(278) Exploring Novel Genes Associated in the Death of Primordial Follicle Oocytes Upon Cyclophosphamide Treatment

Amirhossein Abazarikia¹, Wonmi So¹, Shuo Xiao², So-Youn Kim^1,3

¹Olson Center for Women’s Health, Department of Obstetrics and Gynecology, College of Medicine, University of Nebraska Medical Center, Omaha, NE

²Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ

³Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE

Abstract Text:

TAp63α, a transcription factor related to the tumor suppressor p53, is highly expressed in oocytes of primordial follicles in an inactive form. Our previous studies have demonstrated that TAp63α serves as a central regulator in triggering the death of primordial follicle oocytes, leading to primary ovarian insufficiency (POI). Following DNA damage induced by chemotherapeutic agents, TAp63α accumulates and becomes activated through hyperphosphorylation, inducing oocyte apoptosis by upregulating pro-apoptotic genes such as Puma and Noxa. However, the mechanisms underlying oocyte death in primordial follicles remain poorly understood.

To elucidate the underlying mechanism of TAp63α-induced oocyte apoptosis, we conducted RNA sequencing (RNA-seq) analysis using oocytes from wild-type (WT) and oocyte-specific Trp63 conditional knockout (p63 cKO) mice following cyclophosphamide treatment. WT and cKO mice at postnatal day 3 (PD3) received a single injection of either solvent or 150 mg/kg cyclophosphamide. Ovaries were harvested 18 hours post-injection, and the total RNA of oocytes from primordial follicles was extracted for RNA-seq. Gene Ontology (GO) analysis was performed using DAVID v6.8, focused on transcripts with a Log2FoldChange >1, which were considered differentially expressed and included in further analyses.

Differentially expressed genes (DEGs) in the WT control group compared to the CPA-treated WT group revealed 1,457 upregulated and 860 downregulated genes. In contrast, the p63 cKO control group, compared to the CPA-treated p63 cKO group, exhibited 182 upregulated and 47 downregulated genes. To investigate key processes triggered by CPA and mediated by TAp63α, we selected the GO term ‘apoptotic pathway’. A significant enrichment of genes associated with apoptotic pathways (GO:0006915) was observed in both the CPA-treated WT and CPA-treated p63 cKO groups. However, significant differences were noted in the magnitude of enrichment. Comparative analysis revealed that 48 genes were uniquely associated with the CPA-treated WT group. In contrast, only two genes, Id1 and Tnfrsf10b, were enriched exclusively in the CPA-treated p63 cKO group. A heatmap of log2 fold changes illustrated the overlap of DEGs between the two groups, highlighting sixteen genes. These include key regulators of apoptosis, such as Mdm2, Pmaip1 (Noxa), and Bbc3 (Puma), which, unexpectedly, were upregulated in both the CPA-treated p63 cKO and CPA-treated WT groups. This suggests that Pmaip1 (Noxa), and Bbc3 (Puma), previously considered downstream targets of TAp63a, are not direct targets of TAp63α. Notably, extrinsic apoptotic signaling was activated exclusively in the WT group but not in the p<em>63 cKO group. Importantly, both Casp8 and Fas were upregulated in the WT group, indicating that the extrinsic apoptotic mechanism is related to oocyte death in primordial follicles in response to cyclophosphamide. Immunofluorescence staining revealed FAS expression was specifically upregulated in the oocytes of primordial follicles. These findings suggest that Fas may be a target gene involved in oocyte death in primordial follicles when oocytes are exposed to cyclophosphamide.

This study provides valuable insights into the molecular dynamics of TAp63α-mediated apoptosis, with potential implications for developing targeted therapeutic strategies to protect the ovarian reserve from the adverse effects of chemotherapy.