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

## TL;DR

This study identifies HNRNPR mutations as a cause of male infertility and shows that treatments like artificial oocyte activation can restore fertilization.

## Contribution

HNRNPR mutations are newly linked to infertility, with a novel mechanism involving PLCζ regulation and therapeutic interventions.

## Key findings

- HNRNPR mutations disrupt PLCζ expression and oocyte activation in both human and mouse models.
- Artificial oocyte activation and NusA-PLCζ treatment effectively restore fertilization in HNRNPR-mutant models.
- Loss of hnRNPR function leads to abnormal calcium oscillations in oocytes following ICSI.

## Abstract

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.

Pathogenic HNRNPR mutations were identified as a novel cause of sperm-borne oocyte activation failure. Mechanistic and therapeutic insights reveal RNA splicing-dependent regulation of PLCζ and potential clinical interventions.

Pathogenic HNRNPR mutations were discovered in infertile men whose partners repeatedly failed to achieve fertilization after IVF and ICSI.A knock-in mouse model carrying the human Hnrnpr mutation recapitulated sperm-borne oocyte activation failure.Disrupted Plcz1 splicing and reduced PLCζ expression in sperm were observed due to loss of hnRNPR function.Aberrant calcium oscillations in oocytes were demonstrated following ICSI with Hnrnpr-mutant sperm.Fertilization was effectively restored by artificial oocyte activation (Srcl2) or treatment with recombinant NusA-PLCζ protein.

Pathogenic HNRNPR mutations were discovered in infertile men whose partners repeatedly failed to achieve fertilization after IVF and ICSI.

A knock-in mouse model carrying the human Hnrnpr mutation recapitulated sperm-borne oocyte activation failure.

Disrupted Plcz1 splicing and reduced PLCζ expression in sperm were observed due to loss of hnRNPR function.

Aberrant calcium oscillations in oocytes were demonstrated following ICSI with Hnrnpr-mutant sperm.

Fertilization was effectively restored by artificial oocyte activation (Srcl2) or treatment with recombinant NusA-PLCζ protein.

Pathogenic HNRNPR mutations were identified as a novel cause of sperm-borne oocyte activation failure. Mechanistic and therapeutic insights reveal RNA splicing-dependent regulation of PLCζ and potential clinical interventions.