GLUCOCORTICOID REPRESSION OF GONADOTROPIN-RELEASING-HORMONE GENE-EXPRESSION AND SECRETION IN MORPHOLOGICALLY DISTINCT SUBPOPULATIONS SF GT1-7 CELLS

Two morphologically distinct subpopulations of GT1-7 cells have been c haracterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy proces ses, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit few er processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I an d type II GT1-7 cells. For secretion studies, cells were attached to C ytodex beads and perifused with control medium or medium containing de xamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and pro foundly decreased the already low level of GnRH secreted by type II ce lls. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreact ivity in type II cells. Both the endogenous mouse GnRH mRNA and the tr anscriptional activity of a mouse GnRH promoterluciferase reporter gen e plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no differ ence between type I and type II nuclear extracts in the pattern of pro tein-DNA complexes formed on two previously identified negative glucoc orticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid recep tors (CR) by Western blot analysis. Cytosols from type I or type II ce lls were incubated with [H-3]dex to obtain GR binding parameters. Bind ing data were consistent with a one-site model for dex binding in each case. Small differences in K-d(1.7 nM, type I; 3.1 nM, type II) or B- max (similar to 3600 sites/cell, type I; similar to 1800 sites/cell, t ype II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells le ading to greater transcriptional susceptibility to dex, coupled with l ow GnRH storage levels, may be reflected in exquisite sensitivity of G nRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells t o alter GnRH secretion. (C) 1997 Elsevier Science Ireland Ltd.