Pig Seminal Plasma Extracellular Vesicles Enhance Motility During Preservation of Epididymal Spermatozoa but Impair Capacitation and IVF Parameters

Abstract

BACKGROUND: Extracellular vesicles (EVs) produced by accessory sex glands play a key role in sperm functionality, influencing motility, viability, acrosome integrity, metabolic activity, and capacitation. Most studies on the effects of EVs on spermatozoa have focused on ejaculated spermatozoa, which have already been exposed to their own native EVs. This prior exposure may contribute to the variability in reported effects of EVs on sperm function. OBJECTIVES: To evaluate the role of EVs on the function of pig epididymal spermatozoa and their potential use for improving sperm preservation and in vitro fertilization (IVF). MATERIALS AND METHODS: EVs were isolated from ejaculates of boars with proven fertility using ExoQuick ULTRA and subsequently characterized according to the guidelines of the International Society for Extracellular Vesicles (ISEV). Epididymal spermatozoa were collected from the cauda epididymis and diluted in MR-A medium before being incubated with EVs for 72 h. Sperm motility, viability, acrosome integrity, and mitochondrial activity were assessed at 0, 24, 48, and 72 h. These parameters were also evaluated at 24 and 48 h after sperm incubation at 38.5°C for 5 h. To evaluate the role of EVs on sperm function, PKA activity (pPKA) and tyrosine phosphorylation (Tyr-P) were assessed in capacitated spermatozoa by Western blot. IVF assays were also performed at 0 and 48 h of storage to evaluate sperm quality. The IVF parameters evaluated were oocyte penetration rate, sperm count per oocyte, and sperm adhesion to the zona pellucida. RESULTS: Incubation of spermatozoa with EVs improved motility, but impaired acrosome integrity and viability. Incubation of spermatozoa with EVs also caused a reduction in pPKA activity. Regarding Tyr-P, it was observed that EVs prevented the phosphorylation of 20 kDa proteins. IVF parameters decreased when fertilization was performed with EV-incubated spermatozoa. CONCLUSIONS: Supplementation with EVs improves sperm motility, modulates capacitation by reducing protein phosphorylation, and reduces fertilization parameters.