The Genetics And Mechanism Study Of Asthenoteratospermia Caused By CFAP65 Mutations

Background:Infertility remains a widespread issue affecting10–15%couples worldwide,and male factors account for about 50%of the whole infertility cases.The asthenoteratospermia due to disorders of spermiogenesis is a common cause of male infertility,whose pathogenesis is closely related to genetic factors.Spermiogenesis is the final phase of sperm development that involves complex and highly ordered spermatid differentiation,including three key events,namely the acrosome biogenesis,the flagellum assembly and nuclear condensation accompanied by the specie-specific nuclear shaping.Defects in genes involved in these processes contribute to sperm deformity with highly reduced motility,resulting in male infertility characterized by severe asthenoteratospermia.However,the genetic causes for most asthenoteratospermia remain unclear.Identifying the pathogenic genes of asthenoteratospermia and elucidating how these genes function during these key events of spermiogenesis are the fundamental for the prevention and treatment of these diseases in the future,and are the window into understand the complex spermiogenesis itself.Purpose:Our objective was to identify novel pathogenic genes causing severe asthenoteratospermia,and to explore the potential molecular mechanisms of these genes during spermiogenesis,so as to provide helpful theory foundation for genetic counseling and clinical therapy for these patients in the future,and to provide new insights into the molecular mechanisms during spermiogenesis.Methods:We performed whole exome sequencing of 47 individuals with severe asthenoteratospermia from 45 unrelated families.Mutation screening was performed in a control cohort of 637 individuals,including219 with oligoasthenospermia,195 with non-obstructive azoospermia and223 fertile controls.Ultrastructural and immunostaining analyses of patients’spermatozoa were performed in the affected individuals to characterize the effect of CFAP65 variants.Following,we generated the Cfap65 knockout mice model to explore the role of CFAP65 during spermiogenesis by using multiple strategies,such as the histopathological analysis,immunostaining,Western blotting,proteomic analysis and endogenous immunoprecipitation.Results:One homozygous nonsense mutation（NM＿194302,c.G5341T:p.E1781X）,two pairs of compound heterozygous mutations（c.C2284T:p.R762Xandc.1751del C:p.P584fs;c.5714＿5721del:p.L1905fs and c.C3021A:p.N1007K）in CFAP65 were identified in 3 individuals with completely immotile spermatozoa,respectively.No bi-allelic deleterious variants of CFAP65 were detected in the control cohort of 637 individuals.Analyses of spermatozoa from CFAP65 mutant patients showed highly aberrant sperm morphology,such as misshapen sperm heads,curved/coiled sperm tails.Ultrastructural and immunostaining analyses further showed severe structural defects,such as acrosome hypoplasia,disruption of the mitochondrial sheath（MS）,and absence of the central pair complex.CFAP65 was shown to localize at the acrosomal region and the mid-piece of the spermatozoa from normal control,but was absent in the spermatozoa from CFAP65 mutant patients.By using Cfap65 knockout mouse model,we found that Cfap65^-/-male mice were completely infertile and exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella.In addition,64.3%（18/28）of the Cfap65^-/-mice also showed phenotypes of hydrocephalus and rhinosinusitis.We focused on the spermitogenesis process caused by Cfap65 deletion.In Cfap65^-/-mouse testes,sperm heads were hyper-constricted accompanied by abnormal manchette development and acrosome biogenesis.Meanwhile,assembly of sperm anxoneme and the MS were also severely affected in Cfap65^-/-male mice.Furthermore,the proteomic analysis of Cfap65^-/-mouse testes revealed that the proteostatic system during acrosome formation,manchette organization and MS assembly was disrupted when CFAP65 was lost.Importantly,endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 could form a cytoplasmic protein network comprising MNS1,RSPH1,TPPP2,ZPBP1 and SPACA1;CFPA65 deletion caused abnormal expression and localization of these proteins during spermiogenesis,leading to abnormal spermiogenesis.Conclusion:In this study,we found that bi-allele mutations in CFAP65 could cause severe human asthenoteratospermia characterized by defective sperm head and abnormal flagellum morphology.CFAP65deficiency could cause disorders of spermitogenesis by disrupting the interactions of proteins associated with acrosome formation and flagellar assembly,which manifested as severe asthenoteratospermia.Our findings have important clinical significance for genetic counseling of these patients,and also provide insights into the molecular mechanisms involved in acrosome biogenesis and flagellum assembly during spermiogenesis.