ACTRT3 deficiency causes ruffled acrosomes in human and mouse spermatozoa as a novel sperm-derived factor inducing fertilization failure

Background Fertilization failure has always been a significant challenge for clinical treatment, with a few genetic factors previously revealed in sperm-derived fertilization failure cases. To date, its pathogenesis remains incompletely understood. Methods Whole-exome sequencing was performed to detect potentially pathogenic mutations in the three infertile male patients with unexplained fertilization failure. The pathogenicity of the identified ACTRT3 mutations was assessed and validated in affected individuals and mouse models recapitulating the phenotypic features observed in the probands using transmission electron microscope (TEM), western blotting, co-immunoprecipitation (Co-IP), liquid chromatography-tandem mass spectrometry (LS-MS), and immunofluorescence. Artificial oocyte activation (AOA) treatment was employed to overcome the phenotype of fertilization failure in affected patients and mice. Results Novel homozygous and compound heterozygous mutations were identified in ACTRT3 (c.712 C > T, p.Gln238*; c.813dupT, p.Asp272* and c.892G > C, p.Gly298Arg) in three unrelated probands with unexplained fertilization failure. TEM revealed an ultrastructural anomaly in ACTRT3 -mutant spermatozoa, characterized by the acrosomal ruffling, a pathological change that cannot be observed by a conventional optical microscope. Subsequently, the phenotypic features observed in affected families were recapitulated in Actrt3- knockout (KO) mice, including acrosomal ruffling resulting from disrupted acroplaxome architecture, and failure of pronucleus formation. Furthermore, co-immunoprecipitation revealed that ACTRT3 interacts with ACTRT1, ACTRT2, ACTL7A and ACTL9, and they assemble to form a sperm-specific actin-related protein (ARP) complex critical for anchoring the acrosome to the nucleus. Finally, spermatozoa from probands and KO mice both showed decreased expression in PLCZ1, which causes oocyte activation deficiency and consequent fertilization failure. With the help of AOA, the fertilization failure phenotype was successfully reversed, enabling the successful delivery of healthy offspring in both probands and KO mice. Conclusions This study provides comprehensive evidences that bi-allelic pathogenic ACTRT3 variants cause sperm-derived fertilization failure, and establishes AOA as a viable therapeutic strategy for ACTRT3-mutated male infertility.