Integrative analysis of proteomic and phosphoproteomic of granulosa cells during follicular atresia in porcine

Ovarian follicular atresia is a natural physiological process, but its mechanism is not fully understood. In this study, a quantitative proteomic and phosphoproteomic of granulosa cell (GC) in the healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides and LC-MS/MS analysis. Altogether, 6,201 proteins were quantified, and 4,723 phosphorylation sites of 1,760 proteins were quantified. There are 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with FC>5 in H/SA and H/A, respectively. In addition, there are 20 (H/SA, up) and 39 (H/A, up) phosphosites with FC>7, which could serve as potential biomarkers to distinguish different quality categories of follicles. The results of western blotting and immunofluorescence indicated the reliability of the proteomic analysis. Further analysis of the differential expressed proteins (DEPs) and phosphorylated proteins (DEPPs) revealed some key proteins (e.g. MIF, beta catenin, integrin β2), key phosphosites (e.g. S76 of Caspase6, S22 and S636 of Lamin A/C), pathways (e.g. apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g. STAT5A, FOXO1, BCLAF1), and kinases (e.g. PBK, CDK5, CDK12, AKT3) that involved in atresia process. Our proteomic and phosphoproteomic profiling and functional research comprehensively analyze the dynamic changes of protein expression and phosphorylation during follicular atresia, and gave a new explanation for the regulation of this process.