Loss of SPACAI function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex

STUDY QUESTION: Is the sperm acrosome membrane-associated protein I (SPACAI) gene critical to human globozoospermia? SUMMARY ANSWER: The biallelic loss-of-function (variant of SPACAI) causes globozoospermia as a result of acrosome-acroplaxome complex damage. WHAT IS KNOWN ALREADY: SPACAI expression decreases in patients with globozoospermia. Spaca I gene-disrupted mice have abnormally shaped sperm heads that resemble those of human globozoospermia. STUDY DESIGN, SIZE, DURATION: We recruited a consanguineous family with two brothers affected by infertility as a consequence of globozoospermia. The semen analysis data and ART outcomes were collected. Exome sequencing (ES) was used to identify potential pathogenic variants. Protein-protein interaction (PPI) technologies and proteomic analysis were utilized to explore the pathogenic mechanism. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two globozoospermic brothers and their consanguineous parents were recruited to identify the potential pathogenic variant through ES. A homozygous nonsense variant in the SPACAI gene in both brothers inherited from the heterozygous parents was identified. Twenty normal fertile males were recruited as controls. Sperm ultrastructure was observed with transmission electron microscopy. Western blotting was performed to measure SPACAI expression level in the sperm from the patients. Mass spectrometry (MS) analyses were used to identify differentially expressed proteins and to investigate proteins that interact with SPACAI. Coimmunoprecipitation (co-IP), yeast two-hybrid (Y2H) and immunofluorescence colocalization assays were used to confirm the PPI. MAIN RESULTS AND THE ROLE OF CHANCE: A nonsense variant (NM_030960.2: c.53G>A; p. Trp18*) in the SPACAI gene was identified as the pathogenic variant in a family with globozoospermia. Patient IV: I and Patient IV:2 had a phenotype very similar to that of SPACAI gene-disrupted mice. The nonsense variant in SPACAI led to premature transcriptional termination in the signal peptide, which was confirmed by western blotting. MS-based proteomics analysis showed that eight interactors of SPACAI were differentially expressed in the patients' sperm, including actin-like Protein 7A (ACTL7A), an important component of the acrosome-acroplaxome complex. The PPI of SPACAI and ACTL7A was confirmed via co-IP and Y2H assays. Immunofluorescence showed that SPACAI and ACTL7A colocalized in mature sperm, revealing that these proteins were coexpressed spatially. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of globozoospermia, only two patients from one family harbouring the SPACAI variant were found. Future studies should evaluate SPACAI variants in larger cohorts to corroborate this finding. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that the SPACAI gene was critical for globozoospermia, which expanded the spectrum of causative genes for globozoospermia. This study also provided evidence for ICSI clinical outcomes for patients with SPACAI-deficient globozoospermia, which may guide clinical treatment strategies. Furthermore, this study explored the pathogenesis of globozoospermia caused by SPACAI deficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Precision Medical Research of National Key Research and Development Program (2018YFC 1002400), National Natural Science Foundation of China (81873724), and Natural Science Foundation of Shanghai (20ZRI472700). The authors have no conflicts of interest to disclose.

Automated summary

The study identified a critical role of the SPACAI gene in globozoospermia, expanding the spectrum of causative genes. Additionally, evidence was provided for ICSI clinical outcomes in patients with SPACAI deficiency, potentially guiding treatment strategies. The study also explored the pathogenesis of globozoospermia caused by SPACAI deficiency.