Nrf2 regulates female germ cell meiosis initiation

Abstract

Meiosis initiation of the female germ cell is critical for primordial follicle formation and ovarian reserve. Nuclear factor E2-related factor 2 (Nrf2) is a vital transcriptional factor for antioxidation. However, little is known about the role of Nrf2 in female fertility. Our aim of this study was to investigate the effect of Nrf2 on meiosis initiation and early oogenesis, hoping to find a new way to improve ovarian reserve. CD-1 mice were maintained in a temperature- and light-controlled facility with free access to water and food. Mating was timed overnight and the appearance of the vaginal plug was considered as E0.5 in the next morning. Female genital ridges (GRs) were collected from E11.5 to E14.5 fetal mice. GRs were either used directly to analyzed Nrf2 expression or incubated with inhibitors for 48 h to test the effect of Nrf2 on meiosis initiation and cell cycle. After additional culture with normal medium, the effect of Nrf2 on oocyte progression and primordial follicle formation was tested. Then, p53 inhibitor was used to analyzed the effect of p53-p21 pathway in this process by retesting the above indexes. Brusatol was used as Nrf2 inhibitor, while PFTa as p53 inhibitor. mRNA and protein levels of genes were tested by real-time PCR and western blot, respectively. Meiotic prophase I stages of oocytes were evaluated by oocyte cytospread. The expression pattern of Scp3 was used as a marker of different oocyte progression stages. Serial paraffin section and HE staining was used for the analysis of primordial follicle formation. Consistent with the previous study1, Brusatol was also confirmed as an effective inhibitor of Nrf2 in GR, as protein level of Nrf2 and mRNA levels of the downstream factors were all inhibited by Brusatol. Nrf2 was dynamically expressed in E11.5-14.5 GR, and reached peak in E12.5, suggesting Nrf2 might be involved in meiosis initiation. Inhibiting the expression of Nrf2 with Brusatol downregulated mRNA levels of meiosis genes (Stra8, Scp3, Dmc1, Dazl, Rec8), changed the expression of cell cycle-related genes (p53, p21, cyclin E), delayed oocyte progression in lepetotene, and decreased the number of primordial follicles. Further studies showed the effect of Brusatol on meiosis initiation and follicle formation was significantly rescued by the addition of p53 inhibitor. Nrf2 regulated meiosis initiation and early oogenesis in female germ cell via p53-p21 pathway.