ABSTRACT: Follicular fluid (FF) fills the interior portion of the ovarian antral follicle and provides a suitable microenvironment for the growth of the enclosed oocyte through molecular factors that originate from plasma and the secretions of follicular cells. FF contains extracellular nanovesicles (ffEVs), including 30-100-nm membrane-coated exosomes, which carry different types of RNA, proteins, and lipids and directly influence oocyte competence to develop embryo. In the present study, we aimed to characterize the protein cargo of EVs from the FF of 3-6-mm follicles and uncover the origins of ffEVs by assessing expression levels of corresponding mRNAs in bovine follicular cells and oocyte and cell proteomes. Isolated exosome-like ffEVs were 53.6 + 23.3 nm in size and could be internalized by cumulus-oocyte complex. Proteomes of ffEVs and granulosa cells (GC) were assessed using nanoflow liquid chromatography coupled with high-resolution tandem mass spectrometry after the gel fractionation of total proteins. In total, 460 protein isoforms corresponding to 322 unique proteins were identified in ffEVs; among them, 190 were also identified via GC. Gene Ontology terms related to the ribosome, protein and RNA folding, molecular transport, endocytosis, signal transduction, complement and coagulation cascades, apoptosis, and developmental biology pathways, including PI3K-Akt signaling, were significantly enriched features of ffEV proteins. FfEVs contain numerous ribosome and RNA-binding proteins, which may serve to compact different RNAs to regulate gene expression and RNA degradation, and might transfer ribosomal constituents to the oocyte. Majority of genes encoding ffEV proteins expressed at different levels in follicular cells and oocyte, corroborating with numerous proteins, which were reported in bovine oocyte and cumulus cells in other studies thus indicating possible origin of ffEV proteins. The limited abundance of several mRNAs within follicular cells indicated that corresponding ffEV proteins likely originated from circulating exosomes released by other tissues. Analysis of bovine ffEV transcriptome revealed that mRNAs present in ffEV accounted for only 18.3% of detected ffEV proteins. In conclusion, our study revealed numerous proteins within ffEVs, which originated from follicular and other cells. These proteins are likely involved in the maintenance of follicular homeostasis and may affect oocyte competence.
