Deficiency Of ACTL7A Leads To Male Infertility And Mechanism Research

Infertility is defined as a couple’s inability to conceive after a period of twelve months of regular unprotected intercourse and male infertility is caused by male factors.Any factor that affects spermatogenesis,maturation,capacitation or fertilization may lead to male infertility.According to statistics,genetic factors account for approximately 15% of male infertility.Although assisted reproductive technologies,such as in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI),can treat some male infertility patients,others with genetic factors are still unable to pregnant after repeated treatment.China is a large population country and the incidence rate of male infertility is increasing.It not only brings enormous psychological pressure to patients and families,but also causes serious social problems.Therefore,it is of great significance to study the causes of male infertility and further find effective treatment methods.In this study,a family with male infertility was collected.Two patients were clinically diagnosed as teratospermia.The two infertile couples were treated with IVF and rescue intracytoplasmic sperm injection(R-ICSI),which showed failed fertilization.Whole exome sequencing(WES)and genetic analysis of the genomic DNA of the patients revealed that ACTL7 A gene was the candidate disease-causing gene of the family,both patients carried a homozygous ACTL7 A missense mutation(c.224A>C,p.D75A),parents carried the heterozygous mutation,and alignment of amino acid sequences of ACTL7 A protein shows that D75 is highly conserved during evolution.Protein structure prediction showed that a segment of α-helix in the wild-type ACTL7 A protein transited to random coil in the mutant protein and mutant amino acid lost the hydrogen bond with Ser170,which may affect the structural stability of ACTL7 A protein.Immunofluorescence analysis showed that ACTL7 A protein was almost not expressed in patients’ sperms.Transmission electron microscopy revealed that the acrosome was folded and detached from the nuclear membrane.The CRISPR-Cas9 technology was used to construct Actl7 a knockout mice and fertility assessment showed that homozygous male knockout mice were infertile.There were no significantly abnormal in the size,weight,structure of testis and epididymis,sperm number,total motility and forward motility of homozygous male mice.However,the acrosomal morphology changed from the sickle shape in wild mice to the cap-like structure in Actl7 a homozygous knockout mice.Transmission electron microscope analysis demonstrated the acrosome of sperm detachment from the nuclear envelope and showed a shedding and folding form,which were similar to the abnormalities in the patients’ sperms.Eggs from normal mice and sperms from homozygous knockout male mice were fertilization failure by IVF and ICSI,and the number of sperms binded to zona pellucida(ZP)were decreased significantly during IVF.Further studies showed that the expression of zona pellucida binding protein(ZPBP)was not affected in the testis of Actl7 a homozygous knockout male mice,but its localization in sperm was changed,and co-immunoprecipitation showed that ACTL7 Acould interact with the ZPBP,suggesting that the alterant localization of ZPBP might affect the sperm ZP-binding ability and result in the number of sperms bind to the zona pellucida of eggs were obviously reduced.Immunofluorescence showed that PLCZ1,an important protein related to calcium oscillations in oocytes,localization was significantly changed and and the expression level was decreased in the sperms of patients and Actl7 a homozygous knockout male mice.Oocyte activation is a key step for successful fertilization,therefore,calcium oscillation was further detected after IVF and ICSI in this study,results displayed that sperms of Actl7 a homozygous knockout male mice could not induce calcium oscillations in oocytes of wild-type female mice.These findings suggest that acrosome malformation,reduced sperm ZP-binding ability and failed calcium oscillation in oocytes may be the causes of male infertility in humans and mice caused by ACTL7 A gene deficiency.In conclusion,in this study,genetic studies were cariried out in a male infertility family,and the candidate disease-causing gene ACTL7 A was identified.The Actl7 a knockout mice model was further constructed,homozygous knockout male mice were sterile,sperm acrosome was severely deformed,and the sperm of the Actl7 a homozygous knockout male mice failed to fertilize with oocytes of wild-type mice after IVF and ICSI,which were consistent with the phenotypes of patients.The binding ability of sperms from Actl7 a homozygous knockout male mice and zona pellucida in oocytes of normal female mice was significantly decreased,which may be caused by the changed localization of zona pellucida binding protein ZPBP and destructive interaction between ACTL7 A and ZPBP,then will affect ZPBP bind to zona pellucida in eggs.Furthermore,deficiency of ACTL7 A gene in human and mice significantly altered the localization and expression of PLCZ1 protein in sperms,which caused the failure calcium oscillations in oocytes and resulted in fertilization failure after ICSI.In this study,we found that human ACTL7 A gene mutation led to male infertility and deciphered pathogenesis through the Actl7 a knockout mice model,which not only provides new genetic and mechanistic explanations for male infertility,but also provides theoretical basis for genetic counseling,prenatal and clinical diagnosis of male infertility.