Androgen rapidly activates phosphorylation signaling pathways in Sertoli cells.

Androgen-activated phosphorylation signaling pathways are of critical importance during spermatogenesis. The present study comprehensively analyzed androgen-activated phosphorylation signaling pathways in testicular Sertoli cells. Primary cultured mouse Sertoli cells were treated with testosterone for varying durations. Using tandem mass tag-labeled phosphoproteomics technology combined with Mfuzz clustering and functional enrichment analysis, key phosphorylation signaling pathways were identified. Mass spectrometry revealed 13,015 quantifiable phosphorylation sites, and Mfuzz clustering of expression pattern categorized 330 proteins into 6 clusters. Functional enrichment analysis highlighted key pathways, including Rap1 and MAPK kinases. Inhibitor experiments confirmed that the disruption of phosphorylation signaling impaired the integrity of the blood-testis barrier. Collectively, these findings demonstrated that androgens rapidly activate the protein phosphorylation signaling network in Sertoli cells, regulating critical processes such as blood-testis barrier formation, thereby providing new insights into male fertility and potential therapeutic strategies for spermatogenic disorders.