Ptbp1 facilitates cytoskeletal organization through the mTOR-PKCa pathway in Sertoli cells thereby supporting spermatogenesis

Spermatogenesis is a highly coordinated process to produce mature spermatozoa. Somatic lineage Sertoli cells located in seminiferous tubule provide structural and paracrine support for spermatogenesis. Proper regulation of the Sertoli cell cytoskeleton dynamics is essential for this function; however, its regulatory mechanisms remain unclear. We show that the RNA-binding protein PTBP1 maintains cytoskeletal integrity in Sertoli cells via mTORC2 signaling. Sertoli cell–specific Ptbp1 deletion in mice results in germ cell loss, disrupted blood–testis barrier, and male infertility. Transcriptomic analysis of purified Sertoli cells revealed the dysregulation of cytoskeletal- and adhesion-related genes. RIP-seq demonstrated that PTBP1 binds to Rictor mRNA, which is a core mTORC2 component. PTBP1 loss reduced RICTOR protein levels and downstream PKCa activation, impairing the cytoskeletal structure. Rescue of cytoskeletal defects in PTBP1-deficient TM4 cells by constitutively active PKCa confirmed the functional relevance of the mTORC2–PKCa axis. These findings identify PTBP1 as a post-transcriptional regulator of mTORC2 signaling that supports Sertoli cell architecture and spermatogenesis, providing mechanistic insights into the regulation of male fertility. Overall design: RNAseq of FACSed Sertoli cells; PTBP1-immunoprecipitated RNAseq