Small and Large Seminal Extracellular Vesicles Differentially Modulate Capacitation‐Related Parameters in Frozen–Thawed Porcine Epididymal Spermatozoa

ABSTRACT

Seminal extracellular vesicles (sEVs) have been proposed as modulators of sperm function, but their exact role remains unclear. Most studies use ejaculated sperm for clarification. This approach has limitations; nevertheless, as ejaculated sperm have already been exposed to their own native sEVs, which hinders the assessment of their specific roles. This study aimed to evaluate the potential impact of small (S‐) and large (L‐) sEVs on capacitation‐related parameters of epididymal porcine spermatozoa. Frozen–thawed epididymal sperm were incubated in a non‐capacitating medium with or without S‐ or L‐sEVs at 37°C under 5% CO ₂ for 3 h. Acrosome integrity, membrane lipid disorder, intracellular Ca ²⁺ and protein tyrosine phosphorylation (pTyr) were assessed by flow cytometry at 30 min and 3 h of incubation. Both S‐sEVs and L‐sEVs decreased ( p  < 0.0001) membrane lipid disorder at 30 min, but not at 3 h. L‐sEVs increased ( p  < 0.05) intracellular Ca ²⁺ levels at 30 min and decreased ( p  < 0.01) pTyr at 3 h. These results demonstrate a differential, time‐dependent modulation of capacitation‐related parameters by S‐ and L‐sEVs in porcine epididymal spermatozoa. This provides direct evidence of their primary effects on spermatozoa not exposed to seminal plasma.