CCAAT/enhancer-binding proteins alpha and beta directly regulate ovulation and selectively regulate gene expression in granulosa cells by preovulatory stage- and dose-dependent mechanisms

The preovulatory luteinizing hormone surge orchestrates complex cellular and molecular events leading to ovulation. CCAAT/enhancer-binding proteins alpha and beta (C/EBPα/β) are transcription factors acutely induced by the luteinizing hormone surge and crucial for ovulation and granulosa cell luteinization. However, biological processes and their regulatory mechanisms downstream of C/EBPα/β in the preovulatory ovary are not completely understood. To address this knowledge gap, we generated Cebpa/bfl/fl;Pgr-Cre mutants and compared them with Cebpa/bfl/fl;Cyp19a1-Cre mutant female mice. The Cebpa/bfl/fl;Cyp19a1-Cre mutants have undetectable levels of C/EBPα/β throughout preovulatory stages and no ovulation, aligning with previous reports, while Cebpa/bfl/fl;Pgr-Cre mutants presented gradual deletion of C/EBPα/β during late preovulatory stage and reduced ovulation rate. Comparison of these two models indicates that sustained expression of C/EBPα/β throughout preovulatory stages is necessary for successful ovulation. This provides a unique opportunity to interrogate gene regulatory mechanisms by C/EBPα/β during different preovulatory time windows and the impact of dysregulating C/EBPα/β on ovulation-associated biological processes. Our study revealed that C/EBPα/β regulate gene expression and distinct biological functions via preovulatory stage-, dose-, and/or potentially isoform ratio- dependent mechanisms. These findings shed new light on the intricate gene regulation mechanisms by C/EBPα/β downstream of the LH surge. To understand biological processes and their regulatory mechanisms downstream of C/EBPα/β in the preovulatory ovary, we we conducted Bulk RNA sequencing analysis on granulosa cells isolated from WT, Cebp-C, and Cebp-P mutants at 8 h and 12 h post-hCG.