Azoospermia phenotype and scRNA-seq reveal hnRNPK as a factor essential for male germ cell development in mice

The process of male germ cell development is a central determinant of spermatogenesis. Nevertheless, the genetic regulatory mechanisms underlying male germ cell development in mammals remain largely unclear. In this study, employing a germ cell-specific Hnrnpk knockout mouse model combined with multi-omics analyses, we identified hnRNPK as a key factor necessary for maintaining normal development in differentiating spermatogonia. Phenotypically, adult mice with germ cell-specific hnRNPK deletion exhibited infertility, characterized by a near-complete absence of spermatocytes in the seminiferous tubules. Single-cell RNA sequencing (scRNA-seq) analysis revealed that hnRNPK deletion induced cell-cycle dysregulation in differentiating spermatogonia, triggering apoptotic cell death. As a consequence, the population of differentiating spermatogonia in the testes is markedly diminished, and these cells fail to undergo proper maturation or successfully enter meiosis. Mechanistically, cytoplasmic hnRNPK exerts its regulatory function at the post-transcriptional level, regulating the translation efficiency (TE) of genes involved in meiosis, the cell cycle, and transcriptional regulation. Furthermore, hnRNPK interacts with and colocalizes with DAZL at the 40S ribosome, thereby modulating the initiation of target messenger RNA translation. In the nucleus, hnRNPK interacts with splicing factors and participates in the splicing of target genes related to germ cell differentiation and meiosis. Collectively, these findings emphasize the functional role and mechanistic involvement of hnRNPK in differentiating spermatogonia, providing valuable insights into the post-transcriptional regulatory mechanisms that govern male germ cell development.