CAPZA1 deficiency disrupts sperm flagellar structure and motility, potentially involving the p300/SLC7A11 pathway.

OBJECTIVE: To investigate the genetic and molecular role of CAPZA1 in asthenozoospermia and its impact on sperm motility and flagellar integrity. METHODS: Whole-exome sequencing (WES) was first performed in an infertile family with asthenozoospermia to identify candidate variants. The CAPZA1 variant was further screened by Sanger sequencing in 20 infertile men with asthenozoospermia and 20 age-matched fertile controls. CAPZA1 expression and sperm motility parameters were assessed by Western blot and computer-assisted semen analysis, respectively. Structural abnormalities were examined using transmission electron microscopy (TEM). In vitro CAPZA1 knockout (KO-CAPZA1) was achieved in isolated mouse round spermatids using CRISPR-Cas9, followed by RT-qPCR, Western blot, ELISA for cystine levels, and thiol quantification to assess downstream effects. Protein localization of DNAH9 and FSCN1 was analyzed by immunofluorescence. In vivo CAPZA1 deletion was induced via adeno-associated virus (AAV)-mediated CRISPR-Cas9 delivery into mouse testes, and subsequent sperm motility, protein expression, and ultrastructure were evaluated. RESULTS: A rare homozygous missense mutation in CAPZA1 (c.11T>C, p.Phe4Ser) was first identified by WES in the proband of an infertile family and was subsequently detected by Sanger sequencing in 3 of 20 asthenozoospermic patients. CAPZA1 protein expression was significantly reduced in mutant sperm, with a strong positive correlation to progressive motility (r = 0.849, p < 0.001). TEM revealed disorganized flagellar ultrastructure, including asymmetric fibrous sheath and partial dynein arm loss. In KO-CAPZA1 mouse spermatids, p300/CBP, SLC7A11, H3K27ac expression were decreased. Reduced cystine content and increased DTNB-reactive thiol groups after TCEP reduction indicated disrupted thiol/disulfide homeostasis. DNAH9 and FSCN1 expression and localization were disrupted in KO-CAPZA1 cells. KO-CAPZA1 in mice resulted in significantly decreased sperm progressive motility (p < 0.001) and abnormal axonemal structure, without affecting testicular morphology or sperm count. CONCLUSION: CAPZA1 deficiency impairs sperm motility and flagellar architecture through disrupted cytoskeletal protein regulation and redox imbalance, and represents a novel genetic contributor to asthenozoospermia.