas2、Ddx5、Srsf10、Uhrf1及Bud31。此外，多组学分析提示，SRSF1可通过直接结合并调控Tial1的可变剪接，进而调控精原干细胞的自我更新与分化过程。综上，本研究揭示了SRSF1介导的可变剪接在精原干细胞自我更新与分化中的关键作用，该发现可为解析精原干细胞转录后调控网络的分子机制提供研究框架。本研究从出生后5天（5 dpp）的小鼠体内分离制备睾丸组织，并从中提取总RNA。