(228) Polypyrimidine tract binding protein 1 (Ptbp1) facilitates cytoskeletal organization through the mTOR-PKCα pathway in Sertoli cells thereby supporting spermatogenesis

Abstract Authors: Manabu Ozawa¹, Nobuaki Yoshida¹, Masahito Ikawa^1,2
1. Laboratory of Reproductive Systems, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
2. Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Abstract Text:

Spermatogenesis relies on the structural and biological support of Sertoli cells, which regulates germ cell maintenance and differentiation. However, the molecular mechanisms controlling Sertoli cell dynamics remain largely unknown. Here, we identified PTBP1, an RNA-binding protein highly expressed by Sertoli cells, as a critical regulator of spermatogenesis through its role in maintaining Sertoli cell function. Sertoli cell-specific deletion of Ptbp1 in AmhCre;Ptbp1 flox/flox (Ptbp1 cKO) mice results in severe spermatogenic defects and complete male infertility. Histological analysis revealed that in Ptbp1 cKO mice, Sertoli cells detached from the basement membrane, leading to disruption of the blood-testis barrier (BTB). Transcriptomic analysis of purified Sertoli cells from Ptbp1 cKO testes revealed significant dysregulation of genes involved in cytoskeletal organization and cell adhesion. RNA immunoprecipitation sequencing (RIP-seq) demonstrated that PTBP1 protein binds to key components of mTORC2, including Rictor mRNA. To further investigate the impact of PTBP1 deficiency on mTORC2 signaling in Sertoli cells, we analyzed its downstream effects and found that PTBP1 loss led to a marked reduction in RICTOR protein levels and impaired mTORC2-PKCα signaling, thereby disrupting cytoskeletal organization. Functional rescue experiments using a hybrid gene-inducible system, which included Ptbp1 knockdown and PKCα activation in TM4 cells, a murine Sertoli cell-derived line, showed that restoring PKCα activity partially rescued the cytoskeletal defects caused by PTBP1 loss, highlighting the role of mTORC2-PKCα signaling in Sertoli cell function. These findings reveal a novel mechanism by which PTBP1 post-transcriptionally regulates Rictor to maintain mTORC2 activity, ensuring Sertoli cell cytoskeletal integrity and supporting continuous spermatogenesis. This study provides new insights into the molecular regulation of Sertoli cell function and identifies PTBP1 as a potential target for therapeutic intervention in male infertility.