Loss of ACTL7A causes small head sperm by defective acrosome-acroplaxome-manchette complex.

BACKGROUND: Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms. METHODS: Nuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a. RESULTS: The new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice. CONCLUSIONS: Loss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

Novel bi-allelic variants in ACTL7A are associated with male infertility and total fertilization failure.

STUDY QUESTION: What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? SUMMARY ANSWER: Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. WHAT IS KNOWN ALREADY: ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. STUDY DESIGN, SIZE, DURATION: We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. WIDER IMPLICATIONS OF THE FINDINGS: : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER: N/A.

Loss of SPACA1 function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex.

STUDY QUESTION: Is the sperm acrosome membrane-associated protein 1 (SPACA1) gene critical to human globozoospermia? SUMMARY ANSWER: The biallelic loss-of-function (variant of SPACA1) causes globozoospermia as a result of acrosome-acroplaxome complex damage. WHAT IS KNOWN ALREADY: SPACA1 expression decreases in patients with globozoospermia. Spaca1 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 SPACA1 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 SPACA1 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 SPACA1. Co-immunoprecipitation (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 SPACA1 gene was identified as the pathogenic variant in a family with globozoospermia. Patient IV:1 and Patient IV:2 had a phenotype very similar to that of Spaca1 gene-disrupted mice. The nonsense variant in SPACA1 led to premature transcriptional termination in the signal peptide, which was confirmed by western blotting. MS-based proteomics analysis showed that eight interactors of SPACA1 were differentially expressed in the patients' sperm, including actin-like Protein 7A (ACTL7A), an important component of the acrosome-acroplaxome complex. The PPI of SPACA1 and ACTL7A was confirmed via co-IP and Y2H assays. Immunofluorescence showed that SPACA1 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 SPACA1 variant were found. Future studies should evaluate SPACA1 variants in larger cohorts to corroborate this finding. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that the SPACA1 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 SPACA1-deficient globozoospermia, which may guide clinical treatment strategies. Furthermore, this study explored the pathogenesis of globozoospermia caused by SPACA1 deficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Precision Medical Research of National Key Research and Development Program (2018YFC1002400), National Natural Science Foundation of China (81873724), and Natural Science Foundation of Shanghai (20ZR1472700). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.