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Regulation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene Expression in Sheep: Interaction of GnRH and Estradiol¹

Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Dr. Terry Nett, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523. E-mail: tnett{at}colostate.edu

	Abstract

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GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 µg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 µg im) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone.

Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHß and FSHß subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHß subunit (P < 0.005) and FSHß subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHß subunit mRNA (P < 0.01) but did not affect amounts of LHß subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHß subunit (P < 0.01) and FSHß subunit (P < 0.005).

From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell’s ability to respond to estradiol.

	Introduction

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BINDING OF GnRH to GnRH receptors on gonadotropes initiates a cascade of events necessary for the synthesis and secretion of LH and FSH. Regulation of GnRH receptors is important because the quantity of LH released in response to a physiological challenge with GnRH is dependent on the concentration of GnRH receptors on the plasma membrane of gonadotropes (1). In the ewe, GnRH is released in a pulsatile fashion from the hypothalamus (2) and is a homologous regulator of its own receptor. Stimulation of the pituitary gland by pulsatile GnRH is required to maintain steady-state concentrations of GnRH receptor messenger RNA (mRNA) and numbers of GnRH receptors. Depriving the pituitary gland of GnRH by hypothalamic-pituitary disconnection in ewes results in decreased numbers of GnRH receptors (3, 4, 5), which can be restored to control values by replacement of pulsatile GnRH (3, 6). Concentrations of mRNA encoding GnRH receptor are also increased by pulsatile GnRH (7). In contrast to the effects of pulsatile GnRH, continuous infusion of GnRH leads to a desensitization of gonadotropes (8, 9). This desensitization is characterized by a decrease in the number of GnRH receptors by approximately 50%. However, it is not known whether the reduction in numbers of GnRH receptors is caused by receptor internalization, mediated at the level of expression of the GnRH receptor gene, or both.

Another important endocrine regulator of ovine GnRH receptors is estradiol. Treatment with estradiol consistently increases GnRH receptor gene expression and numbers of GnRH receptors in vivo (4, 10, 11) and in cultured pituitary cells (12, 13, 14). In the ewe, estradiol stimulates expression of the GnRH receptor gene and numbers of GnRH receptors in the absence of GnRH (5), thus indicating that this effect of estradiol is exerted directly at the pituitary level. Because GnRH and estradiol both regulate concentrations of GnRH receptor, it is likely that the interaction of these hormones has important effects on the sensitivity of gonadotropes and, ultimately, the regulation of gonadotropin secretion. However, the interaction of GnRH and estradiol in regulating GnRH receptor is not well understood. Because estradiol affects its target cells via an intracellular receptor, whereas GnRH binds to receptors on the plasma membrane, it is assumed that these hormones influence GnRH receptor synthesis by different cellular mechanisms. In the present study, the potential for estradiol to override the negative effect of continuous infusion of GnRH on expression of GnRH receptors was addressed. Our objectives were to: 1) characterize changes in steady-state concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors following continuous infusion of GnRH; and 2) measure effects of estradiol on concentrations of GnRH receptor mRNA and numbers of GnRH receptors after desensitization of the anterior pituitary gland by GnRH.

	Materials and Methods

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Animals and treatments
Sexually mature, western range ewes that had been ovariectomized for at least 6 weeks were used in this experiment. Twenty ewes were randomly assigned to 4 treatment groups so that each treatment group contained 5 ewes. Two groups (10 ewes) were administered saline and 2 groups (10 ewes) were administered GnRH (10 µg/h) continuously for 136 h via Alzet osmotic minipumps (2ML4; Alza Corp., Palo Alto, CA). Minipumps were placed sc in the back of the neck at time 0. At 124 h, 5 ewes receiving saline and 5 ewes receiving GnRH were given an intramuscular injection of 25 µg estradiol-17ß (estradiol; Sigma Chemical Co., St. Louis, MO) dissolved in safflower oil. At 136 h (12 h after treatment with estradiol), anterior pituitary glands were collected from all 20 ewes after anesthesia with sodium pentobarbital (29 mg/kg BW, iv) and exsanguination. Pituitary glands were divided midsagitally, frozen immediately on solid CO₂, and stored at -80 C. Jugular blood samples were collected every 4 h for 120 h and then every 30 min for the last 16 h of the experiment. All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee, and were in compliance with NIH guidelines.

Analysis of mRNA
Polyadenylated (poly A+) RNA was prepared from one half of each pituitary gland (15). Integrity of this RNA was verified with Northern blot analysis of mRNA encoding GnRH receptor, and steady-state concentrations of mRNA encoding GnRH receptor and gonadotropin subunits were quantified by slot blot analysis as described previously (11). Briefly, 1.5 µg of poly A+ RNA were immobilized in duplicate on a nylon membrane (Hybond; Amersham, Arlington Heights, IL), cross-linked by exposure to UV light (Stratagene, La Jolla, CA), and probed with complementary DNAs for ovine GnRH receptor (11), bovine LHß subunit (16), bovine FSHß subunit (17), and bovine -subunit (18). After hybridization, the final washes were in 0.5 x SSC (750 mM NaCl and 75 mM Na citrate), 0.1% SDS at 65 C. The nylon membrane was exposed to Hyperfilm MP (Amersham) for 4 days (GnRH receptor and FSHß subunit), 7.5 h (LHß-subunit), or 8 h (-subunit). After quantities of GnRH receptor and gonadotropin subunit mRNAs were analyzed, the membrane was stripped of radiolabeled complementary DNA and reprobed with radiolabeled dT (18 mer) as described by Juengel et al. (19). Autoradiographs were analyzed with the NIH 1.52 Image Analysis Program. Concentrations of mRNA were normalized to the amount of poly A+ RNA in each sample and expressed as percent of control values.

Hormone and receptor assays
Circulating concentrations of LH (20) and FSH (21) were determined by RIA using NIH-oLH-S24 and NIH-oFSH-S12 as reference preparations, respectively. Mean limit of detection, intraassay coefficient of variation (CV), and interassay CV were 127 pg/ml, 11.4% and 11.4%, respectively, for the LH assay and 6.0 ng/ml, 10.3%, and 10.8%, respectively, for the FSH assay.

Numbers of GnRH receptors were quantified using a standard curve technique (8). The concentration of GnRH receptors in a pool of bovine pituitary membranes was determined by Scatchard analysis. A standard curve was generated by incubating increasing quantities of the membrane pool with 0.2 nM [¹²⁵I]D-Ala⁶-GnRH-Pro⁹-ethyl-amide ([¹²⁵I]D-Ala⁶). Partially purified membranes prepared from each experimental pituitary gland were incubated with 0.2 nM [¹²⁵I]D-Ala⁶ in assay buffer [10 mM Tris base, 1 mM CaCl₂, 0.1% BSA] for 4 h on ice. At the end of the incubation, tubes were centrifuged immediately after the addition of 3 ml of ice-cold assay buffer. Amounts of specifically bound [¹²⁵I]D-Ala⁶ in the pellets were compared with the standard curve, and the number of GnRH receptors in each sample was calculated. All samples were quantified in a single assay.

Statistical analyses
Differences in serum concentrations of LH and FSH were determined by ANOVA for repeated measures. Steady-state concentrations of mRNAs for GnRH receptor, gonadotropin subunits, and numbers of GnRH receptors were analyzed using one-way ANOVA. Heterogenous variances were corrected by log-transformation of data before ANOVA. When significant effects of treatment were observed, means were separated using least significant differences. Pearson correlation coefficient (r) was computed to describe the relationship between concentrations of mRNA for GnRH receptor and numbers of GnRH receptors. All analyses were performed using SAS (22). Data are presented as mean ± SEM.

	Results

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Effects of GnRH and estradiol on serum concentrations of gonadotropins
Before administration of GnRH, the mean concentration of LH in serum was 4.2 ± 0.9 ng/ml. Treatment with GnRH caused serum concentrations of LH to rise, and the maximal mean concentration (71.8 ± 16.7 ng/ml) was observed 4 h after insertion of minipumps. Serum concentrations of LH then decreased in GnRH-treated ewes and were not different from those in saline-treated controls from 8 h to 124 h of the experiment (P > 0.6; data not shown). In saline-treated ewes, injection of estradiol at 124 h was followed by an increase in LH secretion. Compared with ewes treated only with saline, concentrations of LH in estradiol-treated ewes were elevated (P < 0.05) from 133 h until pituitary collection at 136 h (Fig. 1A). There was also an increase in LH secretion in GnRH-treated ewes in response to estradiol, but this increase occurred sooner; serum concentrations of LH in this group were higher (P < 0.05) than those in ewes treated only with GnRH from 130 until pituitary collection at 136 h (Fig. 1A).

View larger version (28K): [in this window] [in a new window]   Figure 1. Mean concentrations of LH (A) and FSH (B) in serum collected from ovariectomized ewes at 30-min intervals during the 12-h period preceding pituitary collection. Ewes received either saline (control, E; 10 ewes total) or GnRH (GnRH, GnRH + E; 10 ewes total) continuously for 136 h. As shown by the arrows, a single injection of estradiol was administered at 124 h to 5 ewes pretreated with saline (E) and 5 ewes pretreated with GnRH (GnRH + E). Estradiol caused serum concentrations of LH to increase sooner (P < 0.05) in GnRH-treated ewes compared with saline-treated ewes. For LH concentrations, SEM averaged 0.4 (range = 0.1–1.5) for the control group; 0.8 (range = 0.4–1.5) for the GnRH group; 3.1 (range = 0.6–20.4) for the E group; and 2.2 (range = 0.1–8.2) for the GnRH + E group. For FSH concentrations, SEM averaged 56.3 (range = 34.3–104.2) for the control group; 56.3 (range = 47.8–75.3) for the GnRH group; 114.1 (range = 57.5–159.1) for the E group; and 47.4 (range = 28.1–104.0) for the GnRH + E group.

GnRH receptor mRNA and GnRH receptor concentrations
Figure 2 illustrates mean concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. Relative to control values, amounts of GnRH receptor mRNA and concentrations of GnRH receptor in ewes treated continuously with GnRH decreased by 48% (P < 0.01) and 69% (P < 0.05), respectively. Treatment with estradiol increased the amount of mRNA encoding GnRH receptor and concentrations of GnRH receptor by 1.6-fold (P < 0.05) and 1.9-fold (P < 0.01), respectively. Concentrations of GnRH receptor mRNA in ewes treated with GnRH and estradiol were similar to those in controls but elevated 2.2-fold relative to amounts in ewes treated only with GnRH (P < 0.001). Similarly, numbers of GnRH receptors in ewes treated with GnRH and estradiol were elevated by 4.6-fold relative to ewes treated only with GnRH (P < 0.005). Concentrations of GnRH receptor were correlated to amounts of GnRH receptor mRNA (r = 0.78, P < 0.0001).

View larger version (18K): [in this window] [in a new window]   Figure 2. Mean relative concentrations of GnRH receptor mRNA and GnRH receptors in anterior pituitary tissues of ovariectomized ewes (n = 5 per group). Ewes were treated continuously for 136 h with either saline (Control, E) or GnRH (GnRH, GnRH + E). At 124 h, 5 ewes pretreated with saline (E) and 5 ewes pretreated with GnRH (GnRH + E) received a single injection of estradiol. Significant differences among means (P < 0.05) are indicated by lowercase letters for GnRH receptor mRNA and uppercase letters for GnRH receptors.

View larger version (26K): [in this window] [in a new window]   Figure 3. Mean relative concentrations of mRNA encoding LHß subunit, FSHß subunit, and -subunit in anterior pituitary tissues of ovariectomized ewes (n = 5 per group). Ewes were treated continuously for 136 h with either saline (Control, E) or GnRH (GnRH, GnRH + E). At 124 h, 5 ewes pretreated with saline (E) and 5 ewes pretreated with GnRH (GnRH + E) received a single injection of estradiol. Significant differences among means (P < 0.05) are indicated by lowercase letters for LHß subunit mRNA and uppercase letters for FSHß subunit. Amounts of subunit mRNA were similar among groups.

	Discussion

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Pituitary desensitization induced by continuous treatment with GnRH is marked by decreased tissue concentrations of GnRH receptors (8, 9, 23). To determine whether this reduction is mediated by decreased expression of the GnRH receptor gene, we measured steady-state concentrations of mRNA encoding GnRH receptor and found that amounts of GnRH receptor mRNA were decreased by continuous GnRH treatment. Reduced GnRH receptor gene expression has also been observed following treatment of sheep with synthetic GnRH agonists (14, 24). However, these agonists are much more potent than GnRH, and the present study is the first to characterize GnRH receptor gene expression following continuous treatment with native GnRH. GnRH receptors are internalized after binding GnRH (25, 26), and the reduction in numbers of GnRH receptors caused by continuous GnRH treatment likely reflects this internalization. However, evidence that amounts of mRNA encoding GnRH receptor also decrease following continuous treatment with GnRH indicates that desensitization of gonadotropes is not due solely to GnRH receptor internalization, but is also mediated by decreased expression of the GnRH receptor gene.

In accordance with previous reports, treatment of ovariectomized ewes with estradiol increased concentrations of GnRH receptor (4, 10, 27) and amounts of GnRH receptor mRNA (5, 11, 28). Concentrations of GnRH receptor mRNA and GnRH receptors in ewes pretreated with GnRH were not increased by estradiol compared with saline-treated controls but were significantly elevated over values in ewes treated only with GnRH. These observations indicate that continuous delivery of GnRH resulted in a new, lower baseline of GnRH receptor gene expression and numbers of GnRH receptors. From this new baseline, treatment with estradiol was still effective in stimulating GnRH receptor gene expression and numbers of GnRH receptors despite desensitization of the pituitary gland to GnRH. Increased numbers of GnRH receptors have been observed previously in estradiol-treated ewes following infusion of GnRH (29), and the present study extends those observations by showing that estradiol also stimulates GnRH receptor gene expression in the face of pituitary desensitization by GnRH. Similarly, treatment with estradiol increased amounts of GnRH receptor mRNA and restored GnRH receptor concentrations in ewes chronically treated with GnRH agonist (24). Because estradiol is effective in overriding negative effects of continuous GnRH on GnRH receptor gene expression and numbers of GnRH receptors, we suggest that estradiol increases synthesis of GnRH receptors faster than continuous infusion of GnRH causes receptor internalization, resulting in a net increase in receptor numbers.

As we have reported previously (29), increased secretion of LH in response to treatment with estradiol occurred sooner in GnRH-treated ewes compared with saline-treated ewes. In both saline- and GnRH-treated ewes, estradiol stimulated GnRH receptor gene expression and (presumably) de novo synthesis of GnRH receptors. Once inserted into the plasma membrane of gonadotropes, these receptors could immediately bind the excess exogenous GnRH present in GnRH-treated ewes and initiate LH secretion. In contrast, newly synthesized receptors in saline-treated ewes did not have immediate access to elevated concentrations of GnRH because of the time required for estradiol to stimulate endogenous GnRH secretion from the hypothalamus. Therefore, the more rapid response to estradiol following treatment with GnRH can be explained by the fact that the pituitary glands in GnRH-treated ewes were exposed to high concentrations of exogenous GnRH at the time of estradiol injection.

In addition to effects on GnRH receptor gene expression, treatment with GnRH and/or estradiol also affected amounts of mRNA encoding the gonadotropin subunits. In a recent study, we found that quantities of LHß and FSHß subunit mRNA, but not -subunit mRNA, were reduced in cows by continuous infusion of GnRH (23). Similarly, chronic treatment of ewes with GnRH agonist decreased amounts of LHß subunit mRNA (24). Therefore, decreased expression of genes encoding LHß and FSHß subunit following continuous treatment with GnRH in the present study agrees well with previous results in domestic ruminants. Treatment with estradiol decreased expression of the FSHß subunit gene but did not affect quantities of subunit mRNA or LHß subunit mRNA. Suppressive effects of estradiol on FSHß subunit gene expression in ewes have been observed previously (30, 31, 32). Although decreased expression of and LHß subunit genes after acute treatment of ovariectomized ewes has been reported (32), Hamernik and Nett (30) found that expression of these genes was reduced during the decreasing portion of the LH surge but not the increasing portion. In the present study, serum concentrations of LH were rising in estradiol-treated ewes at the time of pituitary collection. Therefore, the lack of effect of estradiol on quantities of and LHß subunit mRNA can be explained by the fact that tissues were collected during the period of positive feedback by estradiol. In the absence of an effect of estradiol on LHß subunit gene expression, mean quantities of LHß subunit mRNA were similar in ewes treated with GnRH and those treated with GnRH and estradiol. The greater magnitude of decrease in amounts of FSHß subunit mRNA in ewes that received GnRH and estradiol likely reflects additive negative regulation by GnRH and estradiol.

Inhibition of the reproductive axis with GnRH agonists is used in clinical settings to treat several diseases including endometriosis, uterine fibroid tumors, precocious puberty, and prostate cancer (33). Unfortunately, long-term administration of GnRH agonists has serious side effects such as accelerated bone loss and menopausal-like symptoms. To combat these side effects, estrogen replacement therapy is often prescribed (33, 34). Results of the present study indicate that although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell’s ability to respond to estradiol. Consequently, it is possible that therapy with exogenous estradiol may undermine the desired effects of continuous treatment with GnRH analogs. This interaction of estradiol and GnRH on GnRH receptors may have important implications and should be considered in current clinical applications of GnRH agonists and estrogen replacement therapy.

	Acknowledgments

 
The authors wish to thank K. Rollyson, D. Haworth, M. Allen, J. Juengel, P. Silva, E. McIntush, M. Royals and M. Gallegos for their help collecting blood samples. We also thank T. Garner and X. Sha for their technical assistance.

	Footnotes

 
¹ A preliminary report has appeared in the abstracts of the 30th Annual Meeting of The Society for the Study of Reproduction. This work was supported by USDA Grant 95–37203-1997 (to T.M.N.).

² Supported by the United States Air Force.

Received June 11, 1998.

	References

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