Genetic Study And Pathogenesis Exploration Of The Newly Causative Gene-DNALI1 In Human Asthenozoospermia

Object: asthenozoospermia（AZS）,defined as decreased sperm motility（progressive forward motility < 32% or total motility < 40%）,is one of the most common types of male infertility.In this study,we used whole exon sequencing to screen DNALI1 mutations in consanguineous families with asthenospermia.We generated the knockout gene models by CRISPR-Cas9 technology to confirmed the association of these genes with AZS.Furthermore,based on the explorative studies in these patients and the corresponding KO mice,we primarily elucidated the molecular mechanism of this gene in sperm flagellogenesis.Methods: Overall,idiopathic ASZ patients,from 65 consanguineous Chinese families,were recruited from the First Affiliated Hospital of Anhui Medical University.Possible pathogenic genes were identified by WES from blood samples,and further verified by Sanger.Western Blot（WB）and immunofluorescence（IF）experiments were used to detect the effect of mutant genes on the expression and distribution of encoded proteins.transmission electron microscopy was used to observe the ultrastructural changes of spermatozoa.We also generated the knockout models by CRISPR-Cas9 technology to further confirmed the association of this gene with AZS.Subsequently,DNALI1 interacting proteins were identified by human sperm co-immunoprecipitation and mass spectrometry,and differential proteomics in mouse testis was detected to analyze the pathogenesis of AZS caused by DNALI1 mutation.The patient received ICSI assisted pregnancy therapy,the ICSI fertilization rate and cleavage rate were statistically compared,and a pregnancy outcome after transplantation was followed up.Results: We identified etected a homozygous frameshift mutation in DNALI1[c.663₆66del（p.Glu221fs）] in a subject（AC050）by whole-exome sequencing（WES）.Sanger sequencing confirmed that these biallelic mutations originated from heterozygous asymptomatic parents,suggesting an autosomal recessive mode of inheritance.Spermatozoa of the otherwise healthy proband were entirely immotile,but exhibited normal flagellar morphology.WB and IF showed that the DNALI1 mutation caused a complete deletion of the DNALI1 protein in the patient’s sperm,which initially identified DNALI1 mutation as a potential new pathogenic gene for male AZS.Relative to the symmetrical FS in the control sperm,asymmetric FS was frequently observed in the mutant flagella.This was further confirmed by our IF data,which revealed that the AKAP4-immunostained foci were relatively absent in the flagella lacking DNALI1.We established the Dnali1-KO mice using the CRISPR-Cas9 genome editing technique to confirm that DNALI1-deficiency is indeed a novel causative mutation for human AZS.CASA analyses revealed that all epididymal sperms were immotile in the Dnali1-/-mice.Furthermore,evidence from TEM revealed typical FS abnormalities in a majority of spermatozoa,similar to the proband.Overall,the sperm phenotypes of the Dnali1-/-mice were fully consistent with those of the DNALI1663₆66del patient,thereby allowing us to declare that DNALI1 deficiency indeed caused male infertility with AZS.To explore the pathogenic mechanism of AZS caused by DNALI1 mutation,in this study,we performed co-immunoprecipitation and proteomic analysis of human sperm DNALI1,and found that DNALI1 was linked to dynein heavy chains DNAH1 and DNAH12.IF detected some dynein heavy chain deletions such as DNAH1 and DNAH7 in the patient’s sperm.IF detected the presence of DNALI1 in sperm from previously studied DNAH1\7\10 deficient patients.These suggest that DNALI1 is indispensable for flagellar assembly of DNAH1,DNAH7,and DNAH12.In contrast,DNAH1,DNAH7,and DNAH10 are not required for DNALI1 flagellar distribution.These were also observed in sperm of Dnali1-KO mice.To elucidate the significance of DNALI1 in flagellar assembly,we assessed DNALI1 expression during spermatogenesis.DNALI1 was expressed in the cytoplasm of round spermatids prior to flagellar formation.Then we found strong DNAH1 signals were detected in the cytoplasm of Dnali1-/-round spermatids,however,this signal did not extend to the flagella.That was to say,the loss of DNALI1 did not affect DNAH1 expression,however,it hindered its anchoring to the flagella.This study has clarified the pathogenic mechanism of DNALI1 mutation causing asthenospermia: loss of DNALI1 leads to failure of dynein anchoring to the flagella,which affects sperm motility and leads to AZS.Furthermore,evidence from TEM revealed typical FS abnormalities in Dnali1-/-spermatozoa,similar to the proband.The expression and localization of the fibrous sheath protein AKAP3 or AKAP4 were also abnormal in both proband and Dnali1-/-mouse sperm.To further explore the mechanism of the abnormal assembly of flagellum,we identified the DNALI1-interacting protein in the testes using MS.Gene ontology（GO）analysis identified enrichment of cellular components of the microtubule cytoskeleton,sperm flagellum,sperm fibrous sheath,and dynein complex.Combined with the abnormal localization of AKAP3 and AKAP4,we hypothesized that DNALI1 may be involved in the regulation of cytoplasmic dynein,which,in turn,played an indispensable role in flagella,especially FS assembly.Conclusions:（1）we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice,which possesses multiple crucial roles in modulating flagellar assembly and motility.（2）DNALI1 possesses multiple crucial roles in modulating flagellar assembly and motility: the loss of DNALI1 hindered the anchoring of DHC to the flagella;DNALI1 may be involved in the regulation of cytoplasmic dynein and loss of DNALI1 leads to abnormal symmetry of the fibrous sheath in sperm.