Estrogen Receptors and in Rat Decidua Cells: Cell-Specific ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 10 Printed in U.S.A.

Estrogen Receptors ␣ and ␤ in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin* C. TESSIER, S. DEB, A. PRIGENT-TESSIER, S. FERGUSON-GOTTSCHALL, G. B. GIBORI, R. P. C. SHIU, AND G. GIBORI Department of Physiology and Biophysics, University of Illinois College of Medicine (C.T., S.D., A.P.-T., S.F.-G., G.B.G., G.G.), Chicago, Illinois 60612; and Department of Physiology, Faculty of Medicine, University of Manitoba (R.P.C.S.), Winnipeg, Manitoba, Canada R3E 0W3 ABSTRACT Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), ␣ and ␤. In this investigation, we analyzed the expression of decidual ER␣ and ER␤, studied their regulation by PRL and steroid hormones and examined the ability of decidual ER␤ to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ER␣ and ER␤ by PRL and ovarian steroid hor-

mones. Whereas PRL, estradiol, and progesterone all increased ER␤ messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ER␣. Estradiol had no effect on ER␣ expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ER␣ and had no synergistic effect on ER␤ expression. The use of GG-AD cells, which express only ER␤, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ER␤ is expressed in addition to ER␣ in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ER␣, but concomitantly increases the expression of a functional ER␤ that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842–3851, 2000)

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specifically, ER␣ protein (9, 17, 18), but no evidence exists as to whether the rat decidua expresses ER␤ as well. ER␣ and ER␤ are distinct gene products, but have high homology, particularly in the DNA-binding domain (⬎90% amino acid identity) and the C-terminal ligand-binding domain (55% homology) (19). In addition, both ERs have similar binding affinity for physiological ligands and estrogenic substances (20). Differences in the distribution and relative expression of ER␣ and ER␤ isoforms in various rat tissues have been observed (20), although the ovaries and uterus express both receptors. The rat decidua is formed by two major cell populations localized in mesometrial or antimesometrial sites of the uterus (21). The attachment of the blastocyst, an event that is dependent on estradiol in the progesterone-primed uterus of the rat, occurs in the antimesometrium, which develops into the decidua capsularis in the pregnant rats. The mesometrial cells, which form the decidua basalis in the pregnant animal, are the site of trophoblast invasion. Expression of ER␣ has been previously shown to be localized to this cell type (7–9, 16 –18). Two recent investigations using ER␣ knockout (ERKO) mice have demonstrated that progesterone alone is enough to induce stromal cell decidualization in response to artificial stimulation of the uterus (22, 23). However, although Curtis

HE RAT UTERUS undergoes profound changes in response to blastocyst implantation during pregnancy or to an artificial stimulus during pseudopregnancy. One of the most remarkable events is the proliferation and differentiation of the endometrial stromal cells, giving rise to unique cells, termed decidual cells, that differ completely from the original fibroblast cells (1, 2). Estradiol and progesterone are two ovarian steroids necessary for blastocyst implantation and subsequent decidualization. Whereas progesterone is essential for both events, estradiol is required for the initial attachment of the blastocyst in the progesterone-primed uterus (3), the induction of the progesterone receptor (4), the inhibition of interleukin-6 (IL-6) (5), and the maximal growth of the decidua (6). Estrogen-binding sites have been demonstrated in both pregnant (7–9) and pseudopregnant rat decidua (10 –16). Immunochemical analysis has confirmed the presence of estrogen receptor (ER) protein and, more Received March 1, 2000. Address all correspondence and requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. Email: [email protected]. * This work was supported by NIH Grants HD-12356 (to G.G.) and the Ernst Schering Research Foundation (to C.T.).

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ESTROGEN RECEPTORS ␣ AND ␤ IN RAT DECIDUA

et al. (22) found that decidualization is still dependent on estrogen in the wild-type animal, confirming the results reported previously by other groups (24 –27), Paria et al. (23) showed that progesterone alone is able to induce a decidual response in both wild-type and ERKO mice uteri. As no ER␤ messenger RNA (mRNA) could be detected in the uterus of either wild-type or ERKO mice (28), these studies suggest that neither ER␣ nor ER␤ is necessary for decidualization. Because one role of estrogen in uterine decidualization was thought to be the induction of the progesterone receptor (PR), it is not clear how the PR is induced in ERKO mice. Indeed, decidualization of the endometrial stromal cells involves a 4to 5-fold increase in the level of PR in ERKO mice (23). However, the situation seems to be different in the rat, as ER␤ mRNA was shown to be expressed in the uterus (20). This prompted us to examine whether the rat decidua expresses the ER␤ gene. The results of this investigation have revealed for the first time that ER␤, in addition to ER␣, is expressed in the rat decidua. We have also shown a developmental and subcellular localization of both ERs, a differential regulation of ER␣ and ER␤ by PRL and steroids, and the ability of decidual ER␤ to transduce the estradiol signal for the upregulation of the progesterone receptor. Materials and Methods Chemicals Acrylamide and bis-acrylamide were obtained from Accurate Chemical, Inc. (Westbury, NY), and Eastman Kodak Co. (Rochester, NY), respectively; Taq DNA polymerase was purchased from Pan Vera Corp. (Madison, WI); [32P]deoxy (d)-CTP and [␣-32P]dGTP were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL); the oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY); tissue culture medium (RPMI 1640), antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were purchased from Mediatech (Washington DC); FBS was purchased from HyClone Laboratories, Inc. (Logan, UT); trypsin-EDTA was obtained from Life Technologies, Inc.; 17␤-estradiol was obtained from Steraloids, Inc. (Wilton, NH); progesterone, phenylmethylsulfonylfluoride, leupeptin, pepstatin A, aprotinin, and all other reagent grade chemicals were purchased from Sigma (St. Louis, MO); ovine PRL (oPRL; PRL-18; 30 IU/mg), the polyclonal ER-715 antibody and the ER-715 antigenic peptide were gifts from the NIDDK, NIH (Bethesda, MD); the polyclonal ER␤ antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY); and the antigenic peptide was kindly provided by Dr. N. Ben Jonathan (University of Cincinnati, Cincinnati, OH).

Animal model Pseudopregnancy was induced in Holtzman female rats by mating them with vasectomized males at the Harlan facilities (Harlan Sprague Dawley, Inc., Madison, WI). The day a vaginal plug was found was designated day 1 of pseudopregnancy. Rats were kept under controlled conditions of light (14 h/day; lights on, 0500 –1900 h) and temperature (22⫺24 C), with free access to standard rat chow and water. All experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee. Decidualization of uterine endometrium was induced by scratching the antimesometrial surface of both uterine horns with a hooked needle on day 5 of pseudopregnancy under ether anesthesia. For the developmental studies, rats were used at various stages of pseudopregnancy from days 9 –13. Decidualized uterine horns were isolated and washed thoroughly in ice-cold PBS to remove excess blood. The antimesometrial and mesometrial regions were separated as described by Martel et al. (16). All tissue was frozen in liquid nitrogen and stored at ⫺80 C until processed for RNA or protein preparation.

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Primary cell culture Total decidual tissue obtained from three to five rats on day 9 of pseudopregnancy were minced and incubated for 1 h with 50 U/ml collagenase, 2.4 U/ml dispase, and 200 U/ml deoxyribonuclease in a water-jacketed cell stir (Wheaten Scientific, Millville, NJ) at 37 C under mild agitation. At the end of the incubation, dispersed cells were filtered and centrifuged at 200 ⫻ g for 10 min. Cells were gently resuspended in RPMI 1640 medium containing 2 ⫻ antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 ␮g/ml amphotericin B, and 200 ␮g/ml streptomycin), 1 ⫻ nonessential amino acids, 1 mm sodium pyruvate, 0.45% d-glucose, and 10% FBS. Cells (1.2–1.5 ⫻ 106) were seeded in six-well plates and incubated at 37 C in a 95% air-5% CO2 humidified atmosphere. Cells were allowed to attach for 3– 4 h, washed, and then treated for 12 h with different hormones in RPMI 1640 phenol-red free medium supplemented with 1% charcoal dextran-stripped FBS. After treatment, cells were washed twice with ice-cold PBS and stored at ⫺80 C until RNA extraction. Although progesterone is needed for the in vitro differentiation of stromal cells into decidual cells, no progesterone is required for maintaining decidual cells in vitro (29 –32). Our decidual cells cultured in FBS-DCC retained all of the characteristics of decidual cells and never dedifferentiated.

GG-AD cell culture The temperature-sensitive GG-AD cells derived from rat antimesometrial decidual cells (33) and the GG-AD cells stably transfected with the long form of the PRL receptor (5) were grown at 33 C in RPMI 1640 medium supplemented with 10% FBS, 2 ⫻ antibiotic-antimycotic solution (200 U/ml penicillin G, 0.5 ␮g/ml amphotericin B, and 200 ␮g/ml streptomycin), 1 ⫻ nonessential amino acids, 1 mm sodium pyruvate, and 0.45% d-glucose in a 95% air-5% CO2 humidified atmosphere. These cells have retained morphological characteristics of antimesometrial cells; they are polynucleated, large, and have a cytoplasm filled with lipids droplets. They also express the same mRNA as antimesometrial cells, such as activin ␤A and decidual PRL-related protein (dPRP). Before each experiment, cells were cultured for 24 h at 39 C to allow cell differentiation. Cells were then treated with either oPRL (0.01–10 ␮g/ml) or estradiol (0.01–100 ng/ml) for 24 h in RPMI 1640 phenol-red free medium supplemented with 1% charcoal-dextran-treated FBS. At the end of the incubation, the cells were washed with PBS and stored at ⫺80 C for RNA or protein isolation.

RNA isolation and RT-PCR analysis Total RNA from frozen decidual tissue was purified using TriReagent (Sigma) according to the manufacturer’s instructions, whereas total RNA from primary decidual cells was isolated by a one-step guanidinium-thiocyanate-phenol-chloroform extraction procedure (34). For mRNA analysis by RT-PCR, oligonucleotide primer pairs were based on the sequences of the rat ER␣ gene (35) (5⬘-AATTCTGACAATCGACGCCAG-3⬘ and 5⬘-GTGCTTCAACATTCTCCCTCCTC-3⬘), the rat ER␤ gene (19) (5⬘-GTCCTGCTGTGATGAACTAC-3⬘ and 5⬘-CCCTCTTTGCGTTTGGACTA-3⬘), and the rat progesterone receptor gene (5⬘CCCACAGGAGTTTGTCAAGCTC-3⬘ and 5⬘-TAACTTCAGACATCATTTCCGG-3⬘) (36). Primers for either L19 (5⬘-CTGAAGGTCAAAGGGAATGTG-3⬘ and 5⬘-CGTTCACCTTGATGAGCCCATT-3⬘) (37) or S16 (5⬘-TCCAAGGGTCCGCTGCAGTC-3⬘ and 5⬘-CGTTCACCTTGATGAGCCCATT-3⬘) (38) ribosomal proteins mRNA were included to normalize the data. The predicted sizes of the PCR-amplified products were 344, 285, and 325 bp for ER␣, ER␤, and progesterone receptor, respectively. One to 2 ␮g total RNA were reverse transcribed at 42 C using the Advantage TM RT for PCR kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The 20-␮l reaction mixture containing 20 pmol random hexamer primer, 10 pmol oligo(deoxythymidine)18 primer, 1 ⫻ reaction buffer, 0.5 mm dNTP, 20 U RNase, and 200 U Moloney murine leukemia virus reverse transcriptase was diluted to 100 ␮l by adding diethylpyrocarbonate-treated water at the end of the RT reaction. The complementary DNA (cDNA) was either used immediately for PCR or stored at ⫺20 C until use. For PCR amplification, a mixture containing oligonucleotide primers (20 pmol), [␣-32P]dCTP (2 ␮Ci of 3000 Ci/mmol),

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dNTP, and Taq polymerase (0.8 U) was added to 2–10 ␮l cDNA. The total volume was increased to 40 ␮l with 1 ⫻ PCR buffer, and the samples were overlaid with 50 ␮l mineral oil. For PCR amplification of the gene products, cDNA was amplified for five cycles with high annealing temperature (annealing temperature of the primer plus 4 C) to increase specificity, and then amplified for 20 –30 cycles using 94 C for denaturing, 62⫺65 C for annealing depending on the primer, and 71 C for extension in a Robocycler Gradient 40 (Stratagene, La Jolla, CA). The conditions were such that amplification of the product was in the exponential phase, and the assay was linear with respect to the amount of input cDNA. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA).

Molecular cloning of the rat decidual ER␤ by PCR Total RNA obtained from decidua day 13 pseudopregnant rats was isolated, and RT-PCR was performed using oligonucleotides primers as described above for RNA isolation and RT-PCR analysis. The predicted size of the PCR product was 285 bp. The PCR product was electrophoresed on a 0.7% agarose gel. Only one band was detected by ethidium bromide at the expected size (285 bp). The cDNA fragment was extracted from the agarose gel, purified, and subcloned into the pGEM-T Easy vector (Promega Corp., Madison, WI). DH5␣-competent cells (Life Technologies, Inc.) were then transformed with this vector. Three clones were selected and sent to the DNA Sequencing Facility of the University of Chicago for DNA sequencing.

Northern blot analysis Poly(A)⫹ mRNA (10 ␮g) obtained from decidual tissue day 12 pseudopregnancy was isolated by the oligo(deoxythymidine)-cellulose method using an Ambion, Inc., isolation kit (Austin, TX). RNA was fractionated through a 1% agarose gel containing 0.74 m formaldehyde and transferred to a GeneScreen nylon membrane, NEN Research Products (Boston, MA) by overnight capillary blotting with 10 ⫻ sodium chloride-sodium citrate buffer (SSC buffer; 1.5 m sodium chloride and 150 mm sodium citrate, pH 7.0). Membranes were backed at 80 C under vacuum for 2 h. A 750-bp ER␤ fragment kindly provided by Dr. Park Sarge was used to synthesize the ␣-32P-labeled riboprobe with SP6 RNA polymerase as outlined by the vendor (Promega Corp.). RNA blot hybridization with the complementary RNA (cRNA) ER␤ probe was performed at 42 C in 50% deionized formamide, 4 ⫻ SET [600 mm sodium chloride, 80 mm Tris (pH 7.8), and 4 mm EDTA], 0.2% polyvinylpyrrolidone, Ficoll, BSA, and 8% dextran sulfate. The final oligonucleotide probe concentration was 2 ⫻ 107 cpm/ml. Blots were hybridized for 24 h, then washed with 1 ⫻ SSC (containing 0.1% SDS) at 25 C for 15 min, followed by 0.2 ⫻ SSC (containing 0.1% SDS) at 42 C for 15 min and finally 0.2 ⫻ SSC (containing 0.1% SDS) at 55 C for 15 min. The resultant blots were exposed to Kodak X-OMAT film (Eastman Kodak Co.) using intensifying screens at ⫺80 C.

Immunocytochemistry Primary decidual cells obtained from day 9 pseudopregnant rats were grown for 24 h in RPMI 1640 phenol red-free medium supplemented with 1% charcoal-dextran-treated FBS on sterile coverslips (13-mm diameter) in four-well plates (Nunc, Naperville, IL). At the end of the incubation, the cells were washed twice in PBS and fixed for 10 min in PBS-4% paraformaldehyde solution at room temperature. The cells were then washed three times in Tris-buffered saline (TBS; pH 7.6) and permeabilized for 15 min at room temperature in TBS-10% BSA, 0.1% Triton X-100, and 0.2% Tween-20 solution. After one more wash in TBS, the cells were incubated overnight at 4 C with either a polyclonal antibody to ER␣ (1:100 final dilution; ER-715, provided by the NIDDK) or a polyclonal antibody to ER␤ (10 ␮g/ml final dilution; Upstate Biotechnology, Inc., Lake Placid, NY) in TBS-1% BSA. Control cells were treated with TBS-1% BSA alone. The cells were exposed for 3 h at room temperature to a TRITC-conjugated antirabbit IgG (1:200 final dilution). The coverglasses were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA) containing a counterstain for DNA (DAPI-4⬘,6-diamino2-phenylindole) and observed with a Carl Zeiss LSM 510 laser confocal

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microscope (Oberkochen, Germany) equipped with a 40⫻ water immersion objective lens (NA 1.2). The ER␣ polyclonal antibody (ER-715) was raised against a peptide corresponding to a 15-amino acid sequence lying in the hinge region of the rat ER␣. The antirat ER␤ was raised against a synthetic peptide representing amino acids 54 –71 of rat ER␤. It recognizes specifically ER␤ and does not detect ER␣ protein.

Immunoblot analysis To isolate decidual protein, tissue was homogenized in 2 ml ice-cold homogenization buffer containing 25 mm Tris-HCl (pH 7.4), 2 mm MgCl2, 1 mm EDTA, 1 mm phenylmethylsulfonylfluoride, 1 mm dithiothreitol, 1 ␮m leupeptin, 1 ␮m pepstatin A, and 1 ␮g/ml aprotinin in a glass homogenizer. To obtain protein from cultured cells, cells were washed at least twice in cold PBS after treatment and were scraped off the culture dishes using a rubber policeman. The cells were then disrupted with a 26.5-gauge needle/1-cc syringe in homogenization buffer. The protein concentration of the homogenates was determined as described by Bradford (39). Fifty to 100 ␮g of protein were mixed with an equal volume of 2 ⫻ Laemmli buffer and heated for 5 min at 100 C. Equivalent amounts of protein were separated through 12% SDS-PAGE gels under reducing conditions in electrophoresis buffer [25 mm Tris-HCl (pH 8.3), 192 mm glycine, and 0.1% SDS] according to the method of Laemmli (40). Proteins were then electrotransferred to nitrocellulose membranes (0.2 ␮m; Protan, Schleicher & Schuell, Inc., Keene, NH) in cold transfer buffer [20 mm Tris-HCl (pH 8.3), 192 mm glycine, and 20% methanol]. Immunoblotting was performed by first blocking nonspecific binding sites with 5% nonfat milk in TBS buffer [20 mm Tris-HCl (pH 7.6) and 137 mm NaCl] containing 0.1% Tween-20. Blots were then incubated overnight with either ER␤ antibody at a dilution of 2 ␮g/ml or ER␣ antibody at a dilution of 1:750. The membranes were then washed with TBS-Tween 0.1% and incubated with a secondary antibody linked to horseradishperoxidase-labeled antirabbit IgG (Sigma) for 2 h. After extensive washing, blots were developed using an enhanced chemiluminescence Western blotting system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and exposed for 1–5 min to x-ray film (Biomax MR, Kodak). The specificity of the stained protein band was verified by saturating the antisera with excess antigenic peptide (2 and 1.25 ␮g/ml, respectively, for ER␤ and ER␣) overnight before exposure to a blotted membrane. The molecular sizes of the immunoreactive bands were estimated by comigration of prestained SDS-PAGE mol wt standards (Benchmark, Life Technologies, Inc.).

Statistical analysis Data were examined by one-way ANOVA, followed by Duncan’s multiple range test. When appropriate, Student’s t test was used. P ⬍ 0.05 was accepted as statistically different.

Results Immunolocalization of ER␣ and ER␤ in decidual cells in primary culture

To first examine whether decidual cells express ER␣ and/or ER␤, primary cells obtained from total decidual tissue of day 9 pseudopregnant rats were cultured for 24 h as described in Materials and Methods and were subjected to immunocytochemical analysis with either ER␣ or ER␤ antibodies. Although no fluorescence was observed in control cells incubated without primary antibody (Fig. 1A), immunoreactive ER␣ was detected in both cytoplasm and nuclei of decidual cells (Fig. 1B). Immunoreactive ER␤ was observed as punctuate regions only within the nuclei (Fig. 1C). To confirm the nuclear localization of ER␣ and ER␤ proteins, we have performed optical sections in the z-axis of the cells. Immunostaining of both ER␣ (Fig. 1D) and ER␤ (Fig. 1E) was clearly located within the nuclei.


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FIG. 1. Immunolocalization of ER␣ and ER␤ in decidual cells in primary culture. Decidual cells were cultured on sterile coverslips for 24 h in RPMI 1640 phenol-free medium supplemented with 1% charcoal-dextran-treated FBS. Coverslips were prepared for immunocytochemistry as described in Materials and Methods. A, Control cells incubated without primary antibody. B and D, Cells incubated with a polyclonal ER␣ antibody (1:100 final dilution). C and E, Cells incubated with a polyclonal ER␤ antibody (10 ␮g/ml final dilution). All fluorescent images were taken with a ⫻40 objective using a Carl Zeiss LSM 510 laser confocal microscope. D and E, Fourteen optical sections in the z-axis were obtained by scanning from the apical surface of the cell to the adherent base in 0.2-␮m step sizes. One characteristic micrograph of a cell obtained from the middle of the optical sections is presented for ER␣ (D) and ER␤ (E).

Developmental expression of ER␣ and ER␤ mRNA in the rat decidua

To investigate whether ER␣ and ER␤ expression is tissue specific and developmentally regulated, decidual tissue were collected on different days of pseudopregnancy and separated into mesometrial and antimesometrial decidua as previously described by Martel et al. (16). Total RNA was isolated and subjected to RT-PCR with L19 as an internal control. The results shown in Fig. 2 revealed that both forms of the ER mRNA are expressed in the rat decidua and that ER␤ mRNA is preferentially expressed in the antimesometrial tissue, with little expression in the mesometrial decidua throughout pseudopregnancy (Fig. 2A). ER␣ mRNA is also mainly found in the antimesometrial decidua on days 10 and 11 of pseudopregnancy, but becomes expressed in both antimesometrial and mesometrial decidual tissue later in pseudopregnancy (Fig. 2B). Characterization of rat decidual ER␤ expression

To confirm that the immunoreactive protein observed in Fig. 1 and the product amplified by PCR in Fig. 2 was ER␤, RNA was obtained from decidual tissue of day 13 pseudopregnant rats and was amplified by RT-PCR. The PCR product was directly cloned into the pGEM-T Easy vector. Three clones were selected for DNA sequencing. As shown in Fig. 3A, the three clones were similar and identical to the rat ER␤ sequence previously described in the prostate (19). In all three clones, one base was shown to be different from the original published sequence: the A165 was substituted by a G. Northern blot analysis in Fig. 3B showed that the cRNA probe synthesized, as described in Materials and Methods, specifically hybridized to decidual ER␤ mRNA. As previously described in other tissues (41, 42), several transcripts were observed for ER␤ in the rat decidua.

Regulation of ER␣ and ER␤ mRNA expression by PRL, rat placental lactogen I (rPL-I), and steroids in decidual cells in primary culture

Because decidual tissue produces PRL (43) and expresses both types of the PRL receptor (44), and because our laboratory has previously shown that PRL up-regulates the expression of both ER mRNA species in luteal cells (45), we examined the effect of PRL on ER mRNA expression in decidual cells in primary culture. As shown in Fig. 4A, the expression of both ER␣ (upper panel) and ER␤ (lower panel) mRNA was stimulated by PRL treatment. As rPL-I, a trophoblast-produced protein, also binds to the PRL receptor, we examined the effect of this hormone on ER mRNA expression. The results shown in Fig. 3B indicate that within 12 h of culture, rPL-I stimulates both ER␣ and ER␤ mRNA expression. Previous studies of uterine (46) and mesometrial decidual ER␣ (9, 18) regulation by steroids have led to contradictory results. Therefore, we examined the regulation of decidual ER␣ and ER␤ by estradiol and progesterone. Estradiol had no significant effect on ER␣ mRNA levels, but up-regulated those of ER␤ (Fig. 5A). The smallest dose of estradiol (0.1 ng/ml) was highly effective, and no further stimulation was seen at higher doses. Of great interest was the finding that progesterone regulates the expression of ER␣ and ER␤ in an opposite manner (Fig. 5B). Whereas progesterone caused a dose-related down-regulation of ER␣ mRNA expression, it simultaneously induced an increase in the expression of ER␤. Because progesterone enhances the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells (47), we examined the effect of progesterone and PRL cotreatment on ER␣ and ER␤ expression. Progesterone or PRL alone caused an increase in ER␤, but no synergism was observed when both hormones were added to decidual cells

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8, when GG-AD cells were treated with PRL or estradiol. Western analysis, using a polyclonal antibody raised against ER␤ (Fig. 7B), revealed that GG-AD cells express an immunoreactive ER␤ protein that migrates at the same molecular mass (61 kDa) as the protein detected in the decidua. No signal could be detected in either GG-AD or decidual tissue when the antiserum was preabsorbed with an excess of the antigenic peptide, demonstrating the specificity of the reaction. No ER␣ protein could be detected in these cells. We then examined whether in the GG-AD cells ER␤ is regulated similarly by PRL and steroids in the absence of ER␣. The results showed that similar to its effect on primary cells (Fig. 3B), PRL up-regulates, in a dose-dependent manner, ER␤ mRNA levels in GG-AD cells stably transfected with the long form of the PRL receptor (Fig. 8A). In contrast, estradiol regulation of ER␤ in GG-AD cells differed from that in decidual cells in primary culture and clearly showed a biphasic effect (Fig. 8B). At lower concentrations, estradiol stimulated ER␤ mRNA levels as previously observed in primary cells. In contrast, at higher concentrations estradiol induced a marked inhibition of ER␤ expression, suggesting that the presence of ER␣ may prevent estradiol-induced inhibition of ER␤. Biological activity of ER␤ in GG-AD cells

FIG. 2. Developmental expression of ER␣ and ER␤ mRNA in decidual tissue of pseudopregnant rats. Total RNA was isolated from dissected decidual tissue at different stages of pseudopregnancy. RNA samples were subjected to RT-PCR as described in Materials and Methods. Ribosomal L19 mRNA was used in each reaction as an internal standard for normalizing the data. A, Expression of ER␤ in antimesometrial (AM) and mesometrial (M) decidua; B, expression of ER␣. The autoradiograms from one representative experiment are shown (n ⫽ 3).

To determine whether estradiol can transduce its signal through ER␤ in the decidua-derived GG-AD cells, we studied the effects of different doses of 17␤-estradiol on the expression of the PR, a well known estrogen target gene. The results depicted in Fig. 9 clearly indicate that estradiol is able to markedly increase the expression of PR in these cells, which express only ER␤. Interestingly, high doses of estradiol (1 ng/ml) had no stimulatory effect, probably because these levels of estradiol cause a marked down-regulation in the level of ER␤ expression (Fig. 8B), rendering the cells less sensitive to estradiol action. Discussion

in primary culture (Fig. 6B). As shown in Fig. 6A, ER␣ was up-regulated by PRL and down-regulated by progesterone; however, the inhibitory effect of progesterone on ER␣ was not affected by the addition of PRL to the culture, whereas the stimulatory effect of PRL was totally obliterated. ER expression and regulation in the GG-AD decidual cell line

To determine whether estradiol can transduce its signal through the ER␤, we used the GG-AD cell line derived from antimesometrial decidual cells (33). We first characterized these cells and examined whether they express both ER␣ and ER␤ species. RNA and protein isolated from GG-AD cells and from decidual tissue were analyzed by RT-PCR (Fig. 7A) and Western analysis (Fig. 7B). Decidual tissue from day 9 pseudopregnant rats was used as a control. Whereas mRNA species for both forms of ER were readily detectable in the rat decidual tissue, only the mRNA encoding ER␤ could be detected in the GG-AD cells (Fig. 7A). The basal level of ER␤ mRNA expression varied between cell cultures. However, this variation was lower than the regulations observed in Fig.

In the present report we show for the first time by immunofluorescence, PCR, and Northern blot analysis that ER␤, in addition to ER␣, is expressed in the rat decidua. The molecular cloning of our PCR product confirmed that the ER␤ mRNA detected in the decidua is identical to the rat ER␤ previously cloned in the prostate (19). The expression of ER␤ in the rat decidua contrasts with reports from the mouse decidua, where very low, if any, expression of ER␤ could be detected (48). Such a discrepancy in the expression pattern of ER␤ between rat and mouse was also described in the pituitary (49). Previous studies have shown the presence of estrogenbinding sites in the decidua of rats (7–16). This binding activity is probably due to both ER subtypes, which, as shown herein, are largely confined to the antimesometrial cells where blastocyst apposition, a process that depends upon estradiol and progesterone, takes place. The cell specific localization of ER mRNA found in this investigation confirmed previous studies that showed higher levels of estradiol-binding sites in the larger polyploid antimesometrial cells than in the smaller mesometrial cells (14). Interestingly, the same


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FIG. 3. Nucleotide sequence and Northern analysis of ER␤ in the rat decidua. A, A 285-bp PCR product generated from decidual RNA with specific primers (underlined) to the rat ER␤ was sequenced. The results show homology between the decidual ER␤ (1) and the previously cloned rat ER␤ in the prostate (2). The numbers above the nucleotides represent their positions from the ATG initiation codon (A being considered as base 1) in the ER␤ initially cloned in the prostate. The bold underlined bases show the difference from the original published sequence. B, Northern blot analysis. Polyadenylated mRNA was isolated from total decidual tissue on day 12 of pseudopregnancy. Ten micrograms per lane of polyadenylase were electrophoresed on 1% agarose formaldehyde gel, blotted onto GeneScreen nylon membrane, and hybridized with a 32P-labeled cRNA probe synthesized from a 750-bp ER␤ cDNA. The results show an autoradiogram with RNA from two different rats.

FIG. 4. Effects of PRL and rPL-I on ER␣ and ER␤ mRNA expression in decidual cells in primary culture. Decidual cells in primary culture were isolated from pseudopregnant rats (day 9 of pseudopregnancy) and cultured in RPMI 1640 phenol-free medium containing 1% charcoal-dextran-treated FBS for 12 h in presence of different doses of oPRL or rPL-I. Total RNA was prepared and subjected to RT-PCR analysis, as described in Materials and Methods. RT-PCR products were visualized by autoradiography and normalized to the amount of the L19 mRNA internal control. A, Effect of PRL on ER␣ and ER␤ mRNA levels; B, effect of rPL-I. The left panels depict one representative autoradiogram (n ⱖ 3), and the right panels show the densitometric analysis (mean ⫾ SEM of values expressed as a percentage of the control, which was considered 100%). *, P ⬍ 0.05 compared with vehicle-treated controls (by one-way ANOVA, followed by Duncan’s multiple range test).

observations were described in humans, in whom ER immunostaining has been detected in greater amounts in the decidua capsularis than in the decidua parietalis (50). The high expression of the two ER isoforms in this specific cell population may be due to decidual PRL. In the rat, the antimesometrial decidual cells are known to express members of the PRL family such as PRL-like proteins (51, 52) and dPRP (53), which have no PRL-like activity, and PRL (43), which binds to the PRL receptor in decidual cells and affects their function (5, 44). The role of decidual PRL in pregnancy is not yet understood. However, the results of this investigation suggest that one important action of this hormone is

to act locally to up-regulate the expression of both ER␣ and ER␤, rendering the decidua more responsive to estradiol. rPL-I, secreted by the trophoblast later in pregnancy (54), is also able to up-regulate decidual ER expression, suggesting a trophoblast-decidual cell interaction in the expression of this steroid receptor. PRL appears to be a key up-regulator of the ER in many reproductive tissues, such as the ovarian corpus luteum (45) and the mammary gland (55). We have shown that PRL increases mRNA and protein levels of both ER subtypes in the corpus luteum and in a luteal-derived cell line and that this PRL up-regulation of ER is a prerequisite for estradiol

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FIG. 5. Estradiol and progesterone effects on ER␣ and ER␤ mRNA expression in decidual cells in primary culture. Total RNA obtained from decidual cells in primary culture (day 9 of pseudopregnancy) treated 12 h with different doses of estradiol or progesterone was isolated, reverse transcribed into single stranded complementary DNA, and amplified with specific oligonucleotide pairs for ER␣ and ER␤ mRNA, as described in Materials and Methods. A, Effect of estradiol on ER␣ and ER␤ mRNA; B, effect of progesterone. One representative autoradiogram is shown in the left panels. The densitometric analysis from three or more independent experiments (mean ⫾ SEM of values expressed as a percentage of the control, which was considered 100%) is depicted in the right panels. *, P ⬍ 0.05, by one-way ANOVA, followed by Duncan’s multiple range test.

FIG. 6. Effect of PRL and progesterone cotreatment on ER␣ and ER␤ mRNA in decidual cells in primary culture. Decidual cells were isolated from day 9 pseudopregnant rats and cultured in RPMI 1640 phenol-free medium supplemented with 1% charcoal-dextran-treated FBS for 12 h in the presence of PRL (1 ␮g/ml) and/or progesterone (P; 0.1 ␮g/ml). The effects of these hormones on both ER␣ (A) and ER␤ (B) mRNA were determined as described in Materials and Methods. The upper panels show one characteristic autoradiogram (n ⫽ 3), and the lower panels depict the densitometric analysis (mean ⫾ SEM of values expressed as a percentage of the control, which was considered 100%). *, P ⬍ 0.05, by one-way ANOVA, followed by Duncan’s multiple range test.

stimulation of steroidogenesis and corpus luteum hypertrophy (45). PRL was also shown previously to increase the levels of estradiol-binding sites in the mammary gland (55) and to enhance estradiol stimulation of growth in breast tumor-derived cells (56). Whether this effect is due to PRL

up-regulation of gene expression of either one or both ER subtypes remains to be investigated. Whereas PRL up-regulates in concert and in a dose-related manner both ER␣ and ER␤ mRNA expression, estradiol upregulation of ER is subtype specific. Estradiol has no detectable effect on ER␣ expression at any dose used, but increases ER␤ mRNA at low doses in both decidual cells in primary culture and GG-AD cells. In contrast, high doses of estradiol caused a severe down-regulation of ER␤ mRNA expression in GG-AD cells. This inhibitory effect was not seen in decidual cells in primary culture. As the GG-AD cells express only ER␤, whereas decidual cells in primary culture coexpress both subtypes, it is possible that ER␣ in the primary cell culture system may prevent the deleterious effect of high doses of estradiol on ER␤ mRNA. Of great interest was our finding that progesterone, a steroid considered to inhibit ER expression in the uterus (57, 58) and widely used to prevent estradiol-induced endometrial cell proliferation, has opposite effects on the two ER subtypes in the decidua; whereas progesterone decreases ER␣ expression in a dose-related manner, it concomitantly up-regulates that of ER␤. This finding may be of physiological importance and may explain at least in part why some progesterone target cells can remain responsive to estradiol despite progesterone treatment (59, 60). The differential effect of progesterone on the two ER subtypes may also be the reason why the ovaries express high levels of ER␤ and little ER␣, as this gland is subjected to very high levels of locally produced progesterone. As both PRL and progesterone up-regulate ER␤, whereas they have opposite effects on ER␣ and because progesterone was shown to enhance the ability of PRL to signal to a PRL-regulated gene in mammary-gland tumor derived cells (48), we examined the combined effects of PRL and progesterone on ER expression. We showed that in the decidua, progesterone does not stimulate PRL-mediated regulation of either ER␣ or ER␤. Quite to the contrary, PRL stimulation of ER␣ mRNA expression was totally prevented by progesterone, which has, by itself, a strong


FIG. 7. ER expression in GG-AD cells. A, Total RNA was isolated from decidual tissue (DT), day 9 of pseudopregnancy (used as positive control), or GG-AD cells cultured 24 h at 39 C in 1% charcoal-dextrantreated FBS. RNA was subjected to RT-PCR analysis using specific primers for either ER␣ or ER␤, as described in Materials and Methods. Included in each reaction was a pair of oligonucleotide primers for the S16 ribosomal mRNA, used as an internal control. One representative autoradiogram from three different cell cultures is shown. B, Total protein extracts were obtained from decidual tissue (DT) on day 9 of pseudopregnancy or from cultured GG-AD cells. Equal amounts of proteins (50 ␮g) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The left panel shows a representative autoradiogram from a membrane probed with a polyclonal ER␤ antibody (Upstate Biotechnology, Inc.). The right panel depicts the same blot after stripping and probing with an ER␤ antibody saturated with antigenic peptide. The positions of the mol wt markers are shown on the right.

inhibitory effect on this receptor subtype. In addition, whereas both progesterone and PRL each stimulated ER␤ mRNA expression, they had no synergistic effect when added together to the cell culture, suggesting that both hormones may be acting on ER␤ expression by a similar mechanism. The mechanism by which PRL and progesterone signal to ER␣ and ER␤ genes in decidual cells remains to be investigated. The finding that the rat decidua expresses ER␤ in addition to ER␣ led us to examine whether this receptor subtype transduces an estradiol signal to the PR gene. In ERKO mice, the PR is up-regulated in uterine stroma after the induction of decidualization, and the decidua is fully progesterone responsive in terms of gene regulation and morphological changes, suggesting that ER␣ is not necessary for PR expression or function leading to decidualization (22, 23). As estradiol plays a key role in the induction of the PR, it is not clear whether in mice ER␤ is responsible for such an effect. It is clear, however, that in rat decidual cells, estradiol causes a marked stimulation of PR mRNA expression through ER␤. The development of the decidua-derived cell line (GG-AD), which expresses only ER␤, has allowed us to establish that

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FIG. 8. Effect of estrogen and PRL on ER␤ mRNA expression in GG-AD cells. Total RNA, obtained from wild-type GG-AD cells treated with estrogen or from stably transfected PRL-RL GG-AD cells treated with PRL, was subjected to RT-PCR analysis as described in Materials and Methods. A, Effects of different doses of PRL (0.01–10 ␮g/ml) on ER␤ mRNA expression; B, effect of an estradiol dose-response (0.1–10 ng/ml) on ER␤ mRNA levels. The left panels show one representative autoradiogram (n ⱖ 3; the exposure time was 3 h for A and 24 h for B), and the right panels show the densitometric analysis (mean ⫾ SEM of values expressed as a percentage of the control, which was considered 100%). *, P ⬍ 0.05 compared with vehicle-treated controls (by one-way ANOVA, followed by Duncan’s multiple range test).

this receptor type is functional, stimulating gene expression. Decidual ER␤ was shown to transduce a strong estradiol inhibition of IL-6 and gp130 expression (5). This estradiol effect prevents the decidual production and action of IL-6 (5), a cytokine known to be detrimental to the normal progress of pregnancy. The present investigation suggests that ER␤ may also play an important role in sensitizing the uterus to progesterone action by inducing the PR, a process critical for the initiation and maintenance of the decidual reaction. An interesting finding of this investigation is the fact that, in contrast to physiological concentrations, high levels of estradiol totally prevent the expression of PR in GG-AD cells, probably by down-regulating ER␤ and preventing ER signaling. This finding may explain at least in part why high levels of estradiol cause abortion and collapse of decidual tissue. In conclusion, the results of this investigation have revealed for the first time that the rat decidua expresses both ER␣ and ER␤ genes and that these genes are preferentially expressed in a cell population localized in the antimesometrial site of the uterus that forms the decidua capsularis in pregnant rats. The results also indicate that the two ER subtypes are differentially regulated by PRL and ovarian ste-

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FIG. 9. Effect of estradiol on PR mRNA expression in the decidual cell line. Wild-type GG-AD cells were incubated in the presence of different doses of estradiol for 24 h at 39 C. Total RNA was isolated and subjected to RT-PCR analysis as described in Materials and Methods, using specific primers for PR. The upper panels depict representative autoradiograms from one experiment (n ⱖ 3), and the lower panels show the densitometric analysis (mean ⫾ SEM of values expressed as a percentage of the control, which was considered 100%).

roids, and that ER␤ can transduce the estradiol signal to the PR gene in decidual cells. Acknowledgments We are grateful to Dr. Nira Ben-Jonathan for the ER␤ antigenic peptide, Dr. O-Kyong Park-Sarge for the rat ER␤ cDNA, the NIDDK and National Hormone and Pituitary Program (NIH) for the oPRL and ER-715 antibody, Rose Clepper for animal care, and Linda Alaniz-Avila for photography. We also thank Vivian Regala for preparation of the manuscript, and Dr. Catherine Boyer for skillful assistance with the confocal studies. We thank Ying Zhou for her technical assistance with the sequencing of decidual ER␤.

References 1. Weitlauf HM 1994 Biology of implantation. In: Knobil E, Neil JD (eds) The Physiology of Reproduction, ed. 2. Raven Press, New York, vol 1:391– 440 2. Gu Y, Gibori G 1999 Encyclopedia of Reproduction: Deciduoma. Academic Press, New York, vol 1:836 – 842 3. Wu JT, Meyer RK 1970 The effect on implantation of culturing delayed rat blastocysts in medium containing 17␤-estradiol. Biol Reprod 3:201–204 4. Kraus WL, Montano MM, Katzenellenbogen BS 1994 Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene. Mol Endocrinol 8:952–969 5. Deb S, Tessier C, Prigent-Tessier A, Barkai U, Ferguson-Gottschall S, Srivastava RK, Faliszek J, Gibori G 1999 The expression of interleukin-6, IL-6 receptor and gp130 in the rat decidua and a decidual cell line: regulation by 17␤ estradiol and prolactin. Endocrinology 140:4442– 4450 6. Yochim JM, De Feo VJ 1962 Control of decidual growth in the rat by steroid hormones of the ovary. Endocrinology 71:134 –142 7. McCormack SA, Glasser SR 1976 A high-affinity estrogen-binding protein in rat placental trophoblast. Endocrinology 99:701–712 8. McCormack SA, Glasser SR 1978 Ontogeny and regulation of rat placental estrogen receptor. Endocrinology 102:273–280

Endo • 2000 Vol. 141 • No. 10

9. Ogle TF, George P 1995 Regulation of the estrogen receptor in the decidua basalis of the pregnant rat. Biol Reprod 53:65–77 10. Talley DJ, Tobert JA, Armstrong EG, Villee CA 1977 Changes in estrogen receptor levels during deciduomata development in the pseudopregnant rat. Endocrinology 101:1538 –1544 11. Armstrong EG, Villee CA 1978 Estrogen receptor in purified nuclei from deciduomata of the pseudopregnant rat. J Steroid Biochem 9:1149 –1154 12. Peleg S, Bauminger S, Lindner HR 1979 Oestrogen and progestin receptors in deciduoma of the rat. J Steroid Biochem 10:139 –145 13. McConnell KN, Brodie JY, Green B 1980 Nuclear progesterone and estradiol receptors in deciduomata induced in ovariectomised rats. Mol Cell Endocrinol 20:191–204 14. Moulton BC, Koenig BB 1981 Estrogen receptor in deciduoma cells separated by velocity sedimentation. Endocrinology 108:484 – 488 15. Hoshiai H, Uehara S, Suzuki M, Villee CA 1982 The variations of estrogen receptor, progesterone receptor, cAMP, ODC activity and RNA synthesis of deciduomata in pseudopregnant rats. Tohoku J Exp Med 137:349 –360 16. Martel D, Monier MN, Psychoyos A, DeFeo VJ 1984 Estrogen and progesterone receptors in the endometrium, myometrium, and metrial gland of the rat during the decidualization process. Endocrinology 114:1627–1634 17. Ogle TF, Dai D, George P, Mahesh VB 1997 Stromal cell progesterone and estrogen receptors during proliferation and regression of the decidua basalis in the pregnant rat. Biol Reprod 57:495–506 18. Ogle TF, Dai D, George P, Mahesh VB 1998 Regulation of the progesterone receptor and estrogen receptor in decidua basalis by progesterone and estradiol during pregnancy. Biol Reprod 58:1188 –1198 19. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930 20. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ␣ and ␤. Endocrinology 138:863– 870 21. O’Shea JD, Kleinfeld RG, Morrow HA 1983 Ultrastructure of decidualization in the pseudopregnant rat. Am J Anat 166:271–298 22. Curtis SW, Clark J, Myers P, Korach KS 1999 Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor ␣ knockout mouse uterus. Proc Natl Acad Sci USA 96:3646 –3651 23. Paria BC, Tan J, Lubahn DB, Dey SK, Das SK 1999 Uterine decidual response occurs in estrogen receptor-␣-deficient mice. Endocrinology 140:2704 –2710 24. Finn CA 1966 Endocrine control of endometrial sensitivity during the induction of the decidual cell reaction in the mouse. J Endocrinol 36:239 –248 25. Finn CA, Pope M 1986 Control of leucocyte infiltration into the decidualized mouse uterus. J Endocrinol 110:93–96 26. Ledford BE, Rankin JC, Markwald RR, Baggett B 1976 Biochemical and morphological changes following artificially stimulated decidualization in the mouse uterus. Biol Reprod 15:529 –535 27. Milligan SR, Mirembe FM 1985 Intraluminally injected oil induces changes in vascular permeability in the ‘sensitized’ and ‘non-sensitized’ uterus of the mouse. J Reprod Fertil 74:95–104 28. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-␣ (ER␣) and estrogen receptor-␤ (ER␤) messenger ribonucleic acid in the wild-type and ER␣knockout mouse. Endocrinology 138:4613– 4621 29. Irwin JC, Utian WH, Eckert RL 1991 Sex steroids and growth factors differentially regulate the growth and differentiation of cultured human endometrial stromal cells. Endocrinology 129:2385–2392 30. Pippard DF 1987 In-vitro development and degeneration of stromal cells from the uterus of the rat at three stages of pregnancy. J Reprod Fertil 81:249 –257 31. Bell SC, Searle RF 1981 Differentiation of decidual cells in mouse endometrial cell cultures. J Reprod Fertil 61:425– 433 32. Sananes N, Weiller S, Baulieu EE, Le Goascogne C 1978 In vitro decidualization of rat endometrial cells. Endocrinology 103:86 –95 33. Srivastava RK, Gu Y, Zilberstein M, Ou JS, Mayo KE, Chou JY, Gibori G 1995 Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line. Endocrinology 136:1913– 1919 34. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 –159 35. Koike S, Sakai M, Muramatsu M 1987 Molecular cloning and characterization of rat estrogen receptor cDNA. Nucleic Acids Res 15:2499 –2513 36. Park OK, Mayo KE 1991 Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 5:967–978 37. Chan YL, Lin A, McNally J, Peleg D, Meyuhas O, Wool IG 1987 The primary structure of rat ribosomal protein L19. A determination from the sequence of nucleotides in a cDNA and from the sequence of amino acids in the protein.J Biol Chem 262:1111–1115 38. Chan YL, Paz V, Olvera J, Wool IG 1990 The primary structure of rat ribosomal protein S16. FEBS Lett 263:85– 88 39. Bradford MN 1976 A rapid and sensitive method for the quantification of


40. 41. 42.

43. 44. 45.

46. 47.

48. 49.

microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 –254 Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685 Byers M, Kuiper GG, Gustafsson JA, Park-Sarge OK 1997 Estrogen receptor-␤ mRNA expression in rat ovary: down-regulation by gonadotropins. Mol Endocrinol 11:172–182 Maruyama K, Endoh H, Sasaki-Iwaoka H, Kanou H, Shimaya E, Hashimoto S, Kato S, Kawashima H 1998 A novel isoform of rat estrogen receptor ␤ with 18 amino acid insertion in the ligand binding domain as a putative dominant negative regular of estrogen action. Biochem Biophys Res Commun 246:142– 147 Prigent-Tessier A, Tessier C, Hirosawa-Takamori M, Ferguson-Gottschall S, Gibori G 1999 Decidual rat prolactin: identification, molecular cloning and characterization. J Biol Chem 274:37982–37989 Gu Y, Srivastava RK, Clarke DL, Linzer DI, Gibori G 1996 The decidual prolactin receptor and its regulation by decidua-derived factors. Endocrinology 137:4878 – 4885 Telleria CM, Zhong L, Deb S, Srivastava RK, Park KS, Sugino N, Park-Sarge OK, Gibori G 1998 Differential expression of the estrogen receptors ␣ and ␤ in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 139:2432–2442 Hsueh AJ, Peck Jr EJ, Clark JH 1976 Control of uterine estrogen receptor levels by progesterone Endocrinology 98:438 – 444 Richer JK, Lange CA, Manning NG, Owen G, Powell R, Horwitz KB 1998 Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J Biol Chem 273:31317–31326 Tan J, Paria BC, Dey SK, Das SK 1999 Differential uterine expression of estrogen and progesterone receptors correlates with uterine preparation for implantation and decidualization in the mouse Endocrinology 140:5310 –5321 Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? [published erratum appears in Endocr Rev 1999 Aug;20(4):459] Endocr Rev 20:358 – 417

3851

50. Wu WX, Brooks J, Millar MR, Ledger WL, Glasier AF, McNeilly AS 1993 Immunolocalization of oestrogen and progesterone receptors in the human decidua in relation to prolactin production. Hum Reprod 8:1129 –1135 51. Duckworth ML, Peden LM, Friesen HG 1988 A third prolactin-like protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene. Mol Endocrinol 2:912–920 52. Cohick CB, Xu L, Soares MJ 1997 Prolactin-like protein-B: heterologous expression and characterization of placental and decidual species. J Endocrinol 152:291–302 53. Gu Y, Soares MJ, Srivastava RK, Gibori G 1994 Expression of decidual prolactin-related protein in the rat decidua. Endocrinology 135:1422–1427 54. Robertson MC, Croze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid. Endocrinology 127:702–710 55. Sasaki GH, Leung BS 1974 Prolactin stimulation of estrogen receptor in vitro in 7,12 dimethylbenz(A)anthracene-induced mammary tumor. Res Commun Chem Pathol Pharmacol 8:409 – 412 56. Kilic E, Canbay E, Goffin V, McArdle C, Norman M, The proliferative effect of prolactin on breast cancer cells: synergy with estrogen, and abolition by anti-estrogen 80th Annual Meeting of The Endocrine Society, New Orleans, LA, 1998, p 128 (Abstract) 57. Hsueh AJ, Peck Jr EJ, Clark JH 1975 Progesterone antagonism of the oestrogen receptor and oestrogen-induced uterine growth. Nature 254:337–339 58. Evans RW, Leavitt WW 1980 Progesterone inhibition of uterine nuclear estrogen receptor: dependence on RNA and protein synthesis. Proc Natl Acad Sci USA 77:5856 –5860 59. Kumar NS, Richer J, Owen G, Litman E, Horwitz KB, Leslie KK 1998 Selective down-regulation of progesterone receptor isoform B in poorly differentiated human endometrial cancer cells: implications for unopposed estrogen action. Cancer Res 58:1860 –1865 60. Hargreaves DF, Knox F, Swindell R, Potten CS, Bundred NJ 1998 Epithelial proliferation and hormone receptor status in the normal post-menopausal breast and the effects of hormone replacement therapy. Br J Cancer 78:945–949