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Cycles of Transcription and Translation Do Not Comprise the Gonadotropin-Releasing Hormone Pulse Generator in GT1 Cells¹

Departments of Internal Medicine, Cell Biology, and the National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Suzanne M. Moenter, Department of Internal Medicine, University of Virginia, P.O. Box 800578, Charlottesville, Virginia 22908. E-mail: smm4n{at}virginia.edu

Neural control of reproduction is achieved through episodic GnRH secretion, but little is known about the molecular mechanisms underlying pulse generation. The ultradian time domain of GnRH release suggests mechanisms ranging from macromolecular synthesis to posttranslational modification could be involved. We tested if messenger RNA (mRNA) or protein synthesis are components of the pulse generator by determining the effects of transcription and translation inhibitors on episodic GnRH release from immortalized GT1–1 GnRH neurons. Time course and efficacy of transcription and translation blockade were assessed by determining the ability of specific inhibitors to block the robust, rapid induction of c-fos mRNA or protein accumulation by forskolin (10 µM). The transcription inhibitors actinomycin D (ACT-D, 20 µM) or 5,6-dichlorobenzimidazole riboside (DRB, 100 µM), or the translation inhibitors anisomycin (ANI, 10 µM) or puromycin (PUR, 10 µM) were applied to GT1–1 cells 30, 15, or 0 min before forskolin. Northern and Western blots revealed blockade of transcription and translation was rapid and essentially complete. GT1–1 cells were perifused for a 90- to 120-min control period then for 100–130 min with vehicle or inhibitor to examine pulsatile GnRH secretion. GnRH interpeak intervals, peak amplitude, and peak area were not different between control and experimental periods of cells treated with vehicle (n = 15), ACT-D (n = 10), DRB (n = 6), ANI (n = 8), and PUR (n = 6; P > 0.05). This study presents the first clear evidence that the series of reactions resulting in secretion of a GnRH pulse do not include cycles of transcription and translation. Although these mechanisms would be required to replenish components of the pulse generator, they are not integral components of this oscillator. We hypothesize that posttranslational events underlie episodic GnRH release in GT1–1 cells.