Differential distribution of ERalpha and ERbeta mRNA in intrauterine tissues of the pregnant rhesus monkey

Wen Xuan Wu, Xiao Hong Ma, Gordon C. S. Smith, and Peter W. Nathanielsz

Laboratory for Pregnancy and Newborn Research, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Two estrogen receptor (ER) isoforms, ERalpha and ERbeta , have been described. However, no information is available in any species regarding the comparison of ERalpha and ERbeta levels in pregnant intrauterine tissues. We investigated 1) distribution of ERalpha and ERbeta mRNA in myometrium, amnion, choriodecidua, and placenta; 2) their abundance in intrauterine tissues at term not in labor (NIL) and in spontaneous term labor (STL); and 3) immunolocalization of ERalpha and ERbeta in pregnant rhesus monkey myometrium. Myometrium, amnion, choriodecidua, and placenta were obtained at cesarean section from monkeys in STL at 156-166 days gestational age (GA) (n = 4) and from control monkeys NIL at 140-152 days GA (n = 4). RT-PCR was conducted to determine ERalpha and ERbeta and glyceraldehyde-3-phosphate dehydrogenase mRNA abundance in four intrauterine tissues of the pregnant rhesus monkey. The cloned ERbeta PCR fragment was subjected to sequence analysis. ERalpha and ERbeta were localized in the myometrium by immunohistochemistry. We demonstrated that 1) rhesus monkey ERbeta shares >97% identity with human ERbeta in the region sequenced; 2) both ERs were expressed in myometrium, amnion, and choriodecidua but not in placenta in the current study; 3) ERalpha and ERbeta were differentially distributed in myometrium and amnion; 4) ERalpha and ERbeta were immunolocalized in myometrial smooth cells and smooth muscle and endothelial cells of the myometrial blood vessels. The biological significance of these quantitative differences in ER subtypes merits further study.

estrogen receptors alpha  and beta ; myometrium; fetal membrane


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ESTROGEN INFLUENCES the growth, differentiation, and function of many target tissues, especially those in the female reproductive tract. Estrogens play an important role in preparing the myometrium for labor. Estrogens activate myometrial contraction associated proteins (41, 43), thereby increasing the ability of the uterus to contract, promoting the synchronization of the contractile units (5, 9, 22). Target tissue response to estrogen is determined by tissue-specific expression of the estrogen receptor (ER). We have demonstrated that increased ERalpha concentration in the pregnant sheep myometrium and endometrium is associated with the onset of spontaneous term labor (STL) (42) as well as glucocorticoid-induced premature labor (38).

ERalpha cDNA was cloned in 1986 from several species (10), and until recently there has been general acceptance that only a single ER gene existed. At the end of 1995, a second ERbeta was cloned from a rat prostate cDNA library (16), and, subsequently, the homologous human (21) and mouse (36) genes were cloned. The existence of two ERs presents the possibility that variation in the presence and relative extent of ERalpha or ERbeta receptor-specific mechanisms is involved in tissue-specific and physiological state-specific actions of estrogen.

In the rat, ERalpha and ERbeta mRNA differ in their tissue distribution and/or the relative concentration (17). However, no studies have compared levels of ERalpha and ERbeta in nonhuman primate or human tissues. Furthermore, no information is available in any species regarding ERbeta abundance in pregnant intrauterine tissues, which are important targets for estrogen. In particular, the presence of ER in human or nonhuman primate placenta and fetal membranes has been a subject of dispute (2, 6, 15, 31, 33). Using semiquantitative RT-PCR, we investigated 1) differential distribution of both ERalpha and ERbeta mRNA in myometrium, amnion, choriodecidua, and placenta; 2) change of ERalpha and ERbeta mRNA abundance in intrauterine tissues at term not in labor (NIL) and in STL; and 3) immunolocalization of ERalpha and ERbeta in pregnant rhesus monkey myometrium.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and tissue collection. Eight pregnant rhesus monkeys from the California Regional Primate Research Center, Davis, CA, bred only once and of known gestational age (GA) were studied. Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee. The Cornell facilities are approved by the American Association for the Accreditation of Laboratory Animal Care. The pregnant rhesus monkeys were instrumented at 119-136 days GA with electromyogram electrodes sewn in the myometrium (34). Myometrial activity was monitored continuously and evaluated as being either in the low-amplitude, infrequent-contracture mode or in the frequent short-duration labor type contraction mode (13). Myometrium, amnion, choriodecidua, and placenta were obtained at cesarean section from monkeys in STL at 156-166 days GA (n = 4) determined by the occurrence of a switch in myometrial activity from contractures to contractions and from control monkeys NIL at 140-152 days GA (n = 4). Various maternal organs (heart, bladder, kidney, liver, lung, pancreas, spleen, thymus, and thyroid) were also collected from one pregnant rhesus monkey. Collected tissues were frozen in liquid nitrogen for RNA extraction. All tissues were stored at -80°C. One portion of myometrium was frozen in liquid nitrogen-cooled isopentane for ERbeta immunolocalization. A second portion was fixed in 4% paraformaldehyde for ERalpha immunolocalization.

RNA preparation. Total RNA was prepared from individual tissues as previously described (42). Briefly, total RNA was isolated from frozen tissues in 4.2 M guanidinium thiocyanate solution. RNA was pelleted through a 5.7 M cesium chloride cushion. The RNA purity and recovery from each tissue was determined by ultraviolet spectrophotometry (260 and 280 nm). There were no differences in the yield of RNA per milligram of tissue between control and treatment groups or between monkeys in labor and monkeys not in labor. Purified RNA was resuspended in 1 mM EDTA. The poly(A)-enriched RNA was also extracted from multiple rhesus monkey maternal tissues using a Fast-track kit purchased from Invitrogen (San Diego, CA). Total RNA and poly(A)-enriched RNA were stored at -80°C.

RT-PCR. Reverse transcription (RT) was performed in a final volume of 100 µl containing 5 µg total RNA or 1 µg poly(A)-enriched RNA per sample, 5 mM MgCl2, 10 mM Tris · Cl (pH 8.3), 50 mM KCl, 1 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 5 units of RNAse inhibitor, 12.5 units of Moloney murine leukemia virus RT, and 2.5 µM of random hexamers. The RT mixture was incubated at 42°C for 60 min.

PCR primers were as follows: ERalpha forward primer 5'-ACTGCATCAGATCCAAGGGAACG-3'and ERalpha reverse primer 5'-GGCAGCTCTCATGTCTCCAGCAGA-3'were used to amplify an 825-bp fragment of the ERalpha cDNA corresponding to nucleotides 402-1227 in the human ERalpha cDNA (10). ERbeta forward primer 5'-AGCAGCTGCACTGTGCCGGCAAG-3'and ERbeta reverse primer 5'-CCTCTGCCGGGCTGCACTCGGA-3' were used to amplify an 839-bp fragment of the ERbeta cDNA corresponding to nucleotides 569-1408 bp of human ERbeta cDNA (21). The primers for ERbeta were designed from the regions with no homology with ERalpha cDNA sequence. Primers for ERalpha and ERbeta had similar melting temperatures. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as a normalization control gene) forward primer 5'-GGAAGGTGAAGGTCGGAGTCAACG-3'and GAPDH reverse primer 5'-TGGATGAC- CTTGGCCAGGGGTGC-3' were used to amplify a 556-bp fragment of GAPDH cDNA corresponding to nucleotides 1457-3981 bp of human GAPDH gene (8). This pair of primers was separated by five introns.

For each PCR sample, 50 µl of a PCR mixture, comprising 2 mM MgCl2, 1× PCR buffer, 1.25 units AmpliTaq DNA polymerase/50 µl, 0.125 µM of each downstream and upstream primer, 1 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), and 5 µl RT templates were used. The PCR reaction was performed in a DNA thermal cycle (Gene Amp PCR system 2400; Perkin Elmer-Cetus, Norwalk, CT). PCR was conducted at three cycle lengths that were within the linear range for each message. For ERalpha cDNA, each cycle consisted of 94°C for 1 min, 63°C for 1 min, and 72°C for 1.5 min. For ERbeta cDNA, each cycle consisted of 94°C for 1 min, 65°C for 1 min, and 72°C for 1.5 min. For GAPDH cDNA, each cycle consisted of 94°C for 1 min, 66°C for 1 min, and 72°C for 3 min. Amplification products were analyzed by electrophoresis in 1.2% agarose gel. The specificities of PCR products for ERalpha and ERbeta were further analyzed by nested PCR.

Semiquantitative RT-PCR measurement of ERalpha and ERbeta mRNA. In our previous study, expression of ERalpha mRNA was very low in pregnant rhesus monkey intrauterine tissues (39). ERbeta mRNA is not detectable by Northern blot analysis in the rhesus monkey myometrium using 5 µg poly(A) RNA (data not shown). In the present study, we chose semiquantitative RT-PCR. Because myometrium contained the highest concentration of ERbeta mRNA in the various intrauterine tissues studied, we first determined whether the detection of the ERbeta PCR product was linear in the myometrium. We chose three cycle numbers (25, 30, and 35 cycles) and determined the abundance of PCR product in six samples of myometrium collected from animals NIL and STL. Twenty-five cycles were insufficient to produce a readily detectable signal and 30 cycles gave barely visible signals when 5 µg of RT reaction mixture were used (Fig. 1). In contrast, at 35 cycles there were distinct signals for ERbeta in different samples. A similar approach was used to determine the linear cycle range for ERalpha and GAPDH. As a result of this preliminary validation, we chose 35 cycles for ERalpha and ERbeta and 30 cycles for GAPDH.


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Fig. 1.   Establishment of conditions for semiquantitative RT-PCR. Total RNA was isolated from myometrial tissues of pregnant rhesus monkeys and cDNA was produced using random hexamer and RT. RT reaction mixture (5 µg) was subjected to 25 (lanes 1-6), 30 (lanes 7-12), and 35 (lanes 13-18) cycles of PCR using specific primers to amplify an 839-bp product encoding the rhesus monkey estrogen receptor beta  (ERbeta ). M, molecular size markers.

Each sample underwent RT reaction in a total volume of 100 µl reaction mixture containing 5 µg total RNA, which was then quantified by ultraviolet spectrophotometry. Two and five micrograms of RT reaction mixture were used to further determine the linear range over a wide range of different samples. For ERalpha and ERbeta mRNA detection, 5 µg RT reaction mixture were used throughout the study, while 2 µg RT reaction mixture were used for GAPDH mRNA detection. Each set of PCR samples was analyzed on a single gel stained with ethidium bromide and quantified by scan densitometry.

Subcloning and sequencing of ERbeta cDNA. PCR products containing a single band were cloned directly in the pCR 2.1 vector using the TA cloning kit (Invitrogen, San Diego, CA) under the conditions recommended by the manufacturer. The plasmid DNA was purified using the Biorobot system (QIAGEN), and the sequencing was done by Taq cycle sequencing using DyeDeoxy terminators in an Applied Biosystems automated sequencer (ABI 377 DNA sequencer) at the core sequencing facility of Cornell University. The cloned fragments of ERbeta were sequenced from at least two different clones and from both the coding and noncoding strands. The DNASTAR program (DNASTAR, Madison, WI) was used for alignments.

Immunolocalization of ERalpha and ERbeta in the myometrium. Because there were no fetal membranes available for immunocytochemical analysis of ERs and placental ERs were too low to be detected by immunostaining, immunolocalization of ERs was only performed in the myometrium. Immunolocalization of ERalpha was performed on floating sections of the myometrium fixed with 4% paraformaldehyde and validated as described previously (38). The use of anti-human ERalpha monoclonal antibody (kindly provided by Dr. Edwards, Colorado University) in the present study was verified as described previously (38).

Frozen sections (4 µm) of myometrial tissue were immunostained for ERbeta using the avidin-biotin immunoperoxidase method. The antibody of ERbeta was purchased from Affinity Bioreagents (Golden, CO; catalogue number PA1-311) and validated for immunohistochemical analysis of ERbeta in various tissues (information provided by vendor). Briefly, frozen sections were thawed and fixed in 4% formaldehyde for 10 min at room temperature and then rinsed twice in 0.05 M TBS (Tris · HCl-buffered saline) for 5 min each. Unless otherwise specified, all slides were sequentially incubated for various times at room temperature with each of the following reagents: step 1) 3% (vol/vol) H2O2 in PBS for 30 min to block endogenous peroxidase activity; step 2) 25% (vol/vol) normal horse serum and 5% (wt/vol) BSA in 0.05 M Tris · Cl with 0.15 M NaCl, pH 7.6, for 1 h; step 3) rabbit polyclonal anti-ERbeta antibody (1:400 dilution) for 20 h at 4°C; step 4) biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) for 0.5 h; step 5) avidin-biotin complex (Vector): 40 µl of each in 5 ml 0.05 M Tris · Cl, pH 7.6, for 0.5 h; step 6) 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) for 10 min: 4 µg/10 ml 0.05 M Tris buffer (pH 7.6) to which was added 0.2 ml 3% H2O2. After each incubation, the slides were washed with TBS for 15 min except for step 2. The slides were then mounted in Permount with or without methyl green counterstaining. Specificity of immunostaining for ERbeta was confirmed by two approaches: 1) omission of the primary antibody and 2) incubation of the slides with normal rabbit serum instead of the primary antibody.

Statistical analysis. After normalization of the content of ERalpha and ERbeta mRNA to GAPDH in individual samples, ERalpha and ERbeta mRNA concentration in each set of PCR was expressed as a ratio of ERalpha and ERbeta to GAPDH mRNA. Differences between different groups (NIL vs. STL) or between different tissues were subjected to two-way ANOVA followed by multiple comparison using a Tukey-Kramer procedure. Data throughout are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of ERalpha mRNA in intrauterine tissues of the pregnant rhesus monkey. ERalpha mRNA was expressed in the myometrium, choriodecidua, and amnion by quantitative RT-PCR analysis; however, there was no detectable ERalpha mRNA in the placenta when 5 µg RT reaction mixture was used at 35 cycles. Nested PCR further confirmed the specificity of primary ERalpha PCR product (data not shown). Myometrium contained the highest concentration of ERalpha mRNA compared with amnion and choriodecidua (Fig. 2, P < 0.01). There was no significant change of the abundance of ERalpha mRNA in myometrium, amnion, and choriodecidua during spontaneous labor when compared with control monkeys which were not in labor (Fig. 2).


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Fig. 2.   RT-PCR analysis of estrogen receptor alpha  (ERalpha ) mRNA expression in myometrium (MYO; A: lanes 1-6), choriodecidua (CH; A: lanes 7-14), placenta (PLAC; C: lanes 1-8), amnion (AM; C: lanes 9-14) obtained from control rhesus monkeys not in labor (NIL; A: lanes 1-3, 7-10; C: lanes 1-4, 9-11) and monkeys in spontaneous term labor (STL; A: lanes 4-6, 11-14; C: lanes 5-8, 12-14). B and D: abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each corresponding lane. E: ERalpha mRNA was normalized by GAPDH mRNA in each lane and quantified by densitometry and expressed as the ratio of ERalpha to GAPDH mRNA from NIL rhesus monkey (MYO and AM: n = 3; CH and PLAC: n = 4) and STL rhesus monkey (MYO and AM: n = 3; CH and PLAC: n = 4). There were no labor-related changes in the ERalpha mRNA level between NIL and STL. Data are therefore presented as means ± SE after pooling NIL and STL. Abundance of ERalpha mRNA was highest in MYO (*P < 0.01) compared with CH and AM. ERalpha mRNA was not detected in the placenta in eight animals examined.

Expression of ERbeta mRNA in intrauterine tissues of the pregnant rhesus monkey. Quantitative RT-PCR analysis of ERbeta mRNA in intrauterine tissues of the pregnant rhesus monkey revealed that myometrium contained the highest concentration of ERbeta mRNA compared with the three other intrauterine tissues examined (Fig. 3, P < 0.05). Nested PCR further confirmed the specificity of primary ERbeta PCR product (data not shown). ERbeta mRNA abundance in amnion is significantly higher than choriodecidua. There were no significant differences in the ERbeta mRNA level during STL compared with control animals NIL (Fig. 3). As with ERalpha , there was no detectable signal for ERbeta mRNA in placental samples. The nucleotide sequence of the 839-bp ERbeta PCR fragments obtained from pregnant rhesus monkey myometrium displayed a 97% homology with the comparable region in human ERbeta cDNA reported by Mosselman et al. (21) (Fig. 4).


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Fig. 3.   RT-PCR analysis of ERbeta mRNA expression in myometrium (MYO; lanes 1-6), amnion (AM; lanes 7-12), and choriodecidua (CH; lanes 13-20) obtained from control rhesus monkeys NIL (lanes 1-3, 7-9, 13-16) and monkeys in STL (lanes 4-6, 10-12, 17-20). B: abundance of GAPDH mRNA in each corresponding lane. C: ERbeta mRNA was normalized by GAPDH mRNA in each lane and quantified by densitometry and expressed as the ratio of ERbeta to GAPDH mRNA from NIL rhesus monkey (MYO and AM: n = 3; CH and PLAC: n = 4) and STL rhesus monkey (MYO and AM: n = 3; CH and PLAC: n = 4). There were no labor-related changes in the ERbeta mRNA level between NIL and STL. Data are therefore presented as means ± SE after pooling NIL and STL. Abundance of ERbeta mRNA was highest in MYO (* P < 0.05) compared with CH and AM. ERbeta mRNA in AM is significantly higher than CH (+P < 0.05). There was no signal for ERbeta mRNA in placenta in eight animals examined (data not shown).



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Fig. 4.   Automated nucleotide sequencing and homology analysis between rhesus monkey ERbeta cDNA and human ERbeta cDNA from nucleotides 569-1408. A homology of 97% was found between the rhesus monkey ERbeta and human ERbeta cDNA. ERbeta sequence reported in this study has been deposited in the GenBank database (accession no. AF119229).

Comparison of ERalpha and ERbeta mRNA abundances in four intrauterine tissues. There was twofold higher concentration of ERalpha mRNA in the pregnant rhesus monkey myometrium than ERbeta mRNA (Fig. 5). In contrast, ERbeta mRNA in amnion is significantly higher than ERalpha mRNA, and there was no significant difference between ERalpha and ERbeta mRNA levels in choriodecidua (Fig. 5). There was no detectable signal for either ERalpha or ERbeta in placenta.


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Fig. 5.   A: representative gel analysis of relative abundance of ERbeta (lanes 1-6) and ERalpha (lanes 7-12) in myometrium and normalized by GAPDH mRNA (B) in each lane. C: comparison of ERalpha (solid bars) and ERbeta (open bars) mRNA in the myometrium (MYO, n = 6), amnion (AM, n = 6) and choriodecidua (CH, n = 8) of the pregnant rhesus monkey (mean ± SE). All the comparisons for ERalpha or ERbeta mRNA across tissues (MYO, AM, and CH) were analyzed in one PCR. In addition, all the comparisons between ERalpha and ERbeta within one tissue were analyzed in a single gel. * P < 0.01, MYO vs. AM and CH; ** and +P < 0.05, ERalpha vs. ERbeta mRNA.

Comparison of ERalpha and ERbeta mRNA distributions in multiple maternal tissues. The relative distributions of ERalpha and ERbeta mRNA in all the maternal tissues examined were very similar, i.e., both ER mRNAs are expressed in atrium, bladder, kidney, liver, lung, pancreas, and thyroid ventricle. The spleen and thymus showed no expression of either ERalpha or ERbeta mRNA. ERalpha mRNA abundance was, in general, higher than ERbeta mRNA in all tissues examined. The atrium and bladder contained less abundant ERbeta mRNA, whereas the bladder, ventricle, and myometrium displayed lower amounts of ERalpha mRNA compared with other tissues studied (Fig. 6). All the comparisons for ERalpha or ERbeta mRNA across tissues (myometrium, amnion, and choriodecidua) were analyzed in one PCR. In addition, all the comparisons between ERalpha and ERbeta within one tissue were analyzed in a single gel.


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Fig. 6.   Distribution of ERalpha (A), ERbeta (B), and GAPDH (C) mRNA in multiple nonreproductive maternal rhesus monkey tissues determined by RT-PCR and analyzed on the ethidium bromide stained gel.

Immunolocalization of ERalpha and ERbeta in the pregnant rhesus monkey myometrium. Positive immunostaining for ERalpha and ERbeta was associated with the myometrial cells in both longitudinal and cross sections (Fig. 7). Both endothelial and smooth muscle cells of blood vessels in the myometrium (Fig. 7) were immunopositive for ERalpha and ERbeta . All specific staining for ERalpha and ERbeta was restricted to cell nuclei.


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Fig. 7.   Immunolocalization of ERalpha (A-D) stained black with nickel-3,3'-diaminobenzidine tetrahydrochloride(DAB) and ERbeta (E-H) stained with DAB in pregnant rhesus monkey myometrium. ERalpha was immunolocalized in myometrial smooth muscle cells (A), endothelial cells (B, arrow), and smooth muscle cells (C, arrows) of blood vessels. D: immunostaining for ERalpha in myometrium was abolished when primary ERalpha antibody was replaced by normal mouse serum. ERbeta was immunolocalized in myometrial smooth muscle cells (E), endothelial cells (F, arrows), and smooth muscle cells (G, arrow) of blood vessels. H: immunostaining for ERbeta in myometrium was abolished when primary ERbeta antibody was replaced by normal rabbit serum. Both ERalpha and ERbeta were present in nuclei of myometrial cells, endothelial cells, and smooth muscle cells of blood vessels in myometrium. Magnification: ×250 (A, D, E, H); ×500 (B, C, F, G).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A pronounced and significant late gestation rise in maternal estrogen concentrations has been demonstrated in pregnant women (3), rhesus monkey (25), and baboons (37). Estrogen also increases immediately before delivery with a varying time course in many other species such as the rat (18) and sheep (4). Maternal administration of androgen results in premature delivery of live young in rhesus pregnancy (20). However, inhibition of aromatization of androgen to estrogen with 4-hydroxyandrostenedione prevents the induction of premature delivery by androgen (23). These studies demonstrate a central role of estrogen including the switch from myometrial contractures to contractions, cervical dilation, and rupture of the fetal membranes. To understand the overall role of estrogen and its receptor in late gestation, it is crucial to obtain information that is precisely documented in relation to the stage of gestation in late pregnancy and parturition. There currently are no human or nonhuman primate studies on intrauterine tissues that have evaluated changes in ER in relation to precise monitoring of the different types of myometrial activity associated with late gestation before and during labor.

Our study is the first to examine the distribution of ERalpha and ERbeta in four different intrauterine tissues in the same animal. Our data clearly demonstrate that both ERalpha and ERbeta are expressed in myometrium, choriodecidua, and amnion, but not placenta. The presence of ER in the primate or human placenta has been a subject of dispute (14). ER protein has been demonstrated by immunocytochemical localization in cultured human placental syncytiotrophoblasts (2). ER mRNA has also been demonstrated at very low levels in the human and nonhuman primate placenta by RNA protection assay and RT-PCR (6, 31). Finally ER has been demonstrated by classic radioligand binding techniques in human placenta extracts (15). However, the absence of ER in the rhesus monkey placenta in the present study is consistent with one recent report (33) that detected progesterone receptor but failed to detect ER in human placenta across different gestational ages using immunocytochemistry, RT-PCR, and receptor binding techniques. Our data also indicate that the absence of ER is a common feature shared by the nonhuman primate and human placenta, which is in contrast with the presence of ERalpha mRNA in the ovine placenta (42). It is unlikely that our failure to detect the ER in rhesus monkey placenta can be attributed to a low ER density, because PCR is sensitive enough to detect one copy of a gene. Alternatively, estrogen's action on the placenta may be mediated by a different unrecognized type of ER, such as a nonclassical membrane-bound receptor or may be exerted through a biophysical change in the cell membrane.

Our observation that ERs remained unchanged in myometrium during spontaneous term labor is consistent with a previous report based on evaluation of estrogen binding (24). Of interest, the current finding contrasts with our previous study of ERalpha in ovine uterine tissues, in which a dramatic increase of ERalpha mRNA is associated with STL (42) as well as glucocorticoid-induced premature labor (38). We and others have clearly demonstrated estradiol-upregulated ERalpha expression and progesterone-downregulated ERalpha expression in nonpregnant uterine tissues in sheep (43), mouse (1), rhesus monkey (26, 27) and human breast cancer cell lines (7). In addition, the observation that uterine ERs are increased by RU-486, which antagonizes progesterone in late gestation in the pregnant rhesus monkey, is another example of progesterone's suppressive effect on uterine ERalpha (11). These results suggest that the control of uterine ERalpha is dependent on the complex interplay of estrogen and progesterone. It is possible that the differences in the balance of maternal plasma estradiol and progesterone levels are responsible for the differential expression of ER in different species during pregnancy. In sheep, the sharply increased maternal plasma estradiol concentration (4) and decreased maternal plasma progesterone (35) that occur immediately before parturition may together be responsible for increased expression of uterine ER during labor. In nonhuman primates and pregnant women, however, the gradual increase of estrogen throughout late gestation and sustained progesterone in maternal plasma during labor constitute a very different hormone environment and may explain the species' differences in ER during labor.

Using RT-PCR, we were able to detect ERalpha and ERbeta mRNA in amnion and choriodecidua. ER was undetectable in the fetal membranes using immunocytochemistry (11) or binding techniques (29). In support of our findings, it should be noticed that ERalpha mRNA was observed in human fetal membrane analyzed by RNA protection assay (6). Our observations represent the first evidence that amnion of nonhuman primate is a target of estrogen. In both the pregnant nonhuman primate (40) and pregnant women (28), the amnion is the major source of prostaglandin (PG) production that is essential to promote parturition. The presence of ER in the amnion further supports a role for estrogen in the stimulation of PG production by amnion. We have previously demonstrated that both estrogen and progesterone are involved in the regulation of PGH synthase 2 in nonpregnant ovine uterine tissues (41). It is possible that the balance between estradiol and progesterone also plays a role in modulating amnion PG production in preparation for and completion of parturition in nonhuman primates and pregnant women.

Our current study represents the first report to evaluate abundance and distribution of the new ER subtype, ERbeta , in pregnant intrauterine tissues in any species. The distribution of ERalpha and ERbeta in intrauterine tissues, as well as the multiple maternal nonintrauterine tissues of rhesus monkey examined are similar, but they differ in their concentration of ERs. It has been shown that ERalpha and ERbeta signal in opposite ways when complexed with the natural hormone estradiol from an AP1 site (30). The ERalpha and 17beta -estradiol complex activated transcription, whereas the ERbeta and 17beta -estradiol complex inhibited transcription. These observations suggest that ERalpha and ERbeta may play different roles in gene regulation. The appearance of both ERalpha and ERbeta in the intrauterine tissues during pregnancy adds another level of complexity to the function of estrogen in promoting parturition.

The nucleotide sequence homology between rhesus monkey of our RT-PCR product containing 839 bp of rhesus monkey ERbeta was 97% identical to the comparable region of the human ERbeta , showing that our amplified cDNA was complementary to the human ERbeta cDNA at nucleotides 569-1408.

We observed that both ERalpha and ERbeta were present in myometrial and vascular smooth muscle cells as well as endothelial cells of the blood vessels in the myometrium, indicating that estrogen may influence myometrial remodeling and uterine blood flow during pregnancy not only through classical ERalpha but also through the newly described ERbeta . A number of previous studies consistently reported the presence of ERalpha in myometrial cells and blood vessels (12, 19, 32); however, the present study is the first to localize ERbeta in the pregnant myometrium in any species.

In conclusion, 1) rhesus monkey ERbeta shares >97% identity with human ERbeta in the region sequenced; 2) ERalpha and ERbeta were expressed in myometrium, amnion, and choriodecidua, but not in placenta, of the pregnant rhesus monkey (myometrium contained the highest concentrations for both ER subtypes); and 3) differential abundance of ERalpha and ERbeta was exhibited in the myometrium and amnion. The biological significance of these quantitative differences in ER subtypes merits further study.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Child Health and Human Development Grant HD-21350.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Dept. of Physiology, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853-6401 (E-mail: pwn1{at}cornell.edu).

Received 30 June 1999; accepted in final form 18 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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