Uptake Assay of Ram Seminal Plasma Extracellular Vesicles to Sperm.

Extracellular vesicles (EVs) are critical mediators of cell-cell communication and play a key role in male reproductive biology by modulating sperm function. This protocol describes a robust and reproducible workflow for isolating EVs from ram seminal plasma using size-exclusion chromatography (SEC) and assessing their uptake by ram spermatozoa. In contrast to ultracentrifugation-based methods, SEC provides a gentle and more efficient isolation approach that preserves EV integrity and functionality. A central innovation of this protocol is the use of carboxyfluorescein succinimidyl ester (CFSE)-labeled seminal plasma EVs (SP-EVs) to evaluate their incorporation into sperm cells through two complementary detection platforms: (i) flow cytometry with standard resolution and (ii) confocal microscopy, for spatial confirmation of EV-sperm interactions. By bridging the gap between EV isolation and functional analysis, this protocol provides a valuable tool for investigating the role of EV-cell interactions. Specifically, it offers potential applications in male fertility preservation, biomarker discovery, and the development of EV-based therapeutic strategies in reproductive medicine. Key features • Provides a gentle, SEC-based EV isolation method optimized for ram seminal plasma, suitable for preserving vesicle integrity in studies of male reproductive biology. • Integrates EV purification with functional assays, enabling direct evaluation of EV-sperm interactions through confocal microscopy and flow cytometry. • Includes a reproducible CFSE-labeling strategy tailored for seminal plasma EVs, ensuring consistent detection of vesicle uptake by ram spermatozoa. • Designed for applications in fertility research, offering a workflow compatible with biomarker discovery, cryopreservation studies, and development of EV-based reproductive interventions.