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Endocrinology Vol. 142, No. 5 1858-1864
Copyright © 2001 by The Endocrine Society

ARTICLES

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

Gilbert R. Pitts, Craig S. Nunemaker and Suzanne M. Moenter

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AFTER INTEGRATING physiological and environmental inputs, the brain regulates reproduction through the secretion of GnRH into the hypophysial portal system. The episodic secretion of GnRH controls both the synthesis and release of LH and FSH from the anterior pituitary. These hormones, in turn, regulate gametogenesis and steroid production. Pulsatile GnRH secretion is crucial for reproductive success for at least two reasons. First, continuous GnRH administration severely reduces LH and FSH secretion (1). Second, variations in GnRH pulse frequency change the relative levels of LH and FSH secretion and synthesis (2, 3, 4), and these changes are critical for normal follicular maturation (5).

Despite the crucial nature of episodic GnRH release, little is known about the underlying mechanisms. This is partially due to the fact that GnRH neurons are sparsely scattered throughout the preoptic area, making in vivo studies problematic. The development of immortalized GT1 cell lines using genetically targeted tumorigenesis helped circumvent this problem by providing pure populations of GnRH-derived neurons (6) capable of secreting GnRH in a pulsatile manner (7, 8, 9). The ability of GT1 cells to release discrete GnRH pulses suggests the oscillatory mechanisms underlying these pulses, often referred to as the GnRH pulse generator, are at least functionally intact in this cell line, making them a useful model for molecular mechanistic studies.

A basic question that remains to be answered with regard to the GnRH pulse generator is the molecular nature of the series of reactions within GnRH neurons that result in a pulse of secretion. The circhoral time domain of the GnRH pulse generator stands at the cusp of mechanisms that are purely posttranslational, such as heart rate (10) and the neural control of respiration (11), and those driven by timed transcription and translation of key molecules such as the circadian clocks (12). This has led to a lack of consensus concerning whether or not transcription and translation are components of the GnRH pulse generator. On the one hand, a mechanism driven by transcription and/or translation would require time for both macromolecular synthesis and degradation, thus may not be able to cycle fast enough to comprise an oscillator that has been reported to cycle as fast as every 15–60 min (13, 14, 15). On the other hand, very rapid control of transcription and translation has been reported (16, 17, 18, 19, 20), raising the possibility the reactions may occur with sufficient rapidity to comprise the GnRH pulse generator and precluding elimination of these mechanisms a priori. The goal of this study was to determine whether macromolecular synthesis, specifically transcription and translation, participate in generating the GnRH secretory rhythm.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
All culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). GT1–1 cells were generously provided by Dr. Richard Weiner (University of California, San Francisco, CA). GT1–1 cells were maintained in DMEM/F12 supplemented with 10% heat-inactivated FBS, penicillin (100 U), and streptomycin (100 µg) in a humidified 5% CO2 environment at 37 C. Cells were passaged when they reached 70–80% confluence and were used between passages 14 and 28. For perifusion experiments, cells were plated onto Matrigel-coated (1:10), 12-well plates and grown to at least 60% confluence (2–4 days) before 24-h serum deprivation in Optimem I supplemented with penicillin/streptomycin.

Efficacy of transcription and translation inhibition
All reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted. The goal of these studies was to determine whether blockade of either transcription or translation inhibited ongoing pulsatile GnRH release. Consequently, it was important to first determine the time course required for inhibition of these processes. Transcription and translation inhibitors were evaluated for their ability to block induction of the immediate early gene c-fos by forskolin. Because c-fos is strongly induced by forskolin (21), the ability to suppress c-fos induction at either the message or protein level provides great confidence that macromolecular synthesis is effectively halted. Further, the very rapid time course of c-fos induction is within the range of that which would be required for transcription or translation to be fundamental components of the GnRH pulse generator. To evaluate the inhibitors, GT1–1 cells were grown to 70% confluence in 100-mm plates as described above, and serum deprived in Optimem I for 24 h. The transcription inhibitors actinomycin D (ACT-D, 20 µM) or 5,6-dichlorobenzimidazole riboside (DRB, 100 µM) or the translation inhibitors anisomycin (10 µM) or puromycin (10 µM) were applied to GT1–1 cells either 30, 15, or 0 min before forskolin (10 µM) to determine how long inhibitors needed to be present before effective suppression of transcription or translation was achieved.

Northern analysis
Cells were washed with ice-cold PBS 30 min after the addition of forskolin and RNA was extracted (RNeasy, QIAGEN, Santa Clara, CA). Total RNA (10 µg) was run on a 1% agarose-formaldehyde gel (22), transferred to Hybond N+ (Amersham Pharmacia Biotech, Arlington Heights, IL) and hybridized to random-primed, 32P-labeled c-fos and GnRH cDNA probes (QuickHybe, Stratagene, La Jolla, CA). After washing, membranes were exposed to film (BioMax, Eastman Kodak Co., Rochester, NY) or a phospho-imager screen (Molecular Dynamics, Inc., Sunnyvale, CA).

Western analysis
GT1–1 cells were treated as above except that cell lysis occurred 60 min after the addition of forskolin to allow time for the possible accumulation of any newly synthesized Fos protein. Following washing, cells were lysed in a hypotonic buffer (20 mM Tris, 137 mM NaCl, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) containing protease inhibitors (1 mM Pefabloc, 2 µM leupeptin, 1 mM vanadate, 1 mM EDTA, and 0.14 U/ml aprotinin). Total protein (50 µg) was run on a 10% acrylamide gel, transferred to Hybond-ECL (Amersham Pharmacia Biotech) and blotted with Fos-specific antibody (1:1000, SC-052, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by goat-antirabbit-HRP secondary (1:50,000, Santa Cruz Biotechnology, Inc.). Antibody binding was visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Perifusion
A Brandel Suprafusion 1000 perifusion system (Brandel, Inc., Gaithersburg, MD) was used to simultaneously perifuse six wells of GT1–1 cells with oxygenated Locke’s buffer (containing in mM NaCl 154, KCl 5.6, CaCl2 2.2, MgCl2 1, HEPES 2, NaHCO3 0.6, D-glucose 2.7, bacitracin 0.02, and 0.05% BSA, pH 7.4) at a flow rate of 100 µl/min. The chamber volume in this system was estimated to be 100 µl. Cells were perifused for a 30-min stabilization period before sample collection at 4-min intervals into ice-cold tubes. Control samples were collected for 90 or 120 min before drug application. ACT-D (20 µM), DRB (100 µM), anisomycin (10 µM), or puromycin (10 µM) were then administered for an additional 100 or 130 min. The control and experimental time periods were chosen to be sufficiently long to discern multiple GnRH pulses before and after inhibition of macromolecular synthesis was achieved. Each perifusion run contained both control and treated cells. Veratridine (50 µM) was administered to the cells at the end of the experiments to test for evoked GnRH secretion; all cultures exhibited increased GnRH release in response to veratridine (data not shown). Viability was also tested by the ability of the cells to exclude trypan blue at the end of the experiment.

RIA and pulse analysis
GnRH levels were determined using a slight modification of a previously described RIA (23). Briefly, sample was incubated with primary antibody (R1245) for 24 h, tracer was added with incubation continuing an additional 24 h, and bound and free tracer were separated by ethanol precipitation. Samples from a single experiment were assayed together in duplicate. Position and amplitude of GnRH peaks were initially estimated using Pulse, or Pulse2 and then determined using deconvolution analysis software (Deconv_s, Ref. 24 , Dr. Michael Johnson, University of Virginia). Interpeak intervals and baseline secretion were compared using two-sample t tests assuming unequal variance (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Efficacy of transcription inhibition
Treatment with forskolin for 30 min markedly increased c-fos messenger RNA (mRNA) accumulation in GT1–1 cells (Fig. 1Go). Addition of the transcription inhibitors ACT-D and DRB blocked forskolin-stimulated c-fos mRNA accumulation whether added 0, 30, or 15 (not shown) minutes before the addition of forskolin, suggesting the inhibition of transcription in response to these treatments is very rapid. GnRH mRNA levels were not influenced by forskolin, ACT-D, or DRB, likely due to the long half-life of GnRH message in GT1 cells (25). These results demonstrate that both ACT-D and DRB quickly and effectively reduce mRNA synthesis, and we thus infer any pulses occurring in a perifusion experiment after addition of these agents must be independent of transcription.



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  		Figure 1. RNA synthesis is rapidly inhibited in GT1–1 cells. Northern blot depicting the effects of actinomycin D (ACT-D) and 5,6-dichlorobenzimidazole riboside (DRB) added either simultaneously or 30 min before forskolin (FSK) on FSK-stimulated cFOS mRNA accumulation over 30 min in GT1–1 cells.

 
Effect of transcription inhibition on episodic GnRH release
Control GT1–1 cells secreted GnRH in a pulsatile manner throughout the perifusion procedure (Fig. 2Go, Table 1Go). The average GnRH interpeak interval of these control cells did not differ between the time corresponding to the control period (first 120 min) and the experimental period (following 100 min, P > 0.05). Treatment of GT1–1 cells during the experimental period with transcription inhibitors did not prevent episodic GnRH release. GnRH clearly continued to be secreted in a pulsatile manner during treatment with ACT-D (Fig. 3Go) or DRB (Fig. 4Go) as evidenced by the presence of several large, distinct GnRH pulses. Neither ACT-D nor DRB altered the interpeak intervals when compared with their respective pretreatment controls (Table 1Go, P > 0.05). In addition to the lack of effect on GnRH pulse frequency, these treatments did not alter either peak pulse amplitude or peak area (P > 0.05). Although it appeared that DRB increased basal secretion, this difference was not significant (P > 0.05). There were no significant between-treatment effects during either the control or experimental periods for any parameter examined.



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  		Figure 2. Episodic GnRH secretion from control GT1–1 cells perifused with Locke’s solution. GnRH values for peaks exceeding the y-axis in the lower right panel at 104 and 128 min were 174.8 and 117.1 pg/ml, respectively. Asterisks denote pulses identified by Deconv_s.

 

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  		Table 1. GnRH interpeak intervals (minutes, mean ± SEM) before and after treatment

 


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  		Figure 3. Episodic GnRH secretion from GT1–1 cells perifused with Locke’s solution for 120 min followed by actinomycin D (ACT-D). GnRH values for the peak exceeding the y-axis in the lower right panel at 8, 12, and 16 min were 68.0, 139.1, and 84.3 pg/ml, respectively. Bar indicates treatment period; asterisks denote pulses identified by Deconv_s.

 


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  		Figure 4. Episodic GnRH secretion from GT1–1 cells perifused with Locke’s solution for 120 min followed by 5,6-dichlorobenzimidazole riboside (DRB). GnRH values for peaks exceeding the y-axis in the lower left panel at 124 and 172 min were 612.3 and 500.1 pg/ml, respectively. Bar indicates treatment period; asterisks denote pulses identified by Deconv_s.

 
Efficacy of translation inhibition
Treatment of GT1–1 cells with forskolin markedly increased Fos protein accumulation (Fig. 5Go). Both anisomycin and puromycin were able to prevent the stimulatory effect of forskolin on Fos protein synthesis, whether given simultaneously or up to 30 min before forskolin. The rapid blockade of protein synthesis by anisomycin and puromycin suggest any pulses occurring in a perifusion experiment after addition of these agents must be independent of translation.



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  		Figure 5. Protein synthesis is rapidly blocked in GT1–1 cells. Western blot depicting the effects of anisomycin (ANI) and puromycin (PUR) added either simultaneously or 30 min before FSK on FSK-stimulated Fos protein accumulation over 60 min in GT1–1 cells.

 
Effect of translation inhibition on episodic GnRH release
Blockade of translation with either anisomycin or puromycin did not prevent the pulsatile secretion of GnRH from GT1–1 cells (Figs. 6Go and 7Go). As was the case with transcription inhibitors, anisomycin and puromycin did not alter the GnRH interpeak interval, peak amplitude or peak area (P > 0.05). Furthermore, there were no significant between-treatment effects on these parameters during either the control or experimental periods (P > 0.05).



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  		Figure 6. Episodic GnRH secretion from GT1–1 cells perifused with Locke’s solution for 120 min followed by anisomycin. Bar indicates treatment period; asterisks denote pulses identified by Deconv_s.

 


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  		Figure 7. Episodic GnRH secretion from GT1–1 cells perifused with Locke’s solution for 120 min followed by puromycin. Bar indicates treatment period; asterisks denote pulses identified by Deconv_s.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, inhibition of either transcription or translation failed to alter episodic GnRH release from GT1–1 cells. Rather, episodic GnRH release continued with an interpeak interval that is consistent with previous reports from this cell line (7, 8, 9). This result contradicts a previous report that demonstrated an inhibitory effect of systemic protein synthesis inhibitors upon pulsatile LH release in rats (26); the site of this inhibitory action could not be determined due to the systemic nature of that study. By using a model system containing only one cell type, the GnRH neuron-derived GT1 cell, the current study importantly extends study of the role of transcription and translation in episodic GnRH release to the level of a single specific cell type. The results support the hypothesis that the oscillatory components of the GnRH pulse generator do not include the classically genomic mechanisms of RNA and protein synthesis.

One possible caveat with regard to these findings is that the treatment duration was insufficient to adequately block transcription or translation. The data obtained in parallel experiments under the same culture conditions argue against this. Although on-going transcription and translation were not directly assessed, the inhibitors were capable of essentially immediate blockade of RNA and protein synthesis as demonstrated by their ability to block forskolin-induced accumulation of c-fos message and Fos protein, respectively. Blockade of the strongly induced fos marker gene and protein strongly suggests the inhibitors used in this study blocked transcription and translation of all genes. All mRNA and protein levels would then decrease in accordance with their respective half-lives, and any cyclic increases would be blocked. Thus, the occurrence of any GnRH pulses after the initiation of treatment suggests that the sequence of steps within the cells leading to a pulse of hormone release does not include either transcription or translation. The observation that pulsatile GnRH release continued with an unchanged interpeak interval, amplitude or area for the duration of the study (nearly 2 h) strengthens this argument.

In vivo, the GnRH pulse generator operates with interpeak intervals ranging from 20 min to over 6 h (13, 14, 15, 27, 28, 29, 30). This falls in the middle range of periodicity for biological oscillators, which extends from sub second to circannual. Comparison of the mechanisms underlying rhythms with different periods may provide clues concerning the construction of the GnRH pulse generator. For example, circadian rhythms, which are longer than the average GnRH interpeak interval, are generated by feedback cycles of mRNA and protein synthesis and degradation (12). The rates of these reactions limit the maximum frequency of these rhythms, suggesting they are less likely to be involved in higher frequency rhythm generation. There are reports, nevertheless, of rapid transcriptional control in the case of immediate early genes (16, 17, 18), long-term depression (19), and independent rapid translational control of long-term potentiation (20). Despite the existence of such rapid control mediated by transcription and translation, the present data suggest these mechanisms do not underlie episodic GnRH release. Interestingly, in addition to differing in underlying mechanism, the function of circadian clocks and the GnRH pulse generator differ. Specifically, the circadian clock keeps constant time, whereas the GnRH pulse generator produces a frequency modulated hormonal signal. Whether or not this fundamental functional difference dictates the underlying mechanisms is an interesting question for further study.

Other rhythmic systems, operating in shorter time domains, use mechanisms that do not include transcription and translation. Respiration and heart rate are two examples of rhythms that operate at higher frequencies than the GnRH pulse generator. These rhythms are generated at a cellular level by oscillating membrane potentials (10, 11). A similar mechanism may also underlie episodic GnRH release as oscillations in GnRH neuron membrane potential could lead to rhythmic firing of action potentials. Consistent with this idea are reports that distinct bursts of hypothalamic electrical activity are correlated with LH release (31), implying the association of these bursts with GnRH secretion (32). Spontaneous action potentials have been reported in whole-cell patch-clamp recordings of green fluorescent protein (GFP)-identified GnRH neurons (33, 34). During prolonged observation, both periods of bursting activity flanked by several minutes of quiescence and subthreshold oscillations in membrane potential were detected in whole-cell patch-clamp recordings of GFP-identified GnRH neurons (33). Although a definitive link between electrical activity and GnRH release remains to be made, evidence from other neuroendocrine systems suggests increased action potential firing rate is associated with peptide secretion (34, 35, 36). It is thus tempting to speculate that membrane oscillations contribute to the generation of episodic GnRH release. Posttranslational modifications such as cycles of phosphorylation and dephosphorylation of ion channels and electrogenic pumps that modify cell excitability and resting potential may thus underlie the GnRH pulse generator. Further, these mechanisms may also help explain release of hormones secreted in rhythmic patterns with frequencies similar to GnRH such as insulin (37, 38) and GH-releasing hormone (39).

Our finding that cycles of transcription and translation are not integral components of the GnRH pulse generator provides new direction for future studies of the mechanisms of GnRH release. These findings should not dismiss the importance of transcription and translation to GnRH neuron function. These mechanisms likely play a role in the modulation of pulse frequency by various inputs and in the synthesis of the molecules that do function as integral components of the pulse generator. These data lead to the hypothesis that posttranslational events are the key components of the series of reactions causing a pulse of GnRH release.


   Acknowledgments
 
We thank Drs. R. Anthony DeFazio, Pei-San Tsai, and Ms. Shannon Sullivan for editorial comments, Dr. Michael Johnson for assistance with pulse analysis, and Dr. Richard I Weiner for the GT1–1 cell line.


   Footnotes
 
1 Research supported by NIH Grants HD-34680, T-32-HD-07382, and the NICHD/NIH through cooperative agreement U-54-HD-28934 as part of the Specialized Cooperative Centers Program in Reproduction Research and by the National Science Foundation Center for Biological Timing. Back

Received November 16, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endogenous Activation of Metabotropic Glutamate Receptors Modulates GABAergic Transmission to Gonadotropin-Releasing Hormone Neurons and Alters Their Firing Rate: A Possible Local Feedback Circuit
J. Neurosci., June 15, 2005; 25(24): 5740 - 5749.
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 J. Neurosci.Home page
P. E. Chappell, R. S. White, and P. L. Mellon
Circadian Gene Expression Regulates Pulsatile Gonadotropin-Releasing Hormone (GnRH) Secretory Patterns in the Hypothalamic GnRH-Secreting GT1-7 Cell Line
J. Neurosci., December 3, 2003; 23(35): 11202 - 11213.
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 EndocrinologyHome page
C. S. Nunemaker, M. Straume, R. A. DeFazio, and S. M. Moenter
Gonadotropin-Releasing Hormone Neurons Generate Interacting Rhythms in Multiple Time Domains
Endocrinology, March 1, 2003; 144(3): 823 - 831.
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 Mol. Endocrinol.Home page
R. A. DeFazio and S. M. Moenter
Estradiol Feedback Alters Potassium Currents and Firing Properties of Gonadotropin-Releasing Hormone Neurons
Mol. Endocrinol., October 1, 2002; 16(10): 2255 - 2265.
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 Mol. Endocrinol.Home page
R. Vazquez-Martinez, G. M. Leclerc, M. E. Wierman, and F. R. Boockfor
Episodic Activation of the Rat GnRH Promoter: Role of the Homeoprotein Oct-1
Mol. Endocrinol., September 1, 2002; 16(9): 2093 - 2100.
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 EndocrinologyHome page
C. S. Nunemaker, R. A. DeFazio, and S. M. Moenter
Estradiol-Sensitive Afferents Modulate Long-Term Episodic Firing Patterns of GnRH Neurons
Endocrinology, June 1, 2002; 143(6): 2284 - 2292.
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 EndocrinologyHome page
R. Vazquez-Martinez, S. L. Shorte, W. J. Faught, D. C. Leaumont, L. S. Frawley, and F. R. Boockfor
Pulsatile Exocytosis Is Functionally Associated with GnRH Gene Expression in Immortalized GnRH-Expressing Cells
Endocrinology, December 1, 2001; 142(12): 5364 - 5370.
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 J. Neurophysiol.Home page
C. S. Nunemaker, R. A. DeFazio, M. E. Geusz, E. D. Herzog, G. R. Pitts, and S. M. Moenter
Long-Term Recordings of Networks of Immortalized GnRH Neurons Reveal Episodic Patterns of Electrical Activity
J Neurophysiol, July 1, 2001; 86(1): 86 - 93.
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