Comparative Proteomic Identification of Ram Sperm before and after In Vitro Capacitation.

Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography-tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein-protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility.

Identification of proteomic markers for ram spermatozoa motility using a tandem mass tag (TMT) approach.

Sperm motility is crucial for ram fertility; however, differences in the proteome of sperm with high- and low- motility in rams (Ovis aries) has yet to be achieved. Thus, the aim of this study was to identify and characterize ram spermatozoa proteins with different abundances between high- and low- motility, and identify of proteomic markers for ram spermatozoa motility using tandem mass tag (TMT) protein labeling and LC-MS/MS. In this study, the abundance of 150 proteins in high-motility (HM) ram sperm was significantly different compared with low-motility (LM) sperm. Proteins involved in sperm motility, mitochondrial activity, and spermatogenesis showed higher abundance in HM ram spermatozoa; proteins involved in protein processing and spliceosome were more abundant in LM ram spermatozoa. In conclusion, Phosphatidylethanolamine binding protein 4 is a potential proteomic marker for ram sperm motility; CCTs/HSPs are hallmarks of the LM spermatozoa in rams. Our findings highlight the functional differences between HM and LM ejaculated spermatozoa and has identified candidate proteins of interest linked to sperm motility in rams. SIGNIFICANCE: Inadequate sperm motility is one of the most important reasons for male subfertility or infertility. In this study, we found that the abundance of 150 proteins in high-motility ram sperm was significantly different compared with low-motility sperm. Proteomic biomarkers were discovered to reflect the motility variation in ram spermatozoa; these biomarkers may assist in illuminating the molecular mechanisms underlying sperm motility. This study expands the potential direction for sperm quality screening and animal breeding.