Regulatory Mechanism of Gene Expression by Estrogen in Chicken Granulosa Cells of Pre-Hierarchical Follicles.

The difference in chicken egg production is closely related to the efficiency of follicle selection, which is marked by granulosa cell differentiation and progesterone production with cholesterol as the substrate. The conversion of 7-dehydrocholesterol to cholesterol catalyzed by 7-Dehydrocholesterol reductase (DHCR7) is the rate-limiting step in cholesterol synthesis. Our previous study revealed that estrogen enhanced the mRNA expression of three transcript variants (T1, T3, and T4) in a dose-dependent manner in the granulosa cells of chicken pre-hierarchical follicles (Pre-GCs). This study investigates the molecular mechanisms through which estrogen regulates in chicken Pre-GCs. At the transcriptional level, through CUT&RUN-qPCR, we found that under basal conditions, sterol-regulatory element binding protein 2 (SREBP2) bound to the promoters of three transcript variants to promote cholesterol synthesis in Pre-GCs to maintain low cholesterol levels; meanwhile upon estrogen treatment, estrogen receptors α and β bound to the regulatory regions of three chicken transcript variants, leading to a reduction in the interaction between SREBP2 and . At the translational level, the upstream open reading frames (uORFs) and N6-methyladenosine (mA) modification in the 5'UTR of different transcripts differentially regulate the expression of T3 and T4, as detected by dual-luciferase reporter assays, but this regulation is not affected by estrogen. This study systematically explores the molecular mechanisms through which estrogen upregulates expression in chicken Pre-GCs and provides a clue for understanding the molecular mechanisms underlying cholesterol synthesis in chicken ovarian follicles.