Regulation and Function of the Rhox8 homeobox gene in granulosa cells

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DESCRIPTION (provided by applicant): Ovulation is a complex and intriguing biological process that is essential for mammalian reproduction. The actions of granulosa cells in the ovary are central to successful ovulation. Initially the follicle consists of a single layer of granulosa cells surrounding each primordial oocyte. Upon stimulation by gonadotropins, granulosa cells rapidly divide and the follicle grows until proliferation is terminated by a surge in luteinizing hormone (LH) which initiates a developmental switch to luteal cells and initiates the signaling cascade that ultimately leads to ovulation. An essential mediator of this process is the steroid hormone progesterone. Knockout studies have identified several progesterone-regulated genes that contribute are necessary for folliculogenesis and ovulation. However, virtually none of these genes are transcribed due to direct progesterone-receptor interaction with their promoters. The Rhox homeodomain protein family has several members that are excellent candidates to regulate events in granulosa cells. While expression of the Rhox genes is restricted to reproductive tissues, ovary, testis, epididymis, and placenta, very few have been examined for function. We have recently discovered that Rhox5-null male mice exhibit lower epididymal sperm numbers, have sperm possessing motility defects, and are subfertile. However, Rhox5- null female mice have no apparent complications in ovulation. Like Rhox5, Rhox8 is highly expressed in a developmentally regulated manner in granulosa cells in a similar "window" of expression. Thus, RHOX8's continued production in Rhox5-null animals may explain why they do not exhibit more severe constraints on their fertility. Furthermore, Rhox8 is unique among the Rhox genes in that it remains expressed through the periovulatory window, suggesting it may be uniquely situated to regulate luteal cell differentiation and the final steps of ovulation. In this application, I propose to determine the regulation and function of Rhox8 in granulosa cells. I propose to accomplish this task by verifying the importance of a putative progesterone response element within Rhox8's promoter as well as investigating the other cis-acting factors that contribute to Rhox8's unique window of expression during folliculogenesis. The timing of Rhox8 induction in the follicle suggests that it may regulate granulosa cell proliferation, survival, and differentiation (functions attribute to progesterone signaling in general). Thus, we will begin to characterize RHOX8's role in these processes using cultured granulosa cells. The elucidation of Rhox8's function in granulosa cells is important because it will provide an important "building block" towards our long-term goal of learning the independent and collaborative functions of all the Rhox genes in the gonads. PUBLIC HEALTH RELEVANCE: Mammalian reproduction requires successful ovulation. While knockout studies have provided definitive proof that progesterone signaling is required for ovulation, the critical downstream effectors of progesterone signaling which have been identified are not directly regulated by progesterone. Currently, there is a gap in our fundamental knowledge concerning transcription factors which translate the progesterone receptor activation signal to the expression of target genes that modulate ovulation. Preliminary studies suggest that Rhox8 is regulated by the coordinated efforts of progesterone receptor and homeobox factors. We believe characterization of the involvement of these cis-acting factors on the Rhox8 promoter and the consequences of Rhox8 disruption in granulosa cells will begin to eliminate some of the "black box" within the progesterone signaling pathway.