Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance.

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either self-renew or produce undifferentiated progenitors. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 is essential for sustaining spermatogonial populations in mice. Its deletion in adult germ cells resulted in germ cell loss and infertility, and its deletion in spermatogonia in the developing testis resulted in SSC depletion and compromised spermatogenesis. Consistent with the in vivo results, deletion of Cnot3 in cultured SSCs caused a reduction in cell proliferation and viability, and downregulation of SSC markers. 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 crucial for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - promotes the degradation of transcripts encoding differentiation factors to maintain the SSCs in the stem cell state, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation in SSCs and male germ cell development.