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and ß in the Rat Uterus1
a Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E2), progesterone (P4), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ER is the dominating subtype in the rat uterus. E2 seemed to increase the ER mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P4 reduced ER expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E2 or P4 did not alter the expression of ERß, on either the mRNA or protein level. In conclusion, the distribution and regulation of ER and ERß differ in the different compartments of the rat uterus. The complex uterine responses to E2 and P4 are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERß probably plays a minor role in the regulation of uterine physiology.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two subtypes of estrogen receptor (ER) have been described: the "classical" now-named ER, and a newly discovered ERß [7]. The rat ERß is a protein of 485 amino acids that is smaller than ER, which consists of 595 amino acids [7, 8]. Since ER was first reported as a mediator of estrogen hormone action, it has been characterized as a ligand-activated transcription factor, a member of the nuclear hormone receptor family, and as involved in many important physiological processes [9]. ER is expressed in a wide range of tissues, and the basal ER levels vary substantially between different cell types [9]. Although ERß is highly homologous to the rat and mouse ER protein, particularly in the DNA-binding domain and in the C-terminal ligand-binding domain [7, 10], and although it shares many functional characteristics of ER, the relative level of ERß mRNA expression and the tissue location of ERß are distinct from those of ER [7, 11].
ER turns on target gene expression and functions as a regulator of ligand-activated transcription in E2-responsive tissues [12], whereas P4 attenuates cell sensitivity to E2 by decreasing ER levels [1]. It has been shown that nuclear ER levels decrease as serum P4 levels increase in the rat uterus [13], and that P4 decreases sensitivity of cells to estrogens by inhibiting ER-mediated transactivation through direct interactions of ligand-bound progesterone receptor (PR) and ER [14]. Interestingly, in the human uterus, ER levels decrease equally in all the cell types in the P4-dominated luteal phase of the menstrual cycle [15]. An explanation for this reduction could be that P4 eliminates the influence of E2 from the cells and thereby antagonizes estrogen-mediated events, including cell proliferation [1].
In rat reproductive tissues, a reverse transciption-polymerase chain reaction study showed that ER expression is moderate to high in the uterus, testis, ovary, and epididymis, whereas ERß expression is high in prostate, ovary, uterus, and testis [11]. This is contradictory to results obtained by in situ hybridization showing ERß expression in the rat uterus to be undetectable or very low [11, 16]. Telleria et al. [17] have demonstrated that mRNAs for ER and ERß are differentially coexpressed in the rat corpus luteum during pregnancy, and the expression of both mRNAs in luteal cells is up-regulated by prolactin and placental lactogens. In both wild-type mice (wt) and ER knockout mice (ERKO), the expression of ERß mRNA differs between the ovary and the uterus [18]. The high ovarian expression of ERß seen in the wt mice is slightly reduced in the ERKO mice [18]. Expression of ERß mRNA variants has been found in breast cancer cell lines and tumors; thus ERß may play a role in the acquisition of antiestrogen resistance [19]. Although both mRNA and protein of ERß have been detected in the rodent uterus [11,18], a specific function of ERß in the uterus has not been established [12]. We have examined the expression and regulation of ERs in the OVX rat uterus after E2 and/or P4 treatment. A solution hybridization method was used to measure the levels of the mRNAs for ER and ERß. In situ hybridization and immunohistochemistry techniques were used to detect the distribution of the two mRNAs and proteins in the various cell types of the rat uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animal studies were approved by the Committee on Animal Care in Sweden and in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Hormones
E2 and P4 were purchased from Sigma Chemical Co. (St. Louis, MO). The hormones were dissolved in 99.5% ethanol at a high concentration and then diluted with propyleneglycol to the proper concentration. The final concentration of ethanol in the injections was less than 5%.
Tissue Collection
During ether anesthesia, the rat uterus was removed, stripped of fat and connective tissue, weighed, and cut into one half piece and two quarter pieces. One quarter was immersion fixed in 4% formaldehyde at 4°C for 8 h and stored at 4°C in 0.1 M NaPO4 + 0.4% formaldehyde and thereafter embedded in paraffin. The rest of the tissue was immediately frozen in liquid nitrogen and stored at -70°C until analyzed.
Preparation of Total Nucleic Acids
Total nucleic acids (TNA) were prepared as described by Sahlin et al. [20]. Briefly, after thawing, one half of the uterus was homogenized and digested with proteinase K (Merck, Darmsladt, Germany) in an SDS-containing buffer, followed by subsequent extraction with phenol-chloroform as described by Durnam and Palmiter [21]. The DNA content of the TNA samples was determined by a fluorometric assay at the wavelength 458 nm with Hoechst Dye 33258 [22].
Hybridization Probes
The in vitro synthesis of radioactive cRNAs was performed essentially as described by Melton et al. [23] using reagents supplied by Promega Biotech (Madison, WI). The probes were radiolabeled with [35S]UTP (Amersham, Bucks, UK).
The antisense ER probe used for mRNA determinations was derived from pMOR101, an EcoRI fragment of 1.9 kilobases containing the whole open reading frame of the mouse ER [24]. It was subcloned into the RNA expression vector pGEM-3. Restriction of this vector with BglII allows the synthesis of a probe corresponding to nucleotides 1470–2062 that encodes the C-terminal half of the steroid-binding domain E and all of domain F. The protein sequence of the mouse ER is 97% homologous to the rat ER [25]. The sense cRNA probe was prepared from a plasmid containing the same cDNA in opposite orientation. Cross-reactivity between the mouse-derived probe and rat ER mRNA has been previously shown [26].
The 300-base pair probe used for ERß mRNA determinations corresponds to an XbaI-EcoRI fragment of the 3' untranslated region, derived from an ERß clone from rat prostate. It was subcloned into a Bluescript KS plasmid. After linearization with XbaI, T3 RNA polymerase was used to transcribe the antisense probe. The sense probe was transcribed from a template linearized with EcoRI using T7 polymerase.
Solution Hybridization Analysis of mRNA
A solution hybridization analysis of specific mRNA was carried out as previously described in Sahlin et al. [20], with the following modification in order to be quantitative. The labeled hybrids protected from RNase digestion were precipitated by addition of trichloroacetic acid and collected on the filters. The radioactivity on the filters was monitored in a scintillation counter, and the results were compared with a standard curve of known amounts of in vitro-synthesized mRNA complementary to the probe used. Results are expressed as amol (10-18) mRNA/µg DNA in the TNA samples.
In Situ Hybridization
Two paraffin sections (5 µm) from each treatment group were used for uterine mRNA examination of ER and ERß by in situ hybridization. A normal rat ovary was used as a positive control of ERß expression. The 35S-labeled sense probes were used as controls to define background levels of silver grains on sections adjacent to those probed with the antisense probes.
Tissue sections were dewaxed in Bioclear (Aduso Esclusivo Di Laboratorio, Milano, Italy) and rehydrated in descending concentrations of ethanol, followed by a 5-min wash in PBS buffer (0.01 M KPO4/0.15 M NaCl, pH 7.4). The sections were treated with proteinase K (1 µg/ml) in PBS at 37°C for 30 min. The slides were washed in 0.1 M triethanolamine buffer (pH 8.0) for 5 min at room temperature (RT) and acetylated for 10 min with 0.25% acetic anhydride. After two 5-min washes in double-strength SSC (single-strength SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) and a rinse in RNase-free H2O, the slides were dehydrated in ascending concentrations of ethanol and air dried at RT for 30 min. Slides were put in a 60°C oven for 30 min in order to linearize the RNA. A denaturing hybridization mixture solution (50% formamide, double-strength SSC, single-strength Denhardt's, 20 mM Tris-HCl, 1 mM EDTA, 10% dextrane sulfate, 0.5 mg/ml yeast tRNA, and 0.1 M dithiothreitol) containing 25 x 106 cpm/ml probe was preheated and put on each section. The slides were covered with Parafilm (American Can Co., Greenwich, CT) and incubated in a humidified container at 55°C overnight (16–20 h). After the slides were washed in 4-strength SSC (containing 10 mM dithiothreitol), 0.5-strength SSC, and 0.1-strength SSC, treatment with RNase A (20 µg/ml) for 30 min at 37°C was followed by a wash in RNase buffer (2 M Tris-HCl, 0.5 M EDTA, 5 M NaCl, pH 8.0) for another 30 min. The slides were treated with a series of double-strength SSC and 0.1-strength SSC washes at RT. After dehydration in ethanol, slides were air dried at RT for at least 30 min. The slides were dipped in 1:1 autoradiography emulsion (NTB-2; Eastman Kodak, Rochester, NY) and exposed for 6 wk at 4°C before developing. Kodak Developer D19 and fixer were used for processing the slides. Thereafter sections were counterstained with hematoxylin and eosin Y, dehydrated, and mounted with Pertex (Histolab, Göteborg, Sweden). Background hybridization signals were detected on control slides that were incubated with the respective sense cRNA probe.
Immunohistochemistry
Paraffin sections (5 µm) from 5 uteri of each experimental group were used. A standard immunohistochemical technique (avidin-biotin-peroxidase) was used to visualize ER and ERß immunostaining intensity and distribution. A monoclonal mouse anti-human antibody was used for detection of ER (Zymed Laboratories, San Francisco, CA). It recognizes the N-terminal domain (A/B region) of ER. A polyclonal rabbit anti-rat ERß antibody (PA1–310; Affinity Bioreagents, Golden, CO), which corresponds to the C-terminal amino acid residues 467–485, was used for detection of ERß.
After the tissue sections were dewaxed and rehydrated, an antigen retrieval procedure was performed. Sections were pretreated in a microwave oven at high power, in 0.01 M sodium citrate buffer (pH 6.0) for 10 min, and then allowed to cool for a further 20 min. After washing in buffer (0.1 M PBS, pH 7.6, for ER and 0.1 M Tris-buffered saline [TBS], pH 7.4, for ERß), nonspecific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide (Merck) in methanol for 10 min at RT. After a 10-min wash in buffer, sections were exposed to a 30-min non-immunoblock using diluted normal horse serum (Vectastain; Vector Laboratories, Burlingame, CA) in PBS for ER immunostaining and normal goat serum (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS containing 5% (w:v) BSA for ERß, in a humidified chamber at RT. The tissue sections were thereafter incubated with the primary antibody. The ER antibody was diluted 1:6 in PBS and incubated on sections at 4°C overnight. The ERß antibody was diluted 1:200 in TBS with 5% BSA and incubated at 4°C for 48 h. Negative controls were obtained by replacing the primary antibody with non-immunoserum of the equivalent concentration. In addition, the ERß antibody was preabsorbed with neutralizing synthetic ERß peptide (PEP 007; Affinity Bioreagents) overnight to demonstrate antigen specificity. After primary antibody binding, the sections were incubated with the appropriate second antibody: for ER, a biotinylated horse anti-mouse IgG (Vectastain; Vector) diluted in normal horse serum was used for 60 min at RT; for ERß, a biotinylated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology) diluted in normal goat serum was incubated for 30 min at RT. Thereafter the tissue sections were incubated for 30–60 min at RT with a horseradish peroxidase-avidin-biotin complex (Vectastain Elite; Vector). The site of the bound enzyme was visualized by the application of 3,3'-diaminobenzidine in H2O2 (DAB kit; Vector), a chromogen that produces a brown, insoluble precipitate when incubated with enzyme. The sections were counterstained with hematoxylin and dehydrated before they were mounted with Pertex.
Image Analysis
In order to more accurately estimate the expression of ER mRNA in a particular cell type of the uterus, a Leica microscope and Sony (Park Ridge, NJ) video camera connected to a computer using an image analysis system (Leica Imaging System Ltd., Cambridge, England) were used to quantitate the number of silver grains per measuring field in sections from control and treated animals. Quantification was performed on the digitized images of systematic randomly selected fields of stromal, epithelial, and glandular cells of the endometrium. Ten fields were analyzed from each section (2 sections per uterus) in 2 rats from each group, and the result obtained by the antisense probe was compared to the background signal obtained from the sense probe on adjacent sections. Results are expressed as the ratio of silver grain numbers obtained from the antisense and sense probes. Comparisons were performed between cell types and between treatment groups.
A Leica microscope connected to a computer using Colorvision software (Leica Imaging System Ltd.) was used to assess immunostaining quantitatively by a computer imaging analysis system. Quantification of immunostaining was performed on the digitized images of systematic randomly selected fields of endometrial stroma, from which non-stromal elements (e.g., luminal and glandular epithelium) were interactively removed and analyzed separately. Twelve fields were analyzed and measured separately in each section of luminal and glandular epithelia and stromal cells (2 sections/uterus). With use of color-discrimination software, the total area of positively stained cells (brown reaction product) was measured and expressed as a ratio of the total area of cell nuclei (brown reaction product + blue hematoxylin). Furthermore, the density of the positive staining was semiquantitatively measured by three different color discriminations: strong (+++), moderate (++), or faint (+) brown reaction.
Statistics
The results of the uterine mRNA levels from the solution hybridization assays are presented as mean ± SEM. The evaluation was made by one-way ANOVA, and significance was determined by Scheffés test [27]. The results of in situ hybridization and immunohistochemistry as measured by image analysis are presented as median and range. The statistical calculation was performed by ANOVA on ranks followed by Kruskal-Wallis test. Significance was determined by Dunn's test. Values with different letter designations are significantly different (P < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The uterine wet weight was highest in the 48EE group and was significantly increased in all groups treated with E2 as compared to the OVX control (data not shown).
Expression of ER and ERß in Rat Uterus
To ascertain the relative distribution of the two subtypes of ER (ER and ERß) in the rat uterus, a solution hybridization assay was performed to quantitatively determine the mRNA levels, and in situ hybridization was carried out to localize the two ER mRNAs. In addition, immunohistochemistry was performed to localize and semiquantitatively measure ER and ERß protein concentrations in the various cell types of the uterus.
Solution Hybridization
The results revealed that both types of ER mRNAs were expressed in the rat uterus.
With respect to ER, the group treated with E2 24 h before animals were killed showed an increased level of ER mRNA as compared to that in the OVX control. P4 had no effect on the ER mRNA level on its own (Fig. 1a).
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With respect to ERß, ERß mRNA expression in the rat uterus was lower than that of ER (Fig. 1b). The ERß mRNA level was highest in the 24E group, in which the level was significantly different from that in the 48PP and 48EP groups.
The data from the solution hybridization assays present an average of the mRNA level among the various cell types existing in the uterus. For a preliminary estimation of the distribution of the mRNAs between the various cell types, see in situ hybridization data (Fig. 2 and Table 2).
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In Situ Hybridization
ER mRNA was expressed at a high level in stromal and epithelial cells of the OVX rat uterus (Fig. 2a). ER mRNA was also expressed in myometrium (Fig. 2b). The minimal background signal on adjacent sections for endometrium and myometrium is shown in Figure 2, c and d, respectively. To compare ER mRNA expression in particular cell types of the uterus, graphic representation of silver grain counts per field, obtained with an image analysis system, is shown in Table 2. ER mRNA expression in stromal cells was slightly lower in the 24P and 48EE groups as compared to the OVX control rats. The 48PP, 48EP, and 48PE groups all tended to have an even more decreased ER mRNA level as compared to the OVX controls (Table 2). In glandular epithelium (GE) and luminal epithelium (LE), the trend was a slightly increased level of ER mRNA in the 24E group as compared to the OVX control group. The ER mRNA level in the LE showed a tendency to decrease in the groups receiving P4 the last 24 h before death. In GE, only the 48PP group seemed to have a decreased ER mRNA level as compared to the OVX controls. These results were obtained from two portions of two rat uteri from each group, and were included in order to demonstrate the trends of localization of ER mRNAs in support of the data obtained by solution hybridization and immunohistochemistry.
The ERß mRNA detected in the stroma and epithelium of the uterus as found by in situ hybridization was low as compared to the expression of ERß in preovulatory follicles of the rat ovary (Fig. 2, e–g). Minimal background levels were observed on adjacent sections of uterus and ovary after hybridization with ERß (Fig. 2, h–j) sense probe. No differences in the ERß mRNA levels were seen between the experimental groups when comparison was within the various cell types (data not shown).
Immunohistochemistry
ER. ER immunoreactivity was confined to the nuclei of endometrial epithelium and stroma, as well as myometrium. In OVX control uteri, a high intensity of ER immunostaining was seen in the stroma and GE (Fig. 3a and Table 3), with 65% of stromal nuclei and virtually all nuclei of GE staining strongly positive (Fig. 4, a–b). In the LE, 90% of the cell nuclei stained positive (Fig. 4c), but less intensely than in the GE (Fig. 3a and Table 3). In the groups treated with E2 alone, no significant changes in the numbers of cells expressing ER in the stroma, GE, and LE (Fig. 3, d–e, and Fig. 4) were observed. The stronger immunostaining (++/+++) of ER was significantly decreased in the GE. No significant changes were observed in the intensity of ER staining in the LE and stroma after single E2 treatment (Table 3). In the groups treated with P4 alone, both cell numbers and intensity of ER immunostaining were significantly decreased in stromal cells of the 24P group (Fig. 3g, Fig. 4a, and Table 3). A significant decrease was observed in the intensity and cell numbers for ER immunoreactivity in the LE after 24-h or 48-h P4 treatment (Fig. 3, g–h, and Fig. 4c), which was not seen in the GE (Fig. 3, g–h, and Fig. 4b). After E2+P4 treatment, the group receiving E2 first (48EP) showed no significant changes in cell numbers or intensity of ER expression in the stroma, GE, and LE as compared to the OVX control group (Fig. 3f, Fig. 4, and Table 3). In the group receiving P4 first (48PE), the stronger intensity (++/+++) of ER staining was significantly decreased in the stroma and GE as compared to that in the OVX group (Fig. 3i and Table 3). The cell numbers for ER immunoreactivity in GE were also significantly decreased (Fig. 4b). There were no significant changes in cell numbers or intensity of ER staining in the LE in the 48PE group, as compared to the OVX group (Fig. 4c).
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In addition, a difference of the intensity of ER immunostaining in the LE was found between the 24P and 48EP treatment groups, the latter showing higher immunoreactivity (Fig. 4c and Table 3).
The presence of ER was observed in both circular and longitudinal myometrium in the OVX control (Fig. 3c) and treated groups. Positive ER immunoreactivity was also observed in vascular smooth muscle cells in the endometrium and myometrium (Fig. 3c). Most of the endothelial cells of the vessels were negative (Fig. 3c). No immunostaining was observed in the negative controls (Fig. 3b).
ERß. As with ER, ERß immunoreactivity was also confined to the cell nuclei of the endometrium and myometrium. ERß immunostaining in stroma, GE, and LE was faint (+), with 51–62% of cell nuclei staining positive in OVX rats (Fig. 5a and Table 4). Results from image analysis (Table 4) showed that about 48–64%, 35–50%, and 56–68% of cells in the stroma, GE, and LE, respectively, stained positive in the treatment groups. The cell numbers and intensity of immunostaining of ERß seemed slightly higher in the stroma of the groups treated with E2 the first 24 h (Fig. 5, d–f, and Table 4). However, no significant differences in immunoreactivity were observed in the OVX control as compared to the treated groups (Fig. 5 and Table 4). Consistent with results from in situ hybridization, ERß immunoreactivity was intensely expressed in granulosa cells of preovulatory follicles in the rat ovary (data not shown). Positive immunostaining for ERß was observed in the myometrium and vessels. Most endothelial cells in arteries stained positive for ERß (Fig. 5c).
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No specific nuclear staining was found in the negative control sections after incubation with serum (data not shown) or the peptide corresponding to the epitope of the antibody (Fig. 5b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was first suggested as a mediator of estrogen action by Jensen and Jacobson in 1962 [28]. A second subtype of ER was discovered in 1996 by Kuiper et al. [7] and named ERß. No comparison of ER and ERß expression in the uterus on both mRNA and protein levels has been performed previously. In this paper, we present the results on both ER and ERß expression in the rat uterus after steroid hormone treatment.
The present results demonstrate that ER is the dominating subtype in the rat uterus. The uterine ER mRNA level was significantly up-regulated by E2 in OVX rats after 24-h treatment. This is consistent with the results from previous studies [16, 20, 29]. The distribution of ER mRNA as determined by in situ hybridization indicated that the effect of estrogen was transient and was predominant in the GE and LE. Our results from immunohistochemistry showed that P4 down-regulated the ER level in the LE after 24-h and 48-h treatment, with a corresponding trend for the ER mRNA level. In the stroma, ER immunoreactivity was decreased after 24-h P4 treatment, but the ER mRNA level seemed to have decreased after 24 h and even more so after 48-h treatment. In the GE, P4 seemed to reduce the ER mRNA levels only after 48-h treatment.
The mechanism of E2-mediated up-regulation of ER is still unclear, but the most well-supported mechanism of estrogen up-regulation of target gene expression involves enhanced rates of transcription [30]. E2 may also up-regulate endometrial ER gene expression posttranscriptionally [31]. It has been shown that E2 induces DNA synthesis 12–16 h after injection, and mitosis follows at 18–24 h in OVX rats [4, 5]. A single injection of E2 stimulates mitosis in the LE and GE, but not in the stroma [5]. In addition, it is well known that P4 strongly opposes the proliferative effect of estrogens in the uterine epithelium [32], and P4 inhibits E2-induced uterine epithelial proliferation through PR [33]. There is considerable biological evidence for cross-talk between ER and PR signaling pathways [14]. E2-induced epithelial proliferation in the uterus is mediated indirectly via stromal ER [34]. P4 selectively decreases ligand-bound ER and inhibits recovery of ER at transcriptional and translational levels in the rat uterus [1]. The repression of ER-mediated transcriptional activity and the inhibition of ER retention by P4 are mediated by the ligand-bound PR [14, 35]. The present study demonstrates that P4 significantly decreased the ER protein level in the LE, but not in the GE. A recent study showed that the LE and GE display different regulation of ER expression [36]. Thus, the GE and LE are differently regulated both by E2 and P4.
In contrast to some of ER mRNA trends, the stronger immunoreactivity of ER showed the highest concentration in the stroma and GE in the OVX rats (Table 3). E2 decreased the intensity of ER immunoreactivity in GE, whereas the ER mRNA level seemed to increase. This negative effect on the intensity of ER immunostaining in GE agrees with a previous study in the mouse [36]. It has been shown that pretreatment with P4 suppresses epithelial mitosis and instead causes a mitotic response to E2 in the stroma [5]. In the present study, no regulatory effects of single E2 treatment on the ER level were observed in the LE or the stroma. However, when E2 was given 24 h prior to P4, ER immunoreactivity increased in the LE as compared to the level after treatment with a single injection of P4, with a similar trend seen for the ER mRNA level. E2 stimulates and P4 inhibits proliferation of the uterine epithelium via the stromal ER and PR, respectively [33]. P4 appears to selectively inhibit E2-dependent down-regulation of ER in the GE in a PR-dependent manner while having no significant effect on ER expression of stroma or LE [36]. Our results confirm a complex regulation of E2 and P4 in the rat uterus that may be directly or indirectly mediated by ER and PR, the effects varying between the cell types [33].
The contrasting results between mRNA and protein levels of ER in GE could be explained by a temporally different regulation by E2. It has been shown that an increase in endometrial ER mRNA levels precedes enhanced ER protein concentration during the preovulatory estrogen surge in ewes [37]. The E2-suppressed steady state of ER mRNA caused a decrease in ER protein levels in the rat uterus [38]. The short half-lives of both mRNA and protein products of the ER gene make ER levels susceptible to transcriptional and/or post-transcriptional regulation [38,39].
ERß expression in the rat uterus was much weaker than that of ER in all groups. Our unpublished data were consistent with reports showing that OVX slightly up-regulated ER mRNA [29] and decreased ERß mRNA expression [16] in the rat uterus. We found that the distribution of ERß mRNA detected by in situ hybridization was very weak in the stroma, epithelium, and myometrium of the OVX uterus. E2 and/or P4 had no effect on the ERß mRNA expression in the various uterine compartments, although the total mRNA level in the rat uterus was higher in the 24-h E2 treatment group as compared to the 48-h P4 and E2+P4 treatment groups. A recently published study by Shughrue et al. [16] using in situ hybridization showed only a weak ERß mRNA signal present in the intact uterus and undetectable levels of ERß mRNA in the OVX rat. E2 did not regulate ERß expression in the rat uterus [16]. This low ERß expression is in agreement with our in situ hybridization results, although we did find weak ERß expression also after OVX.
The present study showed that the pattern of immunolocalization of ERß in the uterine compartments is similar to that of ER. ERß was observed in the LE and GE and was distributed throughout the stroma with weaker positive immunostaining than for ER in all groups. The immunolocalization of ERß is in agreement with the recent report by Saunders et al. [40] and shows partial disagreement with a report by Hiroi et al. [41]. The latter group detected ERß immunostaining only in the nuclei of the GE, the ERß protein level being too low to detect in the LE of the intact rat uterus [41].
It has been shown that ERß mRNA is expressed in very low levels in the ERKO and wt mouse uterus and vagina [18]. These findings may explain why characteristic estrogen-induced responses in the ERKO mice are not observed when these animals are treated with E2 [42]. It is uncertain whether ERß plays any role in the E2 responses in rodent uterus [16, 18]. Our results, together with those of others from studies in the rat and mouse [16, 18], show that ERß is less prominent than ER in the uterus. ERß does not appear to mediate any of the known actions of E2 on the uterus [16, 43, 44], but it may still have an unknown function in the regulation of uterine physiology. Krege et al. [44] have published a study on ERß knockout mice indicating that since female ERß knockouts are fertile, ERß deficiency does not seem to preclude successful gestation and production of viable pups, although the litters are fewer and the litter size is smaller. Therefore, uterine function must not be too compromised in these animals—indicating that ERß is essential for normal ovulation efficiency but not for female fertility [44].
In addition, we found that endothelial cells in vessels (artery) were positive for ERß; these findings differed from those for ER immunoreactivity, which was mostly seen in vascular smooth muscle cells. Responses to estrogens have been found in both smooth muscle cells and endothelium of the vessels, which could be directly mediated by ERs [45]. Our results suggest that ERß and ER may play different roles with regard to the effects of E2 on vessels in the rat uterus and thereby regulate uterine function also indirectly.
The biologic significance of two ERs is still unclear. However, the differential distribution of these two ERs among the tissues and within the compartments of different tissues provides some understanding of how estrogens can have tissue-specific and species-specific actions [46, 47]. Since the two ER subtypes have been shown to heterodimerize in vitro, and the transcriptional activity of this complex is different from that of the homodimers [10], the effects of estrogens may differ depending on whether a cell expresses ER, ERß, or both [16].
The discovery of ERß [7] raises the possibility of reevaluating the functions of ERs in mediating E2-induced epithelial proliferation and differentiation in reproductive tissues. The identification of ERß will help to elucidate the actors that mediate the diverse and important pleiotropic actions of estrogens in its target tissues.
In conclusion, we have shown that the expression and regulation of ER and ERß on both mRNA and protein levels differ in the various cell types of the rat uterus, and that ER is the predominant ER. Furthermore, our results support the concept that E2 and P4 have both positive and negative effects on ER expression in the epithelial and stromal compartments. E2 enhances ER mRNA synthesis in the rat uterus, and the complex uterine responses to E2 and P4 are directly or indirectly mediated by differential cell-specific expression of their receptors. We present evidence that functional responses to P4 treatment in relation to ER expression differ between LE and GE. E2 and/or P4 did not show any prominent effects on ERß expression, which suggests that ERß alone may not be sufficient to play any major role in the regulation of uterine physiology; still, ERß could be regulated by factors other than steroid hormones and therefore be of importance in the uterus.
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ACKNOWLEDGMENTS |
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was kindly supplied by Prof. M.G. Parker, Imperial Cancer Research Fund, London, UK. The cDNA for ERß was a generous gift from Dr. G.G.J.M. Kuiper and Prof. J-Å Gustafsson, Karolinska Institutet, Huddinge, Sweden. |
FOOTNOTES |
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2 Correspondence: Lena Sahlin, Division for Reproductive Endocrinology, Karolinska Hospital, L5:01, S-17176 Stockholm, Sweden. FAX: 46 8 51773485; lena.sahlin{at}kbh.ki.se
Accepted: May 11, 1999.
Received: February 2, 1999.
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  H. H Ortega, N. R Salvetti, and V. Padmanabhan Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance Reproduction, May 1, 2009; 137(5): 865 - 877. [Abstract] [Full Text] [PDF]
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 M. V.T. Lobo, L. Huerta, M. I. Arenas, R. Busto, M. A. Lasuncion, and A. Martin-Hidalgo Hormone-sensitive Lipase Expression and IHC Localization in the Rat Ovary, Oviduct, and Uterus J. Histochem. Cytochem., January 1, 2009; 57(1): 51 - 60. [Abstract] [Full Text] [PDF]
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 N. H. Ing, D. A. Massuto, and L. A. Jaeger Estradiol Up-regulates AUF1p45 Binding to Stabilizing Regions within the 3'-Untranslated Region of Estrogen Receptor {alpha} mRNA J. Biol. Chem., January 18, 2008; 283(3): 1764 - 1772. [Abstract] [Full Text] [PDF]
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 M. Heneweer, R. Houtman, J. Poortman, M. Groot, C. Maliepaard, and A. Peijnenburg Estrogenic Effects in the Immature Rat Uterus after Dietary Exposure to Ethinylestradiol and Zearalenone Using a Systems Biology Approach Toxicol. Sci., September 1, 2007; 99(1): 303 - 314. [Abstract] [Full Text] [PDF]
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 D. Stygar, B. Masironi, H. Eriksson, and L. Sahlin Studies on estrogen receptor (ER) {alpha} and {beta} responses on gene regulation in peripheral blood leukocytes in vivo using selective ER agonists J. Endocrinol., July 1, 2007; 194(1): 101 - 119. [Abstract] [Full Text] [PDF]
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 H. Zang, L. Sahlin, B. Masironi, E. Eriksson, and A. Linden Hirschberg Effects of Testosterone Treatment on Endometrial Proliferation in Postmenopausal Women J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2169 - 2175. [Abstract] [Full Text] [PDF]
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 J. C. Kwekel, L. D. Burgoon, J. W. Burt, J. R. Harkema, and T. R. Zacharewski A cross-species analysis of the rodent uterotrophic program: elucidation of conserved responses and targets of estrogen signaling Physiol Genomics, November 17, 2005; 23(3): 327 - 342. [Abstract] [Full Text] [PDF]
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J Varayoud, J G Ramos, L Monje, V Bosquiazzo, M Munoz-de-Toro, and E H Luque The estrogen receptor {alpha} {Sigma}3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus J. Endocrinol., July 1, 2005; 186(1): 51 - 60. [Abstract] [Full Text] [PDF]
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 A. Guzeloglu, T. R. Bilby, A. Meikle, S. Kamimura, A. Kowalski, F. Michel, L. A. MacLaren, and W. W. Thatcher Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy J Dairy Sci, October 1, 2004; 87(10): 3268 - 3279. [Abstract] [Full Text] [PDF]
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W. Dietrich, A. Haitel, J. C. Huber, and W. J. Reiter Expression of Estrogen Receptors in Human Corpus Cavernosum and Male Urethra J. Histochem. Cytochem., March 1, 2004; 52(3): 355 - 360. [Abstract] [Full Text] [PDF]
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 H. Cardenas and W. F. Pope Attenuation of Estrogenic Effects by Dihydrotestosterone in the Pig Uterus Is Associated with Downregulation of the Estrogen Receptors Biol Reprod, February 1, 2004; 70(2): 297 - 302. [Abstract] [Full Text] [PDF]
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R. M. Bigsby, A. Caperell-Grant, N. Berry, K. Nephew, and D. Lubahn Estrogen Induces a Systemic Growth Factor Through an Estrogen Receptor-Alpha-Dependent Mechanism Biol Reprod, January 1, 2004; 70(1): 178 - 183. [Abstract] [Full Text] [PDF]
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E. M. Jablonski, N. A. McConnell, F. M. Hughes Jr., and Y. M. Huet-Hudson Estrogen Regulation of Aquaporins in the Mouse Uterus: Potential Roles in Uterine Water Movement Biol Reprod, November 1, 2003; 69(5): 1481 - 1487. [Abstract] [Full Text] [PDF]
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K. C. Fertuck, J. E. Eckel, C. Gennings, and T. R. Zacharewski Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol Physiol Genomics, October 17, 2003; 15(2): 127 - 141. [Abstract] [Full Text] [PDF]
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 D. C. Mitchell and N. H. Ing Estradiol Stabilizes Estrogen Receptor Messenger Ribonucleic Acid in Sheep Endometrium via Discrete Sequence Elements in Its 3'-Untranslated Region Mol. Endocrinol., April 1, 2003; 17(4): 562 - 574. [Abstract] [Full Text] [PDF]
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 P. Dan, J. C. Y. Cheung, D. R. L. Scriven, and E. D. W. Moore Epitope-dependent localization of estrogen receptoralpha , but not -beta , in en face arterial endothelium Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1295 - H1306. [Abstract] [Full Text] [PDF]
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S. B. Pillai, J. M. Jones, and R. D. Koos Treatment of Rats with 17{beta}-Estradiol or Relaxin Rapidly Inhibits Uterine Estrogen Receptor {beta}1 and {beta}2 Messenger Ribonucleic Acid Levels Biol Reprod, December 1, 2002; 67(6): 1919 - 1926. [Abstract] [Full Text] [PDF]
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C. E. Gargett, M. Zaitseva, K. Bucak, S. Chu, P. J. Fuller, and P. A. W. Rogers 17{beta}-Estradiol Up-Regulates Vascular Endothelial Growth Factor Receptor-2 Expression in Human Myometrial Microvascular Endothelial Cells: Role of Estrogen Receptor-{alpha} and -{beta} J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4341 - 4349. [Abstract] [Full Text] [PDF]
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J. G. Ramos, J. Varayoud, V. L. Bosquiazzo, E. H. Luque, and M. Munoz-de-Toro Cellular Turnover in the Rat Uterine Cervix and Its Relationship to Estrogen and Progesterone Receptor Dynamics Biol Reprod, September 1, 2002; 67(3): 735 - 742. [Abstract] [Full Text] [PDF]
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 A. Stavreus-Evers, B. Masironi, B.-M. Landgren, A. Holmgren, H. Eriksson, and L. Sahlin Immunohistochemical localization of glutaredoxin and thioredoxin in human endometrium: a possible association with pinopodes Mol. Hum. Reprod., June 1, 2002; 8(6): 546 - 551. [Abstract] [Full Text] [PDF]
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M. V.T. Lobo, F. J. M. Alonso, A. Latorre, and R. M. del Rio Immunohistochemical Localization of Taurine in the Rat Ovary, Oviduct, and Uterus J. Histochem. Cytochem., September 1, 2001; 49(9): 1133 - 1142. [Abstract] [Full Text] [PDF]
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D. Stygar, H. Wang, Y. S. Vladic, G. Ekman, H. Eriksson, and L. Sahlin Co-localization of oestrogen receptor {beta} and leukocyte markers in the human cervix Mol. Hum. Reprod., September 1, 2001; 7(9): 881 - 886. [Abstract] [Full Text] [PDF]
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H. O. D. Critchley, R. M. Brenner, T. A. Henderson, K. Williams, N. R. Nayak, O. D. Slayden, M. R. Millar, and P. T. K. Saunders Estrogen Receptor {beta}, But Not Estrogen Receptor {{alpha}}, Is Present in the Vascular Endothelium of the Human and Nonhuman Primate Endometrium J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1370 - 1378. [Abstract] [Full Text]
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H. Wang, Y. Stjernholm, G. Ekman, H. Eriksson, and L. Sahlin Different regulation of oestrogen receptors {{alpha}} and {beta} in the human cervix at term pregnancy Mol. Hum. Reprod., March 1, 2001; 7(3): 293 - 300. [Abstract] [Full Text] [PDF]
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 J. S. Krumenacker, S. M. Hyder, and F. Murad Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus PNAS, January 16, 2001; 98(2): 717 - 722. [Abstract] [Full Text] [PDF]
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T. Kurita, K.-j. Lee, P. T.K. Saunders, P. S. Cooke, J. A. Taylor, D. B. Lubahn, C. Zhao, S. Mäkelä, J.-A. Gustafsson, R. Dahiya, et al. Regulation of Progesterone Receptors and Decidualization in Uterine Stroma of the Estrogen Receptor-{{alpha}} Knockout Mouse Biol Reprod, January 1, 2001; 64(1): 272 - 283. [Abstract] [Full Text]
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L. Sahlin, H. Wang, Y. Stjernholm, M. Lundberg, G. Ekman, A. Holmgren, and H. Eriksson The expression of glutaredoxin is increased in the human cervix in term pregnancy and immediately post-partum, particularly after prostaglandin-induced delivery Mol. Hum. Reprod., December 1, 2000; 6(12): 1147 - 1153. [Abstract] [Full Text] [PDF]
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H. Wang, H. Eriksson, and L. Sahlin Estrogen Receptors {alpha} and {beta} in the Female Reproductive Tract of the Rat During the Estrous Cycle Biol Reprod, November 1, 2000; 63(5): 1331 - 1340. [Abstract] [Full Text]
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Z. Weihua, S. Saji, S. Makinen, G. Cheng, E. V. Jensen, M. Warner, and J.-A. Gustafsson Estrogen receptor (ER) beta , a modulator of ERalpha in the uterus PNAS, May 23, 2000; 97(11): 5936 - 5941. [Abstract] [Full Text] [PDF]
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 D. M. Duffy, C. L. Chaffin, and R. L. Stouffer Expression of Estrogen Receptor {alpha} and {beta} in the Rhesus Monkey Corpus Luteum during the Menstrual Cycle: Regulation by Luteinizing Hormone and Progesterone Endocrinology, May 1, 2000; 141(5): 1711 - 1717. [Abstract] [Full Text] [PDF]
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 S Dupont, A Krust, A Gansmuller, A Dierich, P Chambon, and M Mark Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes Development, January 10, 2000; 127(19): 4277 - 4291. [Abstract] [PDF]
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L. Sahlin, H. Wang, B. Lindblom, H. Eriksson, A. Holmgren, and A. Blanck Thioredoxin expression in human myometrium and fibroids Mol. Hum. Reprod., January 1, 2000; 6(1): 60 - 67. [Abstract] [Full Text] [PDF]
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