Ovarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GCs) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides, and liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. In total, 6,201 proteins were quantified, and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7 that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin β2), 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, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, and AKT3) involved in the atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Further study showed that mutant caspase6 Ser76 to Ala increased the ratios of cleaved caspase6/caspase6 and cleaved caspase3/caspase3 and dephosphorylation of caspase6 at Ser76 increased cell apoptotic rate, a new potential pathway of follicular atresia. Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

卵巢卵泡闭锁是一种自然的生理过程；然而，该机制尚不完全清楚。在本研究中，通过 TMT 标记、磷酸肽富集和串联液相色谱对猪健康 (H)、轻度闭锁 (SA) 和闭锁卵泡 (A) 中的颗粒细胞 (GC) 进行定量蛋白质组和磷酸蛋白质组分析。质谱 (LC-MS/MS) 分析。总共对 6,201 个蛋白质进行了定量，并对 1,760 个蛋白质的 4,723 个磷酸化位点进行了定量。总共，在 H/SA 和 H/A 中分别鉴定了 24 个（11 个向上，13 个向下）和 50 个（29 个向上，21 个向下）倍数变化 (FC) > 5 的蛋白质。此外，还有 20 个（H/SA，向上）和 39 个（H/A，向上）FC > 7 的磷酸位点可以作为区分不同质量类别的卵泡的潜在生物标志物。蛋白质印迹和免疫荧光证实了蛋白质组分析的可靠性。一些关键蛋白（例如，MIF、β连环蛋白、整合素β2）、磷酸位点（例如，caspase6的S76、核纤层蛋白A/C的S22和S636）、通路（例如，细胞凋亡、肌动蛋白细胞骨架通路的调节）、转录因子（例如， 、STAT5A、FOXO1 和 BCLAF1）以及参与闭锁过程的激酶（例如 PBK、CDK5、CDK12 和 AKT3）被揭示通过进一步分析差异表达蛋白（DEP）和磷酸化蛋白（DEPP）。进一步研究表明，Caspase6 Ser76 突变为 Ala 增加了 cleaved caspase6/caspase6 和 cleaved caspase3/caspase3 的比率，并且 caspase6 Ser76 去磷酸化增加了细胞凋亡率，这是滤泡闭锁的新潜在途径。 总的来说，本研究的蛋白质组和磷酸化蛋白质组谱及功能研究全面分析了卵泡闭锁过程中蛋白质表达和磷酸化的动态变化，并为这一过程的调控提供了一些新的解释。