Estrogen plays an important role in regulating gonadotropin- releasing hormone (GnRH) neuronal function in the female brain during pubertal development and through the reproductive cycle. GnRH is a decapeptide produced by GnRH neurons located in the hypothalamus. GnRH plays a major role in regulating the development of puberty and reproduction. Estrogen has a bimodal effect on GnRH. Although its stimulatory effect has been well established, its inhibitory effect is not well understood. In addition, estrogen was thought to indirectly regulate GnRH gene expression through estrogen receptors (ER) expressed by cells afferent to GnRH neurons. Preliminary studies presented in this proposal indicate expression of functional ER in a GnRH- expressing neuronal cell line NLT, that mediate negative estrogen regulation of the GnRH gene. This study proposes that ER expressed by GnRH neurons play an important role in regulating GnRH gene expression during pubertal development and across the estrous cycle. The first specific aim will study the mechanism of negative regulation of GnRH gene expression by estrogen in vitro in NLT cells by: 1) identifying GnRH promoter region(s) that mediate negative regulation by estrogen, 2) identifying functional ER domains required for negative regulation of GnRH, 3) studying binding of the estrogen-ER complex to putative estrogen response sites in the GnRH promoter, and 4) identifying cofactors involved in mediating negative regulation of GnRH by estrogen. The second major aim will explore the physiologic significance of the ER in GnRH neurons in vivo by developing and studying transgenic and knock-out mouse models. Three mouse models will be generated: 1) a model of targeted inhibition of ER in GnRH neurons using a GnRH targeting construct fused to a dominant negative mutant of the ER that inhibits ER alpha and beta isoforms, 2) a model of targeted overexpression of ERalpha in GnRH neurons using a GnRH targeting construct fused to wild type ERa, 3) targeted ERa knock-out in GnRH neurons using a Cre- loxP binary system. These mice will be studied using physiologic and molecular approaches to determine the physiologic significance of ER in GnRH neurons. These in vitro and in viva studies will help define the contributing role of ER in GnRH neurons to the development of puberty and reproduction.