Characterization and therapy of fertilization failure in murine and human models with HNRNPR mutations

Oocyte activation is essential for successful fertilization and subsequent embryonic development. However, only a few disease-causing genes have been associated with sperm-derived oocyte activation failure, and the underlying molecular mechanisms and therapeutic approaches remain largely unknown. Here, we identified pathogenic mutations in HNRNPR from three infertile patients whose partners repeatedly failed to achieve transferable embryos despite undergoing both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Remarkably, artificial oocyte activation (AOA, Srcl₂) combined with ICSI successfully restored fertilization. Whole-exome sequencing revealed HNRNPR mutations shared among affected families. To establish causality, we generated a knock-in mouse model, in which males exhibited phenotypes consistent with those observed in patients. Mechanistically, ICSI with sperm from Hnrnpr-mutated mice was unable to induce normal calcium oscillations in oocytes, while spermatozoa from both humans and mice exhibited reduced expression and mislocalization of phospholipase C zeta (PLCζ). Further analyses demonstrated that hnRNPR regulates Plcz1 splicing in an m6A-dependent manner. Beyond Srcl₂ treatment, we also developed NusA-PLCζ to effectively restore oocyte activation. Collectively, these findings reveal a previously unrecognized molecular mechanism by which HNRNPR mutations cause sperm-borne oocyte activation failure and male infertility, while highlighting targeted therapeutic strategies to restore fertilization.