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

Abstract

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare

“…The identification of the uEVs cargo in different species has revealed key molecules involved in these events, pointing them as valuable diagnostic tools for fertility status 26 – 28 . In the mare, only one recent study revealed the protein composition of uEVs in cyclic mares so far 29 .…”

Comparison of the Variability of Small Extracellular Vesicles Derived from Human Liver Cancer Tissues and Cultured from the Tissue Explants Based on a Simple Enrichment Method

“…Although the purity of EVs obtained with DGU methods is high, the purification process is time-consuming and requires complex experimental procedures and techniques, which make a challenge to transform towards clinical applications. The commercial preparation kits from different manufacturers and batch numbers vary in quality, causing variations of harvested EVs [22,28,29]. In the present study, we developed a simple method (method 3) for isolation of sEVs with high purity using human liver cancer tissues.…”

Extracellular vesicles activate ATM‐Chk2 signaling pathway through the intercellular transfer of mitochondrial DNA in HBV‐infected human hepatocytes

“…Total protein concentration was measured by using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). CD9, HSP70, ANXA1 and ANXA2 were used for known positive markers of EVs 22 . Flotillin‐2 23 and Calnexin 24 were also used for known positive markers of MVs.…”

“…CD9, HSP70, ANXA1 and ANXA2 were used for known positive markers of EVs. 22 Flotillin-2 23 and Calnexin 24 were also used for known positive markers of MVs. Calnexin 24 and Fiblillarin were used for negative markers of EVs.…”