Proteomic analysis of iTRAQ in melatonin-treated sheep epididymal epithelial cells

During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. Melatonin is a lipophilic hormone with multiple functions in regulating the fertility. Previous studies have shown that melatonin affected the capacitation or maturation of sperm in the epididymis. The aim of this study was to investigate the effects of melatonin on epididymal caput epithelial cells in sheep. In the study, we used iTRAQ labelling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in melatonin-treated sheep epididymal caput epithelial cells. We identified 69 differentially expressed protein; 41 were upregulated and 28 were downregulated in samples from sheep in melatonin treated. We validated the differential expression of a subset of these proteins using qPCR and Western blot. Gene ontology annotation identified that the differentially expressed proteins function in cellular processes and metabolic processes. Notably, five of the differentially expressed proteins as SOD1, COL1A1, PRM1, NQO2, and FN1 are involved in sperm migration and sperm maturation. KEGG enrichment analysis demonstrated significant enrichment in several cardiac-related pathways, such as "PI3K-Akt signaling pathway", "AGE-RAGE signaling pathway in diabetic complications", "ECM-receptor interaction", and "Ribosome". Our results suggest that candidate biomarker (SOD1, COL1A1, PRM1, NQO2, and FN1) discovery can aid in understanding sperm development and maturation in sheep. These results provide insights into the potential mechanisms of melatonin regulation of sperm maturation in epididymal caput epithelial cells.