Protein Cargo of Extracellular Vesicles From Bovine Follicular Fluid and Analysis of Their Origin From Different Ovarian Cells

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.

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

“…Nordin et al, [ 38 ] have shown that UC can induce some EV disruption after UC for 70 min at 120,000 g and recommended that the recovery of intact EVs by UC should be done at a maximum speed of 100,000× g . In our laboratory, we have used UC protocols based on 100,000× g for this study, and also in previous studies to isolate oviductal and follicular EVs from different species with good results based on TEM observations [ 15 , 16 , 17 , 51 , 52 , 53 ]. However, considering the possibility that UC or CE processes could affect the EVs morphology, we used trehalose during the CE wash and dissolved the pellet after UC in PBS/25 mM trehalose.…”

Protein Palmitoylation in Bovine Ovarian Follicle

“…We then compared the lists of the proteins identified from bovine follicular fluid exosome-like EVs (294 IDs) [ 45 ], cumulus cells (1381 IDs) [ 52 ], granulosa cells (418 IDs) [ 45 ], and bovine oocytes (1936 protein IDs) [ 53 ] with the list of follicular palmitoyl-proteins ( Figure 2 a). 32 IDs, 48 IDs and 43 IDs of follicular palmitoy-proteins were shared with GC, CC and oocyte proteomes, respectively.…”

“…To investigate expression of palmitoylation and depalmitoylation enzymes in bovine follicular cells and oocytes, we have compared the list of 41 known enzymes with the lists of proteins identified from bovine oocytes [ 53 ], cumulus cells [ 52 , 56 ], mural GC, and extracellular exosome-like EVs from follicular fluid [ 45 ]. Indeed, LYPLA1 and PPT2 were found among 593 proteins identified in bovine GC [ 45 ]; PPT1, PPT2 and LYPLA2 were found among 2018 proteins identified in bovine oocytes [ 53 ], and PPT1, LYPLA1 and LYPLA2 proteins were reported in the list of 1703 identified proteins from CC surrounding immature oocytes [ 52 ]. PPT1 and LYPLA2 were also identified from CC surrounding in vivo and in vitro mature oocytes [ 56 ].…”

“…However, only one palmitoyltransferase, ZDHHC13, was identified from bovine mature oocytes [ 53 ], and from CC of in vivo mature COCs [ 56 ]. Neither palmitoylation nor depalmitoylation enzymes were detected in the list of proteins identified in follicular fluid exosome-like EVs [ 45 ].…”

“…In bovine ovary, size and concentration of follicular fluid EVs (ffEVs) significantly varied between the follicles at different growth stages [ 42 , 43 ]. Small exosome-like ffEVs, which are released to follicular fluid by follicular cells and the oocyte, transport molecular cargo of RNAs, proteins and lipids [ 42 , 44 , 45 ], and are involved in molecular exchanges between an oocyte and surrounding cumulus cells (CC), and granulosa cells (GC) [ 46 ]. At the ovarian level, small ffEVs mediate molecular signaling in the follicle and affect oocyte competence to develop embryo [ 47 ].…”