SAT-419 Mechanisms Underlying Glucocorticoid-Induced Suppression of Pulsatile LH: Insights from Immortalized Kisspeptin Cells

Abstract

Stress inhibits fertility at least in part by inhibiting gonadotropin secretion. Part of the stress response is an increase in circulating glucocorticoid concentrations, specifically corticosterone in mice. We have demonstrated that continuously elevated corticosterone concentrations can inhibit ovarian cyclicity and suppress luteinizing hormone (LH) pulse frequency in female mice (ICN 2018, 310). Since the gonadotropin releasing hormone (GnRH) and LH pulse generator is likely formed by the kisspeptin neuron population in the arcuate nucleus of the hypothalamus, this suppression by corticosterone may be mediated by kisspeptin cells. Our immunohistochemical data indicating that arcuate kisspeptin neurons contain glucocorticoid receptor (GR) in mice (ICN 2018, 312), provides evidence in support of this hypothesis. To investigate direct actions of glucocorticoids in kisspeptin neurons, we utilized an immortalized mouse arcuate kisspeptin neuron cell line (KTaR cells). RT-PCR data indicated the presence of mRNA for GR in KTaR cells. KTaR cells were subsequently transfected with a mouse mamillary tumor virus (MMTV)-luciferase plasmid, which contains a positive glucocorticoid response element, and treated with varying concentrations of a synthetic glucocorticoid, dexamethasone. The MMTV promoter exhibited a dose-dependent increase in luciferase activity, with doses in the μM to nM range producing a significant increase in promoter activity compared to vehicle treated samples (n = 3 independent experiments). Together, these data indicate that functional GR is present in KTaR cells and that this cell type could be valuable in understanding glucocorticoid effects on LH pulse dynamics. Arcuate kisspeptin cells in mice (and other mammals) also contain neurokinin B and dynorphin, and are termed KNDy cells. The KNDy hypothesis for GnRH/LH pulse generation posits that each pulse of GnRH is caused by a pulse of kisspeptin secretion and that each pulse of kisspeptin is turned on (stimulated) by neurokinin B (NKB; encoded by Tac2) and terminated (inhibited) by dynorphin (encoded by pDyn). To determine if these genes are regulated by glucocorticoids, we treated KTaR cells with 1μM dexamethasone for 24 hrs and quantified transcript abundance with qPCR. Dexamethasone treatment did not alter mRNA abundance of Kiss1 or pDyn themselves or the receptors for NKB or dynorphin (Tacr1, Tacr2, Tacr3, and Oprk1). However, dexamethasone treatment caused a significant decrease in Tac2 mRNA abundance compared to vehicle treatment (51.7 ± 10.0% reduced). These experiments demonstrate that KTaR cells have functional GR and that dexamethasone treatment suppresses NKB transcript abundance. These data raise the possibility that the inhibitory actions of glucocorticoids on LH pulse frequency that we recently demonstrated in mice are mediated by an inhibition of NKB, a factor implicated in GnRH pulse onset.