Advancing Understanding of the Protein Composition of Human Seminal Extracellular Vesicles

Seminal extracellular vesicles (SEVs) carry a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids, which influence sperm function and have potential to modulate the female reproductive tract immune response after intromission. However, the full spectrum of SEV cargo involved in these processes remains incompletely defined. Here, we employed label-free quantitative high-resolution mass spectrometry to characterize the human SEV proteome, identifying 5079 associated proteins. These proteins were shown to likely originate from multiple regions of the male reproductive tract, notably the seminal vesicles and prostate, providing evidence for heterogeneous tissue origins of SEVs. Bioinformatic analysis revealed enrichment in sperm- and immune-related functions, as well as functions linked to protein translation. Notably, we identified several proteins with established roles in sperm physiology and immune signaling that had not previously been linked with SEV function. These included; Adenylate kinase isoenzyme (AK)2/9, and Calcium-binding tyrosine-phosphorylation regulated protein (CABYR), implicated in sperm motility, and immune regulators such as the Toll-like receptor 4 ligand, High mobility group protein B1 (HMGB1), and the Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibitor epsilon (NFκBIE). Altogether, these findings expand the known SEV proteome and highlight proteins that may influence both male and female reproductive capacity.