The Role of Core Binding Factors (CBFs) in the periovulatory process

A. SPECIFIC AIMS The preovulatory gonadotropin surge initiates the final process of follicular maturation that culminates in ovulation of an expanded cumulus-oocyte complex (COC) and formation of the corpus luteum (CL). This periovulatory process is requisite for female fertility. One essential change induced by the gonadotropin surge required for final follicular maturation is the expression of specific transcription factors. However, our knowledge of the identity and regulatory actions of LH-iriduced key transcription factors remains very limited. Recent studies (1-3) by our laboratory and others shed light on a small family of nuclear transcription factors, Core Binding Factor (CBF), as a key transcriptional regulator involved in the periovulatory process. CBF functions as a heterodimer comprised of the DNA binding RUNX protein(s) and non-DNA CBF binding partner, CBFI3 (4-6). Our preliminary data showed the rapid induction of CBF - components (RUNX1, RUNX2, and CBFf3) by the LH surge in periovulatory follicles. Using an in vitro model we further demonstrated that CBF5 (RUNX1/CBFI3 and RUNX2/CBFj3) regulate RtMJX the expression of periovulatory genes that are known to be critical for COC expansion, ovulation, and luteinization. Moreover, our pilot study revealed that inhibition of RUNX activity ft blocked COC expansion in vitro. Based on these novel findings, we hypothesized that CBFs are key transcriptional regulators necessary for successful COC expansion, ovulation, and luteinization. In this RO3 pilot proposal, we will establish a novel transgenic mouse model in which the physiological significance of CBF expression in the ovary can be examined for the first time and begin to define transcriptional regulatory pathways essential for the final stage of periovulatory follicle development. The long term goal of this project is to identify and define the actions of LH-induced/activated transcriptional regulatory machinery that controls the periovulatory process. Specific Aim #1: To demonstrate the functional significance of CBFs in the ovary Hypothesis: Targeted inactivation of CBFs results in defective COC expansion, ovulation, and luteinization. Purpose: CBFs are highly induced by the LH surge in periovulatory follicles in rodents and humans (1-3). Studies using anin vitro model demonstrated that CBFs are important for up-regulation of key periovulatory genes and COC expansion (12:7-9). But, the challenge in demonstrating the physiological importance of CBFs in vivo is the overlapping expression of Runxl and Runx2 and their functional redundancy in periovulatory follicles. To circumvent this problem, we propose to establish ovarian cell specific CBF$ knockout mouse model using Cre!lox technology. Since CBFj3 is a binding partner for both RUNXI and RUNX2, targeted deletion of CBFf3 abrogates the activity of all CBF5 (RUNX1/CBFI3 and RUNX2/CBFI3) in the ovary. This aim will be accomplished by fertility assessment of tránsgenic mice lacking functional CBFs in the ovary and thorough characterization of their periovulatory ovarian phenotype with particular emphasis on COC expansion, ovulation and CL formation. Specific Aim #2: To identify downstream targets of CBFs and determine the transcriptional regulatory actions of CBFs in periovulatory follicular cells Hypothesis: CBFs regulate the expression of key periovulatory genes by acting as direct transcriptional regulators. Purpose: CBFs function as transcriptional regulators. Although our in vitro studies have identified a few target genes for CBFs (1 ;2;7-9), these data needs to be verified in vivo. More importantly, comparison of the gene expression profile between mice deficient in functional CBF5 and genotype-matched control mice will identify transcriptional regulatory pathways essential for final maturation of periovulatory follicles. This approach is invaluable for identifying direct transcriptional targets of CBFs, thus determining the transcriptional regulatory actions necessary for successful ovulation of an expanded COC and formation of the CL.