The extremely low levels of GnRH expression in the ovary along with the paucity and scattered distribution of GnRH neurons have limited the study of the differential regulation of GnRH gene expression. In preliminary in vitro and in vivo studies, my laboratory has localized a mouse GnRH (mGnRH) DNA sequence (-2444 to -2078 bp) that mediates this differential gene expression. In hypothalamic GnRH neurons, this region functions as an enhancer and has been termed a Hypothalamic GnRH Enhancer Element (HGEE). In the ovarian granulosa cells, this same region functions as a repressor, or Ovarian GnRH Repressor Element (OGRE). Within this region is a well-defined Oct-1 binding site, which when mutated results in a loss of hypothalamic expression in a GnRH neuronal cell line (NLT). In contrast, this same mutation did not increase expression in an ovarian cell line (KK). Thus DNA sequences responsible for the function of the HGEE can be dissociated for those necessary for the OGRE. In addition, in vitro and in vivo studies have localized a proximal element in the mGnRH promoter (-356 to -249 bp) apparently important for cell-specific expression in the hypothalamus and a second element necessary for cell-specific expression in the ovary. We hypothesize that these proximal elements in concert with the corresponding distal elements mediate either hypothalamic or ovarian expression. This proposal will directly test this central hypothesis in three specific aims: 1) To characterize enhancer/repressor and cell-specific elements in the mGnRH gene in vitro using GnRH neuronal and ovarian granulosa cell lines; 2) To identify the Hypothalamic GnRH Enhancer Element (HGEE) and the Ovarian GnRH Repressor Element (OGRE) in the mouse GnRH promoter; and 3) To determine the role of Oct-1 in the GnRH neuron in vivo by generating a cell-specific knockout of Oct- 1. These studies will employ both functional and structural studies of the mGnRH gene in vitro and in transgenic animals. The overall goal of this proposal is to examine the role of Oct-1 in hypothalamic and ovarian mGnRH gene expression in order to elucidate the molecular mechanisms by which hypothalamic and ovarian mGnRH gene expression are differentially regulated.
