The Effect Of Vitamin C On Cell Cycle And Apoptosis By DNA Methylation On Bovine Granulosa Cells

In the mammalian ovary, orderly cell cycle can protect cells from apoptosis. Granulosa cell is an important kind of somatic cell in the mammalian ovary, it play roles of support, regulation and nutrition on oocyte development by gap junction or paracrine etc. Vitamin C(VC) also called L-ascorbic acid, as an important vitamin, it also can prevent cell aging and apoptosis. The latest study found that VC not only can effectively remove Reactive Oxygen Species(ROS) and its products, but also can regulate intracelluar epigenetic modifications by participating in DNA demethylation and histone modification processes. The study showed that VC can inhibit bovine granulosa cell apoptosis, but the mechanism except remove intracellular ROS, whether affect related genes DNA methylation and cell cycle is not clear.This experiment was first isolated granulosa cells and cultured by ITS+ culture system in vitro, cleared VC and Hydroxyurea(HU) optimal concentration, effect of VC on cell cycle and effect of VC and HU on intracellular ROS level detected by FCM. Results showed that, with increasing concentrations of VC, granulosa cell apoptotic rate decreased, in the range of acceptable, 100 μM VC can effectively inhibit apoptosis; 1 m M HU could significantly inhibit cell cycle. In addition HU while adding VC can effectively alleviate granulosa cell cycle inhibition caused by HU, but the role of VC protect cell cycle progression and inhibit apoptosis did not cause by reducing intracellular ROS level.We examined the role of VC on cell cycle and apoptosis related genes m RNA expression levels by q RT-PCR, then tested the effect of VC on key genes DNA methylation levels by Bisulfite Sequencing PCR(BSP), therefore investigated the mechanism of VC on granulosa cell cycle and apoptosis. Results showed that VC play a role in promoting cell cycle by regulating the expression of CDK4、CDK6、CCNA、CCNB、P21 and RRM1. VC could inhibit apoptosis by regulating the expression of BCL2, BAX and P53. In this process, VC affected the DNA methylation levels of CCNB and CDK4. This study provided a new idea for further understanding the biological function of VC and how to use VC effectively and provided a theoretical basis for further understanding mechanism of granulosa cell apoptosis.