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Isolation and characterization of equine uterine extracellular vesicles: a comparative methodological study


Almiñana, Carmen; Rudolf Vegas, Alba; Tekin, Muhittin; Hassan, Mubbashar; Uzbekov, Rustem; Fröhlich, Thomas; Bollwein, Heiner; Bauersachs, Stefan (2021). Isolation and characterization of equine uterine extracellular vesicles: a comparative methodological study. International Journal of Molecular Sciences, 22(2):979.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Veterinary Clinic > Department of Farm Animals
Dewey Decimal Classification:570 Life sciences; biology
630 Agriculture
Scopus Subject Areas:Physical Sciences > Catalysis
Life Sciences > Molecular Biology
Physical Sciences > Spectroscopy
Physical Sciences > Computer Science Applications
Physical Sciences > Physical and Theoretical Chemistry
Physical Sciences > Organic Chemistry
Physical Sciences > Inorganic Chemistry
Uncontrolled Keywords:Physical and Theoretical Chemistry, Inorganic Chemistry, Organic Chemistry, Spectroscopy, Molecular Biology, Catalysis, General Medicine, Computer Science Applications
Language:English
Date:19 January 2021
Deposited On:10 Feb 2021 15:37
Last Modified:13 Feb 2021 20:49
Publisher:MDPI Publishing
ISSN:1422-0067
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.3390/ijms22020979

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