Restoration of Spermatogenesis is Dependent on Activation of a SPRY4-ERK Checkpoint Following Germline Stem Cell Damage

Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury in adult mice, germline-specific Spry4 gene deletion (Spry4G-KO) reduces stem cell regeneration with an enhanced genotoxic stress response and differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs). Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor rapamycin, was sufficient to promote spermatogonial proliferation in Spry4G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, reducing spermatogonial genome integrity, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.