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Endocrinology Vol. 139, No. 5 2432-2442
Copyright © 1998 by The Endocrine Society

ARTICLES

Differential Expression of the Estrogen Receptors {alpha} and ß in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens1

C. M. Telleria, L. Zhong, S. Deb, R. K. Srivastava2, K. S. Park, N. Sugino, O.-K. Park-Sarge and G. Gibori3

Department of Physiology and Biophysics (C.M.T., L.Z., S.D., R.K.S., N.S., G.G.), College of Medicine, University of Illinois, Chicago, Illinois 60612; and Department of Physiology (K.S.P., O.-K.P.-S.), University of Kentucky, Lexington, Kentucky 40536

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. E-mail: ggibori{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ER{alpha} and ERß) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER{alpha} and ERß messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER{alpha} and ERß) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER{alpha} mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ERß mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER{alpha} and ERß mRNA expression at the cellular level, by in situ hybridization, showed ER{alpha} expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ERß mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypo- physectomy, performed early in pregnancy, induced a sharp decline in ER{alpha} mRNA expression but a less-marked reduction in ERß mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER{alpha} and ERß mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ERß mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ERß mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ROLE of estradiol in luteal function has been extensively investigated in the pregnant rat. Intraluteal estradiol stimulates steroidogenesis, increasing the supply of cholesterol substrate by mobilizing cholesterol storage and enhancing its transport to the mitochondria (1, 2). Estradiol also stimulates cholesterol synthesis (3, 4) and luteal cell content of lipoprotein receptors for the uptake of circulating cholesterol (5, 6). In addition, estradiol mediates luteal cell hypertrophy, contributing to the increase in the size of the corpora lutea that occurs at midpregnancy. Estradiol elicits this action by stimulating overall protein biosynthesis (7) through a mechanism that involves an increase in the levels of elongation factor-2, a 100-kDa protein that participates in peptide elongation, and is an essential component of the protein synthetic machinery (8, 9). Estradiol stimulation of luteal cell hypertrophy is accompanied by a remarkable proliferation of vascular endothelial cells (10, 11), an effect that seems to be mediated by basic fibroblast growth factor (11, 12). The molecular mechanism whereby estradiol controls such a variety of luteal cell regulatory activities remains unknown. Estradiol regulates reproductive functions through the binding of a nuclear protein, the estrogen receptor (ER), which belongs to a superfamily of ligand-activated transcription factors that regulate the expression of target genes by binding to specific response elements (13, 14). The recent cloning of a novel rat ER subtype, named ERß (to distinguish it from the previously cloned ER{alpha}) (15), may provide an explanation for the selective actions of estradiol in the rat corpus luteum.

In the rat, PRL (secreted by the anterior pituitary) and PRL-like hormones from the placenta [placental lactogens I and II (rPL-I and rPL-II)] seem to be required for the luteotropic effect of estradiol in corpora lutea of pregnant rats (11, 16). Experimental evidences indicate that such a synergistic action of PRL with estradiol in the luteotropic process may involve, at least in part, PRL stimulation of luteal receptors for estradiol (17, 18). Despite the fact that binding experiments demonstrated the presence of estrogen-binding sites in the rat corpus luteum of pregnancy (19), no evidence exists as to whether the rat corpus luteum expresses the ER genes. Thus, the aims of the present investigation were to examine: 1) the expression and developmental changes of the two ER (ER{alpha} and ERß) genes in the rat corpus luteum during pregnancy; and 2) the regulation of ER{alpha} and ERß messenger RNA (mRNA) expression by PRL and placental lactogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak (Rochester, NY), respectively. Taq DNA polymerase was purchased from Perkin-Elmer Corp. (Foster City, CA). [32P]-deoxycytidine triphosphate, [32P]-deoxyguanidine triphosphate, and [33P]-uridine triphosphate were from Amersham (Arlington Heights, IL). The oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY). RPMI-1640 medium, antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were from Mediatech (Washington, DC). FBS was from HyClone (Logan, UT). Lipofectin, G418 sulfate (Geneticin) and dithiothreitol (DTT) were from Life Technologies (Grand Island, NY). 17ß-estradiol was from Steraloids Inc. (Wilton, NH). DMEM: Ham’s F12 (DMEM/F12), McCoy’s 5A: Ham’s F12 1:1, human CG (hCG), phenylmethyl sulfonyl fluoride (PMSF), leupeptin, pepstatin-A, aprotinin, and all other reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Ovine PRL (oPRL, PRL-18, 30 IU/mg), the ER-715 antibody, and the ER-715 antigen peptide were a gift from NIDDK (NIH, Bethesda, MD). Purified rat placental lactogen (rPL) I (rPL-I) and recombinant rPL-II were kindly provided by Dr. M. Robertson (Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada).

Animal model and tissue preparation
Pregnant (day 1 = sperm positive) and immature female (day 26 of age) Sprague-Dawley rats were obtained from Sasco Animal Labs. (Madison, WI). They were kept under controlled conditions of light (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.

For the developmental studies, rats were obtained at various stages of pregnancy, from days 4–22 (day of parturition) and at the day after parturition. Corpora lutea were dissected from the ovaries under a stereoscopic microscope. All tissues were frozen in liquid nitrogen and stored at -80 C until processed for RNA or protein preparation. In particular, for the in situ hybridization experiments, whole ovaries obtained from animals at different stages of pregnancy were frozen in dry ice and stored at -80 C until processed.

To determine the effect of PRL on ER mRNA expression in corpora lutea, pregnant rats were hypophysectomized, using a transauricular approach, on day 3 of pregnancy. Surgery was performed under ether anesthesia, with minimal stress to the animals. Completeness of hypophysectomy was evaluated by examination of the pituitary, removed at the time of operation, and visualization of the pituitary fossa at autopsy. Hypophysectomized rats were injected sc with 125 µg PRL (NIDDK oPRL-18, 30 IU/mg) twice daily in 50% polyvinylpyrrolidone, pH 9.0, for 4 days. Control rats were treated with vehicle. An additional group of intact pregnant rats was included in the study.

Granulosa cell culture
Maturation of preovulatory follicles was stimulated by treatment of immature rats at day 28 of age, with 0.15 IU hCG sc, twice daily for 2 days (20). Luteinization of these preovulatory follicles was subsequently achieved by an ovulatory dose (10 IU) of hCG on the third day, via the tail vein. Luteinized granulosa cells were harvested from preovulatory follicles 7 h after the iv injection of hCG. Briefly, follicles were incubated sequentially in DMEM/F12 (1:1), containing 6 mM EGTA and 0.5 M sucrose, respectively; and granulosa cells were harvested by needle-pricking the follicles. The cells were plated in 60-mm culture dishes at 8 x 105 cells/ml and were incubated at 37 C, under 95% air-5% CO2 atmosphere, in DMEM/F12 containing 15 mM HEPES, 1% FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 mg/ml amphotericin B. After 72 h of incubation, the medium was changed, and the cells were treated, during 12 h, with different doses of oPRL (0.01–10 µg/ml), rPL-I (0.01–1 µg/ml), or rPL-II (0.1–1 µg/ml). Cells were washed with PBS several times after treatment, and they were stored at -80 C until RNA isolation.

Generation of the GG-CL cell culture
The luteal cell line, termed GG-CL, was developed from large luteal cells that were purified to homogeneity by flow cytometry from corpora lutea of day-14 pregnant rats, as reported previously (21). Cells were cultured in McCoy’s 5A: Ham’s F12 (1:1) containing 25 mM HEPES and 2% antibiotic-antimycotic solution, and 5% FBS for 24 h, washed several times, and then infected with the SV-40tsA209 mutant virus, as reported (22). The transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of the transformed cells were isolated and passed, and one clone (designated GG-CL cells) was used in this study.

Transfection of GG-CL cells with the long form of the PRL receptor
For stable transfection of GG-CL cells with the long form of PRL receptor (PRL-RL), we adopted the procedure of Felgner et al. (23), with slight modification. The complementary DNA (cDNA) of the PRL-RL was subcloned into pMT2poly and transformed to prepare large quantities of plasmid DNA. We used another vector pSV2neo to confer the neomycin resistance to the cells. Plasmid DNA was purified after transformation by equilibrium centrifugation in cesium chloride-ethidium bromide gradient. GG-CL cells were plated in 6-well dishes and grown to 33% confluency in RPMI-1640 medium containing 5% FBS. Cells were washed with serum-free medium without antibiotics, and 1.8 ml of this medium was added to each well. Meanwhile, for each well, 10 µl lipofectin reagent was mixed with 10 µg plasmid DNA (isolated from pMT2poly and pSV2neo) and 180 µl of RPMI-1640 (without serum and antibiotics) and incubated for 15 min at room temperature. This mixture of lipofectin and DNA complex was laid on the cells in 1.8 ml of medium and was incubated for 24 h at 37 C, under an atmosphere consisting of 95% air-5% CO2. Medium was replaced with the growth medium containing 5% FBS and antibiotics and was incubated for 48 h. After 48 h, medium was again replaced with fresh growth medium and treated with 100 µg/ml G418 sulfate. G418 sulfate addition was continued every alternate day until G418 sulfate resistant colonies were identified. These colonies were picked and cultured in the growth medium containing 5% FBS until cells were confluent. For identifying the successful stable transfection with the PRL-RL, cells were grown and passed at several times, and the presence of PRL-RL mRNA was identified in the extracted RNA by RT-PCR using specific primers, as previously described (24).

Untransfected GG-CL cells, as well as GG-CL cells expressing the PRL-RL (GG-CL PRL-RL), were cultured at 33 C under 95% air-5% CO2 in RPMI-1640 medium supplemented with 1 x glutamine, 2 x antibiotic-antimycotic solution, 1 x nonessential amino acids, 1 x sodium pyruvate, 0.45% D-glucose, and 1% FBS. The cells were treated with different hormones, and either RNA or proteins were extracted.

To study the effect of PRL on ER mRNA expression, GG-CL PRL-RL cells were treated for 8 h with different doses of oPRL (0.01–1 µg/ml). Cells were washed with PBS several times, after treatment, and were stored at -80 C until RNA isolation. When the effect of PRL on ER immunoreactive proteins was investigated, GG-CL PRL-RL cells were cultured for 8 h in the presence of either 0.1 µg/ml oPRL or vehicle, washed with PBS, and immediately harvested for protein isolation. For the studies involving the interaction of protein extracted from GG-CL cells and DNA, GG-CL PRL-RL cells were cultured for 48 h in the presence of 17ß-estradiol (10 ng/ml) and/or oPRL (0.1 µg/ml). Cells were then washed, and purified nuclear extracts were obtained (see below).

To test the effect of estradiol on protein kinase C delta isoform (PKC-{delta}) mRNA expression, GG-CL cells were cultured with different doses of 17ß-estradiol (10-11-10-8 M) for 6 h, then washed with PBS, and stored at -80 C until RNA extraction.

RNA isolation and RT-PCR analysis
Total RNA from frozen corpora lutea was purified by homogenization in guanidinium thiocyanate and centrifugation through a cesium chloride cushion (25), whereas total RNA from cultured cells was isolated by a one-step guanidinium-thiocyanate-phenol-chloroform extraction procedure (26).

Oligonucleotide primer pairs were based on the sequence of the rat ER{alpha} gene (27) (5'-AATTCTGACAATCGACGCCAG-3' and 5'-GTGCTTCAACATTCTCCCTCCTC-3') and rat ERß gene (15) (5'-AAAGCCAAGAGAAACGGTGGGCAT-3' and 5'-GCCAATCATGTGCACCAGTTCCT T-3'). For the PKC-{delta} isoform, the following primers were used: 5'-CACCATCTTCCAGAAAGAACG-3' and 5'-CTTGCCATAGGTCCCGTTGTTG-3', as previously reported (28). In each reaction, an additional pair of oligonucleotides specific to the rat ribosomal protein S16 (29) was included for use as an internal control (5'-TCCAAGGGTCCGCTGCAGTC-3' and 5'-CGTTCACCTTGATGAGC CCATT-3'). The predicted sizes of the PCR-amplified products were 344 bp for ER{alpha}, 204 bp for ERß, 352 bp for PKC-{delta}, and 100 bp for S16. One to three micrograms of total RNA were reverse transcribed at 42 C using random hexamer primers (Pharmacia, Piscataway, NJ) and Maloney murine leukemia virus RT (Life Technologies, Gaithersburg, MD) in a 20-µl reaction mixture. The reaction mixture was added to tubes containing specific oligonucleotide primers (50 pmol each) for amplification of either form of the ER or PKC-{delta} cDNAs. A mix containing the oligonucleotide primers for S16 mRNA (50 pmol each), Taq DNA polymerase (2.5 U), and [32P]-deoxycytidine triphosphate (2 µCi of 3000 Ci/mmol) was added to each tube; and the final vol was increased to 90 µl with 1 x PCR buffer (20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 mM MgCl2). The samples were overlaid with light mineral oil, and PCR was carried out for 25 cycles using 95 C for denaturing, 65 C for annealing, and 72 C for extension, in a Perkin-Elmer/Cetus Thermal Cycler (Perkin-Elmer, Norwalk, CT). In particular, when RNA from GG-CL cells was used, 30 PCR cycles were performed. The conditions were such that the amplification of the products was in the exponential phase, and the assay was linear, with respect to the amount of input RNA. Reaction products were electrophoresed on a 8% polyacrylamide nondenaturing gel. After autoradiography, data were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant version 3 sofware (Molecular Dynamic, Sunnyvale, CA). The intensity of the ER{alpha}, ERß, and PKC-{delta} signals was normalized to that of the ribosomal protein S16 internal control.

Immunoblot analysis
Isolated corpora lutea were homogenized in 2-ml ice-cold homogenization buffer containing 25 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 1 µM leupeptin, 1 µM pepstatin-A, and 1 µg/ml aprotinin in a Polytron (Brinkmann Instruments, Rexdale, Ontario, Canada) homogenizer. To obtain the protein extracts from cultured cells after appropriate treatments, cells were washed twice with cold PBS (pH 7.4), scrapped off the dishes using a rubber policeman, and disrupted with a 25-gauge needle/1-cc syringe in the same homogenization buffer. To obtain the nuclear and cytosolic fractions, the total homogenates were centrifuged at 1,000 x g for 10 min. The pellets containing the crude nuclear fraction were resuspended in homogenization buffer. The supernatants were centrifuged at 105,000 x g during 30 min, and the new supernatants were considered the cytosolic fraction. Homogenates were assayed for protein content (30), appropriately diluted in 6 x concentrated electrophoresis sample buffer, boiled for 10 min, and stored at -80 C until electrophoresed. Equivalent amounts of protein (50 µg) were separated through 10% SDS-PAGE gels and electrotransferred to nitrocellulose membranes (Amersham). Immunoblotting was performed by blocking nonspecific binding with 5% nonfat milk in TBS buffer containing 0.1% Tween 20. Blots were then incubated overnight with a 1:750 dilution of the polyclonal antibody ER-715 (kindly provided by the NIDDK). This polyclonal rabbit antiserum was raised against a synthetic peptide corresponding to the hinge region of the rat ER{alpha} molecule (31). The membranes were washed and incubated with a secondary antibody linked to horseradish-peroxidase labeled antirabbit IgG (Sigma) for 2 h. After extensive washing, blots were developed using an enhanced chemiluminescence Western blotting detection system (Amersham Corp., Arlington Heights, IL) and exposed for 15–120 sec to x-ray films. Data were analyzed using a Molecular Dynamics Densitometer and ImageQuant version 3 software (Molecular Dynamics).

Specificity of the stained protein band was ascertained by exposing the antiserum to excess antigen peptide (1.25 µg/ml) overnight before exposure to a blotted membrane. The molecular size of immunoreactive bands was estimated by the comigration of a prestained SDS-PAGE molecular mass standard of proteins ranging from 10–200 kDa, approximately every 10 kDa (Benchmark, Gibco/BRL, Gaithersburg, MD).

Nuclear extract preparation and electrophoretic mobility shift assay (EMSA)
Crude nuclear extracts, obtained as reported above, were subjected to a further purification and concentration. Briefly, crude nuclear extracts were resuspended in high-salt buffer (20 mM HEPES (pH 7.9), 350 mM KCl, 0.2 mM EDTA, 20% glycerol, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) and incubated for 1 h on ice. Insoluble proteins and particles were removed by centrifugation at 10,000 x g for 15 min at 4 C. The nuclear extracts were dialyzed overnight against dialysis buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 0.1 mM PMSF, and 20% glycerol) at 4 C. Typically, 10 µg of purified nuclear protein extract were used in each binding reaction. DNA-binding reactions were carried out in binding buffer (5 mM Tris (pH 8.0), 40 mM KCl, 6% glycerol, 1 mM DTT, and 0.05% Nonidet P-40), 2 µg of poly(deoxyinosinic-deoxycytidylic)acid, 0.1 µg of denatured salmon sperm DNA, and 10 µg BSA with 10,000 cpm of oligonucleotide probe that was labeled by end-filling with Klenow (Life Technologies, Gaithersburg, MD) in the presence of [32P]-deoxyguanidine triphosphate. Preincubation with unlabeled probe was conducted on ice for 15 min, then the labeled probe was added and allowed to bind at room temperature for 30 min. The entire reaction was loaded onto a 4% polyacrylamide gel and electrophoresed at 160 V at room temperature. Gels were dried and autoradiographed with intensifying screens. The following oligonucleotide and its complement were used as probe and competitor: 5'-GATCCGTCAGGTCACAGTGACCTGATG-3'(32). The underlined portions represent the palindromic sequence of the estrogen response element (ERE) reported in the vitellogenin A2 gene (33).

In situ hybridization
Ovaries were removed from storage at -80 C and brought to -20 C, and 20-µm sections were cut using a Zeiss cryostat. Sections were mounted onto positively charged glass slides, fixed in 5% paraformaldehyde (pH 7.5) for 5 min, washed in 2 x saline sodium citrate (SSC) for 5 min, rinsed in distilled deionized water, washed in 0.1 M thiethanolamine (pH 8.0), and incubated in 0.25% acetic anhydride in 0.1 M thiethanolamine (pH 8.0) for 10 min. Sections were dehydrated through an ethanol series and vacuum dried until hybridization. Sense and antisense [33P]-uridine triphosphate RNA probes were synthesized using SP6 or T7 RNA polymerase. Templates were an EcoRI/Pstl (750 bp) subclone of the rat ERß cDNA encoding the 5'-UTR and N-terminal A/B region (34), and a PCR clone encoding the hormone-binding domain of the rat ER{alpha} cDNA (34). The RNA probe [1 x 107 cpm/ml in hybridization buffer: 50% formamide, 5 x SSPE (750 mM NaCl, 50 mM NaH2PO4 (pH 7.4), and 1 mM EDTA), 2 x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, and 100 mg/ml yeast tRNA] was applied to the tissue sections, and the sections were overlaid with coverslips. Slides were hybridized in a humidity chamber at 47 C for 12 h. After hybridization, the coverslips were removed and sections were treated with ribonuclease A (20 µg/ml) at 37 C for 30 min, washed in increasingly lower concentrations of SSC down to 0.1 x SSC at 60 C, and dehydrated through an ethanol series. The slides were exposed to Kodak XAR-5 film (Eastman Kodak) for 2–3 days at room temperature and were then processed for liquid emulsion autoradiography using NTB-2 emulsion (Eastman Kodak). Slides were developed three weeks later using Kodak D-19 developer and fixer (Eastman Kodak) and stained with hematoxylin.

Statistics
Data were examined by one-way ANOVA followed by Duncan’s multiple-range test. When appropriate, the Student’s t -test was used. A level of P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental expression of ER{alpha} and ERß mRNA species in the rat corpus luteum during pregnancy
To examine whether the two ER genes (ER{alpha} and ERß) are expressed in the pregnant rat corpus luteum, and to determine whether such expression is temporally associated with particular stages of pregnancy, we examined the ER{alpha} and ERß mRNA levels by in situ hybridization and RT-PCR analysis.

Our results, employing in situ hybridization followed by emulsion autoradiography performed in whole ovaries taken from rats at different days of pregnancy, show that ER{alpha} mRNA is expressed within the ovary in small and growing follicles, as well as in corpora lutea, with maximal expression observed at midpregnancy (Fig. 1Go, upper panel). Despite the fact that ERß mRNA was detected in both follicles and corpora lutea, the signal was much stronger in the follicles than in the luteal structures. On the other hand, the ovarian expression of ERß mRNA remained more or less similar throughout pregnancy in both the follicles and the corpora lutea (Fig. 1Go, lower panel).



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  		Figure 1. Localization of ER{alpha} and ERß mRNAs in rat ovaries obtained at different stages of pregnancy, as determined by in situ hybridization. Sections shown with darkfield photographs (50 x amplification) were hybridized with 33P-labeled antisense RNA complementary to either ER{alpha} mRNA (upper panel) or ERß mRNA (lower panel), whereas their respective adjacent sections were hybridized with labeled sense cRNA, as indicated. The days of pregnancy studied are also displayed.

 
The RT-PCR studies shown in Fig. 2AGo revealed that both forms of the ER mRNA are expressed in the corpus luteum throughout pregnancy, with, however, a different pattern of expression. ER{alpha} mRNA was expressed at low levels early in pregnancy; it increased thereafter and declined just at the day of parturition, remaining at low levels 1 day after parturition. In contrast, ERß mRNA remained constant throughout pregnancy, with a significant decline at parturition (Fig. 2BGo).



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  		Figure 2. Developmental expression of ER{alpha} and ERß mRNA in the rat corpus luteum during pregnancy. Corpora lutea were dissected from ovaries of rats at different stages of pregnancy, and total RNA was isolated and analyzed by RT-PCR, as described in Materials and Methods. Data were quantified by densitometry and corrected using S16 as an internal standard. The mRNA levels for each day of pregnancy are graphically represented in panel B as the mean ± SEM (n = 3); to compare results from separate experiments, values were also normalized within each experiment to the maximum ratio of receptor/S16 products, which was considered 100%. a, P < 0.01, compared with days 12, 15, 17, 18, 19, and 20; b, P < 0.05, compared with days 12, 15, 17, 18, 19, and 20 (one-way ANOVA, followed by Duncan’s multiple-range test); pp, post parturition.

 
Expression of ER immunoreactive proteins in the pregnant rat corpus luteum
Immunoblot of electrophoresed proteins from a pool of corpora lutea, obtained at different stages of pregnancy, are shown in Fig. 3AGo. Also shown in Fig. 3AGo is an extract from uteri taken from cycling rats. Two ER isoforms were found in luteal tissue: a high-molecular-mass moiety of 67 kDa and a smaller ER isoform of 61 kDa, both forms being located mainly in the nuclear cell fraction (Fig. 3AGo, left panel). When antiserum ER-715 was preabsorbed with excess antigen peptide before incubation on the blot, both bands observed in the corpus luteum extracts did not show immunoreaction ascertaining the specificity of the immunoreactive proteins (Fig. 3AGo, right panel). Fig. 3BGo depicts a representative autoradiograph of an immunoblot of ER protein prepared from corpora lutea obtained at different stages of pregnancy. The high-molecular-mass ER immunoreactive protein (67 kDa) was highly expressed at midpregnancy (days 12, 17, and 20), but it was barely detectable in early (day 7) and late (day 22) gestation. In contrast to the high-molecular-mass isoform, the smaller ER moiety of 61 kDa was abundantly expressed throughout pregnancy.



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  		Figure 3. Cellular localization and changes in ER immunoreactive proteins in the rat corpus luteum during pregnancy. A, Corpora lutea (CL) were isolated and pooled from animals at different days of pregnancy; T, total extract; N, nuclear extract; C, cytosolic extract. Total extract from uteri (Ut), taken from cycling rats, was used as control. Peptide +, ER probed with the primary antibody previously saturated with antigen peptide; B, total protein extract was obtained from corpora lutea at the indicated days of pregnancy. Equal amounts of proteins (50 µg) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using the polyclonal antibody ER-715, as described in Materials and Methods. Depicted are autoradiographs from a representative experiment. Positions of the molecular weight markers (Mr) are shown on the left.

 
PRL regulation of luteal ER{alpha} and ERß mRNA expression in vivo
Because the corpus luteum is exposed to high levels of pituitary PRL in early pregnancy, and to even higher concentrations of placental PRL-related hormones later on in gestation, and because PRL is a prerequisite for estradiol action in the corpus luteum (16), we examined the effect of PRL in vivo on luteal ER mRNA expression. Rats were hypophysectomized on day 3 of pregnancy and treated with or without PRL for 4 days. As shown in Fig. 4AGo, hypophysectomy caused a remarkable decrease (P < 0.01) in the expression of ER{alpha} mRNA, an effect that was totally reversed by treatment with PRL. In contrast to the marked effect on ER{alpha}, hypophysectomy had a less marked, but significant (P < 0.05), inhibitory effect on ERß mRNA expression, which also was reversed by PRL treatment (Fig. 4BGo).



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  		Figure 4. Effect of PRL on ER{alpha} and ERß mRNA expression in rat corpora lutea. Pregnant rats were hypophysectomized on day 3 and treated either with PRL (+) or vehicle (-) for 4 days, as described in Materials and Methods. Intact rats, on day 7 of pregnancy, were used as controls. Corpora lutea were isolated, and total RNA was prepared and subjected to RT-PCR. Results were quantified by densitometry and corrected using S16. The autoradiograms from one representative experiment are shown for the ER{alpha} (A) and ERß (B) mRNA. Normalized mRNA levels are graphically represented in the right panel as the mean ± SEM (n = 3). Columns with the same letter differ significantly (a and b, P < 0.01; one-way ANOVA, followed by Duncan’s multiple-range test).

 
ER{alpha} and ERß mRNA species in cultured luteinized granulosa cells: regulation by PRL and placental lactogens
To examine further the regulation of the ER mRNA expression by PRL, we used highly luteinized granulosa cells in culture. These cells express both the short and the long forms of the PRL receptor (data not shown). Because, in the pregnant rat, PRL ceases to be secreted at midpregnancy, whereas the placenta produces sequentially the PRL-like hormones rPL-I and rPL-II (16), we also examined the effect of both these hormones on ER mRNA expression. Results indicated that within 12 h of culture, PRL, rPL-I, and rPL-II stimulated both ER{alpha} (Fig. 5Go, A and C) and ERß (Fig. 5Go, B, and C) mRNA expression in a dose-dependent manner.



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  		Figure 5. Effect of PRL and placental lactogens on ER{alpha} and ERß mRNA expression in luteinized granulosa cells. Granulosa cells were isolated from preovulatory follicles and cultured in DMEM/F12 (1:1) containing 1% FBS. After a 72-h incubation, the medium was changed, and cells were incubated for an additional 12 h in the presence of different doses of oPRL (PRL), rPL-I, or rPL-II in the absence of serum. 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 S16 mRNA internal control. The autoradiograms from one experiment are shown for the ER{alpha} (A) and ERß (B) mRNA. Panel C depicts the densitometric analysis from three independent experiments (mean ± SEM of values expressed as percentage of the control, which was considered 100%). a, P < 0.01, compared with vehicle-treated controls (one-way ANOVA, followed by Duncan’s multiple-range test).

 
ER expression in the GG-CL luteal cell line: effect of PRL
To develop an experimental model to further study the hormonal regulation of ER expression in luteal cells, we investigated the expression of ER in a rat luteal cell line termed GG-CL, derived from the large luteal cells (35). Total RNA was extracted from GG-CL cells and corpora lutea and was subjected to RT-PCR analysis by using specific primers for ER{alpha} and ERß. As shown in Fig. 6Go, whereas both ER{alpha} and ERß mRNA were detected in the corpora lutea, only the mRNA encoding for ERß could be detected in the GG-CL cells. When we examined for ER immunoreactive isoforms in the corpus luteum and in the GG-CL cells (Fig. 7AGo), we detected both the 67-kDa and the 61-kDa proteins in the corpus luteum, but only the 61-kDa form in the GG-CL cells, which was localized in the nuclear cell fraction. No signal could be detected when the antiserum was absorbed with the antigen peptide used to develop the antibody (Fig. 7BGo).



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  		Figure 6. Expression of ER{alpha} and ERß mRNA in the GG-CL cell line. Total RNA was isolated from GG-CL cells, reverse transcribed into single-stranded cDNA, and amplified with specific oligonucleotide primers for ER{alpha} (A) and ERß (B) mRNA, 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 standard. Corpora lutea of day 15 pregnant rats (CL) were used as positive controls in A and B, respectively. The data are representative of three different experiments.

 


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  		Figure 7. ER immunoreactive proteins in the GG-CL cells. A, Total protein extracts were obtained from a pool of pregnant rat corpora lutea (CL) and from cultured GG-CL cells; B, protein extracts were obtained from cultured GG-CL cells; T, total extract; N, nuclear extract; C, cytosolic extract. Total extract from uteri (Ut), taken from cycling rats, was used as control. Peptide +, ER probed with the primary antibody saturated with antigen peptide. Equal amounts of proteins (50 µg) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using the polyclonal antibody ER-715, as described in Materials and Methods. Depicted are autoradiographs from a representative experiment. Positions of the molecular weight markers (Mr) are shown on the left.

 
To study whether PRL is able to regulate ERß mRNA expression in the absence of ER{alpha} mRNA, the luteal cell line (GG-CL) that apparently expresses ERß mRNA (but not ER{alpha} mRNA) was used. However, because this cell line does not express the PRL receptor, we stably transfected these cells with the long form of the PRL receptor (36). Culture of the transfected GG-CL PRL-RL cells in the presence of PRL resulted in a dose-related up-regulation of the ERß mRNA expression within 8 h of incubation (Fig. 8Go). The increase in the ERß mRNA levels induced by PRL treatment was accompanied by a significant increase (P < 0.05) in the expression of the 61-kDa immunoreactive protein detected by immunoblot (Fig. 9Go).



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  		Figure 8. Effect of PRL on ERß mRNA expression in the luteal cell line. GG-CL cells, stably transfected with the long form of the PRL receptor, were incubated in the presence of different doses of oPRL (PRL) for 8 h. Total RNA was isolated 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 S16 mRNA internal control. The autoradiogram from one representative experiment is shown in panel A. The densitometric analysis from three independent experiments (mean ± SEM of values expressed as percentage of the control, which was considered 100%) is depicted in panel B. a, P < 0.01, compared with vehicle-treated controls (one-way ANOVA, followed by Duncan’s multiple-range test).

 


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  		Figure 9. Effect of PRL on the ER immunoreactive protein in the luteal cell line. GG-CL cells, stably transfected with the long form of the PRL receptor, were treated with 0.1 µg/ml oPRL (+) or vehicle (-) for 8 h. Total extract was obtained, and equal amounts of proteins (50 µg) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using the polyclonal antiserum ER-715, as described in Materials and Methods. The left panel shows a representative autoradiograph. The right panel depicts the densitometric analysis from three independent experiments (mean ± SEM). a, P < 0.05, compared with vehicle-treated controls (Student’s t test).

 
Once we established the expression of ERß mRNA and its regulation by PRL in the luteal cell line, we examined by EMSA the DNA binding activity of the ER immunoreactive protein found in these cells. A double-stranded DNA probe, containing the ERE reported in the vitellogenin A2 promoter (32, 33), was subjected to a binding experiment with the nuclear extract obtained from cells treated with either estradiol alone or estradiol plus PRL. Results shown in Fig. 10Go indicated the formation of several shifted complexes that are specific, given that the interaction disappeared when an excess of the unlabeled probe was added to the reaction mixture. Interestingly, the nuclear extract, obtained from cells treated with estradiol plus PRL, showed an increased interaction with the DNA, when compared with that exhibited by the cells treated with estradiol alone.



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  		Figure 10. Effect of PRL on ERE-binding activity in GG-CL cells. A, Sequence of the coding strand of the ERE located in the vitellogenin A2 gene used in the EMSA. Underlined are the palindromic EREs. B, EMSA were performed using a double-stranded oligomer containing the sequence reported in panel A, and nuclear extracts from cells stably transfected with the long form of the PRL receptor and treated for 48 h with 17ß-estradiol (10 ng/ml) plus vehicle (E + Veh) or 17ß-estradiol plus oPRL (0.1 µg/ml) (E + PRL), as described in Materials and Methods. A 200-fold excess unlabeled probe was added to each extract to determine the specificity of the shifted complexes formed. The positions of the shifted complexes and the free probe are indicated. The autoradiograph is representative of three separate experiments.

 
Effect of estradiol on PKC-{delta} mRNA levels in the GG-CL cell line
Finally, to examine whether the GG-CL luteal-derived cells, which apparently express only ERß mRNA, can be a target of estradiol action, we studied the effect of different doses of 17ß-estradiol on the expression of an estrogen target gene. We used the PKC-{delta} isoenzyme as a target gene, because it was previously shown to be highly up-regulated by estradiol in the rat corpus luteum of pregnancy (37, 38) and because the GG-CL cells express this PKC isoform. Results depicted in Fig. 11Go indicate that estradiol induced a dose-dependent up-regulation of PKC-{delta} mRNA expression in the luteal-derived cells within 6 h of culture.



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  		Figure 11. Effect of estradiol on PKC-{delta} mRNA expression in the luteal cell line. GG-CL cells were incubated, in the presence of different doses of 17ß-estradiol, for 6 h. Total RNA was isolated 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 S16 mRNA internal standard. The autoradiogram from one experiment is shown in panel A. The densitometric analysis from three independent experiments (mean ± SEM of values expressed as percentage of the control, which was considered 100%) is depicted in panel B. a, P < 0.01, compared with vehicle-treated controls (one-way ANOVA, followed by Duncan’s multiple-range test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Synergism between intraluteal estradiol and pituitary PRL or PRL-like hormones from placenta is essential for the control of corpus luteum function in pregnant rats (11, 16). Because apparently unrelated luteal cell functions, such as steroidogenesis and cell hypertrophy, are controlled by the synergism between estradiol and PRL (11, 16), most probably, multiple mechanisms are involved in the cross-talk between these two luteotropins. We recently reported that two forms of the PRL receptor, the long and the short subtypes, are coexpressed in the rat corpora lutea of pregnancy (24). In the present investigation, we demonstrate that the two known ER species, ER{alpha} and ERß, are also expressed in the luteal tissue throughout pregnancy, whereas PRL and placental lactogens up-regulate their expression. Taken together, these data indicate that multiple signaling steps, triggered through different types of PRL receptor and ER, could be involved in the synergism between PRL and estradiol, to regulate luteal function, and that the up-regulation of ER by PRL is one such mechanism.

Ovarian estradiol regulates reproductive and nonreproductive tissue function, generally by modulating gene transcription through the ER (39). The ER-mediated gene transcription is initiated by binding of the DNA binding domain of the dimerized receptor to a consensus palindromic DNA sequence, the ERE (32). Until 1995, only one ER type was cloned (27), but the fact that disruption of the ER gene did not completely eliminate the ability of small ovarian follicles to grow, as evidenced by the presence of secondary and antral follicles in the knock-out mouse ovary (40, 41), suggested the possibility that the ovary expresses an estrogen-binding molecule(s) other than the classical ER. An important approach to answering such a question came from the cloning of a novel ER cDNA from rat prostate (15). This ER subtype was named ERß to distinguish it from the previously cloned ER, now called ER{alpha}. A recent study revealed that ER{alpha} and ERß are both expressed in the rat ovarian follicles and that ERß is the more abundant subtype in this ovarian tissue (34). In the present study, we report that both ER subtypes are expressed also in the corpus luteum of pregnancy, but displaying a different pattern of expression depending on the day of pregnancy studied. The pattern of ER{alpha} expression in the corpora lutea of pregnancy follows that of the serum progesterone levels and corpora lutea growth (11, 16), i.e. low at the beginning of pregnancy, with a sharp increase at midpregnancy and a decline at the time of parturition. In contrast, the ERß expression does not seem to change substantially throughout pregnancy, displaying only a slight decline at the end of the gestational period.

It is difficult to determine how estradiol signals in a tissue coexpressing both receptor subtypes. No significant differences in the affinity for the ligand have been measured between the two ER subtypes (42). Two recent and independent studies demonstrated that homodimers ER{alpha}/ER{alpha} and ERß/ERß, or heterodimers ER{alpha}/ERß, can be formed in vitro, can bind to the EREs in the DNA, and can stimulate the transcription of a reporter gene (43, 44). The DNA binding affinity of the heterodimer is similar to that of the ER{alpha} homodimer but greater than that of the ERß homodimer (44). Therefore, the relative expression of ER{alpha} and ERß in the corpus luteum and the difference in DNA binding activity between heterodimers and homodimers could be a mechanism for differential responsiveness of the luteal tissue to estrogens at different stages of pregnancy.

Two ER immunoreactive proteins, of 67-kDa and 61-kDa, are detected in the corpus luteum of pregnancy by using the polyclonal antibody ER-715, which was raised against a peptide corresponding to a 15-amino-acid sequence lying in the hinge region of the rat ER{alpha} (27, 31). The high-molecular-mass 67-kDa isoform is the expected full-length ER{alpha} protein demonstrated in several reports by using polyclonal and monoclonal antibodies (31, 45, 46, 47). Accordingly, the pattern of the 67-kDa protein expression in the corpora lutea during pregnancy mirrors the ER{alpha} mRNA expression. In contrast, it is difficult to explain the identity of the 61-kDa ER immunoreactive protein recognized by the ER-715 antibody. Our observation that under the RT-PCR condition of the linear range of amplification, only ERß mRNA could be detected in the GG-CL cells, together with the fact that these cells express only the 61-kDa ER immunoreactive isoform, suggest that this protein could be ERß. Also, the size of this protein (61-kDa) corresponds to that reported for the ERß synthesized in an in vitro translation system (15). However, it is possible also that the 61-kDa isoform detected in the rat corpus luteum and in the luteal cell line GG-CL represents either an ER{alpha} degradation product, an ER{alpha} isoform caused by alternative splicing, or a protein that is not related to ER{alpha} but that cross-reacts with the ER-715 antibody. Therefore, because the expression of this 61-kDa ER immunoreactive isoform is up-regulated by PRL in the GG-CL cells, it will be of interest to reveal its identity.

Estrogen binding activity has been observed in the nucleus of the rat corpus luteum throughout pregnancy, with maximum levels at midpregnancy (19), and a PRL stimulation of such activity has been proposed (17, 18). PRL and GH also were shown to stimulate estradiol binding activity in the rat liver (48). In the present investigation, we demonstrated clearly that PRL and placental lactogens up-regulate the luteal estradiol receptors by stimulating both ER{alpha} and ERß mRNA expression. In the GG-CL cells that seem to express only ERß, we have shown that not only the specific ERß mRNA, but also the 61-kDa immunoreactive protein, were increased by PRL treatment. This increase in the levels of the protein could explain the increased interaction between nuclear extracts obtained from GG-CL cells and the labeled DNA probe containing the palindromic ERE. However, we cannot rule out the possibility that other factors, such as coactivators and corepressors, could be affected by PRL treatment and therefore modulate the interaction with the response element.

The luteotropic action of PRL and PRL-related hormones is mediated through two subtypes of the PRL receptor that bind PRL and placental lactogens (49, 50) and are classified as long or short, depending upon the length of the cytoplasmic domain (51, 52, 53). The expression of the two PRL receptor forms remains elevated in the corpora lutea during most of the period of pregnancy, with, however, a dramatic decline occurring before parturition (24). Interestingly, despite the existence of high levels of placental lactogen in the circulation before parturition (16), a drop in the luteal ER expression was observed. Most probably, as a consequence of the dramatic decline in the luteal content of PRL receptor mRNA and protein observed at the end of pregnancy (24), the corpora lutea become unresponsive to the circulating lactogen, which can no longer stimulate the expression of the ER genes. Results from this investigation indicate that PRL and placental lactogen stimulation of ER mRNA expression occurs through the long form of the PRL receptor, because the GG-CL cells used express only the PRL receptor long-form mRNA (36). In the rat corpus luteum, PRL can signal through the Janus tyrosine kinase JAK2 and members of the Stat5 family of transcription factors, Stat5a and Stat5b (54, 55, 56). Whether the Stat5 family of proteins is essential for the PRL-mediated stimulation of ER expression in the corpus luteum remains to be investigated.

The fact that 17ß-estradiol is able to stimulate the mRNA levels of an estrogen target gene in the GG-CL cells expressing ERß mRNA, and apparently lacking ER{alpha} mRNA, suggests a mechanism of action involving ERß in this luteal cell line. One classical estrogenic effect reported in several estrogen target tissues is the increase in the expression of the progesterone receptor-gene (57). However, the rat corpus luteum does not seem to express the progesterone receptor (58). Therefore, as an estrogen-target gene, we used the {delta} isoform of the PKC, because it was shown to be strongly up-regulated by estradiol in rabbit and rat corpus luteum (38, 59, 60). Although the role of PKC-{delta} in luteal function is still unknown, the increase in its expression before parturition suggests a possible participation of this kinase in the luteolytic process (38).

In conclusion, the results of this investigation have established that: 1) ER{alpha} and ERß mRNAs are differentially coexpressed in the rat corpus luteum during pregnancy; 2) PRL and placental lactogens up-regulate the expression of both ER mRNA species in luteal cells; and 3) the GG-CL luteal-derived cells, expressing ERß mRNA and apparently lacking ER{alpha} mRNA, respond to estrogen in functional studies. This luteal cell line could prove to be an important tool with which to study the regulation of ERß gene expression.


   Acknowledgments
 
We are grateful to Dr. M. C. Robertson for the purified rPL-I and the recombinant rPL-II; Dr. D. I. H. Linzer for the PRL receptor expression vector; the NIDDK and National Hormone and Pituitary Program (NIH) for the oPRL and the ER-715 antibody; R. Clepper for animal care; L. Alaniz-Avila for photography; and V. Rogala for the preparation of the manuscript. We especially wish to thank Dr. J. Ou for his technical assistance.


   Footnotes
 
1 This work was supported by NIH Grants HD-11119 (to G.G.), FIC 1F-05TW-05241 (to C.M.T.), and HD-30719 (to O.-K.P.-S.). Back

2 Present address: Faulkner Centre for Reproductive Medicine, 1153 Centre Street, Boston, Massachusetts 02130. Back

3 NIH Merit Awardee (HD-11119). Back

Received November 10, 1997.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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