Characterization of large and small extracellular vesicles from follicular fluid in cow

BACKGROUND Follicular fluid extracellular vesicles (ffEVs) participate in cell communications inside the follicle by exchange of different RNAs, proteins, and lipids between different follicular cells and enclosed oocyte, and thus affect cellular functions and signaling in target cells. We aimed to compare ffEVs of different size by comparative analysis of their morphology and protein cargo.METHODS Follicular fluid (FF) was aspirated from antral follicles of ovaries from slaughtered cows. Fractions of microvesicles (MV) were separated by centrifugation of cleared FF 30min at 12,000g. Fractions of small ffEV (exosomes, Exo) were obtained by ultracentrifugation of MV-depleted FF 90 min at 100,000g. Transmission electron microscopy (TEM) was performed on intact ffEVs and 1µM sections. Presence of EV markers was analyzed by Western blot. Peptide/protein profiles of ffEVs samples (n=24) were acquired by MALDI-TOF mass spectrometer (MS) RapifleX Tissuetyper (Bruker Daltonics) in positive linear ion mode in the 2,000 – 30,000 m/z range, in 9 technical replicates. Spectral processing and statistical analyses were performed using home R software based on MALDIquant & MALDIquantForeign packages (v1.19.3 & v0.12). Peaks annotation were obtained from Top Down proteomics database.RESULTS CD81 and HSPA8 markers were detected in both MV and Exo fractions. By TEM, the mean diameter of MV and Exo was 170±72 nm and 60±22 nm, respectively. The ffEVs contain inclusions of different electron density. MV fractions are more heterogeneous than Exo and contain the debris of different organelles like mitochondria, endoplasmic reticulum and lipid droplets. Exo fractions contain protein agglomerates and ribosomes. By MS, 366 m/z peaks were detected from ffEVs mean spectra. Differential analysis of normalized peak height values between MV and Exo revealed overabundance of 46 m/z in MV and 31m/z in Exo (p2). Their annotation revealed both small proteins and proteolytic fragments.CONCLUSIONS Morphology, vesicular cargo, and peptide-protein fingerprints significantly differed between small ffEVs and MVs that revealed their different origin and potential roles in functional activity inside the follicle.Funds: INRAE France; Russian Science Foundation (project 19-16-00115)