The proteome of large or small extracellular vesicles in pig seminal plasma differs and defines sources and biological functions
Seminal plasma contains many and morphologically heterogeneous extracellular vesicles (sEVs) which, released by cells of the testis, epididymis and accessory sex glands, are involved in male and female reproductive processes. The goal of this study was to describe the proteome of porcine sEVs isolated by a size exclusion chromatography (SEC)-based procedure and size-classed as small (S-EVs) or large (L-EVs). The sEV-subsets were characterized for their total protein concentration, morphology, size distribution, EV-specific protein markers and purity. A total of 988 proteins were identified by LC-MS/MS and 737 of them were quantified by SWATH in S-EVs, L-EVs and EVs-free samples. The differential expression analysis revealed 197 differentially abundant proteins between both EV-subsets, and 37 and 199 between S-EVs and L-EVs vs EVs-free samples, respectively. The gene ontology (GO) enrichment analysis of differentially abundant proteins suggested, by the type of protein detected, that S-EVs would be released mainly through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, the protein types detected might imply L-EVs are released by fusion of multivesicular bodies with the plasma membrane and would be involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating of subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV-subsets, suggesting different sources and biological functions.