Date of Award
Fall 12-20-2024
Level of Access Assigned by Author
Open-Access Dissertation
Language
English
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical Sciences
First Committee Advisor
Ewelina Bolcun-Filas
Second Committee Member
Laura Reinholdt
Third Committee Member
James Coffman
Additional Committee Members
Lucy Liaw
Jennifer Trowbridge
Abstract
CHEK2 kinase and its target TAp63 are key mediators of the DNA damage response (DDR) of the immature oocyte found in primordial follicles (PFs). TRP53, a target of CHEK2 in all cell types, had been previously considered non-essential for oocyte elimination. To identify additional factors contributing to the oocyte's DDR, we conducted a transcriptional analysis of irradiated and non-irradiated ovaries from wild-type and Chek2-/- mice to identify factors involved in radiation- induced oocyte elimination. Gene Set Enrichment Analysis revealed activation of the p53 pathway, interferon response, apoptosis, and TNFα signaling via NFkB in irradiated ovaries. Transcriptomic analysis at the single-cell level identified CHEK2-dependent differentially expressed genes, including novel oocyte-specific candidates (Cbr2 and Ankrd65) and known DDR genes (p53). To determine if Cbr2, Ankrd65, and p53 contributed to the oocyte's DDR, we generated or obtained knockout mouse models to quantify PF survival in response to ionizing radiation (IR). Ankrd65 and CBR2 are regulated by p63; therefore, PF survival after IR cannot be quantified. At wean age, Cbr2-/- but not Ankrd65-/- ovaries have reduced PF numbers, suggesting a role for CBR2 in detoxifying oxidative stress. To determine if p53 contributes to PF elimination during IR treatment, TAp63A/ATrp53-/- ovaries were treated with a high dose of IR, at which loss of TAp63 activity does not prevent oocyte death. TAp63A/ATrp53-/- ovaries had improved PF survival compared to controls, confirming that TRP53 is required for PF elimination at higher loads of DNA damage. Using ovaries lacking p53 exclusively in the oocytes, we showed that p53 expression in the oocyte is responsible for PF elimination. Analysis of p53 activation in the ovary after high-dose radiation revealed a larger form of p53 expressed in irradiated oocytes but not in somatic cells. This suggests an oocyte-specific mechanism that regulates p53 pro-apoptotic activity in response to IR-induced damage. Follow-up studies will define mechanisms regulating TAp63 and TRP53 activity and downstream targets (CBR2 and ANKRD65) of the ovarian response to DNA damage. Thus, improving our understanding of how genotoxic treatments lead to PF loss.
Recommended Citation
Mills, Monique, "The Role of TAP63 and P53 in the Primordial Oocyte's Response to DNA Damage" (2024). Electronic Theses and Dissertations. 4108.
https://digitalcommons.library.umaine.edu/etd/4108