Regulation of gonadotropin-releasing hormone (GnRH) gene expression by 5alpha-dihydrotestosterone in GnRH-secreting GT1-7 hypothalamic neurons.
Hypothalamic GnRH secretory neurons are precisely regulated by circulating gonadal steroids. However, the question of whether these cells are directly responsive to steroid hormones remains a central and controversial issue in reproductive science. In the present study, we demonstrate the expression of androgen receptor (AR) in a mouse hypothalamic GnRH-secreting cell line, GT1-7. AR messenger RNA was detected by Northern blot analysis of 10 microg total cellular RNA. Western blot analysis revealed a 110K AR immunoreactive band, and saturation binding analysis confirmed the presence of a high affinity low capacity androgen binding entity (Kd = 0.06 nM; Bmax = 12.4 fmol/mg protein). In addition, GT1-7 cells were found to express ARA70, an AR-specific coactivator that has been reported to enhance transactivational activity of the AR. GT1-7 cells transiently transfected with an androgen responsive MMTV-luciferase reporter construct displayed a 4.2-fold induction of luciferase reporter gene activity by 1 nM 5alpha-dihydrotestosterone (DHT), further demonstrating the presence of a functional AR. Treatment of GT1-7 cells with 1 or 10 nM DHT resulted in approximately 55% reduction in GnRH messenger RNA measured at 24 and 36 h after treatment. This repression was completely blocked by hydroxyflutamide, an AR antagonist. These results provide the first demonstration that androgen acts directly through an AR-mediated pathway to repress GnRH gene expression in hypothalamic GnRH-secreting neurons.
Belsham
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DD
Department of Physiology, University of Toronto and Toronto Hospital Research Institute, Ontario, Canada. d.belsham@utoronto.ca
Evangelou
A
A
Roy
D
D
Duc
V L
VL
Brown
T J
TJ
D000818:Animals; D002478:Cells, Cultured; D013196:Dihydrotestosterone / Q000494:pharmacology*; D005485:Flutamide / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011944:Receptors, Androgen / Q000032:analysis
D012333:RNA, Messenger; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; C014290:hydroxyflutamide; D007987:Gonadotropin-Releasing Hormone; D005485:Flutamide