DIS3 mediated RNA decay is required for maintenance of spermatogenic lineage in mice

Spermatogonial stem cells (SSCs) self-renewal and differentiation are the foundation for continious spermatognenesis in mice. Here, we investigate the essential role of DIS3 in maintaining SSC homeostasis and facilitating germ cell differentiation to ensure male fertility. Conditional inactivation of DIS3 in male germ cells have severely impaired SSC self-renewal and differentiation, which results in the failure of spermatogenesis associated with a Sertoli cell-only syndrome and adult sterility. RNA-seq analysis reveals that Dis3 deficiency abolishes its nucleolytic activity and causes significant dysregulation of the expression of transcripts in Dis3 mutant testes. We have also found that the pervasive transcription products described previously, such as Promoter Upstream Transcripts (PROMPTs), accumulate robustly upon DIS3 dysfunction in Dis3 cKO testes. In addition, scRNA-seq analysis indicates that DIS3 mutation significantly impairs germline stem cell development that blocks stem cell proliferation and differentiation. Overall, we show that DIS3 ribonuclease plays a critical role in the maintenance of spermatogenic lineage during spermatogenesis in mice. scRNA-seq was performed using P6 testicular cells from control and DIS3 knockout mice to identify different subpopulations. Re-clustering of germ cells was performed to determine the germ cell distribution and differentially expressed genes in knockout mice.精原干细胞（Spermatogonial stem cells, SSCs）的自我更新与分化，是维持小鼠精子发生持续进行的核心基础。本研究探究了DIS3在维持精原干细胞稳态、促进生殖细胞分化以保障雄性生育能力中的关键作用。雄性生殖细胞中DIS3的条件性灭活会严重损害SSCs的自我更新与分化能力，进而引发精子发生障碍，表现为唯支持细胞综合征（Sertoli cell-only syndrome）并导致成年雄性不育。RNA测序（RNA-seq）分析显示，Dis3缺陷会使其丧失核酸酶活性，并导致Dis3突变小鼠睾丸中转录本的表达出现显著失调。此外，本研究还发现此前已有报道的普遍性转录产物（如启动子上游转录本Promoter Upstream Transcripts, PROMPTs），在Dis3条件性敲除小鼠的睾丸中会因DIS3功能异常而显著富集。单细胞RNA测序（scRNA-seq）分析进一步表明，DIS3突变会严重损害生殖系干细胞的发育进程，阻断干细胞的增殖与分化。综上，本研究证实DIS3核糖核酸酶在小鼠精子发生过程中，对生精谱系的维持发挥着至关重要的作用。本研究利用对照组与DIS3敲除小鼠出生后第6天的睾丸细胞开展单细胞RNA测序，以识别不同的细胞亚群；并对生殖细胞进行重新聚类，明确了敲除小鼠体内的生殖细胞分布特征及其差异表达基因。