Vitamin D3 and insulin treatment affects porcine follicular fluid-derived extracellular vesicles characteristics and proteome cargo

Abstract

The study aimed to investigate whether vitamin D₃ and insulin alone or in co-treatment can influence characteristics and protein cargo of the porcine follicular fluid (FF)-derived extracellular vesicles (EVs). To this end, medium antral follicles were cultured in vitro alone (C; control) or with 1α,25(OH)₂D₃ (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination (VD+I). The properties of the FF-EVs were assessed by transmission electron microscopy, nanoparticle tracking analysis, electroforetic light scattering, and flow cytometry, while the global proteomic analysis was conducted by liquid chromatography-tandem mass spectrometry coupled with the TMT-isobaric mass tag labeling. In all groups, particles represented a typical cup-shaped morphology, ranged between 50 and 450 nm in diameter, and expressed marker proteins, such as CD63 and CD81. The I treatment decreased the concentration and average size of the FF-EVs, while VD reversed only the effect on the particle concentration. Proteomic analysis revealed 48 differentially abundant proteins (DAPs) between examined groups, whilst greater amount of DAPs was identified following VD and I treatment alone than in co-treatment. Functional analysis showed that VD alone or in combination with I decreased predominantly the abundance of ribosomal proteins. In the I group, proteins involved in oxidative stress were down-regulated. We also found that the FF-EVs are carriers of adiponectin, which was up-regulated in the VD+I group. To sum up, VD and I seem to be novel modulators of the porcine FF-EVs characteristics and protein cargo, and thereby could modify ovarian follicle function via the EV-mediated pathway.