Proteome Analysis of Porcine Follicular Fluid During Folliculogenesis.

Mammalian oocytes progressively acquire their full developmental competency or quality within the growing follicle. It is well-known that poor quality oocytes are generally derived from small compared to large follicles. Additionally, oocytes matured in the presence of follicular fluid of different follicle sizes display distinct developmental competency. Follicular fluid (FF) contains various molecules whose composition is influenced by external factors having beneficial or harmful effects on oocyte quality. Full characterization and evaluation of FF predisposition to such factors would make FF optimal sources for detecting suitable predictors of oocyte quality. In this study, we compared the proteome profiles of FF at different follicular developmental stages. Follicles were dissected from healthy sow ovaries, measured and classified as small (SFF;<3mm), medium (MFF;3-6mm), and large (LFF;6-12mm). Follicular fluid was aspirated from individual follicles, centrifuged and collected for protein quantification and estradiol assay. All procedures were performed at 4°C and repeated four times. Samples with highest estradiol content were selected for protein digestion (trypsin) and LC-Nano-ESI-MS/MS analyses. Database searches were performed using the SEQUEST algorithm in Bioworks 3.3, and peptides detected at p≤0.05 were matched to Sus scrofa NCBI RefSeq or Ensembl databases for protein identification. Results were filtered using a decoy and proteins detected with p≤0.05 were selected for functional annotation using the Agbase algorithm. In each FF group, approximately two thousands proteins were detected, of which 23 to 26% (558-570) of total proteins were shared between groups. Quantitative and qualitative protein variations were observed, and gene ontology (GO) analyses indicated protein associations with various cellular component, molecular function and biological process. Higher GO annotation numbers were found in LFF compared to other groups, but were significantly lower in LFF when considering the highest abundant proteins. Regardless of groups, FF comprised of various proteins involved in lipid transportation, storage and metabolism. Large amounts of lipid binding proteins were detected in MFF and LFF (57% and 44%) compared to SFF (33%), while proteins binding retinoids and glycoproteins were specific to SFF and LFF, respectively. In summary, we show that FF contains various proteins that are putatively active during folliculogenesis and oogenesis. Perturbations of these proteins may predispose oocytes to poor developmental competency. This work was supported by USDA-ARS Biophotonics Initiative #58-6402-3-0120. (poster)