Functional Study Of The Deubiquitinase USP42 During Mouse Spermatogenesis

Spermatogenes is a well-regulated biology process,which contains self-renew of the spermatogonia stem cells(SSCs),meiosis of the spermatocytes and spermiogenesis.In recent years,various studies regarding the important roles of protein posttranslational modifications(such as phosphorylation,acetylation,glycosylation and ubiquitination)in spermatogenesis have been reported,which span the whole cycle of spermatogenesis.Ubiquitination is a reverse biology reactions mediated by ubiquitinase and deubiquitinase.Under E3 ligase,ubiquitin(Ub)is added to the amino acid residues of substrate,while deubiquitinase can remove the ubiquitin chain.To date,the functions of ubiquitinase in spermatogenesis have been widely studied,while little is known about the relationship between deubiquitinase and spermatogenesis.To better understand the functions of deubiquitinase in spermatogenesis,we firstly analyzed the reported deubiquitinase and screened USP42 as a candidate male fertility essential protein.USP42 is a testes-riched deubiquitinase,which has been indicated to deubiquitinate P53 and histone H2B in vitro and cell line,while its role in testes is not clear.Usp42 knock out(KO)mice were generated,and we found male Usp42 KO mice were completely infertile,while the fertility of female KO mice seemed normal.Morphological examination showed that Usp42 KO testes had spermatogenic arrest,displaying the disordered testis-specific junction,ES(ectoplasmic specialization),abnormal spermatid head shap and spermiation failure,as well as the final cell apoptosis.Whereas no measurable difference could be detected about the other cell lineages(such as SSCs,spermatocytes,Sertoli cells,endothelial cells and Leydig cells)in KO testes.Due to both Sertoli cells and germ cells contributing to the ES junction,we constructed Usp42 germ cell specific KO mice(g-KO)and Sertoli cell specific KO mice(s-KO),respectively.We found that the phenotypes of g-KO testes were similar to the findings in global KO,while s-KO showed no obvious phenotype,suggesting Usp42 specifically function in germ cells.To uncover the molecular mechanism of Usp42 in spermatogenesis,we generated Usp42-flag knock in mice.Co-IP assay showed that USP42 could interact with ES associated protein,RAI14,in testes.Moreover,the protein level of RAI14 in KO testes was significantly decreased,whereas the mRNA level was not altered.Furthermore,the poly-ubiquitination level of RAI14 in KO testes was obviously elevated.The above data demonstrated that USP42 could act as a deubiquitinase to bind to its substrate,RAI14,and remove the ubiquitin chain,thus,maintaining the stabilization of RAI14 protein.Via a series of cellular studies in virto,we showed USP42 could extend the half-life of RAI14 protein and deubiquitinate RAI14 in a K48-linked style.In the further study,we found that the deubiquitinating RAI14 by USP42 was dependent on the binding of KK domain of USP42 to RAI14.In addition,in vivo study revealed that the loss of RAI14 could lead to the disarrangment of F-actin,which caused the decay of ES structure and finally spermatogenic arrest.Overall,USP42 could regulate the stabilization of RAI14 by its deubiquitination activity,thus,maintaining the ES structure and regulating spermatogenesis.The present study demonstrated a crucial role of deubiquitinase in spermatogenesis and provided new target for genetic testing and treatment of patients with non-obstructive azoospermia(NOA).