Insights into the role of hnRNPK in spermatogenesis via the piRNA pathway

Deletion of hnRNPK in mouse spermatogonia leads to male sterility due to arrest permatogenesis, yet the underlying molecular mechanisms remain elusive. This study investigated the testicular proteome on postnatal day 28 (P28) to elucidate the infertility associated with Hnrnpk deficiency, identifying 791 proteins with altered expression: 256 were upregulated, and 535 were downregulated. Pathway enrichment analysis demonstrated that the downregulated proteins are primarily involved in spermatogenesis, fertilization, and piRNA metabolic processes. In Hnrnpk cKO mice, key proteins essential for piRNA metabolism, such as PIWIL1, TDRD7, DDX4, and MAEL, exhibited reduced expression, resulting in impaired piRNA production. Mechanistic studies employing RNA immunoprecipitation (RIP), dual-luciferase reporter assays, and fluorescence in situ hybridization/immunofluorescence (FISH/IF) assays demonstrated that hnRNPK directly interacts with the 3'UTR of piRNA pathway transcripts, enhancing their translational efficiency. These results establish that Hnrnpk deficiency disrupts the piRNA pathway by diminishing the expression of essential regulatory proteins, thereby impairing piRNA production and spermatogenesis. Our findings elucidate a novel molecular basis for infertility linked to hnRNPK dysfunction and advance understanding of post-transcriptional regulation in male germ cell development.