Mechanism Study Of USP26 During Mouse Spermatogenesis

Spermatogenesis is the process by which diploid germ cells eventually form functional haploid sperm through meiosis.It mainly includes the initiation of meiosis,spermatocytes meiosis and spermatids formation.The process of sperm production plays a decisive role in male reproduction,and abnormalities at any stage are likely to cause defects in spermatogenesis and lead to male infertility.Previous studies have shown that the expression of various of specific proteins during spermatogenesis is involved in the regulation of spermatogenesis,a delicate and complex process.In this study,an optimized chromatin purification method was used to purify the chromatin of mouse testes at different stages and mass spectrometry was performed to screen potential proteins that regulate spermatogenesis.In the mass spectrometry results,Ubiquitin-specific protease 26(USP26)has drawn our attention.It has been reported that deubiquitinating enzymes,as negative regulators of protein ubiquitination,play an important role in the regulation of ubiquitination,and USP26 is a member of this family.Studies has shown that USP26 gene mutations in male patients with non-obstructive azoospermia can cause male spermatogenesis disorders,however,the molecular mechanism remains unclear.Therefore,this subject was conducted to explore the function and possible mechanism of USP26 in mouse spermatogenesis.Firstly,we made the rabbit polyclonal antibody of USP26 through constructing the recombinant plasmid of Pet-42b-USP26,performing prokaryotic expression and purification and immunizing the rabbit with the purified protein.Secondly,the expression and distribution of USP26 was examined by western blot and immunofluorescent assay.The results showed that USP26 is testis-specific and mainly located in sperm acrosome.Afterwards,the USP26 gene was knocked out through the CRISPR-CAS9-based gene editing technology,with the gene edited mouse the mechanism of USP26 in mouse spermatogenesis was studied.The knockout efficiency of USP26 was confirmed by Western blot,and then the knockout mouse phenotypes were analyzed.It was found that knocking out USP26 had no effect on testicular weight of mice,however,the number of sperm decreased,and in vitro fertilization analysis revealed there was no obvious difference in the development of mouse embryos between wild type and USP26 knockout mouse.Meanwhile,there was no significant difference in the littersize of the knockout mice compared with wild-type mice.The above results showed that knockout of USP26 has no significant effect on male fertility in mice,though the mutation in men cause male infertility.Therefore,although the knockout of USP26 in mice may cause abnormal ubiquitination to some sperm-related proteins,and affect the spermatogenesis partially,but this regulation is not necessary for spermatogenesis.