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Article

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

1
Functional Genomics Group, Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, 8315 Lindau, Switzerland
2
UMR85 PRC, INRAE, CNRS 7247, Université de Tours, IFCE, 37380 Nouzilly, France
3
Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse-Faculty, University of Zurich, 8057 Zurich, Switzerland
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Laboratoire Biologie Cellulaire et Microscopie Electronique, Faculté de Médecine, Université de Tours, 37032 Tours, France
5
Faculty of Bioengineering and Bioinformatics, Moscow State University, 119992 Moscow, Russia
6
Gene Center, Laboratory for Functional Genome Analysis, LMU Munich, 81377 Munich, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(2), 979; https://doi.org/10.3390/ijms22020979
Received: 8 January 2021 / Revised: 15 January 2021 / Accepted: 16 January 2021 / Published: 19 January 2021
(This article belongs to the Special Issue Extracellular Vesicles in Reproduction)
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. View Full-Text
Keywords: extracellular vesicles; exosomes; uterus; mare; Equus caballus; EVs protein cargo; mass spectrometry; uterine fluid; uterine lavage extracellular vesicles; exosomes; uterus; mare; Equus caballus; EVs protein cargo; mass spectrometry; uterine fluid; uterine lavage
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MDPI and ACS Style

Almiñana, C.; Rudolf Vegas, A.; Tekin, M.; Hassan, M.; Uzbekov, R.; Fröhlich, T.; Bollwein, H.; Bauersachs, S. Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study. Int. J. Mol. Sci. 2021, 22, 979. https://doi.org/10.3390/ijms22020979

AMA Style

Almiñana C, Rudolf Vegas A, Tekin M, Hassan M, Uzbekov R, Fröhlich T, Bollwein H, Bauersachs S. Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study. International Journal of Molecular Sciences. 2021; 22(2):979. https://doi.org/10.3390/ijms22020979

Chicago/Turabian Style

Almiñana, Carmen, Alba Rudolf Vegas, Muhittin Tekin, Mubbashar Hassan, Rustem Uzbekov, Thomas Fröhlich, Heinrich Bollwein, and Stefan Bauersachs. 2021. "Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study" International Journal of Molecular Sciences 22, no. 2: 979. https://doi.org/10.3390/ijms22020979

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