Extracellular vesicles in ram seminal plasma: transport and transfer of capacitation regulating factors to sperm

ABSTRACT In mammals, seminal plasma is a complex fluid surrounding spermatozoa, composed of secretions from the male reproductive tract. It plays a crucial role in modulating sperm function, but it remains unclear whether the components that regulate sperm physiology, travel freely or within extracellular vesicles secreted by the reproductive tract and accessory glands. This study evaluated three methodologies—ultracentrifugation (UC), size-exclusion chromatography (SEC), and polyethylene glycol precipitation (PEG)—for isolation of ram seminal plasma extracellular vesicles enriched fractions (SP-EVs), assessing their efficiency in terms yield, morphology, protein profile and functionality. Western blot confirmed the presence of EV-specific markers (CD9, CD63, HSP70), minimal cytoplasmic and lipoprotein contamination. SEC, particularly the second fraction (P2), yielded SP-EVs with conserved morphology, apparently reduced aggregation, and a unique protein profile enriched in low molecular weight proteins, compatible with most capacitation-modulating proteins. In contrast, UC and PEG resulted in higher particle concentration and aggregation. By CFSE labeling of SP-EVs, all preparations exhibited a targeted binding pattern to spermatozoa, with distinct patterns localized to midpiece, head and post-acrosomal regions. Additionally, western blot analysis showed that SP-EVs transport and transfer binder of sperm proteins (RSVP20 and RSVP14) to spermatozoa, with RSVP20 showing the highest incorporation, particularly from the P2. SPINK3, despite being detected in SP-EVs, was not incorporated, indicating selective protein delivery. These findings may be important to understand the role of seminal plasma extracellular vesicles on sperm, and significant for improving the efficiency of reproductive biotechnologies, as these ram SP-EVs enrichment fractions can deliver functional proteins to spermatozoa.