A Novel DNAH1 Mutation And A Novel Reciprocal Translocation T(9;22)Underlying Human Male Infertility,and Deficiency Of Exonuclease 5 Is Dispensable For Male Fertility In Mice

Infertility is one of the most important and burning issues of the present world as there is a marked increase in the incidence rate of infertility.Infertility affects 10-15%of couples worldwide and nearly half of the infertility is contributed by male factors.There are many genetic factors have been reported to be potential causes of infertility.This study was designed to identify and investigate the(a)genetic basis of infertility in a consanguineous Pakistani family with oligoasthenoteratozoo sperm ia,(b)to determine the effects of a novel chromosomal translocation on spermatogenesis in an infertile man,(c)and to determine the function of Exo5 gene in mouse spermatogenesis.Part 1:A consanguineous Pakistani family suffering primary infertility was recruited in this study.The family comprises three infertile brothers.Semen analyses of patients revealed mild to severe oligospermia and complete immotility of spermatozoa.Patients displayed multiple morphological anomalies of flagella but no other symptoms of primary ciliary dyskinesia(PCD).Scanning electron microscopy and transmission electron microscopy revealed missing central pairs of microtubules in patients’ flagella.Whole exome sequencing of this family identified a novel missense mutation in the DNAH1 gene(Chr 3 g.55865T>G),which is predicted to cause substitution of cysteine by tyrosine at amino acid position 1789(p.C1789Y).Subsequent Sanger sequencing confirmed the Mendelian inheritance of the mutation in the family.Hence,here we report a novel DNAH1 missense mutation associated with oligoasthenoteratozoospermia in human.Part 2:We recruited an idiopathic azoospermic patient and karyotype analysis of the patient revealed chromosomal rearrangements 46,XY,t(9;22)(q22;p12).These arrangements were validated in patient’s spermatocytes through immunostaining.Histological investigations of testicular tissue revealed the spermatogenic arrest with no post-meiotic cells identified.Further examination of the meiotic prophase showed multiple defects in spermatocytes,including asynapsis,reduced recombination frequency,extensive foci of DNA damage proteins(γH2AX and RPA2)detected around asynapsed regions of the translocated chromosomes indicative of the failure of DSBs repair,and failure of meiotic sex chromosome inactivation(MSCI)along with aberrant expressions of genes around the chromosomal breakpoints.All these defects collectively led to meiotic arrest and meiocytes degradation that was evident through the TUNEL assay.Altogether,these abnormalities in spermatocytes caused the spermatogenic arrest in the patient,and consequently resulted in male infertility.Part 3:Exonuclease 5(Exo5)belongs to a class of bi-directional,single-stranded DNA-specific exonucleases that encompass an iron-sulfur cluster as a structural motif and mainly play role in the DNA repair pathways.Exo5 has been demonstrated to be essential for a mismatch repair mechanism in various human cell lines.However,it’s in vivo function in mammals remains to be explored.Thus,to investigate the in vivo role of Exo5 in mammalian spermatogenesis,Exo5 knockout mice were generated by CRISPR/Cas9 genome editing technology.Exo5-/-mice exhibited no overt developmental anomalies.Interestingly,we found that,despite having a slight decrease in sperm count,knockout mice were fertile and showed no detectable difference in the testis/body weight ratio,and testicular and epididymal histology.Additionally,cytological analysis of spermatocytes revealed no significant differences in synapsis,recombination,and the prophase I progression of meiosis.Hence,these findings collectively indicate that this gene is dispensable for mouse spermatogenesis.