Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

Area of Science:

• Reproductive Biology
• Biochemistry
• Cell Biology

Background:

• Extracellular vesicles (EVs) are crucial for embryo-maternal communication and pregnancy establishment in various species.
• Knowledge regarding mare uterine EVs and optimal isolation techniques is limited.

Purpose of the Study:

• To characterize uterine EVs from cyclic mares.
• To compare five different isolation methods and their combinations for equine uterine EVs.
• To determine the optimal method for isolating mare uterine EVs based on yield, purity, and protein cargo.

Main Methods:

• Five distinct isolation methods were evaluated: ultracentrifugation (UC), Centricon ultrafiltration (CE), CE with trehalose washing, size-exclusion chromatography (iZON-qEV), and combined approaches.
• EVs were characterized using transmission electron microscopy and Western blotting.
• Protein cargo was analyzed via mass spectrometry.

Main Results:

• All tested methods successfully isolated EVs, but with significant quantitative and qualitative variations.
• Mass spectrometry revealed differences in protein profiles, number of proteins, and protein classes among methods.
• The combination of CE/trehalose/iZON/UC demonstrated superior yield and purity.

Conclusions:

• The combined method (CE/trehalose/iZON/UC) is proposed as an optimal technique for isolating equine uterine EVs.
• This optimized method will facilitate future research into the role of equine uterine EVs in embryo-maternal interactions and pregnancy success.