Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance [RNA-Seq]

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in sustaining spermatogonial populations in mice. We found that Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Furthermore, it is required for SSC self-renewal based on single cell analyses in vivo and SSC culture in vitro. Mechanistically, Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 – likely via the CCR4-NOT complex – actively degrades transcripts encoding differentiation factors to support the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development. Overall design: Testes were dissected from Tamoxifen-treated neonatal mice (Cnot3flox/flox;Id4-eGfp, and Cnot3flox/flox; Ddx4-creER, ID4-EGFP). Tunica were removed and released seminiferous tubules were dissociated with 0.25% Trypsin and Accutase into a single-cell suspension. ID4-EGFP+ cells were collected by FACS sorting. Total RNA was extracted and used for RNA-seq with Illumina TruSeq Stranded Total RNA kit.

精子发生和终身生育能力的核心基础由精原干细胞（spermatogonial stem cells, SSCs）提供。精原干细胞通过不对称分裂，既可以实现自我更新以补充自身种群数量，也可以产生未分化祖细胞，这类祖细胞在定向分化前会进行增殖。然而，目前对于调控精原干细胞维持的分子机制仍知之甚少。本研究证实，CCR4-NOT mRNA去腺苷酶复合物（CCR4-NOT mRNA deadenylase complex）亚基CNOT3在维持小鼠精原细胞群的过程中发挥关键作用。研究发现，Cnot3在未分化精原细胞中呈高表达状态；若在精原细胞中敲除Cnot3，会导致生殖细胞丢失并引发雄性不育。进一步通过体内单细胞分析与体外精原干细胞培养实验证实，CNOT3对于精原干细胞的自我更新是必需的。机制层面研究显示，Cnot3的缺失会导致编码精原细胞分化相关因子的转录本发生去抑制，其中包含谷胱甘肽氧化还原通路中的关键因子，该通路对于精原干细胞的维持至关重要。综上，本研究揭示：CNOT3很可能通过CCR4-NOT复合物，主动降解编码分化因子的转录本，从而维持精原细胞库并保障精子发生的正常进程，凸显了CCR4-NOT介导的转录后基因调控在雄性生殖细胞发育过程中的重要性。

整体实验设计：从经他莫昔芬处理的新生小鼠（基因型为Cnot3flox/flox;Id4-eGfp以及Cnot3flox/flox; Ddx4-creER, ID4-EGFP）中摘取睾丸，去除睾丸被膜后，使用0.25%胰蛋白酶与Accutase将解离出的生精小管消化为单细胞悬液。通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）收集ID4-EGFP阳性细胞。提取总RNA后，采用Illumina TruSeq Stranded Total RNA试剂盒进行RNA测序（RNA-seq）。