| The ovary is a primary reproductive organ, often found in pairs as part of the reproductive system of vertebrate female. Their main functions are the release of fertilizable oocytes and the establishment of a successful propagation of the species. A number of complex transcriptional networks and interactions are involved in the ovarian development. However, tightly controlled ovarian development can be easily disrupted by inner or outside influences which may lead to ovarian dysfunction. Premature ovarian failure (POF) is the loss of ovarian function in women under the age of 40 and it is one of the most important causes of infertility.p53 (Trp53, transformation-related protein 53) was firmly established to be a tumor suppressor, known as the "guardian of the genome". The activity of p53 is tightly regulated by MDM2. This regulation plays an important role both in normal development and tumor suppression. Previous studies in female germ cells have focused on the other member of the p53 family-p63. However, it is difficult to determine whether MDM2-p53 pathway is still conserved and tightly regulated in the female germ cells. Moreover, it is even uncertain whether the p53 pathway can be activated under stresses and whether p53 participates in chemotherapeutic agents induced follicular loss.To address these questions, firstly, we specifically deleted Mdm2 in oocytes at the different stages of folliculogenesis in mice with the Cre-loxP system. Our results showed that absence of Mdm2 in oocytes of primordial follicles (Mdm2Gdf9 cKO) resulted in POF characterized by impeded fertility, reduced ovarian size, exhaustion of the primordial follicle pool and elevated follicle stimulating hormone (FSH) secretion. Absence of Mdm2 in oocytes of primary follicles (Mdm2Zp3 cKO) also resulted in POF. Different from the Mdm2Gdf9 cKO mice, Mdm2Zp3 cKO mice showed intact primordial follicles, but increased degeneration of the growing oocyte and follicular atresia. The progression from primary follicle to secondary follicle was also inhibited. Importantly, all of the defects of follicular development were reversed by concurrent loss of Mdm2 and p53 in oocytes, indicating an essential protective role of MDM2 by inhibiting p53.Further investigation demonstrated that MDM2 was highly expressed and p53 protein was almost undetectable in normal oocytes. With Mdm2 deletion in oocytes in different stages, p53 accumulated in the nucleus, resulting in rapid apoptosis of oocytes in the primordial follicles and degeneration of oocytes in the growing follicles. In addition, we used Nutlin-3 to specificly inhibit the binding of MDM2 and p53 with the ovarian culture assay. Nutlin-3 also successfully induced nuclear accumulation of p53 and p53 dependent oocyte death.Previous studies indicated that under conditions of altered ribosome biogenesis, interaction between the ribosomal protein L5, L11, L23 and MDM2 could inhibit the ubiquitin ligase activity of MDM2, leading to the stabilization and activation of p53. However, in our study, we found that ribosomal stress-inducing agents actinomycin D (Act-D) and 5-fluorouracil (5-FU) did not significantly change the protein levels of p53 in the oocytes. On the contrary, ovarian somatic cells showed a strong p53 response. To further study the reason why the ribosomal stress failed to induce stabilization and activation of p53 in oocytes, we established a mouse model which had weaker inhibition of p53 in oocytes (Gdf9-cre+; Mdm2FM/+; Mdm4+/-).Ovarian development of Mdm2, Mdm4 double heterozygous mice was not affected and p53 protein levels in oocytes were also unchanged. But in the case of 5-FU treatment, p53 protein accumulated in the nucleus of the oocyte. These results indicated that under ribosomal stress, MDM2 and MDM4 played important roles in inhibiting p53 activation. There may be a more restrict and precise regulation mode between MDM2 and p53 in oocytes, which may be distinct from somatic cells.Doxorubicin (DXR) is a commonly used chemotherapy drugs. Previous studies showed that DXR treatment can lead to infertility in female patients due to the rapid follicular loss. In DXR treated mice, impeded fertility was mainly because of rapid granulosa cells apoptosis in secondary and antral follicles. Our study found that p53 protein was increased in the ovarian granulosa cells after a short time of DXR treatment in vivo. We used Hhep53-/-mice for DXR treatment. p53 deletion significantly protected the ovary against DXR induced cell apoptosis and preserved the fertility of the mice.In summary, our study revealed for the first time that the MDM2-p53 network was tightly regulated in oocytes. This regulation is indispensable for oocytes survival, folliculogenesis and fertility in mice. The regulation was insensitive to stress signaling, including ribosomal stress. In addition, p53 participated in the DXR induced ovarian damage. p53 deletion protected the ovary and the fertility of the DXR treated mice. This finding may help us to find new effective treatments to prevent the appearance of POF for the female patients who accept the DXR treatment. |
