(088) The Essential Role of O-GlcNAcylation in the Testis Aging

Abstract

Abstract Introduction With the delay of reproductive age, reproductive disorders related to advanced age have gradually attracted widespread attention. Studies have shown that the decline in male fertility due to aging is manifested as a decrease in semen parameters and spermatogenetic cells histologically. O-GlcNAc (O-acetylglucosamine) glycosylation is a post-translational modification that exists widely in cells. The level of UDP-GlcNAc, which is the substrate of O-GlcNAcylation, changes rapidly with various nutrients and external factors. Therefore, O-GlcNAcylation can respond to glucose, amino acid, fatty acid, and nucleoside metabolism, and is considered to be an important sensor of cellular metabolism. The previous study of our project found that the level of O-GlcNAc modification in aged testis tissue was significantly increased, so this study aimed to explore the specific mechanism of O-GlcNAc modification involved in testicular aging. Objective 1.Explore the modification pattern of O-GlcNAcylation in testis 2.Mimic the high O-GlcNAcylation state of aging mice in young mice, to study the effect of elevated high O-GlcNAcylation level on testis. 3.Reveal the role of key proteins modified by O-GlcNAc in testicular aging Methods Mice were injected intraperitoneally with OGA inhibitor Thiamet-G for 35 days to increase the level of O-GlcNAc to simulate aging. CASA system was used to detect semen parameters, and chromosome spreading technology was used to study mouse testis meiosis. Using protein profiling technology, the differentially modified O-GlcNAc proteins in aged testis and young testis were screened, and point mutations were made at their O-GlcNAc sites to study the specific mechanism of key O-GlcNAc proteins involved in testis aging. Results The O-GlcNAc modification level in the testis of aged mice was significantly increased. After intraperitoneally injecting Thiamet-G into mice for 35 days, the level of O-GlcNAcylation in the testis tissue was significantly increased. High level of O-GlcNAcylation in testis damaged the sperm parameter, decreased the thickness of seminiferous epithelium and increased apoptosis in testis. Chromosome spreading experiments showed that, γH2Ax, which is the marker of DNA double strand breaks, stained unusually at autosomes in pachytene spermatocytes. The elevation of γH2Ax affected the transition of pachytene spermatocytes to diplotene. Importantly, we found that the O-GlcNAcylation of Hyou1 protein was significantly increased in the testis of aged mice. O-GlcNAcylation of Hyou1 promotes the degradation of Hyou1. An O-GlcNAc site of Hyou1 was identified at S634 and mutation of this site decreased the O-GlcNAcylation, increased the protein stability and exerts protective roles in aging testis. Conclusions In the testis of aged mice, the level of O-GlcNAcylation is increased, which leads to the arrest of spermatocyte meiosis by affecting the DNA damage repair pathway. Hyou1 is the key O-GlcNAc protein in testicular aging and mutation of this site exerts protective roles. This study revealed for the first time the specific mechanism of O-GlcNAc involved in testicular aging, and provided a new therapeutic target for clinically delaying testicular aging and treating male infertility. Disclosure No