Protein profile of mouse ovarian follicles grown in vitro

Abstract

STUDY QUESTIONCould the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage?SUMMARY ANSWERFrom a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages.WHAT IS KNOWN ALREADYThe importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells.STUDY DESIGN, SIZE, DURATIONThe experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics.PARTICIPANTS/MATERIALS, SETTING, METHODSSFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 μg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 μg and 170 μg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the ‘Compare gene list’ tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro.MAIN RESULTS AND THE ROLE OF CHANCEIt was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development.LARGE SCALE DATAData are available...

Combined use of Diane-35 and metformin improves the ovulation in the PCOS rat model possibly via regulating glycolysis pathway

“…There is growing evidence showing that granulosa cell energy metabolism disorder is an important cause of abnormal follicles and fertility decline in females. The main energy metabolism of normal folliculogenesis is that granulosa cells take up glucose from surrounding tissues via the glucose transporter (GLUT) on their cell membranes, then glucose producing pyruvic acid and lactic acid through the glycolysis pathway [6,7]. A large number of studies has shown that pyruvic acid and lactic acid are important factors in maintaining follicular growth and development [8].…”

Analysis of the equine “cumulome” reveals major metabolic aberrations after maturation in vitro

“…IPP is condensed with dimethylallyl diphosphate to form geranyl diphosphate (geranyl-PP), a metabolite attributed to a metabolomic compound that was underrepresented in vitro [84]. MVD shows the highest expression in the liver for cholesterol production [84], but its expression was also documented in mouse ovarian follicles in the antral stage [85]. Cumulus cells provide cholesterol to their oocytes, which is transferred through gap junctions.…”

Dynamic genome-scale cell-specific metabolic models reveal novel inter-cellular and intra-cellular metabolic communications during ovarian follicle development

“…Established non-target metabolomic techniques typically require millions of cells [49], and even emerging techniques require on the order of thousands of cells [50], which is prohibited for oocyte isolation. Proteomics approaches that account for post-translational modifications would be desirable, however, non-proteomics data is only available at later ovarian folliculogenesis stages for entire follicles grown in vitro [51]. In fact, the combination of proteomics with the community network approach reported herein would have the potential to reveal the most important systems and key metabolites and enzymes, as it has been done before in human tissues [52].…”