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Endocrinology Vol. 144, No. 5 1802-1811
Copyright © 2003 by The Endocrine Society

Differential Regulation of Gonadotropin Subunit Gene Promoter Activity by Pulsatile Gonadotropin-Releasing Hormone (GnRH) in Perifused LßT2 Cells: Role of GnRH Receptor Concentration

Grégoy Y. Bédécarrats and Ursula B. Kaiser

Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Ursula B. Kaiser, M.D., Endocrine-Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: ukaiser{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pulsatile release of GnRH by the hypothalamus is required to stimulate the pituitary-gonadal axis, and variations in GnRH pulse frequency are associated with differential synthesis and release of LH and FSH by pituitary gonadotropes. How gonadotropes differentiate between GnRH pulse frequencies and subsequently differentially regulate the expression of the LHß and FSHß genes remains to be determined. In the present study, using a perifusion system that allows us to replicate the GnRH pulsatility occurring in vivo, we have systematically characterized the effects of varying GnRH pulse frequencies on LHß, FSHß, {alpha}, and GnRH receptor (GnRHR) gene promoter stimulation in LßT2 cells. We demonstrate that LHß gene promoter activity is stimulated to the greatest extent at higher GnRH pulse frequencies, whereas the FSHß gene promoter is preferentially stimulated at lower GnRH pulse frequencies, reflecting previous observations in primary rat pituitary cells in vivo and in vitro. By measuring GnRH binding, we demonstrate that cell-surface GnRHR number is increased at higher frequencies of pulsatile GnRH and that this increase precedes the differential regulation of LHß and FSHß gene promoter activity. To test the role of GnRHR number in mediating the differential effects of pulsatile GnRH, the rat GnRHR was overexpressed in LßT2 cells, and the response to pulsatile GnRH was again assessed. Interestingly, although overexpression of GnRHR had no effect on the frequency-dependent regulation of LHß, the induction of FSHß gene promoter activity by pulsatile GnRH was reduced, and frequency dependence was abrogated. Our results demonstrate that LßT2 cells represent a suitable model for the study of the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH. Furthermore, our studies indicate that cell-surface GnRHR density is a critical mediator of this differential regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC DECAPEPTIDE, GnRH, is a hormone released in a pulsatile manner into the hypophyseal portal vascular system and delivered to the anterior pituitary. There it binds to specific, high-affinity G-protein-coupled receptors [GnRH receptors (GnRHRs)] on gonadotropes to regulate expression of the gonadotropin subunit genes, as well as the synthesis and release of LH and FSH, which, in turn, direct reproductive development and function. The stimulation of gonadotropin secretion has been shown to be dependent on the pulsatile nature of GnRH release, whereas continuous exposure to GnRH down-regulates LH and FSH secretion (1). In vivo, the pattern of GnRH pulses varies physiologically as a function of hormonal status and reproductive cycle stage (2, 3). Interestingly, these variations in GnRH pulse pattern are associated with differential LH and FSH release, with higher frequencies of GnRH pulses leading to greater LH secretion and, conversely, lower frequencies leading to increases in FSH secretion (4). In the rat, these frequency-dependent effects have been shown to occur in vivo at the level of gonadotropin subunit gene transcription (5, 6). Similarly, in cultured rat primary pituitary cells, the parallel GnRH pulse frequencies also led to differential LHß and FSHß gene expression, indicating that these effects are intrinsic to the anterior pituitary (7). How gonadotropes are able to differentiate GnRH pulse frequencies and activate appropriate signal transduction pathways to differentially stimulate {alpha}, LHß, and FSHß gene expression and release is still unknown but is a question of much interest and investigation.

The response of pituitary gonadotropes to GnRH correlates, at least in part, with the density of GnRHRs on the cell surface (8, 9). A number of factors are known to affect expression of the GnRHR gene; most notably, GnRH itself. We have shown previously that GnRHR gene expression, like the gonadotropin subunit genes, is also dependent on GnRH pulse frequency. The highest levels occur at those GnRH pulse frequencies that preferentially stimulate LHß gene expression, with lower levels occurring at slower frequencies that are associated with preferential FSHß gene expression (10). Similar observations have been made in rats in vivo, with maximal GnRHR numbers occurring after treatment with pulsatile GnRH at 30-min intervals for 48 h (11). These studies indicate a correlation between GnRHR density and the differential gonadotropin responses to varying GnRH pulse frequencies and suggest a possible role for GnRHR levels in mediating these differential responses.

To further evaluate the role of GnRHR number in mediating the differential effects on LHß and FSHß gene expression, we developed a heterologous cell model in which cell-surface GnRHR concentrations could be externally manipulated and controlled. We have previously shown that GnRH is able to stimulate gonadotropin subunit gene promoter activity in a heterologous pituitary somatolactotrope-derived GH3 cell model transfected with the rat GnRHR (rGnRHR; Ref. 12). Moreover, in this model, LHß gene transcription was maximally stimulated at high cell-surface concentrations of GnRHR, whereas lower GnRHR densities were optimal for maximal stimulation of FSHß gene transcription, thus supporting a role for GnRHR levels in mediating differential effects of GnRH on gonadotropin subunit gene expression.

To elucidate the molecular mechanisms underlying the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH, it is essential to develop an experimental model that can replicate these actions in a physiologically relevant system. Recently, several studies have focused on elucidating the signaling pathways involved in mediating the effects of GnRH on LH and FSH subunit gene expression (13, 14, 15, 16). However, these studies have not addressed the differential effects of varying GnRH pulse frequencies, which, to date, have only been studied in vivo and in primary pituitary cell culture models. The use of such models to dissect signal transduction pathways and mechanisms of control of gene transcription present several challenges, because gonadotropes represent only 10–15% of hormone-secreting anterior pituitary cells and are hard to culture and manipulate (17). On the other hand, the use of a heterologous cellular model, such as GH3 cells, is limited in its applicability to gonadotrope physiology, because these cells may not express the necessary gonadotrope-specific transcription factors and may use distinct signal transduction pathways. The recent development and characterization of the LßT2 cell line may provide a more appropriate model for such studies. This immortalized gonadotrope-derived cell line expresses the gonadotropin subunit and GnRHR genes and responds to GnRH with an increase in LHß mRNA levels and LH secretion as well as in rat LHß and ovine FSHß gene promoter activity (16, 18, 19, 20, 21). However, the responses of this cell line to varying frequencies of pulsatile GnRH have not been characterized. In the following studies, we show that pulsatile GnRH differentially regulates gonadotropin subunit gene expression in perifused LßT2 cells in a manner similar to that observed in primary gonadotropes. We also investigate the effects of GnRH pulse frequency on cell-surface GnRHR concentrations, and the consequences of changes in GnRHR number on LHß and FSHß gene regulation by pulsatile GnRH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and perifusion system
The perifusion system used for our studies was designed in our laboratory (Fig. 1Go). The chambers used were built and assembled based on the system described by Jakubowiak et al. (22). All tubing used in the perifusion system was made of Tygon to reduce any binding to GnRH. To test the linear flow rate inside the chambers during perifusion, a dye was run to mimic the delivery of a GnRH pulse. Chambers were placed on an inclined platform to avoid accumulation of air bubbles during perifusion. To verify the stability of GnRH under our conditions, GnRH was assayed by RIA. No changes in GnRH concentration were observed after 24 h incubation at 37 C in media containing 1% fetal bovine serum (FBS; data not shown). To keep the pH constant during perifusion, media was saturated with a gas mixture of 5% CO2-95% O2.



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  		Figure 1. Diagram of the perifusion chamber and the perifusion system. A, Perifusion chambers consist of a silicone rubber gasket glued to a bottom borosilicate glass plate and perforated with Tygon inlet and outlet tubing. A removable top borosilicate glass plate is clamped to the top of the chamber before mounting in the perifusion system. B, Perifusion system: a set of peristaltic pumps (flow rate, 0.25 ml/min) controlled by a time controller, deliver media (DMEM, high glucose, 1% FBS) and/or GnRH (10 nM, continuous or pulsatile, 5 min/pulse) to up to 12 perifusion chambers placed on an inclined platform in a water bath at 37 C. Media was saturated with a mixture of 5% CO2-95% O2 to keep pH constant during the perifusion.

 
LßT2 cells were plated in perifusion chambers previously coated with Matrigel (Becton Dickinson and Co.Labware, Bedford, MA) and incubated for 24 h in static culture in high-glucose DMEM containing 10% heat inactivated FBS and 1% penicillin-streptomycin, at 37 C in a humidified atmosphere of 5% CO2 in air. The chambers were then mounted in the perifusion system and continuously perifused with high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin at a constant flow rate of 0.25 ml/min. During the perifusion, groups of chambers (n = 3) were treated with either media alone (control group), continuous GnRH, or pulsatile GnRH at varying pulse frequencies (1 pulse/30 min, 1 pulse/1 h, 1 pulse/2 h, and 1 pulse/3 h). Up to 12 chambers were run simultaneously (Fig. 1Go). The GnRH pulses were delivered by a set of peristaltic pumps controlled by a time controller (ChronTrol XT; ChronTrol Corp., San Diego, CA). A concentration of 10-8 M GnRH and pulse duration of 5 min were chosen, based on previous studies performed in primary rat pituitary cells (22). Cells were perifused for a total of 20 h, and GnRH stimulation was performed during the final 4, 10, or 20 h in different experiments. Twenty minutes after the last GnRH pulse, chambers were disconnected, and analyses were performed. All cell culture reagents were supplied by Life Technologies, Inc. (Gaithersburg, MD), and GnRH was supplied by Sigma (St. Louis, MO).

Plasmids and transfection methods
The reporter constructs used were generated by fusing -846/0 of the human {alpha} gene ({alpha}Luc), -797/+5 of the rat LHß gene (LHßLuc), -2000/+698 of the rat FSHß gene (FSHßLuc), and -1164/+62 of the mouse GnRHR gene (GnRHR-Luc) to the firefly luciferase cDNA as previously described (12, 23, 24). The rGnRHR-expression vector was prepared by subcloning the rGnRHR cDNA sequence from pcDNA1 (12) into pcDNA3 (Invitrogen, Carlsbad, CA). LßT2 cells were transiently transfected by electroporation (25) with 4 µg/chamber of reporter construct ({alpha}Luc, LHßLuc, FSHßLuc, or GnRHR-Luc) and 2 µg/chamber of RSV or SV40-ß-galactosidase-expression vector and plated in matrigel-coated perifusion chambers. After 24 h of static culture at 37 C in a humidified atmosphere, chambers were mounted in the perifusion system and perifused as described above. In some experiments, rGnRHR (0, 0.5, 2, or 4 µg/chamber) or the empty pcDNA3 control vector was cotransfected as well.

Luciferase assays
At the end of each perifusion experiment, chambers were disconnected, and cells were rinsed twice with ice-cold PBS and lysed with 125 mM Tris-HCl/0.5% Triton. Cell debris was pelleted by centrifugation at 14,000 x g for 10 min at 4 C, and luciferase and ß-galactosidase activities were measured in clear supernatants as previously described (12). Luciferase activity was normalized for ß-galactosidase to correct for transfection efficiency and cell number, and the results were expressed as fold increase, compared with the unstimulated control group.

Binding assay
Approximately 3 x 106 LßT2 cells were plated in each perifusion chamber and incubated in static culture for 24 h, after which chambers were mounted in the perifusion system, and the cells were perifused as described above. At the end of the perifusion experiment, the cells were washed with high-glucose DMEM, 0.1% BSA, and incubated for 60 min at room temperature with 75,000 cpm 125I-Buserelin [generously provided by Dr. P. Michael Conn (26)] in 1 ml of high-glucose DMEM, 0.1% BSA. For measurement of nonspecific binding, one chamber of each group was incubated with 10-6 M GnRH in addition to radiolabeled Buserelin. Cells were rinsed twice with PBS and lysed with 1 ml 0.2-M NaOH, 0.1% SDS, and protein concentration was calculated (Coomassie Plus Protein Assay; Pierce Chemical Co., Rockford, IL). Radioactivity in the lysates was measured in a {gamma}-counter and normalized for protein concentration. Results are expressed as percent specific binding, relative to the unstimulated control group.

Experimental paradigms
GnRH pulse frequency.
To study the effects of varying GnRH pulse frequency on the stimulation of gonadotropin subunit and GnRHR gene promoter activity, LßT2 cells transfected with reporter constructs were perifused for 20 h and stimulated during the final 10 or 20 h of perifusion with either media alone (control), continuous GnRH (10 nM), or GnRH pulses (10 nM, 5 min/pulse) every 0.5, 1, 2, or 3 h. Simulated perifusion experiments with switching, but without GnRH (i.e. with media only in both channels), ruled out any effects of switches in the stream flow on reporter construct activity (data not shown).

Adjusted cumulative dose of GnRH.
To rule out the possibility that differential effects of pulsatile GnRH resulted from a difference in the total cumulative dose of GnRH used during perifusion studies, LßT2 cells transfected with LHßLuc or FSHßLuc were stimulated for 20 h or with pulsatile GnRH (1 pulse/30 min or 1 pulse/2 h), with the concentration of GnRH adjusted to result in an identical final cumulative dose (i.e. 5 nM GnRH per pulse every 30 min, or 20 nM GnRH per pulse every 2 h).

Overexpression of rGnRHR.
To study the effects of GnRHR number on the differential regulation of LHß and FSHß by varying GnRH pulse frequencies, we overexpressed rGnRHR in LßT2 cells. Cells were transfected with 4 µg/chamber LHßLuc or FSHßLuc; 2 µg/chamber SV40-ß-Galactosidase; and 0, 0.5, 2, or 4 µg/chamber rGnRHR (total amount of expression vector was normalized to 4 µg/chamber with the empty pcDNA3 control vector). Cells were stimulated in perifusion for 20 h with media alone (control), with continuous GnRH (10 nM), or with pulsatile GnRH (10 nM, 5 min/pulse every 0.5 or 2 h).

Statistical analysis
All studies were performed in at least three separate experiments, each performed in triplicate. Results were analyzed by two-way ANOVA on repeated measurement followed by a Tukey-Kramer multiple-comparison post hoc test with Instat 3.0 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Varying GnRH pulse frequencies differentially regulate gonadotropin subunit and GnRHR gene expression in LßT2 cells
Previous studies have shown that LHß gene expression is maximally stimulated at a GnRH pulse frequency of every 30 min, whereas FSHß gene expression is maximally stimulated at a slower GnRH pulse frequency of every 2 h, in the rat in vivo (27) as well as in cultured primary rat pituitary cells (7). Our goal was to characterize the LßT2 cell line to determine whether a similar GnRH pulse frequency-dependent mechanism of gonadotropin subunit gene expression was active in this model as well. We thus evaluated the effects of varying GnRH pulse frequencies on LßT2 cells transfected with luciferase reporter constructs ({alpha}Luc, LHßLuc, FSHßLuc, or GnRHR-Luc). Cells were maintained in static culture for 24 h after transfection, to allow adherence to the chambers, and then perifused for 20 h. A control group was perifused with media only, whereas experimental groups were treated with continuous or pulsatile GnRH. To study the temporal effects of pulsatile GnRH stimulation, two sets of experiments were conducted in which cells were stimulated with GnRH for either the full 20 h of the perifusion or for only the final 10 h. Cells were harvested 20 min after the last pulse of GnRH. At the end of the experiment, luciferase activity was measured in cell extracts, and results were expressed as fold increase in activity, compared with the unstimulated control group (Fig. 2Go).



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  		Figure 2. Varying GnRH pulse frequencies differentially regulate gonadotropin subunit and GnRHR gene promoter activity. Luciferase activity was measured in LßT2 cells transfected with (A) LHßLuc, (B) FSHßLuc, (C) {alpha}Luc, or (D) GnRHR-Luc and stimulated with continuous (C) or pulsatile (1 pulse per 0.5, 1, 2, or 3 h) GnRH (10 nM, 5min/pulse) for 10 h (open bars) or 20 h (solid bars). The dotted line represents the relative activity of reporter constructs in perifused cells that received no GnRH stimulation (unstimulated control group). Results are expressed as fold increase over the unstimulated group and represent the mean ± SEM of three separate experiments, each performed in triplicate. a, b, and c, Significantly different fold increase in luciferase activity between GnRH pulse frequencies (P < 0.05).

 
As shown in Fig. 2AGo, stimulation of LßT2 cells transfected with LHßLuc with continuous or pulsatile GnRH resulted in an increase in luciferase activity. All GnRH pulse frequencies stimulated activity after exposure to GnRH for 10 or 20 h. LHß gene promoter activity was stimulated to a greater extent by the higher GnRH pulse frequencies than by the lower frequencies, with a significant difference (P < 0.05) observed between 1 pulse/30 min and 1 pulse/2 h after either 10 or 20 h of GnRH stimulation. Interestingly, continuous stimulation with GnRH for 10 h induced the greatest increase in luciferase activity (11-fold), a significantly higher increase (P < 0.01) than with pulsatile stimulation at any frequency. After 20 h of continuous GnRH, the levels of LHß gene promoter induction were comparable with those of 1 pulse/30 min GnRH. In summary, LHßLuc activity was stimulated by pulsatile GnRH in a frequency-dependent manner similar to that previously reported in primary pituitary cells, with the greatest stimulation occurring at the higher frequencies tested. Surprisingly, continuous GnRH did not down-regulate LHßLuc activity as we had expected.

In contrast to what was observed for LHß, luciferase activity in LßT2 cells transfected with FSHßLuc was preferentially stimulated by lower GnRH pulse frequencies (Fig. 2BGo). After 20 h of GnRH stimulation, the slower frequency of 1 pulse/2 h gave a significantly greater (P < 0.01) increase in activity (3.31 ± 0.14-fold) than observed with 1 pulse/30min (2.54 ± 0.01-fold). The differences were less striking after only 10 h of GnRH stimulation, at which time hourly pulses gave the greatest response. As observed for LHßLuc, continuous GnRH was able to stimulate FSHß gene promoter activity, although to a lesser extent than pulsatile GnRH after 20 h of stimulation. Taken together, like primary pituitary cells, and in contrast to LHßLuc, FSHßLuc activity was preferentially stimulated by the lower GnRH pulse frequency of every 2 h than by the 30-min pulse interval.

As for LHß and FSHß, stimulation of LßT2 cells with continuous or pulsatile GnRH also resulted in an increase in {alpha}-subunit gene promoter activity (Fig. 2CGo). Interestingly, the level of stimulation did not seem to be significantly influenced by GnRH pulse frequency. {alpha}Luc activity was increased by approximately 3.5-fold after 10 h and 4.5-fold after 20 h of stimulation by most frequencies tested, although the lowest frequency was less effective. Continuous GnRH also gave similar levels of stimulation. Thus, the {alpha}-subunit gene promoter activity was stimulated by both pulsatile and continuous GnRH, with less frequency dependence, much as previously reported in rat pituitary cells in vivo and in vitro (7, 27).

Because GnRHR gene expression has been reported to be regulated by pulsatile GnRH in a frequency-dependent manner (7), we also studied the effects of GnRH pulse frequency on GnRHR-Luc activity in transfected LßT2 cells (Fig. 2DGo). Though a modest increase in activity was observed in response to both continuous and pulsatile GnRH and there was a trend toward greater stimulation at higher pulse frequencies, no statistically significant frequency dependence occurred under our experimental conditions.

In summary, our results indicate that in perifused LßT2 cells, the LHß gene promoter is preferentially stimulated by a high GnRH pulse frequency (every 30 min), whereas the FSHß gene promoter is preferentially stimulated by a lower pulse frequency (every 2 h). These results are in agreement with prior observations in vivo and in vitro in primary rat pituitary cells, and they suggest that LßT2 cells share characteristics of GnRH pulse frequency dependence with primary gonadotropes. In addition, in our studies, 20 h of pulsatile GnRH stimulation is necessary to obtain clear differential regulation of gonadotropin subunit gene promoter activity.

Differential regulation of LHß and FSHß gene expression by GnRH pulse frequency is independent of cumulative dose of GnRH
To rule out the possibility that the differential stimulation of LHß and FSHß by pulsatile GnRH resulted from a difference in the total dose of GnRH to which LßT2 cells were exposed during the perifusion study, we performed a set of experiments with a correction for the cumulative dose of GnRH. LßT2 cells were transfected with LHßLuc or FSHßLuc and stimulated for 20 h in perifusion with 5 nM GnRH at 1 pulse/30 min or 20 nM GnRH at 1 pulse/2 h (Fig. 3Go).



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  		Figure 3. Correction for the cumulative dose of GnRH. Luciferase activity was measured in LßT2 cells transfected with (A) LHßLuc or (B) FSHßLuc and stimulated with 5 nM GnRH at 1 pulse per 30 min (0.5h) or 20 nM GnRH at 1 pulse per 2 h (2h) for 20 h. Results are expressed as fold increase over the unstimulated group (-) and represent the mean ± SEM of three separate experiments, each performed in triplicate. a and b, Significantly different fold increase in luciferase activity between GnRH pulse frequencies (P < 0.05).

 
As observed with a fixed concentration of GnRH, LHßLuc was preferentially stimulated by GnRH at 1 pulse/30 min (6.12 ± 0.08-fold), compared with 1 pulse/2 h (4.38 ± 0.01) (P < 0.01; Fig. 3AGo). In contrast, FSHßLuc was again preferentially stimulated by GnRH at 1 pulse/2 h (3.59 ± 0.35-fold), compared with 1 pulse/30min (2.89 ± 0.11) (P < 0.05) (Fig. 3BGo). The fold increases in activity obtained with corrected doses of GnRH were very similar to those observed with a fixed 10-nM GnRH concentration.

GnRH pulse frequency regulates GnRHR number on the cell surface of LßT2 cells
Previous studies of cultured primary rat pituitary cells demonstrated that GnRH binding was stimulated by pulsatile GnRH, with the greatest increase at a 30-min pulse interval (11). To determine whether varying GnRH pulse frequencies induce changes in the number of GnRHRs present on the cell surface of LßT2 cells, we performed GnRH binding assays with LßT2 cells perifused for 20 h and treated with continuous GnRH (10 nM), pulsatile GnRH (10 nM, 1 pulse per 0.5, 1, or 2 h), or media only (control group). At the end of the perifusion, cells were incubated with 125I-Buserelin, and specific binding was measured. To determine the time course of any changes in GnRHR levels, three sets of experiments were performed, corresponding to GnRH stimulation during the final 4, 10, or 20 h of perifusion.

Results of the binding assays are shown in Fig. 4Go. After 4 h of stimulation, all GnRH-treated groups had specific binding below the level of the unstimulated control cells. There was no significant difference between groups treated with pulsatile GnRH at the frequencies tested, or continuous GnRH. After 10 h of GnRH stimulation, the specific binding was increased slightly in all groups treated by pulsatile GnRH, compared with the unstimulated cells; and at a high frequency (1 pulse/30 min), specific binding was significantly greater (P < 0.01), compared with the other frequencies tested. Interestingly, whereas pulsatile GnRH seemed to increase specific binding, continuous GnRH stimulation for 10 h resulted in down-regulation of receptor number (79.9 ± 9.5% of unstimulated controls). After 20 h of GnRH stimulation, the specific binding was further decreased in cells treated with continuous GnRH (59.6 ± 7.0% of unstimulated controls) and was significantly lower than the specific binding of cells treated with pulsatile GnRH. No significant difference in specific binding was observed among the cells treated with varying GnRH pulse frequencies for 20 h.



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  		Figure 4. Higher GnRH pulse frequencies increase the specific binding of GnRH by LßT2 cells. Binding analyses were performed in LßT2 cells after cells were stimulated with continuous (C) or pulsatile (1 pulse per 0.5, 1, or 2 h) GnRH (10 nM, 5min/pulse) for 4 h (open bars), 10 h (striped bars), or 20 h (solid bars). Specific binding was measured after the cells were incubated for 60 min at room temperature with 75,000 cpm 125I-Buserelin with or without 1 µM unlabeled GnRH. The dotted line represents the specific binding of perifused cells that received no GnRH stimulation. Results are expressed as percent of specific binding of the unstimulated group and represent the mean ± SEM of three separate experiments, each performed in triplicate. a and b, Significantly different specific binding between GnRH pulse frequencies (P < 0.05).

 
Taken together, our results indicate that a significant frequency-dependent increase in GnRHR number occurs after 10 h of stimulation with pulsatile GnRH, with the highest binding levels observed with the high frequency of GnRH pulses (every 30 min). This difference in receptor number is followed by frequency-dependent differential regulation of LHß and FSHß gene expression after 20 h of GnRH stimulation.

Overexpression of GnRHR prevents GnRH stimulation of FSHß gene promoter activity in LßT2 cells
Changes in GnRHR number have previously been proposed as a mechanism involved in the differential regulation of gonadotropin subunit gene expression (12). To determine whether such changes, as observed in our binding assay, are involved in mediating the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH, we overexpressed rGnRHR in LßT2 cells. We predicted that the resultant increase in GnRHR number would abrogate the preferential stimulation of the FSHß gene promoter by low GnRH pulse frequencies. To test this hypothesis, LßT2 cells were cotransfected with LHßLuc or FSHßLuc and increasing amounts of an expression vector encoding rGnRHR, and cells were stimulated by GnRH at different pulse frequencies in perifusion.

Because of the low transfection efficiency of LßT2 cells, we were not able to measure and quantify changes in GnRH binding in the subset of cells transfected with the rGnRHR-expression vector. To overcome this limitation, we analyzed the dose dependence of GnRHR plasmid overexpression on the stimulation of LHßLuc and FSHßLuc. The expression of cDNA transfected by electroporation is directly proportional to the amount of plasmid introduced during the transfection; and thus, the GnRHR number present on the cell surface is directly proportional to the amount of rGnRHR plasmid added (12, 28).

The effects of overexpressing rGnRHR are shown in Fig. 5Go. Cells transfected with empty pcDNA3 expression vector only (no rGnRHR) showed the same differential response to pulsatile GnRH as observed in the previous experiments (Fig. 2Go, A and B), with LHß and FSHß preferentially stimulated by high and low GnRH pulse frequencies, respectively. Continuous GnRH stimulation of cells cotransfected with 4 µg rGnRHR expression vector and LHßLuc resulted in a small, but significant, increase in the magnitude of stimulation of luciferase activity (Fig. 5AGo). On the other hand, transfection of LßT2 cells with 2 or 4 µg rGnRHR expression vector had no significant effect on the stimulation of LHßLuc by pulsatile GnRH at either of the frequencies tested (1 pulse/0.5 h and 1 pulse/2 h).



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  		Figure 5. Overexpression of rGnRHR specifically inhibits the GnRH-dependent stimulation of FSHß gene promoter activity. Luciferase activity was measured in LßT2 cells cotransfected with (A) LHßLuc or (B) FSHßLuc and increasing amounts of an expression vector encoding rGnRHR (from 0–4 µg). Cells were stimulated with continuous (C) or pulsatile (1 pulse per 0.5 or 2 h) GnRH (10 nM, 5 min/pulse) for 20 h. Results are expressed as fold increase over the unstimulated control group and represent the mean ± SEM of three separate experiments, each performed in triplicate. *, Significantly different fold increase in luciferase activity between groups (P < 0.05).

 
In contrast, overexpression of 4 µg rGnRHR completely abrogated the stimulation of luciferase activity by pulsatile GnRH in cells transfected with FSHßLuc (Fig. 5BGo). This specific inhibition of FSHß gene expression was dependent on the amount of rGnRHR transfected. In cells transfected with 0.5 or 2 µg rGnRHR, the differential regulation of FSHß gene promoter activity was lost, and no significant difference in luciferase activity could be observed between GnRH pulse frequencies tested (0.5 and 2 h). Moreover, in these cells, the level of FSHß gene promoter activation was not different from that observed in cells transfected with empty pcDNA3 vector and stimulated with 1 pulse GnRH/0.5 h. Interestingly, overexpression of rGnRHR also inhibited the stimulation of FSHßLuc by continuous GnRH.

Thus, increasing the levels of GnRHR in LßT2 cells specifically inhibits FSHß gene promoter stimulation by GnRH and prevents the frequency-dependent differential regulation by pulsatile GnRH, whereas it has no effect on the GnRH induction of the LHß gene promoter.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of pulsatile and continuous GnRH to up- and down-regulate, respectively, the synthesis and secretion of gonadotropins has led to the development of therapies for reproductive disorders. In the rat estrous and human menstrual cycles, GnRH pulse frequency increases during the late follicular phase, to culminate in the preovulatory LH surge, and slows during the luteal and early follicular phase, correlating with higher FSH levels, which permit follicle development (3, 29, 30). The physiological effects of varying GnRH pulse frequency on follicular development and ovulation have been successfully adapted for the treatment of idiopathic hypogonadotropic hypogonadism (31, 32). However, the mechanisms by which gonadotropes differentiate GnRH pulse frequencies and activate appropriate signal transduction pathways to differentially stimulate gonadotropin gene expression and release are still unknown. LßT2 cells, a murine gonadotrope-derived cell line, have been shown to possess many characteristics of a mature gonadotrope, including expression of the {alpha}, LHß, FSHß, and GnRHR genes (18, 19, 20, 21). Thus, LßT2 cells represent the best cell model currently available for the study of mechanisms regulating LH and FSH subunit gene expression. Using an in vitro perifusion system that allows us to replicate the action of pulsatile GnRH, we have investigated the effects of varying GnRH pulse frequencies on the stimulation of {alpha}, LHß, and FSHß gene promoters in LßT2 cells.

In vivo, in the rhesus monkey, high and low GnRH pulse frequencies lead to the preferential release of LH and FSH, respectively (4). Similarly, in the rat, high GnRH pulse frequencies are associated with an increase in {alpha} and LHß subunit transcription rates and mRNA levels and an increase in both LH and FSH secretion, whereas slower GnRH pulse frequencies result in increased FSHß gene transcription and mRNA levels as well as the maintenance of FSH release (5, 6, 27).

In primary rat pituitary cell culture, increases in {alpha} and LHß mRNA levels could be observed after at least 10 and 20 h of hourly pulsatile GnRH stimulation, respectively, whereas an increase in FSHß mRNA could be observed after 4 h of hourly GnRH pulses (33). In addition, after 24 h of stimulation, differential regulation of gonadotropin subunit gene expression by varying GnRH pulse frequencies was observed, with higher and lower GnRH pulse frequencies resulting in higher LHß and FSHß mRNA levels, respectively (7). On the other hand, although levels of {alpha}-subunit mRNA were increased by pulsatile GnRH, little difference was observed between frequencies (7).

In our study, when stimulated with pulsatile GnRH, LßT2 cells responded in a similar manner, with preferential stimulation of LHß and FSHß gene promoter activity by high (one pulse per 30 min) and low (one pulse per 2 h) GnRH pulse frequencies, respectively. In addition, although all genes studied were stimulated after 10 h of pulsatile GnRH treatment, the differential regulation of LHß and FSHß was most apparent after 20 h of stimulation. Again, the {alpha}-subunit gene promoter activity was not differentially regulated by varying GnRH pulse frequencies.

It has been reported previously that GnRH can markedly induce the transcription of its own receptor gene in {alpha}T3-1 cells (34). In addition, in primary rat pituitary cell culture, increases in GnRHR mRNA are GnRH frequency-dependent, with greater stimulation observed under conditions of high pulse frequency (7, 35). In our study, the effects of GnRH on the GnRHR gene promoter were modest, and no significant effects of GnRH pulse frequency were noted. The difference between our results and previous studies may be attributable to unique features of the LßT2 cell line or to differences in the experimental methodologies. Despite the modest increase in GnRHR-Luc activity, GnRH binding was up-regulated by pulsatile GnRH in a frequency-dependent fashion. Possible posttranscriptional effects of GnRH also need to be considered (36).

Interestingly, when LßT2 cells were stimulated with continuous GnRH in our perifusion system, all three gonadotropin subunit and GnRHR gene promoters were stimulated. These effects were particularly notable for {alpha} and LHß. These results were surprising, in view of previous observations in primary rat pituitary cells, in which LHß mRNA was unaffected and FSHß mRNA down-regulated by 12 h or 24 h of continuous GnRH stimulation (33, 37). These results raise the possibility that the receptor and/or postreceptor desensitization of GnRH responsiveness that has been observed in primary pituitary cells may not occur in LßT2 cells. The alternative possibility, that the gene promoter sequences present in our reporter constructs may not be sufficient to mediate down-regulatory effects of continuous GnRH, also needs to be considered.

In vivo, higher amplitude GnRH stimulation, as well as higher GnRH pulse frequency, increases LHß mRNA (38), whereas the increase in FSHß mRNA seems to be independent or inversely proportional to the amplitude of GnRH pulses (39, 40). Although the amplitude of GnRH pulses influences the stimulation of LHß and FSHß gene expression, equalization for the total dose of pulsatile GnRH, administered in vivo in the rat (27) or in vitro in perifused primary rat pituitary cell cultures (7), does not affect the differential regulation of the ß-subunits by GnRH pulse frequency. Similarly, in our perifusion system in LßT2 cells, when the cumulative doses of GnRH were equalized, preferential stimulation of LHß and FSHß gene promoters by high and low GnRH pulse frequencies, respectively, remained similar to the regulation observed with a fixed 10-nM GnRH concentration. These results indicate that in LßT2 cells, GnRH pulse frequency can directly regulate the expression of the LHß vs. the FSHß genes, independent of GnRH pulse amplitude.

There is increasing evidence that, in addition to GnRH, activin is a major stimulator of GnRHR and FSHß gene expression and FSH release (21, 41), with additional modulatory influences by inhibin and follistatin (an activin-binding protein; for review, see Refs. 42 and 43). Like primary pituitary cell culture, LßT2 cells have been shown to express both activin and activin receptors (21). In addition, in vivo in the rat, pituitary activin and follistatin mRNA levels are influenced by GnRH pulse frequency (40). Therefore, possible autocrine or paracrine effects of activin on FSHß gene promoter activity in our experimental system need to be considered. In our studies, we have focused solely on the effects of GnRH pulse frequency, in the absence of exogenous steroids, activin, inhibin, or follistatin. Though changes in activin and/or follistatin production by LßT2 cells, in response to changes in GnRH pulse frequency, are possible mediators of the effects on the gonadotropin subunit genes, our perifusion system would be expected to minimize such contributions by preventing the accumulation of paracrine factors in the perifusion chambers.

GnRHR number has been shown to fluctuate during the estrous cycle in the rat, with an increase in GnRHR observed between metestrus and early proestrus, and a decrease occurring at the time of the preovulatory LH surge (9). Also, in the rat pituitary, GnRHR number fluctuates with changes in GnRH pulse frequency, with the highest receptor concentration observed at a frequency of one GnRH pulse per 30 min, and lower GnRHR densities at higher or lower pulse frequencies (11). Thus, in addition to modulating GnRHR gene expression in perifused rat pituitary cells (7), pulsatile GnRH also controls the receptor number on the cell surface of gonadotropes, with the result that GnRHR levels are highest under conditions in which GnRH stimulation of LH is maximal, and lower under conditions optimal for FSH stimulation by pulsatile GnRH. To explore the role of GnRHR number, we previously demonstrated that cotransfection of increasing amounts of rGnRHR with gonadotropin gene reporter constructs in the heterologous rat somatolactotrope GH3 cell line led to the differential regulation of LHß and FSHß by GnRH, with LHß and FSHß being up-regulated to the greatest extent at high and low cell-surface rGnRHR densities, respectively (12). Based on these results, we hypothesized that the differential regulation of gonadotropin subunit gene promoter expression by frequency of pulsatile GnRH is, in part, regulated via changes in receptor number (44). By measuring GnRH binding in perifused LßT2 cells, we have shown that stimulation with GnRH at a frequency of one pulse per 30 min significantly increased the GnRHR density on the surface of LßT2 cells after 10 h of stimulation. Interestingly, the differential regulation of LHßLuc and FSHßLuc was most apparent after 20 h of pulsatile GnRH stimulation. Thus, an increase in GnRHR number precedes the pulse frequency-dependent preferential stimulation of LHß vs. FSHß gene promoters and may be a mediator of the differential regulation of gonadotropin subunit gene expression by different frequencies of pulsatile GnRH.

We therefore propose that, as summarized in Fig. 6Go (left panel), under conditions of stimulation by low GnRH pulse frequencies, GnRHR density on the surface of LßT2 cells is relatively low; under these conditions, GnRH stimulates all three gonadotropin subunit genes. When the frequency of GnRH pulses increases, the cell-surface GnRHR density increases. This, in turn, is followed by a greater increase in GnRH-stimulated LHß gene expression, but a reduction in the stimulation of FSHß gene expression in response to GnRH. To test our hypothesis (i.e. that the increase in GnRHR number resulting from higher GnRH pulse frequencies is responsible for the reduction in the level of stimulation of FSHß gene transcription by GnRH at such frequencies), we overexpressed rGnRHR in LßT2 cells and assessed the effects on LHß and FSHß responses to pulsatile GnRH [see Figs. 5Go and 6Go (right panel)]. When LßT2 cells, cotransfected with LHßLuc and increasing amounts of rGnRHR cDNA, were stimulated with continuous GnRH, induction of LHßLuc activity was significantly increased, whereas the response to continuous GnRH was significantly reduced in the case of FSHßLuc. Overexpression of rGnRHR in cells transfected with LHßLuc did not affect the responses to pulsatile GnRH, indicating that the stimulation of the LHß gene promoter is directly influenced by the GnRH pulse frequency. On the other hand, when cells were cotransfected with FSHßLuc and rGnRHR (4 µg), GnRH stimulation of the FSHß gene promoter was completely abolished at all pulse frequencies tested. Transfection of as little as 0.5 µg rGnRHR plasmid was sufficient to prevent the preferential stimulation of FSHßLuc at low GnRH pulse frequencies. As observed in GH3 cells (12), our results suggest that the regulation of FSHß gene promoter by GnRH is directly influenced by GnRHR number. Furthermore, receptor number is under the influence of the GnRH pulse frequency.



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  		Figure 6. Model of the differential regulation of gonadotropin subunits by pulsatile GnRH at varying frequencies in LßT2 cells. When LßT2 cells are stimulated by pulsatile GnRH, a low pulse frequency (one pulse every 2 h) stimulates transcription of all three gonadotropin subunit genes. At a higher GnRH pulse frequency (one pulse every 0.5 h), the cell-surface GnRHR concentration increases, resulting in a greater induction of the LHß gene by GnRH but less induction of the FSHß gene. As a result, GnRH stimulation of LHß gene expression increases, whereas stimulation of FSHß gene expression decreases. When rGnRHR is overexpressed in LßT2 cells (right panel), thereby mimicking a high frequency state, frequency-dependent stimulation of FSHß gene expression by pulsatile GnRH is abrogated.

 
Though our studies support a role for GnRH number in contributing to the differential responses to ranging frequencies of pulsatile GnRH, the cellular mechanisms for these effects remain to be elucidated. Our results indicate that an understanding of the effects of GnRH pulse frequency on GnRHR number and, in turn, of GnRHR number on GnRH-stimulated gonadotropin subunit gene expression may provide important insight into these mechanisms. Earlier theoretical work by Goldbeter (45, 46) suggested a role for receptor desensitization and resensitization. More recently, Krakauer et al. (47) have proposed three possible models whereby pulse frequency information is integrated into the GnRH signaling network.

In summary, in the present study, we have shown that LßT2 cells respond to varying GnRH pulse frequencies in a manner similar to that in primary gonadotropes and thus represent a suitable model for identifying the molecular mechanisms underlying the differential regulation of LHß and FSHß gene expression. We have also shown that cell-surface GnRHR density is a critical factor in mediating this differential regulation.


   Acknowledgments
 
We would like to thank Dr. Pamela Mellon for the LßT2 cells, Dr. P. Michael Conn for the radiolabeled 125I-Buserelin, and Dr. Errol Norwitz for critical reading of the manuscript.


   Footnotes
 
This work was supported by NICHD/NIH through cooperative agreement U54-HD-28138 as part of the Specialized Cooperative Centers Program in Reproduction Research, R01-HD-33001 and R01-HD-9938 (to U.B.K.) and The Lalor Foundation (to G.Y.B.).

Abbreviations: FBS, Fetal bovine serum; GnRHR, GnRH receptor; rGnRHR, rat GnRHR.

Received October 30, 2002.

Accepted for publication January 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631–633[Abstract/Free Full Text]
  2. Levine JE, Ramirez VD 1982 LHRH release during the rat estrous cycle and after ovariectomy, as estimated with push-pull cannulae. Endocrinology 111:1439–1448[Medline]
  3. Crowley Jr WF, Filicori M, Spratt DL, Santoro NF 1985 The physiology of gonadotropin-releasing hormone (GnRH) secretion in men and women. Recent Prog Horm Res 41:473–531
  4. Wildt L, Hausler A, Marshall G, Hutchison JS, Plant TM, Belchetz PE, Knobil E 1981 Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology 109:376–385[Abstract]
  5. Haisenleder DJ, Dalkin AC, Ortolano GA, Marshall JC, Shupnik MA 1991 A pulsatile gonadotropin-releasing hormone stimulus is required to increase transcription of the gonadotropin subunit genes: evidence for differential regulation of transcription by pulse frequency in vivo. Endocrinology 128:509–517[Abstract]
  6. Burger LL, Dalkin AC, Aylor KW, Haisenleder DJ, Marshall JC 2002 GnRH pulse frequency modulation of gonadotropin subunit gene transcription in normal gonadotropes—assessment by primary transcript assay provides evidence for roles of GnRH and follistatin. Endocrinology 143:3243–3249[Abstract/Free Full Text]
  7. Kaiser UB, Jakubowiak A, Steinberger A, Chin WW 1997 Differential effects of gonadotropin-releasing hormone (GnRH) pulse frequency on gonadotropin subunit and GnRH receptor messenger ribonucleic acid levels in vitro. Endocrinology 138:1224–1231[Abstract/Free Full Text]
  8. Loumaye E, Catt KJ 1982 Homologous regulation of gonadotropin-releasing hormone receptors in cultured pituitary cells. Science 215:983–985[Abstract/Free Full Text]
  9. Savoy-Moore RT, Schwartz NB, Duncan JA, Marshall JC 1980 Pituitary gonadotropin-releasing hormone receptors during the rat estrous cycle. Science 209:942–944[Abstract/Free Full Text]
  10. Kaiser UB, Conn PM, Chin WW 1997 Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev 18:46–70[Abstract/Free Full Text]
  11. Katt J, Duncan J, Herbon L, Barkan A, Marshall J 1985 The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors. Endocrinology 116:2113–2115[Abstract]
  12. Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM, Chin WW 1995 A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 92:12280–12284[Abstract/Free Full Text]
  13. Yokoi T, Ohmici M, Tasaka K, Kimura A, Kanda Y, Hayakawa J, Tahara M, Hisamoto K, Kurachi H, Murata Y 2000 Activation of the luteinizing hormone ß promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase. J Biol Chem 275:21639–21647[Abstract/Free Full Text]
  14. Ando H, Hew CL, Urano A 2001 Signal transduction pathways and transcription factors involved in the gonadotropin-releasing hormone-stimulated gonadotropin subunit gene expression. Comp Biochem Physiol B Biochem Mol Biol 129:525–532[CrossRef][Medline]
  15. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, Webster NJG 2002 Involvement of both Gq/11 and Gs proteins in gonadotropin-releasing hormone receptor-mediated signaling in LßT2 cells. J Biol Chem 277:32099–32108[Abstract/Free Full Text]
  16. Vasilyev VV, Pernasetti F, Rosenberg SB, Barsoum MJ, Austin DA, Webster NJG, Mellon PL 2002 Transcriptional activation of the ovine follicle-stimulating hormone-ß gene by gonadotropin-releasing hormone involves multiple signal transduction pathways. Endocrinology 143:1651–1659[Abstract/Free Full Text]
  17. Childs GV 1986 Functional ultrastructure of gonadotropes: a review. Curr Top Neuroendocrinol 7:49–97
  18. Turgeon JL, Kimura Y, Waring DW, Mellon PL 1996 Steroid and pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing hormone and GnRH receptor in a novel gonadotrope cell line. Mol Endocrinol 10:439–450[Abstract]
  19. Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122:3319–3329[Abstract]
  20. Graham KE, Nusser KD, Low MJ 1999 LßT2 gonadotroph cells secrete follicle-stimulating hormone in response to activin A. J Endocrinol 162:R1–R5
  21. Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang H-J, Miller WL, Mellon PL 2001 Cell-specific transcriptional regulation of FSH-beta by activin and GnRH in the LßT2 pituitary gonadotrope cell model. Endocrinology 142:2284–2295[Abstract/Free Full Text]
  22. Jakubowiak A, Janecki A, Steinberger A 1989 Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures. Biol Reprod 41:454–463[Abstract]
  23. Jameson JL, Powers AC, Gallagher GC, Habener JF 1989 Enhancer and promoter element interactions dictate cyclic AMP mediated and cell-specific expression of the glycoprotein hormone-{alpha} gene. Mol Endocrinol 3:763–772[CrossRef][Medline]
  24. Albarracin CT, Kaiser UB, Chin WW 1994 Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. Endocrinology 135:2300–2306[Abstract]
  25. Kaiser UB, Halvorson LM, Chen MT 2000 Sp1, SF-1, and Egr-1 binding sites form a tripartite GnRH response element in the LHß gene promoter: an integral role for SF-1. Mol Endocrinol 14:1235–1245[Abstract/Free Full Text]
  26. Marian J, Conn PM 1980 The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding. Life Sci 27:87–92[CrossRef][Medline]
  27. Dalkin AC, Haisenleder DJ, Ortolano GA, Ellis TR, Marshall JC 1989 The frequency of gonadotropin-releasing hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression. Endocrinology 125:917–924[Abstract]
  28. Chu G, Hayakawa H, Berg P 1987 Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15:1311–1326[Abstract/Free Full Text]
  29. Marshall JC, Kelch RP 1986 Gonadotropin-releasing hormone: role of pulsatile secretion in the regulation of reproduction. N Engl J Med 315:1459–1468[Medline]
  30. Reame N, Sauder SE, Kelch RP, Marshall JC 1984 Pulsatile gonadotropin secretion during the human menstrual cycle: evidence for altered frequency of gonadotropin-releasing hormone secretion. J Clin Endocrinol Metab 59:328–337[Abstract]
  31. Martin K, Santoro N, Hall J, Filicori M, Wierman, Crowley Jr WF 1990 Clinical Review 15: management of ovulatory disorders with pulsatile gonadotropin-releasing hormone. J Clin Endocrinol Metab 71:1081A–1081G
  32. Seminara SB, Beranova M, Oliveira LMB, Martin KA, Crowley Jr WF, Hall JE 2000 Successful use of pulsatile gonadotropin-releasing hormone (GnRH) for ovulation induction and pregnancy in a patient with GnRH receptor mutations. J Clin Endocrinol Metab 85:556–562[Abstract/Free Full Text]
  33. Jakubowiak A, Tong D, Janecki A, Sanborn BM, Steinberger A 1991 Pulsatile GnRH stimulation increases steady-state mRNA levels for FSHß, LHß, and {alpha} subunits in superfused pituitary cell cultures. Mol Cell Neurosci 2:277–283
  34. Norwitz ER, Cardona GR, Jeong K-H, Chin WW 1999 Identification and characterization of the gonadotropin-releasing hormone response elements in the mouse gonadotropin-releasing hormone receptor gene. J Biol Chem 274:867–880[Abstract/Free Full Text]
  35. Kaiser UB, Jakubowiak A, Steinberger A, Chin WW 1993 Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro. Endocrinology 133:931–934[Abstract]
  36. Sosnowski R, Mellon PL, Lawson MA 2000 Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone. Mol Endocrinol 14:1811–1819[Abstract/Free Full Text]
  37. Weiss J, Duca KA, Crowley Jr WF 1990 Gonadotropin-releasing hormone-induced stimulation and desensitization of free {alpha}-subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perifused rat pituitary cells. Endocrinology 127:2364–2371[Abstract]
  38. Haisenleder DJ, Katt JA, Ortolano GA, El-Gewely MR, Duncan JA, Dee C, Marshall JC 1988 Influence of gonadotropin-releasing hormone pulse amplitude, frequency, and treatment duration on the regulation of luteinizing hormone (LH) subunit messenger ribonucleic acids and LH secretion. Mol Endocrinol 2:338–343[CrossRef][Medline]
  39. Iliff-Sizemore SA, Ortolano GA, Haisenleder DJ, Dalkin AC, Krueger KA, Marshall JC 1990 Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude. Endocrinology 127:2876–2883[Abstract]
  40. Dalkin AC, Haisenleder DJ, Gilrain JT, Aylor K, Yasin M, Marshall JC 1999 Gonadotropin-releasing hormone regulation of gonadotropin subunit gene expression in female rats: action on follicle-stimulating hormone ß messenger ribonucleic acid (mRNA) involve differential expression of pituitary activin (ß-B) and follistatin mRNAs. Endocrinology 140:903–908[Abstract/Free Full Text]
  41. Norwitz ER, Xu S, Jeong K-H, Bedecarrats GY, Winebrenner LD, Chin WW, Kaiser UB 2002 Activin A augments GnRH-mediated transcriptional activation of the mouse GnRH receptor gene. Endocrinology 143:985–997[Abstract/Free Full Text]
  42. DePaolo LV, Bicsak TA, Erickson GF, Shimasaki S, Ling N 1991 Follistatin and activin: a potential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 198:500–512[Medline]
  43. Welt C, Sidis Y, Keutmann H, Schneyer A 2002 Activins, inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium. Exp Biol Med 227:724–752[Abstract/Free Full Text]
  44. Kaiser UB 1998 Molecular mechanisms of the regulation of gonadotropin gene expression by gonadotropin-releasing hormone. Mol Cells 8:647–656[Medline]
  45. Li Y, Goldbeter A 1989 Frequency specificity in intercellular communication. Influence of patterns of periodic signaling on target cell responsiveness. Biophys J 55:125–145[Abstract/Free Full Text]
  46. Goldbeter A 2002 Computational approaches to cellular rhythms. Nature 420:238–244[CrossRef][Medline]
  47. Krakauer DC, Page KM, Sealfon S 2002 Module dynamics of the GnRH signal transduction network. J Theor Biol 218:457–470[Medline]



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