RNA profiles differ between small and large extracellular vesicle subsets isolated from porcine seminal plasma

BACKGROUND: Extracellular vesicles (EVs) are essential for cell-to-cell communication because they transport functionally active molecules, including proteins, RNA, and lipids, from secretory cells to nearby or distant target cells. Seminal plasma contains a large number of EVs (sEVs) that are phenotypically heterogeneous. The aim of the present study was to identify the RNA species contained in two subsets of porcine sEVs of different sizes, namely small sEVs (S-sEVs) and large sEVs (L-sEVs). The two subsets of sEVs were isolated from 54 seminal plasma samples by a method combining serial centrifugations, size exclusion chromatography, and ultrafiltration. The sEVs were characterized using an orthogonal approach. Analysis of RNA content and quantification were performed using RNA-seq analysis. RESULTS: The two subsets of sEVs had different size distributions (P < 0.001). They also showed differences in concentration, morphology, and specific protein markers (P < 0.05). A total of 735 RNAs were identified and quantified, which included: (1) mRNAs, rRNAs, snoRNAs, snRNAs, tRNAs, other ncRNAs (termed as "all RNAs"), (2) miRNAs and (3) piRNAs. The distribution pattern of these RNA classes differed between S-sEVs and L-sEVs (P < 0.05). More than half of "all RNAs", miRNAs and piRNAs were found to be differentially abundant between S- and L-sEVs (FDR < 0.1%). Among the differentially abundant RNAs, "all RNAs" were more abundant in L- than in S-sEVs, whereas the most of the miRNAs were more abundant in S- than in L-sEVs. Differentially abundant piRNAs were equally distributed between S- and L-sEVs. Some of the all RNAs and miRNAs found to be differentially abundant between S- and L-sEVs were associated with sperm quality and functionality and male fertility success. CONCLUSIONS: Small and large sEVs isolated from porcine seminal plasma show quantitative differences in RNA content. These differences would suggest that each sEV subtype exerts different functional activities in the targeted cells, namely spermatozoa and functional cells of the female reproductive tract.