Extracellular vesicles in follicular fluid of sexually mature gilts' ovarian antral follicles - identification and proteomic analysis.

Ovarian follicular growth and development involves extensive bidirectional cell-to-cell communication between somatic cells and the oocyte. Recently it has been found that extracellular vesicles (EVs) represent a new mechanism of the communication inside the ovarian follicle. The present research shows for the first time the presence of EVs in follicular fluid of small (SFs), medium (MFs) and large (LFs) antral follicles of sexually mature gilts using nanoparticle tracking analysis and Western blot. The highest (P = 0.0338) concentration of EVs was found in follicular fluid of MFs, as compared to LFs and SFs. Furthermore, nanoparticle tracking analysis showed the existence of the population of particles in follicular fluid of all analyzed follicles, which resembles exosomes. That was confirmed by the abundance of exosomal markers, CD9 and CD63, in those samples. The proteomic analysis of EVs from MFs using the nano-liquid chromatography-matrix-assisted laser deposition/ionization time-of-flight mass spectrometry allows to identify 249 proteins that predominantly indicated binding function and catalytic activity. Most of them belong also to the group of cytoskeleton and extracellular matrix proteins (ECM) suggesting their role in the building of cell components. Functional annotation predicted association of identified proteins with processes crucial for follicle development and function, as well as ovulation and corpus luteum formation. Therefore, EVs through their protein cargo might regulate follicle development in sexually mature gilts.