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Endocrinology, doi:10.1210/en.2004-1562
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Endocrinology Vol. 146, No. 6 2760-2765
Copyright © 2005 by The Endocrine Society

Estrogen and Estrogen Receptor-ß (ERß)-Selective Ligands Induce Galanin Expression within Gonadotropin Hormone-Releasing Hormone-Immunoreactive Neurons in the Female Rat Brain

Istvan Merchenthaler, Gloria E. Hoffman and Malcolm V. Lane

Women’s Health Research Institute (I.M., M.L.V.), Wyeth Research, Collegeville, Pennsylvania 19426; and Department of Anatomy and Neurobiology (G.E.H.), University of Maryland, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Istvan Merchenthaler, M.D., DSc, Department of Epidemiology & Preventive Medicine, University of Maryland, Baltimore, 10 South Pine Street, MSTF Room 900F, Baltimore, Maryland 21201. E-mail: imerchem{at}epi.umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Among the many factors that integrate the activity of the GnRH neuronal system, estrogens play the most important role. In females, estrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurons to generate the preovulatory LH surge and ovulation. Until recently, the belief has been that the GnRH neurons do not contain estrogen receptors (ERs) and that the action of estrogen upon GnRH neurons is indirect involving several, estrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurons. Based on our recent findings that GnRH neurons of the female rat coexpress galanin, that galanin is a potent GnRH-releasing peptide, and that ERß is present in GnRH neurons, we have evaluated the effect of 17ß-estradiol and two ERß-selective agonists (WAY-200070, WAY-166818) on the expression of galanin within GnRH neurons. By combining immunocytochemistry for GnRH and in situ hybridization histochemistry for galanin, we demonstrate that 17ß-estradiol (20 µg/kg, sc) stimulates galanin expression within GnRH-immunoreactive neurons in a time-dependent manner. A significant increase was observed 2 h after its administration to ovariectomized rats. However, a more robust expression required 3-d treatment regimen. Treatment with the ß-selective ligands resulted in similar observations, although no statistical analysis is available for the 2 hr survival. These observations strongly suggest that estrogen and the ERß-selective ligands stimulate galanin expression within GnRH neurons via ERß, although an indirect mechanism via interneurons still cannot be ruled out.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH, ALSO CALLED LH-releasing hormone, represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. The release of GnRH into the hypophyseal portal circulation in the median eminence is pulsatile. Cyclic fluctuations in the amplitude and frequency of GnRH release, combined with changes in the secretory capacity of the pituitary gonadotrophs, are responsible for the generation of LH secretion profile observed over the course of the ovarian cycle. The pulsatile pattern of GnRH release is critical for normal ovarian function and, in the female; the massive increase in GnRH release generates the LH surge necessary for ovulation.

Among many factors that integrate the activity of the GnRH neuronal system, estrogens play the most important role. In the male, and for the greater part of the ovarian cycle in females, estrogens exhibit a negative feedback action on LH secretion. In the female, however, estrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurons to generate the preovulatory LH surge and subsequent ovulation [see Herbison 1998 for a recent review (1)]. Despite the critical effect of estrogen in ovulation, up until 2000 the scientific community was exploring the mechanism of estrogen action based on the assumption that the GnRH neurons themselves do not express estrogen receptors (ERs) (2). Therefore, it was postulated that the action of estrogen upon GnRH neurons was indirect involving several estrogen-sensitive neurotransmitter- and neuropeptide-synthesizing systems and glial cells that trans-synaptically regulated the activity of the GnRH neurons [see Herbison 1998 for a recent review (1)].

However, we have recently observed that GnRH neurons of the rat express ERß mRNA, bind 125I-estradiol (3), and contain ERß but not ER{alpha} immunoreactivity (4). In ovariectomized (OVX) rats, approximately 70% of GnRH-immunoreactive (GnRH-i) neurons contained ERß transcripts and 10% accumulated radioactively labeled estradiol (3). Soon after this original report, the presence of ERß and the lack of ER{alpha} within GnRH neurons of rats were confirmed by others (5).

The present studies aimed at providing evidence for the functionality of ERß expressed within GnRH neurons. The low level of binding in these neurons suggests that the expression and functional capacity of ERß protein in GnRH neurons is low, time-dependent, or that estrogens may also elicit their effects via nonconventional (nongenomic mechanisms) (6). Therefore, we selected galanin, a peptide coexpressed with GnRH (7) in a sexually dimorphic manner (8) (9) and dramatically up-regulated by estrogen (10, 11, 12, 13) as an indicator of direct, genomic estrogen action within GnRH neurons.

Our observations show that 17ß-estradiol induces the expression of galanin mRNA in GnRH-i neurons within 2 h after its administration to OVX rats. Chronic treatment (3 d) with 17ß-estradiol and the ERß-selective compounds resulted in a more robust stimulation. These data, therefore, suggest that estrogen, via ERß, may directly regulate GnRH neuronal activity, although indirect actions via interneurons cannot be ruled out.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The ERß-selective ligands (WAY-166818 and WAY-200070) (14) were synthesized by Wyeth Research (Collegeville, PA). Competitive solid phase radioligand binding assay studies indicate that WAY-166818 (no. 81 in Ref. 14) is 55 times more selective for ERß then ER{alpha} in the rat. It is nonuterotropic in the mouse at a dose of 50 mg/kg when administered sc. It is a full agonist on ERß, as measured by its ability to regulate metallothionein-II mRNA in SAOS-2 cells (Heather Harris, Wyeth Research, personal communications; see also Ref. 15). WAY-166818 up-regulates PR mRNA expression in the preoptic area where both ER{alpha} and ERß are expressed, but not the ventromedial nucleus where ER{alpha} is expressed primarily, at a dose of 10 mg/kg sc (Merchenthaler, I., and M. V. Lane, unpublished observation) indicating that WAY-166818 penetrates the blood-brain barrier. Competitive solid phase radioligand binding assay studies indicate that WAY-200070 (no. 92 in Ref. 14) is >100 x more selective for ERß over ER{alpha}. It is nonuterotrophic in both the rat and mouse at sc doses of approximately 50 mg/kg. It does not prevent ovariectomy-induced bone loss at a dose of 10 mg/kg sc. WAY-200070 prevents vasomotor instability in the morphine-addicted model of hot flush at a dose of 15 mg/kg (14). It also up-regulates PR mRNA expression in the preoptic area but not the ventromedial nucleus, at a sc dose of 10 mg/kg (Merchenthaler, I., and M. V. Lane, unpublished observations).

Animals and treatments (coexpression of GnRH and galanin)
Two-month-old, female Sprague Dawley rats (Taconic, Germantown, NY) were OVX by the supplier, shipped to Wyeth, and housed in the animal care facility with a 12-h light, 12-h dark photoperiod and free access to tap water and rodent casein-based diet. On post-ovariectomy d 10, the animals were given a sc injection of either 17ß-estradiol (20 µg/kg; four animals), the ERß-selective ligand (WAY-166818 and WAY-200070) at 20 mg/kg (two animals per compound) or vehicle (1:1 ratio of PBS/dimethylsulfoxide; three animals) in the dorsal cervical region. Because previous studies showed that PR induction in the preoptic area required a dose of 20 mg/kg and a similar dose prevented thermoregulatory dysfunction in the hot flush model (14, 16, 17) without providing uterotropic activity, a similar dose was used in the present experiments.

The animals were overanesthetized with 0.1 ml/100 g body weight of KAX (Ketaset, 100 mg/ ml; Acepromazine, 10 mg/ ml; and Xylazine, 100 mg/ml) and transcardially perfused with 1% and then 4% paraformaldehyde 2.0 or 6.0 h after compound treatment. A second set of animals was treated with either17ß-estradiol (20 µg/kg; four animals), the ERß-selective ligands (20 mg/kg; six animals per compound) or vehicle (1:1 ratio of PBS/ dimethylsulfoxide; three animals) for a consecutive 3 d. The animals were euthanized 6 h after the last injection as described above. The studies described in this paper were reviewed and approved by the Collegeville Animal Care and Use Committee.

Immunocytochemistry (ICC)/in situ hybridization histochemistry (ISHH)
Thirty-micrometer-thick sections were collected in cold PBS containing 3000 U/ml of Heparin (Sigma, St. Louis, MO; catalog no. H3393), transferred to metal mesh baskets (Ted Pella, Redding, CA; catalog no. 4592), rinsed in PBS, and then processed for ISHH with the rehydration steps ending in PBS (18). The processed sections were transferred to 24-well cell culture plates (two to three sections/ well) and hybridized with 300 µl of a galanin probe (19) (35S-uridine triphosphate-labeled galanin; 4 x 106 DPM/ probe/well) in a 50% formamide hybridization mix and incubated overnight at 55 C. The sections were then transferred back to baskets, rinsed [2x standard saline citrate (SSC)/10 mM dithiothreitol], treated with ribonuclease A (50 µg/ml) and washed at 67 C in 0.1x SSC to remove nonspecific label. The sections were transferred to PBS, treated with 5% normal donkey serum, and incubated overnight at 4 C with an GnRH antiserum (a gift from Dr. William Wetsel, Duke University, Durham, NC) diluted 1:5000 in 1% normal donkey serum (20). The sections were washed, incubated with biotinylated donkey antirabbit serum (Jackson ImmunoResearch, West Grove, PA; 1:1500) and the immunoreactivity visualized with a standard ABC (Vector Elite kit, Burlingame CA) method. After the diaminobenzidine (DAB) reaction, the sections were transferred to PBS, mounted on gelatin-coated slides and dipped in NTB-2 (diluted 1:1 with water); air-dried, and stored at 4 C in light-tight desiccator slide boxes. After 4–5 wk of exposure, the slides were developed and coverslipped.

Controls
A number of experimental controls were conducted to validate the methods used in these studies. These included 1) tissue sections carried through the ISHH process, but not hybridized with radiolabeled probe, to demonstrate that the prehybridization process does not interfere with the immunocytochemical method; 2) tissue sections carried through the immunocytochemical process, not hybridized with the radioactive probe but exposed to photographic emulsion to demonstrate that DAB does not induce positive chemography; 3) tissue sections stained with preabsorbed GnRH antiserum; and 4) hybridization with sense probe to galanin.

Evaluation, statistical analysis
The cellular distribution of silver grains, representing galanin mRNA, over DAB-labeled GnRH-i neurons was assessed with high magnification bright-field microscopy to determine if GnRH-i neurons also contained galanin mRNA. The GnRH-i neurons were considered labeled if the number of silver grains localized over the immunoreactive cell was greater than three times the concentration of grains seen over a similar area of neuropil in the lateral preoptic area. The evaluation was based on manual counting of silver grains. The total number of GnRH-i neurons and the number of GnRH-i neurons that concentrated silver grains were also determined manually (visual counts using an eyepiece grid) in 20 sections/ brain at the level of the diagonal band of Broca/medial preoptic area (21) to determine the percentage of GnRH-i neurons that also contained galanin mRNA. The labeling index for the preoptic area/diagonal band of Broca region was computed by dividing the number of double-labeled (galanin mRNA expressing/GnRH-i) cells by the total number of GnRH-i cells. An ANOVA was performed on transformed data {sqrt[arcsin(labeling index)]}, followed by a Tukey-Kramer test of all pairwise treatment comparisons, to determine which treatments were significantly different from the others. A significance level of 0.05 was used for all analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estradiol increases galanin gene expression directly in GnRH-i neurons
The combination of immunocytochemistry and ISHH increased slightly the sensitivity of immunocytochemistry. As a result of removing membrane lipids with rigorous organic solvents for ISHH, the penetration of Igs was elevated resulting in strong signal of GnRH immunocytochemistry (Fig. 1Go, A vs. B). Both GnRH-i cell bodies and processes were strongly stained. Our specificity tests also showed that the DAB chromogen did not induce positive chemography that would have resulted in nonspecific signal (silver grains) for ISHH.



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  		FIG. 1. Immunocytochemical representation of GnRH-i neurons in the preoptic area/diagonal band of Broca region of female rats. The GnRH-i neurons occupy an inverted V-shape area around OVLT (arrow). B, GnRH-i neurons in the same area as shown in panel A taken from a section exposed to ISHH for galanin. Small arrows indicate neurons accumulating silver grains representing galanin mRNA. The intensity of GnRH-i neurons is as intense as of those shown in panel A. C, GnRH immunoreactivity (brown DAB precipitate) and galanin mRNA expression (silver grains) in neurons around the OVLT in OVX rats treated with 17ß-estradiol for 2 h. Arrowhead indicates an GnRH-i neuron expressing galanin mRNA. Open arrow labels an GnRH-i perikaryon not expressing galanin. Galanin expression in GnRH-i perikarya in OVX rats treated with either 17ß-estradiol for 2 h (D) or 3 d (G); with WAY-200070 for 2 h (E) or 3 d (H); or with WAY-166818 for 2 h (F) or 3 d (I). For details, see the text. Magnifications: x200 (A and B); x600 (D and E); x800 (C and F–L).

 
The distribution of GnRH-i neurons in the forebrain followed an inverted V-shape pattern as previously described (22). The vast majority of GnRH-i neurons that expressed galanin were concentrated around the tip of the organum vasculosum of the lamina terminalis (OVLT; Fig. 1Go, A and B). In OXX rats, only 5.4 ± 0.3% of the GnRH neurons expressed galanin mRNA (Fig. 2Go). Estradiol dramatically and time-dependently increased the expression of galanin within GnRH neurons; both the number of GnRH cells coexpressing galanin and the intensity of galanin signal increased with duration of exposure. The shortest exposure to estradiol that resulted in significantly different effect on galanin gene expression (23.55%) was observed 2 h after a single injection of 17ß-estradiol (Figs. 1DGo and 2Go). By 6 h, the number of coexpressing cells further increased and reached 32.1% (Fig. 2Go, D–F). Although only two animals were used for evaluating the effects of ERß-selective compounds 2 h after their administration and therefore, no statistical analysis was performed, an increase in the number of double-labeled cells was similar to the effect of estrogen (Fig. 1Go, E and F).



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  		FIG. 2. Histograms showing the percentage (mean ± SEM) of GnRH-i perikarya coexpressing galanin mRNA on OVX rats or OVX rats treated with 17ß-estradiol for 2 h, 6 h, or 3 d or with the ERß-selective compounds (WAY-200070 and WAY-166818) for 3 d. Sections from the OVX group treated with 17ß-estradiol for 2 or 6 h were pooled from brains of six animals; therefore, no error bars present in these graphs. *, P < 0.05 vs. OVX; sqrt[arcsin(labeling index)] followed by the Tukey-Kramer test.

 
The effect of chronic (3 d) treatment with estrogen or ERß-selective compounds on galanin mRNA expression in GnRH-i neurons
The level of galanin mRNA expression in the preoptic area and individual GnRH-i neurons was significantly higher after 3 d treatment with estrogen (Fig. 1GGo or the ERß-selective compounds (Fig. 1Go, H and I) compared with the effect of a single injection. The vast majority of double-labeled cells were located around the tip of the OVLT in both acutely and chronically treated animals (Fig. 1Go, A and B). Both the smooth and varicose type of GnRH neurons expressed galanin (not shown). The degree of galanin expression in GnRH neurons was 39.2 ± 9.1% in estrogen-treated animals, 16.1 ± 4.7% in WAY-166818-treated animals, and 15.7 ± 6.7% in WAY-200070-treated rats (Fig. 2Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies indicate that estrogen and the ERß-selective ligands induced galanin expression within GnRH-i neurons. The percentage of double-labeled cells varied between 23% and 40% depending on the exposure time and was lower then reported previously (62%) in proestrus rats (7, 8). The discrepancy is probably due to the different techniques employed and the different animal models used. In the original studies (7, 8), single-labeled immunocytochemistry was used to count galanin-i perikarya with GnRH phenotype (fusiform shape) as markers of double-labeled cells. In the present studies, a combination of immunocytochemistry and in situ hybridization histochemistry was used to count DAB-stained, brown GnRH-i that contained silver grains over the cytoplasm. Although the combination of these techniques did not seem to affect the sensitivity of one another, a more thorough quantitative analysis could only provide a definite answer to this question. In the original studies (7, 8), females and male rats were treated with colchicine, a chemical that not only blocks axoplasmic transport and therefore detectability within perikarya, but it may also increase expression of galanin (23). Another factor that could contribute to the differences in the percentages of double-labeled cells is that the in situ hybridization in the present studies used relatively thick sections for hybridization. In doing so, only grains over neurons near the surface can definitively identify cells expressing galanin mRNA. Thus, the low proportions of double-labeled cells may represent the lower proportion of cells near the surface compared with the total cells detected with immunocytochemistry.

The findings that GnRH neurons, although sensitive to estrogens, do not accumulate 3H-estradiol (2) made a major impact during the last 2 decades in our research activity aimed at exploring the mechanisms of estrogen-regulated GnRH neuronal activity. The lack of ERs was reinforced by early immunocytochemical studies using ER{alpha}-selective antisera, such as the H222 and 1D5, and ISHH using ER{alpha}-specific probes (for a review see Ref. 24). Therefore, the recently published observations that GnRH-i neurons of the rat contain ERß mRNA (3) and ERß immunoreactivity (4) and bind 125I- estrogen (3) was surprising and raised the possibility that the GnRH neurons are sensitive directly to estrogen. Because the affinity of 17ß-estradiol to ER{alpha} and ERß is comparable (25), why did those early studies (2) fail to detect 3H-estrogen binding in GnRH-i neurons? The most plausible explanation is that the sensitivity of the in vivo autoradiography using 3H-estrogen was much lower than the in vivo autoradiography we used in our previous studies using 125I-labeled ligand (11-iodovinyl-17ß-methoxy-estradiol; Ref. 3) and thus, the new ligand provided stronger signal even within a shorter exposure time. However, the lack of classical nuclear accumulation of radioactively labeled ligand in GnRH neurons reported by those earlier studies, did not rule out the presence and functionality of membrane or membrane-associated estrogen receptors. In fact, a nongenomic action of estrogen would explain its biological action without being detectable in the nucleus. Indeed, Abraham et al. (6) have reported recently that estrogen increased rapidly the phosphorylation of cAMP response element binding protein in GnRH neurons in vivo and in vitro in a time- and dose-dependent manner. Furthermore, these authors, using ER{alpha} knockout and ERß knockout mice, also showed that the rapid actions of estrogen were mediated via ERß.

Our observations that estrogen increased the expression of galanin within GnRH neurons 2 h after estrogen treatment, is consistent with the notion that estrogen also elicits a classical (genomic) effect in GnRH neurons by increasing the transcription of the galanin gene. In addition to the short onset of estrogen action, the direct, genomic effect of estrogen on galanin expression is supported by the findings that the galanin promoter contains an estrogen response element (ERE) (26), although the presence of ERE on the promoter region of a gene does not prove that the response element is functionally active. For example, the vasopressin gene promoter has two EREs but only one of them is functional during the ligand-dependent trans-activation of the gene by ER{alpha} (27). Moreover, estrogen may also activate galanin expression in GnRH-i neurons indirectly, via neurons expressing ERs and contacting the GnRH neurons. Among others, the anterior ventral paraventricular nucleus (AVPV), a region necessary for estrogen to induce the GnRH and subsequent LH surges and ovulation plays a critical role in mediating estrogen’s action. The AVPV in rats contains neurons that express primarily ER{alpha} and the lesion of this nucleus prevents not only the LH surge and ovulation (28, 29, 30) but the induction of the immediate early gene c-fos expression (31) and estrogen-induced galanin expression in GnRH-i neurons as well (Hoffman, G. E., unpublished observations). Moreover, because blockade of a number of neurotransmitter systems that innervate either GnRH or AVPV also block LH surges and galanin expression in GnRH neurons some aspect of galanin’s expression is thought to be trans-synaptic and activity dependent (10, 13). The AVPV expresses both ER{alpha} and ERß (32, 33) and although ER{alpha} dominates in this nucleus, some contribution of ERß in the effects of ERß-selective agonists (WAY-166818 and WAY-200070) on GnRH galanin expression through the AVPV or other afferents cannot be ruled out. Whether induced by direct action on GnRH neurons or indirectly via ERß expressing afferents to GnRH neurons, the important feature of these ERß effects is that they reflect the changes generally associated with GnRH surges and E-positive feedback mechanisms.

Interestingly, a direct action of estrogen on GnRH neuronal activity was already suggested by Petersen et al. (34) at the time when the notion that the GnRH neurons do not contain estrogen receptors was widely accepted. This group showed that estrogen’s influence on GnRH expression was dependent on the time of the day as well as the location of GnRH neurons in the medial preoptic area-septum continuum. Estrogen exerted a stimulatory effect on GnRH gene expression in only rostral preoptic area neurons in OVX rats. In addition, a gradual increase in GnRH mRNA content, from the low levels found in the morning in estrogen-treated rats, resulted in a peak of expression in the early afternoon before the onset of the LH surge (34). The presence of ERE sequence within the promoter region of the primate GnRH gene could account for direct actions of estrogen on GnRH gene (35).

The theory that estrogen’s negative and positive feedback actions on gonadotropin release are mediated on the same neuronal populations, but a switch from inhibitory to stimulatory feedback occurs during the estrous or menstrual cycle, was popular for decades. However, recent data suggest that the neuronal populations mediating the negative and positive feedback actions of estrogen are different. The brain areas involved in the negative feedback action of estrogen are the preoptic area and the medial basal hypothalamus, whereas the AVPV seems to be responsible for mediating the positive feedback actions of estrogen [for a review see Herbison, 1998 (1)]. In light of the present data, however, the morphological substrate for the positive feedback action of estrogen has to be extended to the GnRH neurons themselves as well.

The sensitivity of galanin gene expression to estrogen seems to be much higher than that of GnRH. As we (7, 8, 12, 36) and others (9, 10, 13) reported, the expression of galanin within a subpopulation of GnRH neurons is sexually dimorphic and depends upon both the organizational effects of testosterone, aromatized to estrogen during development (9, 37) and the activational effects of estrogen in the adult female rat (8, 11, 12, 13). Galanin mRNA expression in GnRH neurons is clearly elevated by the afternoon of proestrus (38), and this has been shown to result principally from a stimulatory action of estrogen (13). A later study aimed at clarifying the temporal relationship among GnRH, galanin, and c-fos expression within GnRH neurons and the LH surge found that c-fos activation occurs about 2 h before a minor elevation in GnRH expression and the preovulatory LH surge. Surprisingly, this study found that galanin’s expression in GnRH neurons does not begin to rise until the time of the LH surge, and it peaks 8–12 h later (39). Because anesthetics, {alpha}-adrenergic, and an N-methyl-D-aspartate (NMDA) receptor blocker all prevent estrogen-induced galanin’s expression in GnRH neurons (10, 13), neuronal activity seems to play a major role in the induction of galanin in these neurons. Thus, ERß signaling resulting in galanin expression may require an additional neuronal signal. The mechanisms of the estrogen-induced and activity-dependent galanin up-regulation might be distinct and studies aimed at identifying these mechanisms are in progress in our laboratories.

Galanin has been shown to dramatically increase GnRH release from nerve terminals in the median eminence (40) suggesting that the site of galanin’s action is likely the GnRH nerve terminals. The observations that galanin antagonists (41) or antisera against galanin (42) can block the preovulatory LH surge and ovulation strongly support this hypothesis. However, the fact that the principal receptor expressed in GnRH neurons is the Gal-R1 (43) makes the stimulation of GnRH release by galanin difficult to explain, particularly because its signaling pathways are inhibitory (44). However, it is possible that when GnRH neuronal activity is low, galanin, coreleased with GnRH, blocks the small amounts of GnRH released; however, when GnRH neuronal activity is elevated, galanin cannot suppress the release of the large bulk of GnRH from nerve terminals in the median eminence but by creating a larger potential difference in the GnRH terminal, it increases the amount of GnRH released. Via this mechanism, as suggested originally by the Steiner’s group (9), galanin may sharpen the pulsatile pattern of GnRH release into the hypophysial portal circulation necessary for induction of an LH surge and subsequent ovulation. It is also possible that the elevated galanin mRNA seen in GnRH neurons 8–12 h after the preovulatory LH surge represents transcripts that are translated later and therefore, the peptide prepares the GnRH neuronal system for the next cycle. The elevated levels of galanin in the GnRH neuronal axis detected in proestrus with immunocytochemistry supports this hypothesis. Galanin, detected with this technology in proestrus morning, is probably the translational product of the transcripts made and stimulated by estrogen during the previous cycle.

In conclusion, these studies provide convincing evidence for the role of ERß as a classical nuclear steroid hormone receptor/transcription factor in stimulating the transcription of the galanin gene within GnRH-i neurons in the rat hypothalamus. Moreover, because galanin has been shown to be involved in the regulation of GnRH release resulting in the preovulatory LH surge and ovulation in the rat, our data suggest that the stimulatory effect of estrogen on GnRH neuronal activity, in addition to the well-known role of ER{alpha} in the AVPV, is also mediated via ERß residing within GnRH-i neurons. Moreover, the stimulatory action of estrogen may involve, among other factors, galanin, as a mediator of estrogen action, though the mechanism of how galanin participates in the feedback regulation of estrogen is not known and requires additional studies.


   Acknowledgments
 
We are truly thankful to Dr. Derek Janssen for his help in the statistical analysis of the data, Drs. Michael Malamas, Michael Collini, and Robert McDevitt for the synthesis of the ERß-selective compounds, Dr. Heather Harris for overseeing the preclinical characterization of the ERß selective ligands.


   Footnotes
 
First Published Online March 24, 2005

Abbreviations: AVPV, Anterior ventral paraventricular nucleus; DAB, diaminobenzidine; ER, estrogen receptor; ERE, estrogen response element; GnRH-i, GnRH-immunoreactive; ISHH, in situ hybridization histochemistry; OVLT, organum vasculosum of the lamina terminalis; OVX, ovariectomized.

Received December 2, 2004.

Accepted for publication March 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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