7895 The accumulation of p63 in oocyte of primordial follicle is essential for triggering apoptosis following cisplatin treatment

Abstract Disclosure: A. Abazarikia: None. W. So: None. S. Kim: None. Background: Women are born with a finite supply of primary oocytes housed in the ovaries, commonly known as the ovarian reserve, which represents their reproductive lifespan. The ovary serves as a crucial organ for production of oocytes and endocrine hormones, including 17β-estradiol and progesterone. However, chemotherapy inevitably leads to the depletion of the ovarian reserve, resulting in primary ovarian insufficiency (POI), which manifests as endocrine dysfunction and infertility. Within the oocytes of primordial follicles, the p63 protein is highly expressed as an inactive, dimeric form, playing a pivotal role in responding to DNA damage, thereby determining the fate of the oocytes. It is well-established that p63 undergoes activation through hyperphosphorylation and a conformational shift to a tetrameric structure. This activation subsequently triggers apoptosis by inducing the expression of proapoptotic genes. Hypothesis: Different chemotherapeutic agents may exhibit distinct mechanisms in the theregulation of p63 in oocytes. Methods: Postnatal day 5 CD-1 mice were utilized in the experimental groups, comprising control (Ctr), CDDP (cisplatin), CHX (cycloheximide), and PYR-41 (a Ubiquitin-Activating Enzyme E1 Inhibitor). We assessed the number of ovarian primordial follicles, the expression of p63, DNA double-strand breaks (DSBs), and apoptosis through immunoblotting and immunofluorescence assays. Additionally, p63 conformation was examined using Native PAGE Gel. Statistical differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test, with significance defined as a p-value less than 0.05. Results: Treatment with CDDP significantly increased p63 levels in oocytes (p<0.05). The combined administration of PYR-41 with CDDP induced a further substantial increase in p63 levels (p<0.001) compared to CDDP treatment alone. Conversely, p63 expression was significantly decreased (p<0.01) with CHX treatment, falling below the levels observed in the CDDP group. Notably, CHX appeared to protect ovarian follicles from the adverse effects of CDDP, as evidenced by the decreased levels of ɣH2AX, cleaved PARP, and PUMA, which is a known target gene of p63 in oocytes. Conclusion: Therefore, our findings strongly indicate that the accumulation of p63 represents the initial step in oocytes preceding CDDP-induced oocyte death, which is subsequently followed by hyperphosphorylation, tetramerization, and degradation of p63. Significance: Comprehending the mechanism behind cisplatin-induced oocyte death will contribute to the development of effective adjuvant therapies aimed at preserving ovarian reserve in young female cancer survivors undergoing CDDP treatment. Presentation: 6/2/2024