Isolation and characterization of seminal extracellular vesicles subsets and their impact on sperm freezability in stallions.

Objective Overview

• This study focused on isolating and characterizing two subsets of small extracellular vesicles (sEVs) from stallion seminal plasma (SP) and examining their role in sperm’s ability to tolerate freezing (freezability).
• The researchers identified differences between sEV subsets in stallions with good vs. poor sperm freezability, suggesting a link between these vesicles and sperm cryotolerance.

Background and Importance

• Sperm cryopreservation is widely used in horse breeding but individual stallions vary significantly in how well their sperm survive freezing and thawing.
• Seminal plasma, the fluid surrounding sperm, contains extracellular vesicles (EVs) that can deliver molecules to sperm, impacting sperm function and health.
• Understanding which components of SP help sperm survive freezing could improve cryopreservation techniques and stallion fertility management.

Study Goals

• To isolate two distinct size-based subsets of small extracellular vesicles (sEVs) from stallion seminal plasma: small (S-sEVs) and large (L-sEVs).
• To characterize these sEV subsets by morphology, concentration, particle size, purity, and presence of known extracellular vesicle markers.
• To compare these characteristics between stallions whose sperm have good freezability (GFE) versus poor freezability (PFE), testing if differences in sEVs correlate with sperm cryotolerance.

Methods

• Collected seminal plasma from 10 stallions and grouped them into good freezability ejaculate (GFE, n=5) and poor freezability ejaculate (PFE, n=5) based on sperm cryosurvival.
• Used size-exclusion chromatography (SEC), a technique that separates particles by size, to isolate the S-sEV and L-sEV subsets from seminal plasma.
• Characterized these sEV subsets following MISEV2023 guidelines, evaluating:
  • Morphology under electron microscopy
  • Concentration and particle size distribution
  • Purity, by assessing contamination such as albumin protein
  • Presence and levels of canonical EV markers: CD9, CD63, CD81, and HSP70, which indicate EV identity and potential function.
• Assessed intactness of sEV particles using the fluorescent dye CFSE to detect viable vesicles.

Key Results

• Stallion seminal plasma contains a heterogeneous population of sEVs, clearly divided into two size-based subsets identifiable via size-exclusion chromatography.
• The small sEV subset (S-sEVs) showed significantly higher expression of certain EV markers (CD9 and HSP70), suggesting these vesicles may have distinct biological roles compared to the larger subset (L-sEVs).
• Stallions with good sperm freezability exhibited a higher proportion of intact sEVs (CFSE+ particles) in both S-sEV and L-sEV fractions compared to those with poor freezability.
• This suggests that the integrity and abundance of sEVs, regardless of size subset, could be contributing to sperm’s ability to survive freezing.

Conclusions and Implications

• The study developed a robust protocol using size-exclusion chromatography to isolate and distinguish two subsets of seminal plasma sEVs in stallions.
• The distinct molecular signatures of these sEV subsets imply different functional roles in sperm physiology.
• Higher levels of intact sEVs in stallions with sperm that freeze well indicate a potential protective or supportive role of these vesicles in sperm cryotolerance.
• These findings open pathways to enhancing sperm cryopreservation by targeting or supplementing specific seminal plasma EV subsets.
• Future research could focus on identifying the specific cargo within these sEVs that modulate sperm freezability and developing EV-based fertility treatments or additives for better cryopreservation outcomes.