Pathogenic variants in ACTRT3 lead to male infertility characterized by fertilization defects

STUDY QUESTION: Are there new genetic factors responsible for male infertility characterized by fertilization defects despite normal sperm motility? SUMMARY ANSWER: We identified the novel pathogenetic gene actin-related protein T3 (ACTRT3) as one of primary causes for male infertility characterized by fertilization defects. WHAT IS KNOWN ALREADY: To date, only a few genes have been reported to be responsible for fertilization defects in male infertility despite normal sperm motility. ACTRT3, as one of the actin-related proteins, is specifically expressed highly in murine testes and interacts with the protein Profilin3 to form a testis-specific complex during spermatogenesis. STUDY DESIGN, SIZE, DURATION: The ACTRT3 variants were identified by whole-exome sequencing in a cohort of 248 infertile couples characterized by fertilization defects (fertilization rate ≤ 30% in their IVF/ICSI attempts) despite normal sperm concentration (≥15 × 106/ml) and normal motility (progressive motility ≥32%; motility of PR (progressive)+NP (non-progressive) ≥40%). Thirty-six hours after cell transfection, immunoprecipitation was performed to detect the interaction between ACTRT3 and actin-like 7A (ACTL7A)/phospholipase C zeta 1 (PLCZ1) (n = 3 biological replicates). A knock-in (KI) mouse model was generated by CRISPR-Cas9. Sexually mature males (10 weeks old) were used for fertility testing (n = 3 mice per group, lasts 6-8 months), semen parameter analysis (n = 3 mice per group), western blot (n = 3 biological replicates), immunofluorescence (n = 3 biological replicates), IVF (n ≥ 3 biological replicates), ICSI (n = 3 biological replicates), scanning electron microscopy (SEM, n = 3 mice per group), transmission electron microscopy (TEM, n = 3 mice per group), Ca2+ oscillations (n ≥ 3 biological replicates), and other functional assays between 2022 and 2025. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT3 variants were identified by whole-exome sequencing and confirmed by Sanger sequencing. ACTRT3 KI mouse was constructed to discover the pathogenic mechanism. We first checked ultrastructure of the sperm through periodic acid-schiff staining, SEM, and TEM. Western blotting, immunofluorescence, and immunoprecipitation were further performed to clarify the molecular mechanism of the ACTRT3 variants. MAIN RESULTS AND THE ROLE OF CHANCE: We identified compound-heterozygous (NM_032487.5: c. C41G (p. Ser14Trp) and c. C687delA (p. Lys229fs)) and homozygous variants (NM_032487.5: c.712C>T (p. Gln238Ter)) in ACTRT3 in two infertile males, and functional studies in transgenic KI mouse models confirmed the pathogenicity of the Actrt3 variants. Defects in ACTRT3 caused decreased protein levels of ACTL7A and disrupted localization of PLCZ1 in sperm, which resulted in acrosome detachment, further impacted the binding between sperm and the zona pellucida of oocytes and caused fertilization failure. In addition, sperm with defective ACTRT3 cannot initiate calcium oscillations, which leads to embryo arrest. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: More cases are needed to confirm the genetic contribution of ACTRT3 variants to male infertility. In addition, it will be more persuasive and informative by adding the rescue experiments of artificial oocyte activation through SrCl2 which is used to active calcium ion oscillation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest the causal relationship between ACTRT3 variants and male infertility caused by fertilization defects, and partially revealing the pathological mechanisms of ACTRT3 variants. This provides new insights into the understanding of male infertility due to fertilization problems and provides references for future clinical treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (82325021, 32130029, and 82201767) and the Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (No. JYB2025XDXM508). TRIAL REGISTRATION NUMBER: N/A.