Protein profile of extracellular vesicles isolated from porcine reproductive fluids

Bc. Polina Tolkachova

Diplomová práce

Abstract:

Extracellular vesicles (EVs) are a promising area of study due to their important role in intercellular communication. These small, membrane-bound particles are present in a wide range of organisms and can be isolated from various body fluids, including semen. In this study, we used ultracentrifugation and filtration techniques to isolate the EVs fraction from boar seminal plasma. To confirm the presence of vesicles, the isolated fraction was subjected to transmission electron microscopy. Using super-resolution microscopy, we were also able to see interactions between isolated seminal plasma EVs (sEVs) and plasma membrane (PM) of boar spermatozoon after co-incubation. With these findings, our next goal was to make a protein profiling of isolated sEVs. For this purpose, we extracted proteins from sEVs under basic and acidic conditions and subjected the samples to mass spectrometry analysis (MS). The results of MS for both conditions showed a high abundance of spermadhesin family proteins. However, the presence of tetraspanins and integrins was not confirmed. Additionally, we employed SDS-PAGE and Western blot with four anti-tetraspanin antibodies, which are the typical markers for EVs detection, three anti-spermadhesin antibodies and an antibody against integrin alpha V. We detected strong signals for all spermadhesins and integrin alpha V for both types of protein extraction conditions. However, only a few tetraspanins showed a very weak signal under acidic conditions, while no signal was ever detected under basic conditions during this study. The potential applications of EVs in reproductive biology are significant, as they have been shown to play important roles in sperm maturation, fertilization, and embryonic development. Our study represents an initial exploration of several methods for the isolation of EVs’ fraction from boar seminal plasma and provides valuable insights into the mechanisms behind sEVs’ formation in this context. However, further research is needed to elucidate the specific functions of EVs in the boar reproductive system and optimize methods for EVs isolation and characterization.

Keywords

spermadhesins, integrin, ultracentrifugation, tetraspanins, exosomes

Jazyk práce: angličtina

Datum vytvoření / odevzdání či podání práce: 16. 4. 2023

Obhajoba závěrečné práce

• Obhajoba proběhla 16. 5. 2023
• Vedoucí: RNDr. Pavla Postlerová, Ph.D.
• Oponent: Katarína Michalková, externi

Česká zemědělská univerzita v Praze

Fakulta agrobiologie, potravinových a přírodních zdrojů

Magisterský studijní program / specializace:
Biotechnologie / Reprodukční biotechnologie