Proteome of bovine cumulus cells as related to oocyte in vitro maturation

The objective of the present study was to evaluate the proteome of bovine cumulus cells (CC) from cumulus-oocytes complexes (COCs) before and after 24 h of in vitro maturation. Ovaries were collected from a near abattoir and COCs were aspirated from follicles 3-8 mm of diameter. Grade I COCs were selected and two groups of 10 COCs were arranged (immature and mature). The CC from the immature group were mechanically removed and frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. The COCs from the matured group were put in TCM199 media supplemented with 10% fetal bovine serum, 0.2 mM Na pyruvate, 50 μg/ml gentamicin, 0.5 μg/ml FSH and 0.5 µg/ml LH under mineral oil and incubated in 5% CO2 and 90% humidity for 24 h. After maturation, CC were collected and processed as described above. Samples were solubilized in 100 µl 1% triton-X 100 and sonicated at 4°C for 15 minutes. For protein electrophoresis, 30 mg of each sample were subjected to 15 % SDS-PAGE and gels were stained with Coomassie Blue G-250, scanned and analyzed with Quantity One software (Bio Rad, USA). Proteins were identified by ESI-MS/MS and Mascot software v 10.0. Protein expression difference was set at 4.0-fold. In silico protein interactions were investigated using Cytoscape v 3.4.0. Major proteins expressed in all CCs were 78 kDa glucose-regulated protein, alpha-2-HS-glycoprotein, alpha-enolase, beta-actin, elongation factor 1-alpha 1, endoplasmin, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein HSP 90- beta, 6-phosphofructo-2-kinase, protein disulfide-isomerase A6, pyruvate kinase PKM, tubulin beta-2B chain and vimentin. Protein 78 kDa glucose-regulated protein (GRP-78) appeared more expressed in immature CC while Alpha-2-HS-glycoprotein (Fetuin-A) and vimentin (VIM) had greater expression in mature CC. As evidenced by in silico analysis, protein GRP-78, which plays a role in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum, interacts with several bone morphogenetic proteins (BMP), which are multifunctional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. Specifically within the interactions, BMP-6 seems to be a potential oocyte secreted factor, involved in the prevention of premature luteinization in cumulus cells via enhancing 17β-estradiol synthesis. Process needed during early stages of maturation, and probably regulated in its early protein assembly by GRP-78. Protein Fetuin seems to interact with bone morphogenetic protein receptor type-1 and 2, BMP and activin membrane-bound inhibitor homolog, a negatively regulator of TGF beta signaling, hemojuvelin, a BMP co-receptor, and bone morphogenetic protein 2, involved in activation of map kinase activity. Such interactions suggest the existence of a well orchestrate process involving cumulus cells proteins during early oocyte and embryo development. In addition, VIM, a class-III intermediate filament, has been reported with greater expression during in vitro development of bovine embryo and it is crucial for nuclear reprogramming in cloned embryos. VIM showed several interactions with proteins involved in structural oocyte and embryo formation. Identification of proteins expressed in cumulus cells will allow the search for molecular markers of oocyte quality. Moreover, this study sets the foundation for finding and understanding the proteins in cumulus cell that modulate oocyte competence and that are potentially related to early embryo development.