The Methylation of the CYP11A1 Gene Affects the Expression Pattern in Different-Diameter Granulosa Cells of Qira Black Sheep

Background: CYP11A1 (a key enzyme in the process of progesterone synthesis) expression is a crucial factor that promotes the proliferation and progressive differentiation of granulosa cells (GCs) to support oocyte maturation and ovulation in livestock. Changes in its expression may be related to DNA methylation, but the specific regulatory mechanism of this remains unclear. Methods: The qRT-PCR, western-blot, CCK-8 assay, RNAi, dual-luciferase activity assay and sulfite sequening were used to explore the effect of the activity and methylation level of the CYP11A promoter on the proliferation of GCs. Results: Our studies first identified that the activity of CYP11A1 promoted the core genes that affected the superior growth of follicles in Qira Black sheep, and activity changes were related to the degree of DNA methylation. We further revealed that the DNA methylation of GCs at different developmental stages was mainly regulated by the promoter region, and also clearly defined the optimal window period for CYP11A1 to affect the proliferation of GCs. Conclusion: In summary, our data reveals the epigenetic regulatory mechanism of GCs in follicle development provides a new perspective for understanding the molecular basis of reproductive cell differentiation, and offers important theoretical support for future research and basic application in the field of livestock reproduction. Discussion: This not only helps to improve the efficient breeding and production system of sheep and the process of genetic improvement, but also provides a theoretical basis for fundamentally accelerating the expansion, breeding, and genetic quality innovation of superior sheep breeds in China, and also offers accurate targets for sheep genetic breeding.