Study On The Function Of High Expression Protein TRIM69 In Testis Tissue In Spermatogenesis

Cell apoptosis is an important biological process to maintain the tissue homeosta-sis.Ubiquitin-mediated protein degradation and modification play crucial roles in the regulation of cell apoptosis.TRIM(tripartite motif) family could be defined as one of the largest subfamily of E3 ubiquitin ligases as they contain a RING-finger domain, which are involved in a broad range of biological processes. In this study we isolated a novel gene Trim69 from human testis cDNA library, which belongs to TRIM protein family.In our previous work, we found that TRIM69 specifically expressed high levels in testis, suggesting that it may play a role in spermatogenesis.However, the biological function of TRIM69 is poorly understood.To learn about the potential roles of TRIM69 in mammalian spermatogenesis, we used Lox-Cre homologous recombination technology and corresponding breeding schemes to generate Trim69 RING domain-knockout mice.Whereas, based on a series of experimental research we found that the deletion of RING domain of Trim69 did not have significant effect on the spermatogenesis and cell apoptosis.Then we investigate the effects of hypoxia injury on testicular reproductive potential in Trim69 RING domain-knockout mice.There is no significant changes in fertility and germ cell apoptosis between control and Trim69 RING domain-knockout mice housed in desig-ned hypoxia chamber for 3 weeks.We also generated complete Trim69-knockout mice by using CRISPR/Cas9 nuclease and truncated guided RNAs. The results suggested that TRIM69 deficiency may have detrimental effects on spermatozoa leading to infertility in mice. In addition, we cloned the gene of human Trim69 into eukaryotic expression vectors which was then transfected into HEK293T cells or HeLa cells. LoVo cell line was chosen as the targeting cell to construct endogenous TRIM69 knockdown cell.We found that the stable cells with ectopically expressed TRIM69 showed more resistance to UV or etoposide-induced apoptosis than the stable control cells while LoVo cell with knock down of endogenous TRIM69 activated apoptosis. Thus our date indicates that Trim69 RING-finger domain may be not necessary in spermatogenesis and fertility of mice while knock out of complete Trim69 may lead to infertility in mice. Expressed human TRIM69 inhibited apoptosis in response to both UV irradiation and etoposide or cisplatin stimulation.