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International journal of molecular sciences2021; 22(2); 979; doi: 10.3390/ijms22020979

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.
Publication Date: 2021-01-19 PubMed ID: 33478136PubMed Central: PMC7835857DOI: 10.3390/ijms22020979Google Scholar: Lookup
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  • Comparative Study
  • Journal Article

Summary

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This research article explains a study that compared five methods of isolating extracellular vesicles (EVs) from equine uterine fluid. The scientists aimed to find out the optimal technique for extracting EVs that could later be studied for their role in pregnancy in mares.

Extracellular Vesicles and Their Role in Pregnancy

  • Extracellular vesicles, or EVs, have been discovered in uterine fluid of various species. These EVs are instrumental in the dialogue between embryo and mother, as well as recognition and establishment of pregnancy.
  • Until now, there is limited information about uterine EVs in horses, specifically mares.
  • The researchers in this study aimed to increase understanding of uterine EVs in mares. They sought to achieve this by employing different methods to isolate these EVs from the uterine fluid, referred to as uterine lavage.

Comparison of Five EVs Isolation Methods

  • The methodology for the study involved the comparison of five different ways to isolate EVs, and also looked at combinations of these methods. These were Ultracentrifugation (UC), concentration of lavage volume by Centricon ultrafiltration (CE), use of CE with different washing steps (using saline with or without an organic compound named trehalose), size-exclusion chromatography (a method to separate particles based on size) with iZON-qEV columns, and the combination of methods based on the best results on EV yield, purity, and protein cargo profiles.

Finding the Most Effective Method

  • Using transmission electron microscopy (a microscopy technique that uses a beam of electrons to create an image of the specimen) and Western blotting (resolving and identifying protein molecules) confirmed that the isolation of EVs was successful with all methods. However, there were variations in the quantity and quality of EVs isolated.
  • Mass spectrometry, a method to sort ions based on their mass to charge ratio, presented differences in protein profiles between methods, and also in the number of defined proteins and protein classes.
  • The study concludes that the combo of CE/trehalose/iZON/UC yielded the best isolation result, providing a good yield and purity of equine uterine EVs. This is the recommended method to be used in future research examining the role of uterine EVs in embryo-mother interaction within mares.

Cite This Article

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

Publication

ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 22
Issue: 2
PII: 979

Researcher Affiliations

Almiñana, Carmen
  • Functional Genomics Group, Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, 8315 Lindau, Switzerland.
  • UMR85 PRC, INRAE, CNRS 7247, Université de Tours, IFCE, 37380 Nouzilly, France.
Rudolf Vegas, Alba
  • Functional Genomics Group, Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, 8315 Lindau, Switzerland.
  • Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse-Faculty, University of Zurich, 8057 Zurich, Switzerland.
Tekin, Muhittin
  • Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse-Faculty, University of Zurich, 8057 Zurich, Switzerland.
Hassan, Mubbashar
  • Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse-Faculty, University of Zurich, 8057 Zurich, Switzerland.
Uzbekov, Rustem
  • Laboratoire Biologie Cellulaire et Microscopie Electronique, Faculté de Médecine, Université de Tours, 37032 Tours, France.
  • Faculty of Bioengineering and Bioinformatics, Moscow State University, 119992 Moscow, Russia.
Fröhlich, Thomas
  • Gene Center, Laboratory for Functional Genome Analysis, LMU Munich, 81377 Munich, Germany.
Bollwein, Heinrich
  • Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse-Faculty, University of Zurich, 8057 Zurich, Switzerland.
Bauersachs, Stefan
  • Functional Genomics Group, Institute of Veterinary Anatomy, Vetsuisse Faculty Zurich, University of Zurich, 8315 Lindau, Switzerland.

MeSH Terms

  • Animals
  • Drainage / methods
  • Drainage / veterinary
  • Extracellular Fluid / cytology
  • Extracellular Vesicles / physiology
  • Extracellular Vesicles / ultrastructure
  • Female
  • Gene Expression Profiling
  • Horses / genetics
  • Horses / metabolism
  • Microscopy, Electron, Transmission
  • Ovulation / physiology
  • Proteome / analysis
  • Proteome / isolation & purification
  • Proteome / metabolism
  • RNA / analysis
  • RNA / isolation & purification
  • RNA / metabolism
  • Therapeutic Irrigation / methods
  • Therapeutic Irrigation / veterinary
  • Transcriptome
  • Uterus / cytology

Grant Funding

  • SNSF grant 31003A_173171 / Schweizerischer Nationalfonds zur Fu00f6rderung der Wissenschaftlichen Forschung
  • Project 2018-03 / Pro Pferd Foundation

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

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