Expression and Intracellular Signaling of Estrogen α- and β-Receptor Subtypes in Native and Immortalized GnRH Neurons

Abstract

Pulsatile GnRH release is an intrinsic property of the GnRH neuron and is modulated by neurotransmitters, pituitary hormones, and gonadal steroids. Despite the prominent role of estrogen in the regulation of hypothalamic-pituitary function, its ability to exert receptor-mediated actions directly on the GnRH neuron has only recently been demonstrated. We and others have demonstrated that estrogen receptors are expressed in both hypothalamic GnRH neurons and their immortalized counterparts, GT1-7 cells. This report describes the roles of estrogen receptor subtypes in GnRH neuronal signaling and secretion. Cell culture; cellular perifusion studies; cAMP and GnRH radioimmunoassay (RIA); patch-clamp recording of action potentials. GT1-7 cells were cultured in DMEM/F-12 medium with 10% heat-inactivated fetal bovine serum, and then in serum- and steroid-free medium prior to stimulation with estradiol or selective ERα and ERβ ligands. The roles of ERα and ERβ in cAMP production and pulsatile GnRH release were determined in perifused GT1-7 cells. cAMP formation and GnRH levels were measured by RIA after treatment with ERα and ERβagonist and antagonist analogs. Action potential firing was recorded by patch-clamping in hypothalamic cell cultures obtained from enzymatically dispersed E18 fetal Sprague-Dawley rat hypothalami. Identified GnRH neurons were subjected to recording in the absence and presence of estradiol, and ERα and ERβ agonist and antagonist analogs. Our previous research showed that physiological estrogen concentrations caused rapid inhibition of spontaneous cAMP production that was prevented by the ER antagonist ICI 182,780. The estrogen receptor that mediates this effect on cAMP could be immunoprecipitated with an antibody to the α-receptor. In the present study, pulsatile GnRH release in cultured hypothalamic cells, cAMP production, and the rate of firing of spontaneous action potentials by identified hypothalamic GnRH neurons, were inhibited during activation of the Gi-coupled ERα receptor. In contrast, selective activation of the Gs-coupled ERβ receptor increased GnRH release, stimulated cAMP production, and increased the rate of firing of action potentials. The ability of estrogen to activate ERα and/or ERβ receptors in a time- and dose-dependent manner regulates the adenylyl cyclase/cAMP signaling pathway, and thereby modulates the frequency and amplitude of pulsatile GnRH release. This process, in conjunction with the modulation of spontaneous electrical activity of the GnRH neuron, is a major factor in the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function.