Molecular Profiling of Extracellular Vesicles Isolated from Boar Reproductive Fluids

Extracellular vesicles (EVs) are small membrane-bound structures that facilitate intercellular communication in the reproductive system, modulating gamete maturation, capacitation, immunomodulation and fertilization. Despite pigs' high relevance as a biomedical model, many aspects of EV biology remain poorly understood. EVs from boar seminal plasma (SP-EVs) are relatively well studied, whereas epididymosomes remain largely uncharacterized. Therefore, this study aimed to isolate and characterize EVs from the caput, corpus and cauda regions of boar epididymis, as well as from SP, with their further precise analysis towards their interaction with sperm. We successfully obtained EVs from all studied fluids with sufficient purity. Importantly, our isolation protocol preserved the EVs' ability to interact with sperm, demonstrated by lipophilic dye staining and biotin labelling experiments, confirming precisely their interaction and cargo transfer to sperm cells. Well-established EV markers, such as Alix and tetraspanins, were detected in the EVs, and additionally, phosphorylated, ubiquitinated and sialylated proteins were uniquely identified. Furthermore, we employed a proteomic approach to characterize EV proteins (mass spectrometry data are available via ProteomeXchange with identifier PXD074929) and investigate their functional roles using the Gene Ontology (GO) database. This study contributes valuable insights into the molecular composition and functional properties of EVs from the male reproductive tract. It may provide a solid framework for further basic and translational research in reproductive biology and biomedicine.