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Regular Article TA |
and ß in the Female Reproductive Tract of the Rat During the Estrous Cycle1
a Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
ABSTRACT
The action of steroid hormones is primarily mediated via a process that involves hormone binding to specific receptors in target cells, which leads to transcriptional activation of steroid-responsive genes and, subsequently, to a modification of cellular responses. The aim of the present study was to obtain information about the dynamics of the two types of estrogen receptors (ERs),
and ß, by comparing their concentration and distribution in the reproductive tract of the rat during the estrous cycle. Twenty-four 55- to 60-day-old female Sprague-Dawley rats were used. The stage of estrous cycle was determined by vaginal smear. ER was the dominating subtype in uterus, oviduct, and cervix/vagina, with the distribution varying in stroma and epithelium during the estrous cycle. A low level of ER mRNA was observed in ovarian stromal cells, with some scattered positive cells found among granulosa cells. ERß expression was observed in the different compartments of uterus and cervix/vagina, but cyclic variation during the estrous cycle was less evident than that of ER. Only a few scattered cells that contained ERß mRNA were observed in oviduct. ERß mRNA was highly expressed in granulosa cells of developing follicles, with a weaker hybridization signal in new corpora lutea. Immunohistochemistry showed that protein levels of ER and ERß have distinct specificity for tissues and cell types, similar to their respective levels of mRNA, as assessed by in situ hybridization. The precise physiological function and importance of ERß is still unclear. The relative physiological and pathological function of each ER subtype in the female reproductive tract remains to be further evaluated.
estradiol receptor, female reproductive tract, hormone action
INTRODUCTION
The female reproductive tract is the major target of estrogens. Their action is primarily mediated via binding to specific intracellular receptors in target cells [1]. The discovery of the ß subtype of estrogen receptor (ER) [2] has led to the assumption that hormonal signals are transduced by both and ß ER subtypes [2–5]. These molecules are members of a superfamily of nuclear-transcription factors with highly homologous DNA binding and ligand binding domains [2–6]. The two receptors bind 17ß-estradiol (E2) with high affinity and specificity [7]. Although ERß shares many functional characteristics with ER, the molecular mechanisms that regulate its transcriptional activity and its tissue location are distinct from those of ER [2, 7].
It is well known that "classic" ER plays a crucial role in the physiological processes of the female reproductive tract. It has been shown that expression patterns of ER and ERß in different uterine compartments are similar, but ERß mRNA and protein levels are much lower than those of ER [8–10]. ERß observed by in situ hybridization was low or undetectable in rat uterus [7], whereas it was widely distributed in endometrium of monkeys [11].
In mammals and rodents, ER activity in uterus and oviduct is regulated by cycling hormone levels [12]. The results from our previous study of hormone-treated ovariectomized rats support the concept that E2 and progesterone (P4) have both positive and negative effects on ER expression in the epithelial and stromal compartments of rat uterus [8]. E2 enhances ER mRNA synthesis in rat uterus 24 h after injection, and the complex uterine responses to E2 and P4 are directly or indirectly mediated by the cell-specific expression of their receptors [8]. Results from our group [8] and others [13] do not indicate any prominent effects on ERß expression after treatment with E2, P4, or both.
In the ovary, ERß is the dominant ER subtype. ERß protein levels in rat ovary are variable during the estrous cycle [14]. Messenger RNAs for ER and ERß are coexpressed in rat corpus luteum during pregnancy, and the expression of both mRNAs in luteal cells is up-regulated by prolactin and placental lactogens [15]. The function of ERß in uterine physiology is not known [13, 16]. When ER-knockout (ERKO) are compared with ERß-knockout (ßERKO) mice, ERKO females are infertile and have hypoplastic uteri and hyperemic ovaries [17], whereas ßERKO females are fertile, but their fertility is reduced, probably due to a direct loss of ERß-mediated estrogen effects in the ovary [18].
In the murine oviduct, a low level of ERß mRNA was detected by reverse transcriptase-polymerase chain reaction [16]. Using an immunohistochemistry method, Saunders et al. [9] reported cells that express ERß, whereas Sar and Welsch found no detectable mRNA expression of ERß [19]. A recent study demonstrated that ERß mRNA was expressed in the vaginal walls of premenopausal women, but it was absent in postmenopausal women, whereas vaginal ER mRNA was found in women at both pre- and postmenopausal stages [20].
Several recent discoveries and comparative studies of ER and ERß have offered new insights into the mechanisms of estrogen action. The physiological role of ERß is still unclear, as is its relationship to ER. It is not known how or if ERß is regulated by endogenous hormones during the estrous cycle. To gain further knowledge about ERß during the estrous cycle, a comparative study of ER and ERß expression in the uterus, vagina-cervix, oviduct, and ovary in the rat was carried out.
MATERIALS AND METHODS
Twenty-four adult female Sprague-Dawley rats were used; each was 55–60 days old and weighed approximately 250 g. The animals were housed in a controlled environment at 20°C on an illumination schedule of 12L:12D. Standard pellet food and water were provided ad libitum. Vaginal smears were taken to determine estrous cycle phase [21].
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.
Tissue Collection
During ether anesthesia, the uterus, ovary, oviduct, and cervix-vagina were removed and stripped of fat and connective tissue, and the organs were weighed. The uterus was cut into one half and two quarter pieces. One quarter of the uterus, one ovary with oviduct, and cervix-vagina were immersion-fixed in 4% formaldehyde at 4°C for 12 h, stored at 4°C in 70% ethanol, and embedded in paraffin. The remainder of the uterus, one of the ovaries, and one piece of vagina were immediately frozen in liquid nitrogen and stored at -70°C until analyzed.
Hybridization Probes
The RNA probes labeled with antisense 35S-uridine 5'-triphosphate (Amersham, Bucks, UK) were the same as those previously described [8]. A second pair of ERß RNA antisense and sense probes corresponding to the 5' untranslated region (400 base pairs, EcoRI-AccI) were also used in this study [2].
Solution Hybridization
Total nucleic acids were prepared as described before [22]. A quantitative solution hybridization analysis of specific mRNA was carried out as previously described [8, 22].
In Situ Hybridization
The in situ hybridization procedure has previously been described [8]. Five paraffin sections (5 µm) from each stage of the cycle were used for mRNA examination of ER and ERß of the uterus, cervix-vagina, oviduct, and ovary. 35S-Labeled sense probes were used as controls to define background levels of silver grains on sections adjacent to those that had been hybridized with antisense probes. Two pairs of antisense and sense ER probes (25 x 106 cpm/ml each) were mixed and hybridized onto each section.
Immunohistochemistry
Paraffin sections (5 µm) from five animals of each cycle stage were used. A standard immunohistochemical technique (avidin-biotin-peroxidase) was carried out as described [8] to visualize ER and ERß immunostaining intensity and distribution. The site of the bound enzyme was visualized by the application of 3,3'-diaminobenzidine in H2O2 (DAB kit, Vector, Burlingame, CA) and the sections were counterstained with hematoxylin.
A monoclonal mouse anti-human antibody was used to detect ER (ZM08-1149, Zymed Laboratories, Inc., 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), that binds to the C-terminal amino acid residues 467–485 was used to detect ERß. Negative controls were obtained by replacing the primary antibody with nonimmuno serum of equivalent concentration. In addition, the ERß antibody was preabsorbed with a twofold excess of neutralizing synthetic ERß peptide (PEP 007, Affinity Bioreagents) overnight to demonstrate antigen specificity. The washing buffer for ERß was Tris-buffered saline (TBS; 0.05 M Tris/0.15 M NaCl, pH 7.5), and for ER, it was PBS (0.01 M PO4/0.15 M NaCl, pH 7.5).
Image Analysis
In order to quantitate the expression of ER mRNA in a particular tissue, a Leica microscope and Sony video camera (Park Ridge, NJ) were connected to a computer using an image analysis system (Leica Imaging System Ltd., Cambridge, UK). The number of silver grains per measuring field in sections from control and treated animals was determined according to the method described by Wang et al. [8]. Results are expressed as the difference in number of silver grains obtained from hybridization by antisense minus sense probes. Comparisons were performed between cell types and between tissues from different stages of the estrous cycle.
The same system was used to assess immunohistochemistry quantitative values. Quantification of immunostaining was performed as described previously [8]. In short, with the use of color discrimination software, the total area of nuclei with a positive stain was measured and expressed as a ratio of the total area of cell nuclei.
Statistics
The results of uterine mRNA levels from solution hybridization assays are presented as the mean ± SEM. Evaluation was performed by one-way ANOVA and significance was determined by the Scheffé test. The results of in situ hybridization and immunohistochemistry measured by image analysis are presented as median and range. Statistical calculations were performed by ANOVA on ranks (Kruskal-Wallis test) and significant differences were evaluated by the Dunn test. Values with different letter designations are significantly different (P < 0.05) (Figs. 1, 4, and 8).
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RESULTS
ER and ERß in Uterus
Levels of ER mRNA as determined by solution hybridization were more than twofold higher in proestrus than in metestrus (Fig. 1A), but no changes in ERß mRNA levels were found during the estrous cycle (Fig. 1B).
To compare ER mRNA expression in particular uterine cell types, graphic representation of silver grain counts per field was measured. The ER mRNA level showed no significant changes during the estrous cycle (data not shown). Figure 2, a and b, shows high ER mRNA expression in the stroma, epithelium, and myometrium during proestrus. No differences in ERß mRNA levels in the stroma, LE (luminal epithelium), and GE (glandular epithelium) were observed during the cycle (Fig. 2, e and f; image analysis data not shown). Background levels are presented on adjacent sections after hybridization with the ER sense probe (Fig. 2, c and d) and the ERß sense probe (Fig. 2, g–h).
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In LE, ER immunoreactivity was higher in proestrus (Fig. 3a) and estrus phases (Fig. 3b) compared with metestrus (Figs. 3c and 4C). Image analysis showed that about 55% of nuclei in the stroma stained positive for ER during the estrous cycle (Fig. 4A). ER levels during estrus were significantly lower than in diestrus (Fig. 4A). More than 90% of cell nuclei in the epithelium were positive for ER (Fig. 4, B and C). In GE, the number of ER-positive cells was lowest in metestrus and significantly higher in diestrus than in metestrus (Fig. 4B). The presence of ER was observed in both circular and longitudinal muscle layers of myometrium (M) and vascular smooth muscle cells (arrow) in all stages of the cycle (Fig. 3d). Most endothelial cells in the vessels were negative (Fig. 3d). For a negative control of ER immunostaining, see Figure 6b.
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Intensity of nuclear ERß immunostaining was weaker than that of ER in the stroma, GE, LE, and myometrium (Fig. 3, e–h and Fig. 4, D–F). Results from image analysis (Fig. 4) showed that about 40%, 35%, and 25% of cells in stroma, LE, and GE, respectively, stained positive during the estrous cycle, but no significant differences were observed between the stages. Immunostaining of ERß tended to be decreased in GE and LE during estrus compared with other stages of the cycle (Fig. 3, e–g; Fig. 4, E and F). Figure 3h shows that smooth muscle cells of the myometrium (M) and most endothelial cells (arrow) in vessels exhibited a positive stain for ERß. No specific nuclear staining was found in negative control sections after incubation with a peptide that corresponded to the epitope of the antibody (Fig. 6f).
ER and ERß in Ovary
Low levels of ER mRNA and protein were found in ovarian stromal cells by in situ hybridization and immunohistochemistry, and some scattered positive cells were observed among granulosa and thecal cells in all stages of the cycle (Fig. 5, a and b; Fig. 6a). Positive immunostaining for ER was also found in germinal epithelium (GeE) and oocytes (Fig. 6a). No ER immunoreactivity was observed in granulosa cells of developing and mature follicles, nor in cells of corpora lutea. No positive ER immunostaining was found in negative controls (Fig. 6b).
Figure 5, c and d, show that ERß mRNA was highly expressed in granulosa cells of developing follicles (arrow) with a weaker hybridization signal in the new corpora lutea. Consistent with results from in situ hybridization, ERß immunoreactivity was expressed in granulosa cells from different follicular stages (Fig. 6c). The most intense staining was observed in preovulatory follicles (Fig. 6d), and the weakest immunostaining was observed in granulosa cells, toward the center of the new corpora lutea (Fig. 6e). In atretic follicles, cells with positive staining for ERß were fewer and less intensely stained. Faint positive immunostaining for ERß was also found in GeE. No ERß immunoreactivity was found in thecal cells or oocytes. Only nonspecific staining for ERß was found in cytoplasm of corpora lutea cells. No specific nuclear staining was found in negative control sections after incubation with the peptide that corresponded to the epitope of the antibody (Fig. 6f). There was some background staining for ERß in cytoplasm and none in nuclei of corpora lutea cells, which was not completely blocked with preabsorbed ERß. Localization of ER and ERß proteins were similar to their respective mRNA assessed by in situ hybridization.
ER and ERß in Cervix-Vagina
In the stroma, as measured by in situ hybridization, ER mRNA levels (Fig. 7c) were increased during metestrus (Fig. 8A). The epithelium exhibited a significant increase in ER mRNA levels from estrus to metestrus (Fig. 8B). ER mRNA was abundantly expressed in the epithelium and stroma during metestrus (Fig. 7c) and diestrus, and in basal/parabasal cells during proestrus (Fig. 7a). Only a few scattered cells containing ERß mRNA in epithelium and stroma were observed, and no difference in expression was observed during the estrous cycle (Fig. 7, b and d). Control sections with minimal background signals after hybridization to ER and ERß sense probes are shown in Figure 7, e and f, respectively.
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The variation in protein levels of ER in LE of cervix/vagina during the cycle was similar to mRNA levels (compare Fig. 8B and 8D). About 60% of nuclei in the stroma stained positive for ER during the cycle, but there were no significant changes during the cycle (Fig. 8C). Cell numbers and intensity of positive staining for ER in the epithelium was low during proestrus (Fig. 7g) and estrus, and significantly increased during metestrus (Fig. 7i) and diestrus (Fig. 8D). Protein levels for ERß (Fig. 7, h and j) were in agreement with mRNA expression. About 40% of the epithelium and stroma were faintly stained for ERß (Fig. 8, E and F). A significant increase of ERß immunostaining in the stroma was found in metestrus compared with estrus (Fig. 8E). No positive staining was observed in negative control sections (Fig. 7, k and l).
ER and ERß in Oviduct
ER mRNA was expressed in oviductal LE, stroma, and smooth muscle cells at proestrus (Fig. 9a). Only a few scattered cells containing ERß mRNA were observed in epithelium and muscle cells (Fig. 9b). The results from immunohistochemistry (Fig. 9, c and d) showed that protein levels of ER and ERß were similar to their respective mRNA levels obtained by in situ hybridization. Intense ER immunostaining was detected in stromal cells, muscle cells, and virtually all epithelial cells during all stages of the estrous cycle (Fig. 9c). In some parts of the oviduct, epithelial cells exhibited negative ER immunostaining, which occurred primarily during diestrus (Fig. 9e). No ERß immunostaining in oviduct LE was observed, but there was background staining in the cytoplasm (Fig. 9d). Only a few cells in muscle cell layers exhibited faint ERß expression.
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DISCUSSION
The present study shows that ER is the predominant ER in rat uterus, oviduct, and vagina/cervix. ERß is abundantly expressed in ovary, weakly expressed in uterus and vagina/cervix, and sparsely expressed in oviduct during the estrous cycle.
In the uterus, the results from solution hybridization are in agreement with our previous study, which showed that ER mRNA levels were highest in proestrus when the plasma E2 level is at its highest, and lowest in the metestrus stage when the plasma P4 concentration starts to increase [22]. It has been demonstrated that E2 causes increased concentrations of both ER and progesterone receptor [23, 24] and it is likely that the rise in circulating E2 levels during the cycle is responsible for triggering the major up-regulation of ER in all cell types. P4 selectively decreased the concentration of ligand-bound ER and inhibited the recovery of ER at transcriptional and translational levels [25]. It has been shown that ER mRNA expression in GE and stroma is markedly changed in human endometrium [10] and monkey uterus [26] during the menstrual cycle. A variation of ER mRNA expression in different uterine compartments was observed in this study, but the difference was insignificant. This may be due to a temporally different regulation of ER mRNA and protein by E2 and P4 [8, 22, 27–31]. In a recent study, the cell-specific response to E2 in rat uterus was shown [31], and the ER mRNA level decreased 6 h after an E2 injection, which agrees well with a previous study of ours that showed a 50% reduction in ER mRNA levels 6 h after an E2 injection to adult rats [22]. Immunohistochemistry showed no difference in positive immunostaining between controls and 24 h after a single E2 injection in neither stroma, GE, or LE [8, 31]. However, when only the stronger ER immunostaining was monitored and analyzed with an image analysis system, we found a major decrease in GE [8]. This implies that the mere existence of ER protein is not enough to determine the effect of estrogens; rather, the level of the ER protein is also important.
In the present study, the pattern of ERß expression was similar to that of ER in endometrium and myometrial smooth muscle cells, but the intensity of ERß immunoreactivity and mRNA hybridization signals were apparently weaker and showed no cyclic changes. These results are consistent with previous studies that demonstrated that ERß expression is much less predominant in rat, mouse, and human endometrium [8, 10, 13], and that cyclic variations in ERß mRNA expression are less evident than that of ER [10].
Previous studies have shown that ovariectomy slightly up-regulates ER mRNA [30] and decreases ERß mRNA levels [13] in rat endometrium. Rats that received P4 for 48 h, or E2 for 24 h following 24 h of P4 treatment, had significantly lower levels of uterine ERß mRNA levels compared with those that had been treated with E2 for 24 h [8]. However, ERß alone may not be sufficient to play a crucial role in the regulation of uterine physiology. The present study and previously published data [8, 10, 29, 32] suggest that ERß may be involved in the effects caused by P4- or E2-responsive gene expression in cycling glandular epithelium cells or that ERß may cooperate with ER [16].
In the ovary, ERß mRNA is highly expressed in small growing follicles but less expressed in larger follicles because of the ability of gonadotropins to down-regulate ERß gene expression [2, 33]. In the present study, ERß protein and mRNA were predominantly observed in granulosa cells of follicles and were detected in all stages of the cycle. The latter is in contrast to a recent study that showed that immunoreactivity of ERß was barely detectable in rat ovary during estrus [14]. Our data agree with other studies in which low ER levels were detected in thecal cells, interstitial gland cells, and germinal epithelium, but not in follicular cells [13, 19].
It has been shown in rats that mRNAs for ER and ERß are differentially coexpressed in the corpus luteum during pregnancy [14, 15] and that ERß was found in the corpus luteum during the estrous cycle [9]. However, the present study and another [14] indicate that the ERß protein and mRNA in corpora lutea were absent or below detection limits. An interesting finding is that both ER and ERß proteins were found in ovarian surface epithelium, which is in agreement with two recent reports that showed the presence of ER mRNA in the germinal epithelium of rat ovary [19] and of ERß mRNA in monkey ovarian surface epithelium [11].
It has been demonstrated that ER is present in both vaginal epithelium and stroma [34] and that the ER level fluctuates more during the menstrual cycle in the epithelium than it does in stroma [35]. Our data showed that ER was abundant in stromal cells and mainly expressed in the basal and parabasal cell layers, with a significant difference in protein and mRNA levels between estrus and metestrus, which is in agreement with previous studies [34–36]. In addition, the present results are in accordance with a study in which immunostaining of ER varied in a similar pattern between the vagina and the ectocervix [36]. The localization of ERß was similar to ER, but ERß signals were much weaker in both epithelium and stroma than those of ER. A significantly different, albeit faint, ERß immunostaining was observed in the stroma between estrus and metestrus stages.
It was recently suggested that E2-induced vaginal epithelial proliferation is indirectly mediated by stromal ER because both epithelial and stromal ER are required for E2-induced vaginal epithelial proliferation, cornification, and normal stratification [37]. However, ERß alone was not sufficient to mediate the E2-stimulated induction of these parameters in the absence of ER [37]. Cooke et al. [38] proposed that whereas the nature of the E2-induced stromal signaling that induces epithelial proliferation is unknown, it is likely to involve growth factors. The effects of E2 on stromal cells could also involve changes in the stromal extracellular matrix, basement membrane, or both, with secondary effects on epithelial cell secretory activity [37]. Taken together with the present study, it is unclear whether ERß may be involved in the paracrine regulation of the response to E2 in the stroma of the vagina/cervix.
In the oviduct, ER protein and mRNA were found in almost all epithelial cells throughout the estrous cycle, with a similar mRNA and protein localization pattern in different cell types. It has been shown that ER immunostaining in ciliated epithelial cells is absent or the intensity of immunostaining is much less than that in nonciliated epithelial cells [36, 38]. ER increased during mid-cycle in the ampulla, whereas ER immunostaining in other segments of the fallopian tube was unrelated to the stage of cycle or to serum E2 levels [39]. The present study was in agreement with these studies. In addition, lack of ER in some parts of the oviduct may support the concept that ER has cyclical fluctuations that accompany the dramatic morphological changes in the reproductive tract during the estrous and menstrual cycles [12], or that ciliated cells predominate in those parts of the oviduct.
We found only a few cells containing ERß mRNA in the oviduct, and faint immunostaining for ERß in some smooth muscle cells. The ERß protein was either absent or too low to be detected by immunohistochemistry in oviduct epithelium. This confirms studies in adult [19] and developing [40] rodents, but is in contrast to another report that showed cells containing ERß protein in the oviduct of adult rats [9]. In addition, no variation in ERß levels was found in the oviduct during the estrous cycle.
The present results show that the two ER subtypes could coexist in the uterus and vagina/cervix, and that this has been shown in some tissues before [8–10]. In conclusion, ERß gene expression was distinct from that of ER in distribution and regulation over the estrous cycle. The relative physiological importance and the role in pathological processes of each ER subtype in the female reproductive tract remain to be further evaluated.
ACKNOWLEDGMENTS
We are grateful for skillful technical assistance from Sonja Åkerberg and Britt Masironi. The cDNA for ER was kindly supplied by Prof. M.G. Parker, Imperial Cancer Research Fund, London, U.K. The cDNA for ERß was a generous gift form Dr. G.G.J.M. Kuiper and Prof. J-Å Gustafsson, Karolinska Institutet, Huddinge, Sweden.
FOOTNOTES
First decision: 10 March 2000.
1 Supported by grants from the Swedish Medical Research Council (3972) (H.E., L.S.), Swedish Society for Medical Research (H.W.), Åke Wibergs Foundation (L.S.), Magn.Bergvalls Foundation (L.S.), and Karolinska Institutet. 
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: June 7, 2000.
Received: February 9, 2000.
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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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Z. Weihua, J. Ekman, A. Almkvist, S. Saji, L. Wang, M. Warner, and J.-A. Gustafsson Involvement of Androgen Receptor in 17{beta}-Estradiol-Induced Cell Proliferation in Rat Uterus Biol Reprod, August 1, 2002; 67(2): 616 - 623. [Abstract] [Full Text] [PDF]
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