Abstract 579 Poster Session I, Saturday, 5/1 (poster 311)

GnRH, a hypothalamic decapeptide produced by GnRH neurons, plays a critical role in regulating the onset and progression of puberty and normal reproductive function. GnRH secretion is regulated by several factors including estrogen. Clinical observations derived from the McCune Albright syndrome and aromatase excess syndrome indicate that prepubertal exposure to estrogen activate hypothalamic GnRH neurons resulting in precocious puberty. Increased GnRH mRNA levels and increased GnRH secretion have been associated with the onset of puberty. In addition, hypothalamic estrogen receptor numbers have been shown to increase at puberty, suggesting a role for the estrogen receptor signaling pathway in regulating GnRH at puberty. Studying factors that regulate GnRH expression has been quite difficult given the paucity of GnRH neurons (800 GnRH neurons were described in the rat hypothalamus) and their scattered distribution in the hypothalamus. GnRH-expressing neuronal cells (NLT) were developed in our laboratory by targeting and neoplastically transforming GnRH neurons in transgenic mice with a promoter fragment of the human GnRH gene fused to a simian virus 40 tumor-producing antigen (SV40-Tag). These NLT cells were shown to express GnRH mRNA and secrete GnRH. In situ hybridization studies and estradiol binding studies indicate the presence of estrogen receptors in this cell line. In addition, an estrogen response element (ERE), that shares 80% homology with consensus ERE sites, was located in the promoter region of the human GnRH gene. Preliminary data indicate that estradiol treatment of NLT cells at 10 nM in stripped serum for 16 hr. induces GnRH gene expression as indicated by increased GnRH mRNA levels by RNAse protection assays compared to untreated cells. Transfection studies in NLT cells using a construct containing two copies of the ERE fused to the minimal thymidine kinase promoter-luciferase reporter gene indicate a 2.5 fold estradiol induced activation of luciferase activity in these cells. These data indicate that estrogen receptors expressed by NLT cells are functional, mediating estradiol activation of the EREtkluc construct, and of endogenous GnRH. These data form the basis for initiating studies of the role of estrogen on pubertal development at the level of the GnRH neuron.