Molecular Human Reproduction, Vol. 7, No. 3, 293-300,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Different regulation of oestrogen receptors 
and ß in the human cervix at term pregnancy
1 Division for Reproductive Endocrinology and 2 Division for Obstetrics and Gynecology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
Abstract
During pregnancy, a cervical connective tissue remodelling takes place, clinically recognized as softening, effacement and dilatation. The role of oestrogens and their receptors (ER) in this process is not clear. ER
is a ligand-activated transcription factor involved in many physiological processes. The identification of a second oestrogen receptor, ERß, has led to a re-evaluation of oestrogen signalling and physiology. The aim of this study was to monitor the expression of the two ERs in the cervix from women at term pregnancy (TP) and after parturition (PP) compared with that of non-pregnant (NP). A solution hybridization assay showed that the level of ER mRNA was significantly decreased in the PP group, when compared with the NP and TP groups. In contrast the ERß mRNA level was increased in the TP group compared with the NP and PP groups. These results were supported by reverse transcription–polymerase chain reaction (RT–PCR). Similar results were observed for the protein with immunohistochemistry. Intense ERß immunostaining was observed in neutrophils and the endothelial cells of blood vessels. In conclusion, this study supports a concept according to which oestrogen might be involved in the final remodelling of the cervix via the modulating effects of the two ERs. Furthermore, oestrogen may mediate some effects on cervical ripening via ERß present in the invading neutrophils. Further studies are needed to elucidate this finding.
cervix/oestrogen/oestrogen receptors/parturition/pregnancy
Introduction
Steroid receptor regulation in the human cervix is similar to that of other organs in the reproductive tract, with oestrogen stimulating, and progesterone antagonizing, oestrogen activity (Gorodeski, 1996
). During pregnancy, the systemic concentrations of oestrogens and progesterone increase up to 100-fold until parturition (Speroff et al., 1989). However, in contrast to other species, the serum concentrations of neither oestrogen nor progesterone are abruptly changed immediately before parturition in humans (Csapo et al., 1971; Turnbull et al., 1974). The precise mechanisms of cervical ripening in humans are not known. The ripening process is associated with an increased collagen turnover resulting in differently organized collagen fibrils (Uldbjerg et al., 1983). This structural change is necessary for a normal onset and progress of parturition.
There has been some debate about whether or not oestrogens stimulate cervical ripening. A study showed an increase in cervical ripening in a group locally treated with oestradiol (150 mg) in a viscous gel, when compared with a control group receiving only the viscous gel (Gordon and Calder, 1977). In another study, oestradiol (200 mg) had no effect on the change of Bishop score and length of labour when given in term pregnancy for induction of labour (Williams et al., 1988). Although oestrogen may not play an active part at parturition, it might influence the gradual cervical connective tissue remodelling during pregnancy (Stjernholm et al., 1997).
Oestrogen action is mediated primarily via binding to specific receptors (ER) in target cells (Gronemeyer, 1992). In previous studies, we have shown that the levels of both ER and the progesterone receptor (PR) in the cervix are significantly lower in term pregnant and post-partal women in comparison with non-pregnant women (Stjernholm et al., 1996, 1997). The decrease in receptor concentrations coincided with biochemical changes in the cervical connective tissue, such as an increased collagen solubility and an altered proteoglycan composition (Stjernholm et al., 1996). The mechanisms behind the decreased levels of ER and PR, and the concomitant biochemical changes in the cervical connective tissue are not known.
Since the second ER subtype (ERß) was discovered in 1996 (Kuiper et al., 1996), a re-evaluation of oestrogen signalling and physiology has been ongoing. It has been shown that the ER and ERß subtypes have opposite effects on gene transcription depending on the ligand and response elements (Paech et al., 1997). ERß can act as a transdominant repressor on ER transcriptional activity at subsaturating concentrations of oestradiol (Hall and McDonnell, 1999). It is not known whether ERß can regulate ER in a similar way during pregnancy when oestrogen concentrations are high. Recently published results from ERß knockout mice (ßERKO) demonstrate that ERß acts as a modulator of ER-mediated gene transcription in the uterus, and is responsible for the down-regulation of PR in the luminal epithelium (Weihua et al., 2000).
Limited information exists of ERß expression in the cervix. In a previous study we have found that ER and ERß co-exist in the rat cervix (Wang et al., 2000) and that there is a significant difference in ERß immunostaining in the stroma between the oestrus and metoestrus stages during the oestrous cycle. Another study, using reverse transcription–polymerase chain reaction (RT–PCR), showed that both ER and ERß mRNAs were expressed in the human cervix and in cultured cervical epithelial cells (Gorodeski and Pal, 2000). The role of ERß in cervical physiology as well as during pregnancy is unknown. Recently, one group (Wu et al., 2000) reported an increased level of ERß in myometrium of term pregnant women and a down-regulation in the expression of labour-associated genes in uterine smooth muscle cells, suggesting that ERß may play an important biological role during term pregnancy. The aim of this study was to monitor the expression of the two ERs in the cervix from women at term pregnancy and immediately post-partum and to compare it with that of the non-pregnant woman in order to find possible changes and relations to cervical ripening.
Materials and methods
Study participants
The non-pregnant group (NP) consisted of six regularly menstruating women with a mean age of 42 years (range 32–49), and a mean parity of 2 (1–3). All underwent hysterectomies due to benign disorders not involving the cervix. The term pregnant group (TP) consisted of eight primiparous women with a mean age of 32 years (range 28–36) and a mean gestational age of 38 weeks (range 37–40). All women had unripe cervices with a Bishop score <5 points and none of them were in labour. Biopsies were obtained during elective Caesarean sections. The post-partal group (PP) consisted of 10 primiparous women from whom biopsies were taken within 15 min after spontaneous vaginal delivery. They had a mean age of 30 years (range 23–35) and a mean gestational age of 41 weeks (range 39–42). All women were healthy with uncomplicated pregnancies, and were without medication. Ethical approval for the study was obtained from the local ethics committee of the Karolinska Hospital (Ref no: 99–099) and all women had given their informed consent.
Tissue collection
Cervical biopsies were obtained transvaginally from the anterior cervical lip at the 12 o'clock position, from 10–20 mm depth. The cervical biopsies were divided and one half was immersion-fixed in 4% formaldehyde at 4°C overnight, stored at 4°C in 70% ethanol and thereafter embedded in paraffin. The other piece was immediately frozen in liquid nitrogen and stored at –70°C until analysed.
Hybridization probes and solution hybridization
The probe used for the ER mRNA determinations was derived from bcpel, a full-length cDNA containing the whole open reading frame of the human ER. The cDNA was inserted in a pGEM7zf vector. Restriction of this vector with BglII allows the synthesis of a probe corresponding to nucleotides 1470–2062 which encodes the C-terminal half of the steroid binding domain (E) and all of domain F.
The probe used for ERß mRNA determinations was derived from a pBS plasmid with an insert of a 187 bp PVUII/EcoRI fragment corresponding to nucleotides 774–979 in the human ERß gene.
Total nucleic acids (TNA) were prepared as described before (Sahlin et al., 1994). A solution hybridization analysis of specific mRNA was carried out as previously described (Sahlin et al., 1994; Wang et al., 1999).
RNA isolation and RT–PCR analyses
Frozen pieces of cervical tissue were processed for isolation of total RNA according to the manual for the SV total RNA isolation system (Promega, Madison, WI, USA). Total RNA (1 µg) was reverse transcribed at 42°C for 45 minutes in a final volume of 40 µl with a reaction mixture containing 50 mmol/l Tris–HCl pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl2, 7.5 mmol/l dithiothreitol, 0.5 mmol/l dNTPs, 1 µg random hexamers and 400 IU of ML-MTV reverse transcriptase (GIBCO, BRL, Paisley, UK). Primer sequences (Arts J et al., 1997) and the predicted product sizes were for the human ER gene: 5'-AATTCAGATAATCGACGCCAG-3'; 5'-GTGTTTCAACATTCTCCCTCCTC-3' and 340 bp; for the human ERß gene: 5'-TAGTGGTCCATCGCCAGTTAT-3'; 5'-GGGAGCCACACTTCACCAT-3' and 395 bp; and for the human ribosomal protein S28, which was used as a control of the amount of RNA added: 5'-GTGCAGATCTTGGTGGTAGTAGC-3'; 5'-AGAGCCAATCCTTATCCCGAAGTT-3' and 552 bp. For PCR amplification, the cDNA (corresponding to 50 ng RNA from the RT-PCR reaction) was added to the reaction mixture containing 20 mmol/l Tris–HCl pH 8.4, 50 mmol/l KCl, 2.5 IU Taq DNA polymerase (Gibco BRL, Paisley, UK), 0.2 mmol/l dNTPs, 1.5 mmol/l MgCl2 and oligonucleotide primer pairs (50 pmol/pair), in a final volume of 50 µl. PCR was performed for 18 cycles for 28S; 28 cycles for ER and 32 cycles for ERß, the amount of PCR products for ER and ERß increased linearly up to 32 and 36 cycles respectively (data not shown). The PCR was performed using 94°C for 1 min (denaturing), 58°C for 1 min (annealing), and 72°C for 1 min (extension), with a final incubation at 72°C for 3 min in a thermal cycler (Perkin-Elmer, Norwalk, CT, USA). The products were subjected to electrophoresis in 2.5% agarose gels; data were analysed using a Fujifilm Las-1000 system (Fujifilm, Japan). The ER and ERß bands are shown together with a S28 band to enable comparison of the amount of RNA loaded to each well.
Immunohistochemistry
Paraffin sections (5 µm) were used and a standard immunohistochemical technique (avidin–biotin–peroxidase) was carried out as described previously (Wang et al., 1999), to visualize ER immunostaining intensity and distribution. A monoclonal mouse anti-human antibody was used for detection of ER (08–1149; Zymed Laboratories, Inc., San Francisco, CA, USA). This antibody recognizes the N-terminal domain (A/B region) of the ER. Replacement of the primary antibody with non-immune serum was used as negative control.
A polyclonal chicken anti human ERß (503) antibody was used for the ERß immunostaining. The preparation of this antibody has been described (Saji et al., 2000). The ERß antibody pre-absorbed with an ERß protein (Panvera, Madison, WI, USA) 1:50 (V/V) overnight at 4°C was used to demonstrate antigen specificity. Tissue sections were incubated with 1:500 dilution of ERß antibody overnight at 4°C in phosphate-buffered saline (PBS) with 3% bovine serum albumin (BSA). For negative controls, incubations were done with the absorbed antibody. After washing, sections were incubated with peroxidase-conjugated rabbit anti-chicken immunoglobulin G (IgG) for 1 h at room temperature. The peroxidase substrate diaminobenzidine (DAB) was used to visualize the reaction (SK 4100; Vector Laboratory, Burlingame, CA, USA). Thereafter the procedure was as described before (Wang et al., 1999).
Another polyclonal rabbit anti-human ERß antibody (PA1-313, Affinity Bioreagents, Inc., Golden, CO, USA) which corresponds to the C-terminal amino acid residues 467–485 of human ERß, was also used for detection of ERß. A standard immunohistochemical technique (avidin–biotin–peroxidase) was used for this antibody immunostaining. The protocol has previously been described (Wang et al., 1999). The washing buffer was Tris-buffered saline (TBS, 0.05 mol/l, pH 7.5) used for ERß (PA1-313) and PBS (0.1 mol/l, pH 7.5) for ER and ERß (503).
We found corresponding immunostaining patterns between the ERß antibodies (PA1 313 versus ERß 503), although PA1 313 displayed more background in the cytoplasm (data not shown). Therefore, the ERß 503 antibody was used for this study.
Image analysis
A Leica microscope and Sony video camera (Park Ridge, NJ, USA) connected to a computer with an image analysis system (Leica Imaging System Ltd, Cambridge, UK) was used to assess quantitative values from immunohistochemistry. The quantification of immunostaining was performed as described previously (Wang et al., 1999). In short, by using colour discrimination software the total area of positively stained nuclei was measured, and expressed as a ratio of the total area of cell nuclei. The intensity of positive staining was also semi-quantitatively measured by three different colour discriminations: strong (+++), moderate (++) or faint (+) brown reaction.
Statistical analysis
Statistical calculations for the solution hybridization results were performed with one way analysis of variance (ANOVA), and evaluation for significance was done with Scheffé's test (Scheffé, 1959). Statistical calculations for the immunohistochemistry results by image analysis were performed by ANOVA on ranks (Kruskal–Wallis test) and significances were evaluated by Dunn's test. Values with different letter designations are significantly different (P < 0.05).
Results
The level of ER mRNA measured by solution hybridization was decreased in the PP group as compared with the NP and TP groups (Figure 1A). This was in contrast to the ERß mRNA level, which was significantly increased in the TP group, compared with the NP and PP groups (Figure 1B).
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Representative gels from RT–PCR analyses are shown in Figure 2
. The ER mRNA bands of the NP and TP groups appeared to be stronger compared with the PP group (Figure 2, top). The ERß mRNA expression seemed to be higher in most women of the TP group as compared with the NP and PP groups (Figure 2, middle). S28 was used as a standard to enable comparison of the amount of RNA loaded to each well (Figure 2, bottom). Thus, the RT–PCR results were in agreement with the solution hybridization results.
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Immunohistochemistry showed that the ER
protein was present in the nuclei of cells in the different compartments of the cervix (Figure 3A,C,E and Figure 4). Of the cell nuclei in the epithelium, >70% were ER positive, but no significant differences were found between the groups (Figure 3A and 4a–c). In the stroma of the NP group, ~50% of the nuclei stained ER positive (Figure 3C). ER immunoreactivity declined in the cervical stroma of the TP group (Figure 3C; 4e and k) and was significantly decreased in the PP group (Figure 3C; 4f and l) compared with the NP group (Figure 3C; 4d and j). When studying only the more intense immunostaining (++/+++), both the TP and PP groups were decreased compared with the NP group (Figure 3E). In the glandular epithelium, ER immunostaining was intensely expressed in the NP group (Figure 4g), but was less strong in the TP group (Figure 4h) and only faint staining was observed in some glandular epithelial cells in the PP group (Figure 4i). Image analysis was not performed for immunostaining of glandular epithelium, since an insufficient number of biopsies contained cervical glands. The immunoreactivity of ER in smooth muscle cells seemed less in the cervix from the TP (Figure 4n) and PP (Figure 4o) groups as compared with the NP group (Figure 4m), indicating a similar pattern to that seen in the stromal connective tissue (Figure 4d–f). ER immunostaining in the vascular endothelial cells was negative (Figure 4j–l, black arrows), although some smooth muscle cells were positively stained in all groups. No ER positive staining could be observed in the neutrophils (white arrows) in the blood vessels (Figure 4j–l). No positive staining was observed in the negative control sections (Figure 4p).
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Positive nuclear ERß immunostaining was found in the cervical epithelium and stroma (Figure 3B,D,F and Figure 5
). The results from image analysis (Figure 3B) showed that 60–70% of the epithelial cells were ERß-positive. There was a significant difference between the TP and PP groups (Figure 3B and 5b-c). In the stroma, the number of ERß positive cells was significantly increased in the TP group (~50% of the cells) (Figure 3D, 5e and k) compared with the NP (Figure 3D, 5d and j) and PP (Figure 3D, 5f and l) groups (~25% of the cells stained). Similar results were obtained when studying only the more intense ERß staining (++/+++) (Figure 3F), although fewer cells were intensely stained. In the glandular epithelium, only faint ERß immunostaining was observed in some epithelial cells in all the groups (Figure 5g–i). The immunoreactivity of ERß in smooth muscle cells showed a similar pattern to that of the stroma in the three groups (Figure 5m–o). The immunoreactivity seemed to be more intense in smooth muscle cells from the TP group (Figure 5n). ERß immunostaining was observed in the endothelial cells (Figure 5j–l, black arrows) and some smooth muscle cells of the vessels in all groups. Neutrophils (defined by the morphology) stained positive for ERß (Figure 5j–l, white arrows), and were more obvious in the TP and PP groups (Figure 5k–l) since the samples contained a higher number of neutrophils. No specific nuclear staining was found in the negative control sections after incubation with a peptide corresponding to the epitope of the ERß antibody (Figure 5p).
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Discussion
Cervical ripening during pregnancy and parturition occurs in distinct stages, including a gradual softening during pregnancy, a latency phase and an active phase of dilatation and cervical protraction at parturition, allowing for expulsion of the foetus (Calder, 1994). It is known that progesterone withdrawal initiates cervical softening (Frydman et al., 1992) even though there is no abrupt fall in peripheral serum progesterone before cervical remodelling.
To further study the potential roles for gonadal steroids in cervical ripening, we measured the two subtypes of ER in the human cervix and found that the ERß mRNA level was increased at term pregnancy, although the ER mRNA level was unchanged. After parturition, ERß expression was decreased again to the level of the non-pregnant group, whereas the ER mRNA level was decreased to a level less than half of that in the non-pregnant group. The protein profile of both ER and ERß showed similar patterns in the stroma as the respective mRNA, suggesting that the regulation of the ERs is primarily at the transcriptional level.
In contrast to spontaneous ripening, antiprogestin treatment at term has been shown to be followed by an increased cervical ER concentration (Stjernholm et al., 1999). These findings indicate that an increase in ER, or a withdrawal of receptor-mediated progesterone inhibition, does not explain the events behind the physiological cervical ripening at parturition. The role of ERß in the cervical ripening during pregnancy and parturition is not known. Results from studies in ßERKO mice showed that ERß could be responsible for the down-regulation of PR in the uterine luminal epithelium (Weihua et al., 2000). The present study suggests a switch from ER to ERß expression in the cervix during pregnancy as could be seen in the TP group. When this occurs during pregnancy is not known. Thus, the increased ERß level could lead to decreased levels of PR in the cervix, leading to decreased progesterone activity similar to antiprogestin treatment. A dramatic switch from ER to ERß expression in the myometrium during pregnancy has been shown previously (Wu et al., 2000). They suggest that ERß inhibits the activity of the transcription factor AP-1 during gestation, thus blocking labour-associated genes, e.g. connexin 43. The AP-1 binding site is activated by 17ß-oestradiol only via ER, whereas oestradiol via ERß inhibits transcription (Paech et al., 1997). The collagenase gene has an AP-1 site in its promoter region (Jonat et al., 1992). Thus, the increased ERß mRNA level in the TP group could indicate a block of AP-1 activity and thereby production of metalloproteinases (MMPs, e.g. collagenase), thus preventing the occurrence of the final cervical ripening. In the PP group, the ERß expression was decreased again to the level of the NP group, indicating a released inhibition. In addition, ER expression had declined compared with the TP group, implying decreased ER activity at the AP-1 site.
Several groups have reported increased concentrations of dihydroepiandrosterone sulphate (DHEAS), a precursor to oestrogen, in women with ripe cervices, compared with those with unripe cervices (Zuidema et al. 1986; Liapis et al. 1993), while serum oestradiol showed no difference between the groups. Administration i.v. of DHEAS to women in late pregnancy was also shown to ripen the cervix (Mochizuki and Maruo, 1985). In-vitro studies on human cervical tissues showed increased collagenase activity in tissue cultures treated with DHEAS, while free steroids had no effect (Yoshida et al., 1993). These results imply that oestrogen, also via its precursor DHEAs, could induce cervical remodelling by regulating collagenase activity.
The endocervical mucosa has been found to contain the highest, while the stroma and the squamous epithelium respectively contain lower and the lowest amounts of ER (Sanborn et al., 1976). The present immunohistochemistry data showed that both ER and ERß were expressed in the squamous epithelium and stroma. Both density and cell numbers of ER and ERß immunostaining were significantly changed in the stroma at term pregnancy and post-partum, whereas in the epithelium only ERß immunoreactivity was decreased after parturition and ER remained unchanged. To our knowledge, cervical squamous epithelium does not play any important role in the cervical ripening process. At present it is difficult to explain the biological significance of the decreased ERß expression in the epithelium after spontaneous delivery. The present study and previous data on the changed local endocrine environment with decreased ER and PR concentrations (Stjernholm et al., 1996, 1997) but increased ERß in term pregnancy with a following decrease in ERß after parturition, suggest that gonadal steroids may exert their main receptor mediated biological effects in the human cervix just before parturition.
It is known that the biochemical events that occur in the cervix during the cervical ripening are similar to those observed in tissue inflammation (Junqueira et al., 1980). Oestrogen has been shown to have a role in leukocyte migration via regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in uterine epithelial cells in mice (Robertson et al., 1996). We found that ERß, but not ER, showed intense immunostaining in neutrophils and the endothelial cells of the vessels. It is tempting to speculate that neutrophils have an ability to respond to oestrogen in the cervix regulated via ERß. Cervical collagenase has been found to originate mainly from neutrophils accumulating in the cervical stroma during parturition (Osmers et al., 1992). Extravasation of neutrophils, mast cells and monocytes with subsequent release of proteolytic enzymes and cytokines may rapidly accelerate the ripening process (Uldbjerg et al., 1983; Kelly et al., 1994). It has been suggested that the oestradiol-regulated, and cytokine-mediated, up-regulation of the adhesiveness of cervical vascular endothelium followed by extravasation and degranulation of neutrophils, may play a crucial role in the initiation of human parturition at term (Winkler et al., 1999). Our data, together to those of others, imply that ERß might be involved in neutrophil invasion of the cervical stroma, and thereby directly or indirectly regulate tissue remodelling at term.
Acknowledgments
The ER cDNA was a generous gift from Donald McDonnell, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina, USA. We gratefully acknowledge Eva Enmark for providing the human ERß cDNA and the ERß 503 antibody was kindly provided by Jan-Åke Gustafson, both from Dept. of Medical Nutrition, Novum, Huddinge, Sweden. The present study received financial support from Magn. Bergvalls stiftelse, the Swedish Medical Research Council (grants 03X-3972 and 04X-9508), Karolinska Institutet and Swedish Society for Medical Research (HW).
Notes
3 To whom correspondence should be addressed at: Division for Reproductive Endocrinology, Karolinska Hospital, L5:01, S-171 76 Stockholm, Sweden. E-mail: Lena.Sahlin{at}kbh.ki.se 
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Submitted on August 29, 2000; accepted on December 20, 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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D. Stygar, H. Wang, Y. S. Vladic, G. Ekman, H. Eriksson, and L. Sahlin Increased Level of Matrix Metalloproteinases 2 and 9 in the Ripening Process of the Human Cervix Biol Reprod, September 1, 2002; 67(3): 889 - 894. [Abstract] [Full Text] [PDF]
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C. E. Gargett, K. Bucak, M. Zaitseva, S. Chu, N. Taylor, P. J. Fuller, and P. A.W. Rogers Estrogen receptor-{alpha} and -{beta} expression in microvascular endothelial cells and smooth muscle cells of myometrium and leiomyoma Mol. Hum. Reprod., August 1, 2002; 8(8): 770 - 775. [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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