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Regulation of Estrogen Receptor-ß Expression in the Female Rat Hypothalamus: Differential Effects of Dexamethasone and Estradiol

Department of Biomedical Sciences (S.S., R.J.H.), Colorado State University, Fort Collins, Colorado 80523; and Neuroscience Program (S.S.), Loyola University Chicago, Maywood, Illinois 60153

Address all correspondence and requests for reprints to: Robert J. Handa, Ph.D., Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State University, 1350 Center Avenue, Fort Collins, Colorado 80523. E-mail: robert.handa{at}colostate.edu.

	Abstract

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Estrogen and glucocorticoids interact in multiple aspects of endocrine regulation by exerting opposing influences on the expression of selective genes. In rats, estrogen receptor (ER)-ß is the predominant form of ER present in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, suggesting its involvement in neuroendocrine regulation. To date, the hormonal regulatory profile of the ERß gene in the rat central nervous system has not been closely elucidated. In the present study, we first examined the effects of dexamethasone (DEX) and estradiol benzoate (EB) on the ERß protein expression in the PVN and SON of ovariectomized female rats. In the SON and parvocellular and magnocellular parts of the PVN, the number of ERß immunoreactive nuclei significantly increased after DEX treatment, compared with the control group, whereas EB treatment caused a significant decrease. The effect of EB was consistent across other brain nuclei such as the anteroventral periventricular nucleus and medial preoptic nucleus. To determine the molecular level at which DEX and EB control ERß expression, we examined the effects of these steroids on ERß mRNA levels using real-time RT-PCR. EB significantly decreased the expression of ERß mRNA in the PVN (P = 0.0006) and SON (P < 0.01). In contrast, DEX did not change ERß mRNA levels. These results indicate that glucocorticoids and estrogen exert opposing regulatory influences on the ERß gene expression. This may represent a mechanism by which these steroids can alter the cellular sensitivity of ERß-expressing neurons to subsequent steroidal activation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE PARAVENTRICULAR (PVN) and supraoptic (SON) nuclei of the hypothalamus control a number of physiological functions, including autonomic and neurohypophyseal regulation as well as several other neuroendocrine operations. In rats, estrogen receptor (ER)-ß is the predominant form of ER present in these brain nuclei, suggesting that it plays a critical role in neuroendocrine regulation. Previous studies demonstrating the presence of ERß in neuropeptide neurons relevant for neuroendocrine function further support the hypothesis that ERß is important in neuroendocrine control (1, 2, 3, 4, 5). Although not always in agreement, these studies have shown that ERß is present to varying degrees in CRH-, arginine vasopressin-(AVP), and oxytocin-expressing neurosecretory neurons of the PVN and SON. In addition, estrogen can regulate the promoter of several neuropeptide genes including CRH (Miller, W. J., S. Suzuki, L. K. Miller, R. J. Handa, and R. M. Uht, unpublished observations) and AVP (6) as well as in vivo neuropeptide expression (7, 8, 9, 10, 11, 12, 13, 14).

The glucocorticoid receptor (GR) is also present in the PVN (15) and SON (16). Furthermore, GR is localized in CRH- and AVP-containing neurons of the PVN (15). At the molecular level, a functional glucocorticoid response element has been identified in the promoter region of the CRH and AVP genes (17, 18). Consistent with these findings, glucocorticoids act to down-regulate expression of both CRH and AVP in the PVN (19, 20, 21). In contrast, there is evidence to suggest that estrogen might up-regulate the expression of CRH (8, 9, 12, 14). Thus, estrogen and glucocorticoids may exert opposing regulatory influences on the expression of neuroendocrine genes through their cognate receptors present in neurons of the PVN and SON.

There are several previous studies that have examined estradiol’s regulation of the ERß gene in the rodent central nervous system. Not all of these studies are in agreement, however (22, 23, 24, 25, 26). It has been reported that estrogen treatment can reduce ERß mRNA levels in the PVN and medial amygdala as well as that of ERß immunoreactivity (ir) in the bed nucleus of the stria terminalis (BNST) and periventricular preoptic area (PVPO) (22, 23). Consistent with this, ERß ir is reduced in the PVPO and BNST after estradiol treatment (24). In contrast, Greco et al. (24) found no changes in ERß ir in the PVN after estrogen treatment, and estrogen is reported to increase ERß mRNA level in the arcuate nucleus (22). These differences may be attributed to the differences in methodology employed (in situ hybridization vs. immunocytochemistry, animal treatment paradigm and species).

Because changing the availability of ERß protein in an individual neuron could influence its sensitivity to estrogen, understanding how circulating hormones regulate ERß gene expression becomes important. In this study, we examined the levels of ERß protein and mRNA in the PVN, SON, and several other hypothalamic nuclei of young adult ovariectomized female rats after dexamethasone, estradiol benzoate, or oil treatments. Our results show that in the PVN and SON, ERß expression is differentially regulated by glucocorticoids and estrogen.

	Materials and Methods

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Animals
A total of 69 adult female Sprague Dawley rats (8 wk of age) were purchased from Charles River Breeding Laboratories (Portage, MI) and housed in the laboratory animal facilities at Colorado State University. Animals were acclimated to standard laboratory conditions (12-h light, 12-h dark cycle with lights on at 0700 h). Rat chow and water were available ad libitum. Three days before being killed, all animals were bilaterally ovariectomized (OVX) through a midventral incision while under isoflurane anesthesia. After surgery, one third received a daily sc injection of 40 µg/100 g body weight of dexamethasone (DEX) in 200 µl sesame oil, one third received 4 µg/100 g body weight of estradiol benzoate (EB) in 200 µl oil, and the remaining third received oil alone. Previously, we determined that such doses of hormone result in saturated occupancy of GR and ER, respectively (27). Our rationale for initiating steroid treatment directly after OVX is due to the possibility of potential changes in sensitivity that might occur with time after OVX. Thus, an approach was taken to deliver steroid immediately after OVX and maintain estrogen-regulated gene expression rather than replace hormone after an extended time after removal of steroid. All animal protocols were previously approved by the Animal Care and Use Committee at Colorado State University. All experiments were carried out in accordance with National Institutes of Health and Institutional Animal Care and Use Guidelines.

Immunocytochemistry
Animals were anesthetized with halothane and rapidly killed by perfusion with approximately 50 ml of 0.9% saline followed by approximately 200 ml of 10% neutral buffered formalin.

We tested two types of fixation (10% neutral buffered formalin and 4% paraformaldehyde), and the most optimal staining for ERß was obtained with 10% neutral buffered formalin (pH 7.0). Thus, all animal tissue used for immunocytochemistry was fixed with 10% formalin in the present study. The brains were removed and postfixed in the same fixative for 2 h at room temperature. Next, the brains were placed in 5% neutral buffered formalin/15% sucrose solution in 0.1 M PBS overnight at 4 C and then transferred to 30% sucrose in 0.1 M PBS at 4 C until permeated. The tissues were subsequently frozen and cut on a cryostat at 35 µm. Tissue was saved in 0.1 M PBS containing 0.1% sodium azide at 4 C for later immunohistochemical processing. Tissue sections from each experimental group were processed at the same time in parallel incubation wells, as previously described (28). Briefly, the tissue was washed three times for 15 min in 0.1 M PBS with 0.1% Triton-X (TX) and then incubated with 0.3% H₂O₂ in 0.1 M PBS with 0.1% TX for 15 min to quench any endogenous peroxidase activity. After standard washes, sections were incubated in 6% normal goat serum in 0.1 M PBS with 0.1% TX to block any nonspecific antibody binding and then incubated for 48 h at 4 C with ERß antiserum (Zymed Laboratories, San Francisco, CA; Z8P; 1:3000) in 0.1 M PBS with TX in the presence of 2% NGS. Next, the tissue was washed three times for 10 min in PBS with TX and incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA; 1:500) in PBS with TX in the presence of 2% normal goat serum for 2 h at room temperature. Sections were subsequently washed and processed according to the avidin-biotin-peroxidase procedure (Vector Laboratories; 1:500). After standard washes, the tissue was rinsed in 0.1 M Tris-buffered saline for 15 min and then developed with nickel intensified 3,3'-diaminobenzidine (0.5 mg/ml; Sigma) in 0.1 M Tris-buffered saline containing 0.03% hydrogen peroxide for 3 min. The reaction was stopped by several washes in 0.1 M PBS. The primary antibody titer (1;1000–10,000), the primary antibody incubation time (24–120 h) and temperature (4–22 C) as well as developing time (1–15 min) were initially varied, and in all subsequent studies, optimal conditions were used to keep staining intensity in the linear part of a staining intensity curve and avoid a ceiling effect. The sections were subsequently mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), air dried for 24 h at room temperature, further dehydrated through a series of increasing alcohols, cleared with xylene, and coverslipped with Permount (Fisher Scientific).

ERß antibody
To test whether alterations in the distribution pattern and/or the intensity of ERß ir after EB treatment is due to preferential recognition of bound vs. unbound forms, we injected EB (10 µg/100 g body weight; in 200 µl sesame oil) to OVX adult female rats and examined staining patterns in these animals 30 min later. We did not observe any differences in the distribution or the intensity of nuclear ERß immunolabeling in animals that were killed acutely after EB injections, compared with those with oil injections [PVN: F (1, 7) = 0.658, P = 0.454, SON: F (1, 6) = 0.699, P = 0.451]. From these data we concluded that the ERß antibody used in the present studies recognizes both the occupied and unoccupied forms of the receptor.

Tissue analysis
Tissue sections were examined using a Axioplan 2 imaging universal microscope (Zeiss, Thornwood, NY), and images were captured with a Zeiss AxioCam digital camera. Appropriate brain nuclei from matched sections (29) were chosen at two different levels (bregma –1.78 and –2.00 for the PVN; bregma –1.33 and –1.53 for the SON) and analysis performed on digitized images by two different approaches. In both cases experimenters were kept blind to experimental groups. First, quantification of nuclei stained with Z8P and evaluation of staining intensity were performed by three independent investigators. These values were subsequently averaged to give a mean number for each brain nucleus per section per animal. Cells were subjectively divided into three groups according to the level of staining intensity (dark, medium, and light). Only the nuclei with clear outlines were considered for quantification. Second, analysis of staining intensity was performed using NIH image analysis software. The mean densities of ERß immunolabeling for a brain nucleus (two sections per animal) were measured (based on a grayscale ranging from 40 to 256 arbitrary units), and these values were averaged to give a mean for a brain area per each animal.

Total RNA isolation
After 3 d of DEX, EB, or oil treatments, OVX female rats were anesthetized with halothane and rapidly killed by decapitation. The brains were quickly removed and frozen in isopentane (–30 C). Tissue was then stored at –80 C until further processing. For dissection of the PVN and SON, a micropunch technique was employed similar to the protocol of Palkovits et al. (30) with modifications described previously (31). Briefly, brain sections (200 µm) were cut frozen on a cryostat and thaw mounted on glass slides (Fisher Scientific) and then kept frozen at –80 C until punching. Micropunching was performed using blunted needles with a diameter of 0.5 or 1.0 mm. Isolation of total RNA was accomplished according to the protocol of Chomczynski and Sacchi (32). Punch-dissected tissues were pooled from two animals and immediately homogenized in a centrifuge tube containing 250 µl guanidinium isothiocyanate buffer [4 M guanidinium isothiocyanate, 0.5% sarcosyl, 25 mM Na citrate (pH 7.0), and 0.1 M ß-mercaptoethanol]. Subsequently, 25 µl of 2 M NaOAc (pH 4.0), 250 µl acid phenol (pH 4.3), and 50 µl chloroform/isoamyl alcohol (49:1) were added, and the mixture was vortexed, incubated on ice for 15 min, and centrifuged at 14,000 x g for 15 min at 4 C. The aqueous phase was recovered; the RNA was ethanol precipitated, washed with ice-cold 70% ethanol, and reconstituted in 25 µl RNase-free water. The RNA concentration was measured with a spectrophotometer, and only those RNA samples with a 260:280 ratio of greater than 1.6 were used.

Reverse transcription (RT)
Equal amounts of total RNA (0.5 µg) were reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Rockville, MD) in the presence of oligo-dT primers, deoxynucleotide triphosphates (100 mM each), first-strand buffer (100 mM Tris-Cl, 900 mM KCl, 1 mM MgCl₂), and 2.5 mM dithiothreitol. The RT reaction was carried out at room temperature for 10 min, followed by a 50-min incubation at 42 C. The reaction was then terminated by denaturing reverse transcriptase at 95 C for 5 min. The RT-generated cDNA samples were stored at –20 C until PCR amplification.

Construction and specificity of ERß primers
A set of specific PCR primers for the rat ERß was designed using commercially available software (Oligo software, version 6.51, Molecular Biology Insights, Cascade, CO). These primers were developed on the basis of established GenBank sequence (accession no. U57439; forward primer position 1414 and reverse primer position 1570; predicted product size 176). The specificity of ERß primer sequences have been previously verified using the BLAST search and by size determination of PCR product after agarose gel analysis and by comparing the melting temperature (Tm) of PCR products to the calculated Tm of the different PCR product species. The Tm of PCR products with the expected size of 176 was determined to be 86.6–86.9 C.

Construction of the ERß standard curve
For the determination of the absolute amount of ERß transcripts in each RT-generated sample, an ERß standard curve was constructed using conventional PCR with the same set of ERß primers. After amplification, PCR products were run on a 1% agarose gel and subsequently purified using MinElute gel extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s directions. Purified products were then diluted and the standard curve generated (ranging from 10 pg to 0.01 fg).

Real-time quantitative PCR amplification
The procedure for real-time quantitative PCR was essentially according to the protocol of Solum and Handa (33). Briefly, hot start PCR was performed using LightCycler DNA Master SYBR Green mix (Roche Molecular Biochemicals, Indianapolis, IN; deoxynucleotide triphosphate mix, Taq DNA polymerase, PCR buffer, SYBR Green I dye), specific primers, 4.0 mM MgCl₂, and 0.5 U Taq antibody (Invitrogen). Samples were subjected to an initial melting step at 95 C for 2 min and amplified at 40 cycles (approximately 5–10 cycles beyond the beginning of the linear phase of amplification) of a 95 C melting step for 2 sec, a 66 C annealing step for 7 sec, and a 72 C elongation step for 10 sec. Samples containing no template were used as negative controls. After PCR amplification, samples were separated on a 1.0% agarose gel with an appropriate molecular weight size marker (Life Technologies, Inc.-BRL, Gaithersburg, MD) to ensure the specificity of the PCR products. The agarose gel was stained using ethidium bromide and visualized under UV light.

Quantitative analysis of ERß cDNA
After real-time PCR, the absolute amount of ERß cDNA that was present in each sample before PCR amplification was calculated using LightCycler data analysis software, as previously described (33). The starting amount of ERß cDNA in each sample should directly reflect the amount of mRNA that was present before RT reaction. Briefly, the standard curve (a series of known amounts of ERß cDNA, ranging from 10 pg to 0.01 fg) was run alongside unknown samples. The cross-line intercepts of these standards are plotted against the known concentrations of standard curve. The cross-line intercept runs parallel to the x-axis on a graph plotting fluorescence intensity (reflecting the absolute concentration of cDNA) vs. cycle number and appears at the beginning of the logarithmic phase of the amplification curve. Those samples containing higher amounts of starting cDNA enter the log phase at earlier cycles than samples with lower concentrations of starting material and thus have smaller cross-line intercept values. The crossing-line intercepts of unknown samples are compared with those of the standard curve to yield the absolute concentration of starting ERß cDNA.

Statistical analysis
Statistical analysis was performed using one-way ANOVA (StatView; SAS Institute Inc., Cary, NC). Post hoc analysis was performed using the Tukey-Kramer test.

	Results

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Specificity of Z8P
Nuclear ERß ir was observed throughout the PVN and SON of DEX-, EB-, or oil-treated OVX female rats. The pattern of nuclear ERß ir in other forebrain regions was also in good agreement with a recent report that used the same ERß antibody (34). Specifically, in addition to the PVN and SON, ERß ir was observed in the nuclei of neurons in the medial septal nucleus, medial preoptic area, anteroventral periventricular nucleus (AVPV), bed nucleus of the stria terminals, nucleus circularis, medial and cortical amygdala, cerebral cortex, and cerebellum. In previous studies, the specificity of this antibody has been reported and verified (34).

DEX and EB differentially regulate the expression of ERß protein in the PVN
In the PVN, the most consistent signals for ERß ir were observed in sections at the levels of bregma –1.78 to –2.00 (Fig. 1). Atlas-matched tissue sections at these two levels were thus chosen from all experimental groups for analysis. The relative staining intensities of nuclear ERß immunolabeling in these sections were graded according to a scale of dark, medium, and light. Labeled nuclei were counted manually and grouped according to staining intensity.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Rat brain illustrations are shown at two different levels coordinated from bregma (A, –1.78; B, –2.00). The maps are modified from Swanson (29 ). The forebrain distributions of nuclear ERß (black dots) ir at these levels in the adult female rat hypothalamus are illustrated. fx, Fornix; dp, dorsal parvicellular part; f, forniceal part; lp, lateral parvicellular part; mpd, medial parvicellular part, dorsal zone; mpv, medial parvicellular part, ventral zone; pv, parvicellular part, ventral zone; pml, posterior magnocellular part, lateral zone; opt, optic tract; SONr, supraoptic nucleus, retrochiasmatic part; v3, third ventricle.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Photomicrographs showing ERß ir nuclei in the PVN at the level of bregma –1.78. Immunocytochemistry was employed using polyclonal Z8P on tissue sections from brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). ERß ir was observed in both the parvocellular and magnocellular parts of the PVN. There were significant increases in ERß ir nuclei in DEX-treated animals, compared with controls, and decreases in the number of ERß-labeled nuclei in PVN of EB-treated animals, compared with controls. V3, Third ventricle. Scale bar, 50 µm.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Differential regulation of ERß ir by DEX and EB in the PVN at the level of bregma –1.78. A, Total number of ERß immunolabeled nuclei was counted from each experimental group in the parvocellular part of the PVN. Each bar represents the mean ± SEM of nuclear ERß ir nuclei (n = 5 per group). #, Significantly greater than control, oil-treated condition (P < 0.03), determined by one-way ANOVA, Tukey-Kramer post hoc. B, The relative staining intensities of nuclear ERß immunolabeling were graded according to a scale of dark, middle, or light. Labeled nuclei were then counted from the parvocellular part of the PVN and placed into each staining intensity category. Each bar represents the mean ± SEM of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than control, oil-treated condition (P < 0.03); *, P < 0.05 vs. oil-treated control, by one-way ANOVA, Tukey-Kramer post hoc. DEX and EB differentially regulate the expression levels of ERß ir in the magnocellular part of the PVN (C and D). C, Total number of nuclear ERß ir in each experimental group. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5 per group). #, Significantly greater than control, oil-treated condition (P < 0.0001); *, P = 0.0003 vs. oil-treated control, by one-way ANOVA, Tukey-Kramer post hoc. D, The relative staining intensities of nuclear ERß ir graded according to a scale of dark, middle, and light. Labeled nuclei were counted and placed into each staining intensity group by three independent investigators. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than control, oil-treated condition (P < 0.002, dark; P < 0.007, medium staining group); *, P < 0.03 (medium), *, P < 0.003 (light staining group) vs. oil-treated control, determined by one-way ANOVA, Tukey-Kramer post hoc. E, The mean densities for each brain nucleus were measured (based on a gray scale ranging from 40 to 256 arbitrary units) using NIH image analysis software, and these values were averaged to give a mean for a brain area per each animal. Values were then normalized to the means of oil-treated control group and are reported in percentages. All values are shown as mean ± SEM (n = 5–6 per group). #, Significantly greater than control, oil-treated group (P < 0.006); *, Significantly less than control group (P < 0.007), as determined by one-way ANOVA, Tukey-Kramer post hoc.

Bregma –2.00.
At level bregma –2.00, at which only the parvocellular divisions of the PVN are present, DEX treatment significantly increased the total number of ERß cells [F (2, 14) = 22.16, P < 0.002], whereas EB treatment caused a significant decrease in the total number of labeled nuclei (P < 0.03; Figs. 4 and 5A). After nuclei were grouped according to staining intensity, DEX treatment significantly increased the number of ERß ir nuclei in the dark staining group only [F (2, 15) = 12.69, P < 0.007; Fig. 5B]. EB treatment, on the other hand, caused a significant decrease in the medium staining group [F (2, 15) = 8.21, P < 0.03]. Similarly, densitometry analysis of ERß immunolabeling revealed that DEX treatment marginally increased the ERß immunostaining (P = 0.052; Fig. 5C). In contrast, EB treatment resulted in a significant decrease in the mean densities of ERß immunostaining [F (2, 14) = 41.11, P < 0.0001)].

View larger version (24K): [in this window] [in a new window]   FIG. 4. Photomicrographs of ERß ir nuclei in the PVN at the level of bregma –2.00. Immunocytochemistry was employed using polyclonal Z8P in brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). Photomicrographs are showing ERß ir observed in the dorsal zone of medial parvocellular part of the PVN. There was a significant increase in ERß ir nuclei in DEX-treated animals and a decrease in the number of ERß-labeled nuclei in EB-treated animals, compared with oil-treated control. V3, Third ventricle. Scale bar, 50 µm.

View larger version (12K): [in this window] [in a new window]   FIG. 5. ERß ir levels in the PVN at the level of bregma –2.00 after ovariectomy and subsequent DEX or EB treatments. A, Total number of nuclear ERß ir neurons counted from each experimental group. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than oil-treated condition (P < 0.002); *, P < 0.03 vs. oil-treated control, as determined by one-way ANOVA, Tukey-Kramer post hoc. B, The relative staining intensities of nuclear ERß ir graded according to a scale of dark, middle, or light. Labeled nuclei were counted and placed into each staining intensity group by three independent investigators. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than oil-treated condition (P < 0.007); *, P < 0.03 vs. oil-treated control, by one-way ANOVA, Tukey-Kramer post hoc. C, The mean densities for each brain nucleus were measured using NIH image analysis software, and these values were averaged to give a mean for a brain area per each animal. Values were then normalized to the means of oil-treated control group and are reported in percentages (n = 5–6 per group). *, Significantly less than control group (P < 0.0001), as determined by one-way ANOVA, Tukey-Kramer post hoc.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Photomicrographs of ERß ir nuclei in the SON at the level of bregma –1.33. Immunocytochemistry was employed using polyclonal Z8P in brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). A significant increase was observed in the number of ERß ir nuclei in DEX-treated animals and a decrease in ERß-labeled nuclei in EB-treated animals, compared with oil-treated control. Opt, Optic tract. Scale bar, 50 µm.

View larger version (13K): [in this window] [in a new window]   FIG. 7. ERß ir levels in the SON at the level of bregma –1.33 after ovariectomy and subsequent DEX or EB treatments. A, Total number of nuclear ERß ir counted from each experimental group. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than oil-treated condition (P < 0.05), as determined by one-way ANOVA, Turkey-Kramer post hoc. B, The relative staining intensities of nuclear ERß ir graded according to a scale of dark, middle, or light. Labeled nuclei were counted and placed into each staining intensity group by three independent investigators. Each bar represents the mean ± SEM number of nuclear ERß immunoreactive nuclei (n = 5–6 per group). #, Significantly greater than control, oil-treated condition (P < 0.0001, dark; P < 0.03, medium staining group); *, P < 0.002 vs. oil-treated control, as determined by one-way ANOVA, Tukey-Kramer post hoc. C, The mean densities for each brain nucleus were measured, and values were averaged to give a mean for a brain area per each animal. Values were then normalized to the means of oil-treated control group and are reported in percentages. All values are shown as mean ± SEM (n = 5–6 per group). #, Significantly greater than control, oil-treated group (P < 0.02). *, Significantly less than control group (P < 0.0003), as determined by one-way ANOVA, Tukey-Kramer post hoc.

View larger version (11K): [in this window] [in a new window]   FIG. 8. Photomicrographs of ERß ir nuclei in the SON at the level of bregma –1.53. Immunocytochemistry was employed using polyclonal Z8P in brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). There was a significant increase in ERß ir nuclei in DEX-treated animals and a decrease in the number of ERß-labeled nuclei in EB-treated animals, compared with control. Opt, Optic tract. Scale bar, 50 µm.

View larger version (14K): [in this window] [in a new window]   FIG. 9. ERß ir levels in the SON at the level of bregma –1.53 after ovariectomy and subsequent DEX or EB treatments. A, Total number of nuclear ERß ir from each experimental group. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5 per group). #, Significantly greater than oil-treated condition (P < 0.02), as determined by one-way ANOVA, Tukey-Kramer post hoc. B, The relative staining intensities of nuclear ERß ir graded according to a scale of dark, middle, or light. Labeled nuclei were counted and placed into each staining intensity group by three independent investigators. Each bar represents the mean ± SEM number of nuclear ERß ir nuclei (n = 5–6 per group). #, Significantly greater than control, oil-treated condition (P = 0.0008); *, P = 0.0003 vs. oil-treated control, by one-way ANOVA, Tukey-Kramer post hoc. C, The mean densities for each brain nucleus were measured using NIH image analysis software. These values were then averaged to give a mean for a brain area per each animal. Values were normalized to the means of oil-treated control group and are reported in percentages. All values are shown as mean ± SEM (n = 5–6 per group). #, Significantly greater than control, oil-treated group (P < 0.008). *, Significantly less than control group (P < 0.002), as determined by one-way ANOVA, Tukey-Kramer post hoc.

View larger version (24K): [in this window] [in a new window]   FIG. 10. Photomicrographs of ERß ir nuclei in the AVPV. Immunocytochemistry was employed using polyclonal Z8P in brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). There was a significant increase in ERß ir nuclei in DEX-treated animals (P < 0.03) and a decrease in the number of ERß-labeled nuclei in EB-treated animals (P < 0.008), compared with control. V3, Third ventricle. Scale bar, 50 µm.

View larger version (19K): [in this window] [in a new window]   FIG. 11. Photomicrographs of ERß ir nuclei in the bed nucleus of the stria terminalis. Immunocytochemistry was employed using polyclonal Z8P in brains of OVX female rats. Animals were treated with DEX (A), oil (B), or EB (C). DEX treatment significantly increased (P = 0.008), whereas EB significantly decreased ERß ir nuclei (P < 0.0001). ST, Stria terminalis. Scale bar, 50 µm.

View larger version (17K): [in this window] [in a new window]   FIG. 12. ERß cDNA levels in the PVN and SON of the hypothalamus of OVX rat after DEX or EB treatments. Real-time RT-PCR was used to quantitatively examine the expression of ERß mRNA levels of the female rat after ovariectomy and subsequent hormone treatments. Animals were given daily injections of DEX, EB, or oil for 3 d. All values are reported as percent of the mean of oil-treated group. Each bar represents the mean ± SEM. A, EB significantly decreased the levels of ERß mRNA (n = 5–6, P = 0.0006) in the PVN, as determined by one-way ANOVA, Tukey-Kramer post hoc. In contrast, DEX did not change ERß mRNA levels (n = 5–6, P = 0.22). B, EB significantly decreased the levels of ERß mRNA (n = 5–6, P < 0.01) in the SON by one-way ANOVA, Tukey-Kramer post hoc. DEX did not induce any changes in the levels of ERß mRNA (n = 5–6, P = 0.96).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our data showing an inhibition of ERß expression in the PVN and SON by estrogen are partly consistent with other studies (22, 23, 24, 25, 26). Estrogen inhibits the expression of ERß mRNA in the PVN and amygdala but not the BNST and MPON (22, 23). Consistent with this, ERß ir is reduced in the periventricular preoptic area and BNST after estradiol treatment (24). In contrast to our findings, Greco et al. (24) found no changes in ERß ir in the PVN. Such differences could be due to animal treatment paradigms. Greco et al. gave a single EB injection before killing animals 48 h later, whereas we injected EB for 3 d before killing. Alternatively, discrepancies might be due to the differences in the types and qualities of ERß antibodies used. The specificity of the Z8P used in this study has been carefully evaluated and verified (34). The distribution of ERß ir we report is also in agreement with previous observations for ERß mRNA (35, 36, 37) as well as ERß protein (34).

The subdivisions of the PVN have been determined based on physiological functions of the neurons in each of the subdivisions (38). In our study, we analyzed the expression of ERß ir at two different levels to examine different subdivisions of the PVN. Although the SON is less heterogeneous in function than the PVN, the distribution of neuropeptides and ERß is not consistent throughout this nucleus. Thus, we examined two anterior-posterior levels of the SON for consistency with the PVN analysis.

EB treatment decreases the ERß ir and mRNA levels in the PVN and SON
EB treatment decreased ERß ir and mRNA in the PVN and SON. A decrease in ERß ir seen after EB treatment is most likely due to down-regulation of receptor expression and not simply changes in circulating hormones because the Z8P ERß antibody recognizes both occupied and unoccupied forms of the receptor.

Similar to ERß, ER is under regulation by estrogen (39, 40, 41, 42). Previous studies have consistently shown that estradiol decreases the levels of ER mRNA as well as protein in the medial preoptic area, AVPV, arcuate nucleus, ventromedial nucleus, bed nucleus of the stria terminalis, hippocampus, and cortical amygdala (22, 43, 44, 45, 46, 47).

The molecular mechanisms underlying the regulation of ER expression by estrogen or any other steroid hormones have not been elucidated. However, Alarid et al. (48) have shown that estrogen induces proteasome-mediated proteolysis of ER, a system responsible for degradation of most proteins in mammalian cells (49). This swift, estrogen-dependent protein degradation process can account for a rapid decline of ER protein after exposure to estrogen, without changes in the levels of ER mRNA. Recently Tschugguel et al. (50) demonstrated that estrogen induces the turnover of ERß protein via a ubiquitin-dependent proteasome pathway in cultured human uterine artery endothelial cells. Whether the decline in ERß ir seen in the PVN and SON of OVX rats after estrogen treatment or the increase after DEX treatment is also due to activation/inhibition of a ubiquitin-dependent proteasome pathway is currently not known.

In most brain areas, the effects of estrogen on ERß gene expression are inhibitory (22, 23, 24, 25, 26). In rats, the predominant form of ER expressed in the PVN and SON is ERß (22, 34, 37, 51), whereas the existence of ER in the PVN and SON is low (our unpublished observations). Thus, if estrogen-dependent down-regulation of ERß mRNA and ir are direct, then they are most likely mediated through ERß itself. Although ER may also regulate ERß in areas in which overlap of expression occurs, Nomura et al. (25) reported that estrogen can reduce ERß protein expression in brain regions that express both ER and -ß at high levels, even in an ER knockout mouse.

We found that the effect of EB on ERß ir was not limited to the PVN and SON. Down-regulation was observed in the AVPV, BNST, PVPO, and MPON as well as the cortex. The molecular mechanism of autologous down-regulation of ERß is not currently known. One means by which estrogen down-regulates ERß protein is by decreasing the levels of ERß mRNA. This could be due to the decreased rate of transcription or shortening of the mRNA half-life.

DEX treatment increases ERß ir in the PVN and SON
Unlike estrogen, DEX treatment increased the number and intensity of immunostained ERß nuclei in PVN and SON neurons. Recently Isgor et al. (52) have shown that adrenalectomy reduces ERß mRNA levels in the PVN of female rat and that corticosterone (CORT) reverses this effect. However, up-regulation of ERß mRNA by adrenal steroids was observed only during proestrus and not at the basal ovarian hormone levels (diestrus 1). In our study DEX treatment of OVX female rats significantly increased the number and staining intensity of ERß ir nuclei in both the parvicellular and magnocellular parts of the PVN as well as in the SON. Such a finding is consistent with the effects of CORT reported by Isgor et al. (52). However, in our study, the up-regulation of ERß protein by DEX was not accompanied by changes in mRNA levels. Taken together, these findings suggest that adrenal hormone regulation of ERß mRNA levels may require the presence of relatively high levels of estrogen (proestrus), whereas DEX can up-regulate ERß protein expression even at the very low levels of circulating estrogen. It should be noted that for our studies, the staining intensity was adjusted to keep it in the linear part of the development curve to prevent a ceiling effect. Thus, the number of ERß-labeled nuclei observed in these studies is relative and do not represent the absolute number of ERß-positive neurons present in these brain areas.

GR is present in the parvicellular and, to a lesser extent, magnocellular parts of the PVN (15) as well as in the SON (16). The presence of GR in neurons within these brain nuclei provides a potential mechanism by which DEX might act to regulate the expression of genes such as ERß. The effect of DEX on ERß gene expression was also observed in the AVPV and extrahypothalamic areas such as the BNST and cortex. However, this effect of DEX is not a general response of the ERß gene because ERß ir in the PVPO and MPON were not DEX responsive. Thus, the regulation of ERß gene expression by DEX seems to be brain region specific.

Two CORT receptor types [GR and mineralocorticoid receptor (MR)] can mediate the effects of DEX (53, 54). The dose of DEX that we administered (40 µg/100 g body weight) is in the range of doses used in previous studies showing that DEX can influence neuropeptide expression in the hypothalamic neurons (55, 56, 57, 58). Our previous studies (27) showed that this dose of DEX can potently inhibit CORT and ACTH secretion to low basal levels. Because we used adrenal-intact animals in these studies and killed them in the morning, animals from non-DEX-treated groups (EB- or oil-treated) should have low circulating levels of CORT. Such levels would predominantly occupy MR but not GR (53, 54, 59). Thus, the observed up-regulation of ERß exerted by DEX is arguably mediated entirely through GR and not MR binding. Furthermore, MR expression is not high in the PVN and SON (54). Additional studies using more precise steroidal manipulations after adrenalectomy would provide a clearer picture of which CORT receptor might regulate ERß and the pharmacodynamics of such a regulatory system. However, because adrenalectomy will obviously remove other adrenal hormones and thus requires animals to be maintained on saline to maintain osmotic balance, coupled with the recent demonstration by Somponpun and Sladek (60) that changes in osmolarity can alter ERß expression, we opted to inject adrenal-intact animals with DEX to prevent this from confounding the results. Physiologically, this treatment paradigm should mimic a chronic stress protocol in which CORT is elevated for several days, and, thus, GR and MR are chronically activated.

DEX treatment does not change the ERß mRNA levels in the PVN and SON
The increase in ERß ir in the PVN and SON after DEX treatment was not accompanied by ERß mRNA up-regulation. Thus, glucocorticoids may be acting posttranscriptionally to up-regulate ERß protein in OVX female rats. The mechanism for this is not currently known. However, if ERß is susceptible to protein degradation in a similar fashion to ER in rodent brain, then one could speculate that glucocorticoids may increase ERß ir by interfering with proteolysis.

Glucocorticoids may do so by hindering ubiquitination of ERß protein or down-regulating the expression of enzymes such as ubiquitin activating enzymes, ubiquitin conjugating enzymes, or ubiquitin-protein ligases. Boehmer et al. (61) demonstrated that, in vitro, glucocorticoids up-regulate protein kinases SGK1 and -3, whose function is to phosphorylate and inactivate ubiquitin ligases. Other studies have shown that DEX will up-regulate UBC mRNA expression in muscle cells (62). Nonetheless, it is possible that glucocorticoids inhibit proteolysis by inducing expression of kinases that inhibit the ubiquitin proteasome pathway.

Physiological significance of differential regulations of ERß expression in the PVN and SON
In light of previous reports demonstrating numerous opposing interactions between estrogen and glucocorticoids at the molecular (63), cellular (44), and physiological levels (27, 64), the ERß gene may represent a nodal point through which these two steroids exert such opposing regulatory influences. By down- or up-regulating the levels of ERß concentrations, estrogen and glucocorticoids may differentially alter the sensitivity of neuroendocrine neurons of the PVN and SON to estrogen, bringing counteracting physiological outcomes. In the present study, the physiological or functional significance of differential regulation of the ERß gene by estrogen and glucocorticoids was not examined. How changes in the cellular concentrations of ERß in response to estrogen or glucocorticoid treatment may affect cellular functions of neurosecretory cells in the PVN and SON that contain ERß and how such alterations in cellular responsiveness can in turn affect physiology and behavior of animals remain to be investigated.

At the physiological level, estrogen and glucocorticoids can differentially regulate the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Estrogen up-regulates, whereas glucocorticoids inhibit HPA function in response to stress (65, 66, 67, 68). To date, the mechanisms underlying the roles of estrogen in enhancement of the HPA function in response to stress are not completely elucidated. Because changes in ERß concentrations in a given cell could influence its sensitivity to estrogen signals, ERß down-regulation by estrogen in neurons of the PVN and SON will likely act to suppress estrogen sensitivity of these neurons. Estrogen has been shown to increase hypothalamic expression levels of two major ACTH secretogogues, CRH (8, 9, 12, 14) and AVP (7, 12), possibly by binding ERß. It is intriguing to speculate that estrogen down-regulation of its receptor could call a stop to its own action in the activation of HPA function. In contrast, increases in cellular ERß levels in response to exposure to glucocorticoids will likely enhance cellular sensitivity to estrogen. This increase in the ERß protein by DEX could therefore potentiate the activational properties of estrogen on HPA function after chronic GR activation.

After stress, levels of glucocorticoids increase, and over time, elevated levels of glucocorticoids will suppress HPA activity via a negative feedback mechanism. In the presence of high levels of estrogen such as during proestrus, however, ERß expression can also be diminished. This could result in a decrease in estrogen sensitivity of CRH and AVP neurons. During stress, however, this may not be the case because levels of estrogen are reported to be decreased in response to elevated levels of CRH (69). Thus, in the absence of high levels of estrogen, glucocorticoids may be able to increase cellular concentrations of ERß in CRH and AVP neurons of the PVN and SON, thereby increasing sensitivity of these neurons to estrogen. Low levels of estrogen may still induce CRH/AVP expression, causing further increases in HPA activity. Thus, an additional underlying mechanism for a known sex difference in HPA regulation might be through up-regulation of ERß by circulating elevated levels of CORT. This could result in a further up-regulation of hypothalamic ACTH secretogogues such as CRH and AVP.

Conclusion
The cellular levels of steroid hormone receptors fluctuate in response to physiological/endocrine status. A major factor governing the cellular responsiveness to steroid hormones may be steroid receptor availability (70). Thus, because ERß is found in neuroendocrine neurons and estrogen can modulate neuroendocrine functions, an understanding of hormone regulatory mechanisms for ERß is important. In summary, the present studies have provided evidence for differential regulation of ERß expression by estrogen and glucocorticoids. Further elucidation of these regulatory pathways may increase our understanding of how these two steroid hormones can interact to regulate various molecular, cellular, and physiological responses in the central nervous system.

	Acknowledgments

 
We thank Dr. J. McNulty for technical assistance and J. Evans and D. Pera for assisting in cell counting.

	Footnotes

 
This work was supported by National Institutes of Health Grants NS39951 and AA12693 (to R.J.H.).

Abbreviations: AVP, Arginine vasopressin; AVPV, anteroventral periventricular nucleus; BNST, bed nucleus of the stria terminalis; CORT, corticosterone; DEX, dexamethasone; EB, estradiol benzoate; ER, estrogen receptor; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; ir, immunoreactivity; MPON, medial preoptic nucleus; MR, mineralocorticoid receptor; OVX, ovariectomized; PVN, paraventricular nucleus; PVPO, periventricular preoptic area; RT, reverse transcription; SON, supraoptic nucleus; Tm, melting temperature; TX, Triton-X; Z8P, ERß antiserum.

Received December 12, 2003.

Accepted for publication April 9, 2004.

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