Analyses of widely targeted metabolic profiling reveals mechanisms of metabolomic variations during Tibetan sheep (Ovis aries) testis development

In mammals, the testis is the organ with the highest transcriptional activity. After gene transcription, translation, and post-translational protein modification, the transcriptional results are finally presented at the metabolic level. Metabolites not only essential for cell signaling and energy transfer, but also directly influenced by the physiological and pathological changes in tissues and accurately reflect the physiological changes. The fact that the testes are oxygen-deprived organs can explain why Sertoli cells and germ cells may use distinctive metabolic pathways to obtain energy in their different stages of development. Therefore, studying metabolic changes during testis development can better elucidate metabolic profile of the testis, which is essential to revealing characteristic metabolic pathways. The present study applied a widely targeted UPLC-MS/MS-based metabolomics approach with large-scale detection, identification and quantification to investigate the widespread metabolic changes during Tibetan sheep testis development. Firstly, a total of 847 metabolites were detected in the sheep testis, and their changes along with the three testis-development stages were further investigated. The results indicated that those metabolites were clustered into amino acids and their derivatives, carbohydrates and their derivatives, organic acids and their derivatives, benzene and substituted derivatives, alcohols and amines, lipids, nucleotides and their derivatives, bile acids, coenzymes and vitamins, hormones and hormone-related compounds, etc. Among them, the most abundant metabolites in the testis were amino acids and lipid metabolites. The results showed that most of the lipids, carbohydrates with their derivatives, as well as alcohol and amines metabolites were high in sexually immature sheep while organic acids, amino acids and nucleotides showed a continuously increasing trend along with testis development stages. Among them, the content of metabolites with antioxidant effects increased along with testis development, while those related with energy synthesis was downregulated with age. Further correlation analyses of each metabolite-metabolite pair emphasized the cross talk between differential metabolisms across testis development, suggesting a significant correlation between lipids and other metabolites. Finally, based on KEGG pathway analysis, we found that the metabolic pathways in Tibetan sheep testis development were mainly clustered into energy metabolism, gonadal development, and anti-oxidative stress. Reactive oxygen species (ROS) are by-products of normal cellular metabolism and are inevitable during testicular energy metabolism. Thus, the anti-oxidative stress function is a key process in maintaining the normal physiological function of testis. These results contributed to a broader view of the testis metabolome and a comprehensive analysis on metabolomic variation among different testis-development stages, providing a theoretical basis for us to understand the sheep testis metabolic mechanism.