H3K9me3 Involved in FOXL2 Regulation of SerpinE2 Expression to Affect Steroid Hormone Synthesis in Follicular Granulosa Cells

ABSTRACT Serine protease inhibitor E2 (SerpinE2), a serine protease inhibitor predominantly expressed in ovarian granulosa cells (GCs), plays a critical role in follicular development and ovulation in mammals. However, its exact role and underlying mechanisms remain unclear. In this study, we used buffalo GCs as a research model to investigate both the effects of SerpinE2 on GCs function and the mechanisms governing its expression. RT‐qPCR analysis revealed that SerpinE2 expression was significantly higher in healthy follicles (HFs) compared to atretic follicles (AFs). Through comprehensive functional assays (CCK‐8, EDU, flow cytometry and ELISA), we demonstrated that SerpinE2 promotes GCs proliferation, enhances steroidogenesis (progesterone and estradiol), and suppresses apoptosis. Mechanistically, dual‐luciferase reporter assays confirmed FOXL2 as a direct transcriptional activator of SerpinE2, with FOXL2 overexpression significantly enhancing both SerpinE2 expression and its steroidogenic effects. Additionally, Western blot analysis demonstrated that H3K9 methylation levels were significantly increased in AFs and FOXL2 knockdown GCs. CUT&RUN‐qPCR further demonstrated that H3K9me3 was highly enriched in the promoter regions of SerpinE2 in AFs and FOXL2 knockdown GCs, leading to reduced SerpinE2 expression. These findings suggest that SerpinE2 promotes steroid hormone synthesis in GCs in a FOXL2‐dependent manner; H3K9me3 is directly involved in FOXL2‐mediated regulation of SerpinE2 transcription in GCs. This study provides new insights into the molecular mechanisms by which SerpinE2 regulates mammalian follicular development.