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Regular Article |
, and the Type II Glucocorticoid Receptor Are Coexpressed in the Same Neurons of the Ovine Preoptic Area and Arcuate Nucleus: A Triple Immunolabeling Study1
a Department of Clinical Veterinary Science, University of Bristol, Langford BS40 5DU, United Kingdom
b Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estrogen (ER) receptors. We sought to determine whether PR, ER, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ER, and about 95% of ER were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ER colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ER-immunoreactive cells contain GR. PR+ER+GR-immunoreactive cells represent 70% of PR, 65% of ER, and 72% of GR in the preoptic area and 70% of PR, 66% of ER, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.
estradiol receptor, glucocorticoid receptor, progesterone receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) [7, 8] have been found in GnRH neurons, which are located predominantly in the preoptic area. Importantly, neither progesterone [9] nor estradiol [10] affect LH or GnRH secretion when administered into the preoptic area. In contrast, these steroids potently modulate GnRH/LH release when administered into the region of the ventromedial/arcuate nucleus [9, 10]. Taken together, these studies suggest that an interneuronal system(s) transduces the steroidal signal to the GnRH perikaryia. Previous studies have shown that ER [11] and PR [4, 6] are expressed in cell populations coexisting in the same hypothalamic areas, but whether these two receptors are present in the same neurons or in phenotypically different subpopulations within the same hypothalamic nuclei has not been established.
Previous studies in our laboratory [5] have provided compelling evidence that a brief 2-wk exposure of long-term ovariectomized sheep to basal levels of estradiol is critical in the ability of progesterone to suppress GnRH secretion. This suggests that the effects of estradiol persist over several weeks in the ewe and provides a model whereby it is possible to investigate what the neuronal-specific differences are between ewes pretreated with estradiol and ewes in which this pretreatment has been omitted and progesterone is incapable of inhibiting GnRH secretion. The first objective of this study, therefore, was to determine whether PR-immunoreactive cells possess ER in the ovine hypothalamus and, importantly, whether estradiol preexposure influences the number of PR-immunoreactive cells.
Reproductive function is also influenced by stress since disruption of ovulation following acute stress has been reported in many species [12–14], including humans [15]. Increased glucocorticoid concentrations are associated with a disruption of the LH surge [15–17]. Although earlier studies [18, 19] reported no effect of cortisol/dexamethasone on the ovine neuroendocrine reproductive axis, more recent investigations have revealed that the ovarian steroid milieu is critical in the efficacy of glucocorticoids to suppress LH release [16, 17]. Preliminary evidence [20] indicated that the distribution of glucocorticoid (GR)-immunoreactive cells overlapped significantly with the distribution with PR- and ER-immunoreactive neurons. It is possible, therefore, that GR-containing cells may also express ER and/or PR.
The second objective of this study, therefore, was to determine whether GR-containing cells may express ER and/or PR and whether the steroidal pretreatment influences coexpression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually mature Dorset Horn ewes (n = 8) were ovariectomized at least 4 mo before the start of the study. Animals were maintained in outdoor barns on natural photoperiod; fed daily with hay, straw, and concentrate; and had free access to water. At the time of the experiments (early breeding season), animals were divided in two groups (n = 4 in each group). In the estradiol- and progesterone-treated group (E+P group), a 1-cm Silastic capsule containing 17ß-estradiol was implanted s.c. for 2 wk to produce basal concentrations of estradiol (approximately 2 pg/ml [21]). No estradiol was administered to the second (progesterone-only) group (P-only). The estradiol implants were removed from the E+P ewes, and at the same time, two progesterone-releasing implants (CIDR, InterAg, Hamilton, New Zealand) were inserted intravaginally for 10 days to both the E+P and P-only ewes to produce mean progesterone concentrations of 2 ng/ml [5]. The progesterone implants were removed for 12 h and then new CIDRs were inserted for 1 h before killing to produce progesterone concentrations of 2.8 ng/ml [5]. Prior to progesterone reinsertion in the E+P-treated ewes, ewes were in an ovarian steroidal state similar to the early follicular phase.
These two treatments groups were chosen because previous studies have shown that progesterone specifically inhibits GnRH section in estradiol- and progesterone-pretreated ewes [5]. In contrast, the suppressive power of progesterone is lost if estradiol pretreatment is omitted. Comparison of the neuronal systems in these two treatment groups provides a unique opportunity to determine why progesterone is capable of suppressing GnRH following exogenous estradiol exposure.
All animal procedures were conducted under Home Office License PPL 30/1670.
Tissue Preparation
Ewes were injected intravenously with 25 000 IU heparin and killed with an overdose of sodium pentobarbitone. Animals were decapitated and the brains were perfused through both carotid arteries with 1 L of 1% sodium nitrite in 0.9% NaCl, followed by 3 L of cold 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB; pH 7.4) and 1 L of 20% sucrose in PB. Following fixation, the brains were extirpated and left in 20% sucrose overnight at 4°C. A brain block containing diencephalon was then dissected out, embedded in Tissue-Tek (Miles Inc., Elkhart, NJ) and frozen by immersion in nitrogen-cooled isopentane. Coronal sections (20 µm) were cut on a cryostat, mounted onto Silane-coated slides [22], and frozen (-30°C) until further processing.
Antibodies
The mouse monoclonal anti-PR antibody (PgRAb8, Neomarkers, Fremont, CA) was raised against the human endometrial carcinoma PR. The antibody used to detect ER was H222, a rat monoclonal antibody raised against human ER; the specifics of the H222 antibody have been previously published [23–25]. GR was detected using a rabbit polyclonal antibody against sequence D (346) to C (367) of the human GR (PA1-511, Affinity Bioreagents, Inc., Golden, CO); characteristics of this antibody have also been published previously [26].
Biotinylated conjugated goat anti-rabbit IgG and alpha methyl coumarin acid (AMCA)-linked streptavidin were purchased from Vector Laboratories (Burlingame, CA). Fluorescein (FITC)-conjugated goat anti-rat IgG and Texas red-conjugated goat anti-mouse IgG were purchased from Jackson Immunoresearch (West Grove, PA).
Triple Immunocytochemical Labeling
Sections were washed three times (10 min) each in PBS (0.01 M, pH 7.4). For PR immunodetection [6], sections were transferred into 10 mM citrate buffer (pH 6) and boiled for three cycles of 3 min in a 500-W microwave oven. After cooling at room temperature (30 min), sections were washed in PBS and incubated for 72 h in 0.3% Triton X100 and 5% normal goat serum in PBS containing anti-PR (0.5 µg/ml), anti-ER (4.5 µg/ml), and anti-GR (1:1000) antibodies (4°C; humid atmosphere). After washing, sections were incubated with Texas red-labeled anti-mouse IgG (1:200, 90 min, 4°C), washed, placed in FITC anti-rat IgG (1:200, 90 min, 4°C), washed, incubated in biotinylated anti-rabbit IgG (1:300, 60 min, 4°C), and finally placed in AMCA-streptavidin (1:200, 60 min, 4°C). Sections were washed and coverslipped with an antifading mounting medium (Vectashield, Vector Laboratories).
Specificity of the Staining
Specificities of the anti-PR [6] and anti-ER [11] antibodies have been previously established in the ovine brain. The specificity of staining for GR in the ewe brain was established after preadsorption of anti-GR antibody for 24 h with synthetic peptide (2 µg/ml; Affinity Bioreagents). Controls included omission of primary antibodies; substitution of primary antibodies with 5% normal mouse, rabbit, and rat sera; and incubation with primary antibodies followed by secondary antibodies raised in inappropriate species. No specific immunoreactivity was observed on sections following any of the control procedures.
Analysis of Results
The observation, evaluation, and image acquisition were made using a Leica DMRB microscope connected to a PC-monitored system equipped with image acquisition software. Three Pleomopak filters were used to detect triple-labeled neurons: AMCA-colored cells were observed through an A filter (excitation wavelength = 340–380 nm), fluorescein-labeled neurons were seen with an L3 filter (excitation wavelength = 450–490 nm), and Texas red staining was detected with an N2.1 filter (excitation wavelength = 515–560 nm). Identification of triple-fluorescent neurons was done by switching from one filter cube to the other during the observation. Observation of triple labeling was made every 200 µm. For each animal, sets of pictures from the same areas, taken with the three different filters, were randomly acquired throughout the rostrocaudal extension of the medial preoptic area (n = 5) and arcuate nucleus (n = 10), delimited following the description of Lehman et al. [11]. Cell counts were performed with software (Adobe Photoshop) allowing superimposition of the three images of the same area. Because no significant differences in the percentages of coexpression between the different populations were obtained between the rostral, medial, and caudal levels of the medial preoptic area or of the arcuate nucleus in each group (data not shown), quantitative data were expressed as the means (±SEM) of single-, double-, and triple-labeled cells/area (medial preoptic area or arcuate nucleus) per animal (Table 1). For each experimental group, colocalization ratios (±SEM) are expressed with respect to the different cell populations in the medial preoptic area and arcuate nucleus (Table 2). Data were analyzed using the Student unpaired t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PR and ER immunostaining were found only in the nucleus, and the distributions of PR- and ER-labeled neurons concur with previous descriptions in the ovine brain [4, 6, 11]. Briefly, PR was found mainly in the preoptic area, the ventrolateral part of the ventromedial nucleus and in the rostrocaudal extension of the arcuate nucleus. ER occupied the same areas and is also found in some limbic structures such as the bed nucleus of the stria terminalis and lateral septum. Extensive overlapping in the distribution of the two receptors was found mainly in the medial preoptic area (Fig. 1, A and B), in the whole rostrocaudal extension of the arcuate nucleus (Fig. 1, D and E), and in the ventrolateral part of the ventromedial nucleus. Although there was no statistical difference between the two steroidal treatments in the number of ER- and PR-immunoreactive neurons (Table 1) and no apparent difference in the visually quantitated intensity of ER-labeled cells, the nucleus of PR-immunopositive neurons appeared to be more distinctly delineated in the E+P-treated animals (Fig. 2, A and B). In contrast, in P-only-treated animals, cell limits were often blurred (Fig. 2, C and D). In this respect, it was easier to see that the nucleolus was free of labeling in the E+P-treated group than in the P-only group (compare Fig. 2, A and B with C and D). In the E+P group, labeled cells also often seemed brighter than in the P-only group (Fig. 2).
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All PR-containing cells possessed ER immunoreactivity in E+P- (Fig. 1, D and E) and in P-only-treated animals. There was no significant difference in the percentage of ER cells that contain PR (Table 2) between E+P- and P-only-treated ewes in the preoptic area (E+P, 95%; P-only, 88%) as well as in the arcuate nucleus (E+P, 96%; P-only, 93%). There were also no significant differences in the colocalization ratios between the different antero-posterior levels of each structure for each group (data not shown).
Colocalization Between PR, ER, and GR
As reported previously [20], GR immunoreactivity was detected in both the nucleus and cytoplasm, although the signal seemed weaker in the cytoplasm (Fig. 1F). The distribution of GR-immunoreactive neurons was consistent with our previous findings [20]. Briefly, GR neurons appear densely grouped in the diagonal band of Broca (Fig. 3C), the medial preoptic area (mainly in its periventricular aspect; Fig. 1C), the suprachiasmatic nucleus, and the rostrocaudal extension of the arcuate nucleus. A more limited concentration of GR-immunoreactive cells was also seen in the other hypothalamic areas, such as the rostromedial part of the ventromedial nucleus (Fig. 3F) and in some limbic structures. The number of GR-immunolabeled cells was higher in animals treated with progesterone alone (Table 1). There was substantial overlap of GR-immunoreactive cells with cells showing PR and ER immunoreactivity, mainly at the level of the preoptic area (Fig. 1, A–C), arcuate nucleus (Fig. 1, D–F), and ventrolateral part of the ventromedial nucleus, but only GR were observed in some areas, such as the diagonal band of Broca (Fig. 3, A–C) and the rostromedial part of the ventromedial nucleus (Fig. 3, D–F). It appears that a subpopulation of GR-immunoreactive cells contains only ER (Fig. 1, E and F) or ER and PR (Fig. 1, D–F) in the preoptic area and arcuate nucleus of E+P- (Fig. 1, D–F) and P-only-treated ewes.
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Quantitative analysis revealed large numbers of neurons expressing both ER and GR in the medial preoptic area and arcuate nucleus (Table 1). These neurons represent between 69% of ER cells in the preoptic area and about 70% in the arcuate nucleus. From the GR neuronal population, 79% in the preoptic area and 66% in the arcuate nucleus possess ER (Table 2). There was no significant difference between treatment groups in any region or between levels of each region in any group (data not shown).
A large subpopulation of neurons triple labeled for PR+ER+GR has been analyzed (Table 1). These neurons represent 71% of PR, 68% of ER, and 75% of GR cell populations in the medial preoptic area of the E+P animals (Table 2). Although no significant difference was found with the P-only-treated group, the percentages were slightly lower: 67% of PR, 60% of ER, 69% of GR (Table 2). Triple-immunofluorescent cells represent 93% of ER+GR cells in the E+P group and 88% in the P-only-treated group (Table 2).
In the entire arcuate nucleus, PR+ER+GR cells represent about 65% of PR, ER, and GR cell populations in E+P-treated ewes and 61–75% of the single-labeled populations in animals receiving only P (Table 2). Triple-labeled cells also represent 96% of ER+GR in E+P-treated ewes and 93% in P-only-treated animals (Table 2). There was no significant difference in the percentages of colocalization between the treatment groups in the arcuate nucleus.
No significant difference in colocalization ratios was obtained between the rostral, medial, and caudal levels of the medial preoptic area and arcuate nucleus in the E+P or P-only group (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the brain of the ewe and, more importantly, is the first to report colocalization of PR, ER, and GR within the mammalian hypothalamus.
The demonstration that all PR-containing neurons in the ovine arcuate nucleus and preoptic area contain ER concurs with previous results obtained in guinea pigs [27, 28]. Similarly, in the rat, data suggest that the estrogen-inducible PR occurs only in cells expressing ER [29] and, in the arcuate nucleus, PR has been taken as a conservative marker for ER [30]. PR labeling was weaker in ewes pretreated only with progesterone, suggesting a lower amount of PR protein in neurons of these ewes. This finding is consistent with reports that estradiol significantly increases PR in the ewe [4] and other species [31]. Perhaps surprisingly, there was no difference in the number of PR-immunoreactive cells between E+P- and progesterone-only-pretreated ewes. Studies on other species have shown that estradiol pretreatment increases the number of detectable PR-immunoreactive cells. However, as our animals were long-term ovariectomized, we have two suggestions, which are not mutually exclusive, explaining why ewes in our study had similar PR levels. First, because ER-labeled cells were as numerous and bright in the E+P-treated group as in the animals receiving only progesterone, it is possible that adrenal sex steroids [32–34] may be sufficient to induce PR expression in sheep. Indeed, a previous study has shown that the difference in mean circulating concentration of estradiol between ovariectomized and ovariectomized plus 1-cm estradiol implant is only 0.8 pg/ml [21]. Second, progesterone itself may be able to induce PR. Indeed, this could explain why, despite estrogen priming, the strength of GnRH suppression by progesterone is dependent on prior progesterone exposure [35]. This hypothesis is also supported by a study on ovariectomized hypothalamic-pituitary disconnected ewes, which reported greater progesterone binding in the pituitary following E+P administration than following estradiol treatment alone [36]. Finally, it is possible that unmasking the PR, which is essential for PR revelation in the ewe using the PgRAb8 antibody, improves the threshold of detection. Recent studies have revealed that high-temperature antigen retrieval also significantly increases the number of detected PR [22] and ER [37] cells in the brain of other species. In the preoptic area of the guinea pig, antigen retrieval increased the number of immunocytochemically detectable PRs by over 100% [22]. Moreover, in the long-term ovariectomized Wistar rat, ER was detectable only after antigen retrieval [37]. We have also found that antigen retrieval augments the number of ER-immunoreactive cells in the sheep brain (unpublished data).
Approximately two thirds of the ER-, PR-, and GR-immunoreactive population was labeled for the two other receptors. Colocalization of ER with GR in the arcuate nucleus of the ewe concurs with results of a preliminary report performed in rats, where 10–20% of ER-immunoreactive cells possessed GR immunoreactivity in this area [38], although, unlike our study, no colocalization was detected in the medial preoptic area. Our data also agree with a study on rainbow trout, which reported colocalization between many ER and GR cells of the preoptic area [39].
The present study suggests strongly that glucocorticoids and ovarian steroids can affect the cellular machinery of the same cell. This hypothesis is strengthened by in vitro investigations revealing that ER, PR, and GR compete and/or interact for the same response elements at the transcription level [40, 41]. Indeed, GR is able to block ER transcriptional activation through the AP-1 response element and PR and ER interact with each other at this response element [41]. Transcriptional interference between the GR and ER has also been noted in rainbow trout, and it has been postulated that this interference explains the negative effects of cortisol on estrogen-mediated effects in this species [42]. These studies demonstrate that ER, GR, and PR influence each other's transcriptional activation properties. Our results provide evidence that such interaction between PR, ER, and GR for the same response element is possible within neurons of the ovine hypothalamus. Interestingly, many studies have shown an up- or down-regulation of the expression of PR, ER, and/or GR by progesterone, estradiol, and the glucocorticoids. For example, estradiol up-regulates PR [4] and down-regulates ER [43, 44] and GR [45] gene expression. Progesterone increases ER levels in monkeys [46] and rats [47], increases or decreases PR levels in rodents [48, 49], but seems ineffective in GR regulation [50, 51]. Moreover, glucocorticoids decrease GR gene expression in the rat brain [50] and have been shown to increase ER mRNA in ovine myometrial cells [52]. The effect of glucocorticoids on PR gene expression has not been investigated. This suggests that cross-talk between one or several steroid receptors is potentially able to modulate the cellular response.
Several neuropeptides have been implicated in both the reproductive and stress axes and have been linked to one or more of these steroid receptors. These neuropeptides are also known to affect reproduction when administered and have fluctuating levels during the stress response. For example, endogenous opioid peptides are strongly implicated in regulating GnRH secretion [53], and in the arcuate nucleus, ß-endorphin coexists with PR in the guinea pig [54] and ER in the sheep [7]. ß-Endorphin expression and release are also increased in vitro by glucocorticoids in rats [55]. Moreover, all ß-endorphin-containing neurons show GR immunoreactivity in the rat [56], and glucocorticoid effects on LH release in this species are thought to be transduced through a neuronal action involving endogenous opioid receptors [57]. Similarly, neuropeptide Y (NPY) is a potent inhibitor of LH release in the ewe [58] and has been implicated in the modulation of GnRH release in other species [59]. Moreover, a small subpopulation of NPY neurons expresses PR in the guinea pig [60] and half of the NPY cells possess GR in the rat [61]. The catecholaminergic system may also be a prime target for these three steroids. ER has been detected in some tyrosine hydroxylase neurons in the ewe [7, 62], as is PR in the guinea pig [63], and some tyrosine hydroxylase neurons of the rat present GR immunoreactivity [64]. Dopamine has been implicated in the regulation of tonic LH release in the ewe [65], and hypothalamic levels of tyrosine hydroxylase increase following a stress in the rabbit [66] and in the rat [67]. Further research is required to establish whether triple-labeled cells occur predominantly in a specific neurotransmitter phenotype.
The question remains as to whether the cells containing ER, PR, and/or GR also express ER in the ewe. The hypothesis suggesting that ER may coexist with PR and ER in some neurons of the ewe brain is attractive because ER immunoreactivity and ER mRNA are coexpressed within most neurons of the medial preoptic area in the rat [68]. Perhaps surprisingly, ER mRNA but not ER mRNA have been detected in rat [69] and mouse [70] GnRH neurons. It is noteworthy that ER and ER mRNA are not cosynthesized in the arcuate nucleus of the ewe [71] and rat [68], but whether ER and ER are colocalized in other hypothalamic regions remains to be determined.
In conclusion, we have demonstrated that all PR-immunoreactive neurons of the ewe preoptic area and arcuate nucleus are also estrogen receptive. Moreover, more than two thirds of the PR- and ER-containing cells express GR, which could explain the close relationships described between stress and impaired reproductive function. Further studies will be necessary to determine the neuropeptidergic content of steroid receptors containing cells and to establish the precise relationship between the steroid receptor-containing neurons and the GnRH system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Laurence Dufourny, University of Wyoming, Department of Zoology and Physiology, Biological Sciences building, Room 428, P.O. Box 3166, Laramie, WY 82071-3166. FAX: 307 766 5625; ldufourny{at}uwyo.edu
Received: 28 February 2002.
First decision: 26 March 2002.
Accepted: 24 May 2002.
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REFERENCES |
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and glucocorticoid receptor in the rat brain. In: Program of the 30th Annual Meeting of the Society for Neuroscience; 2000; New Orleans, LA. Abstract 541.6 This article has been cited by other articles:
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