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Messenger Ribonucleic Acid in Sheep Endometrium by Increasing Its Stability1
a Department of Animal Science and Center for Animal Biotechnology,
b Institute of Biosciences and Technology, and Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-2471
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) gene expression in endometrium in female mammals of all species examined. The purpose of this study was to determine directly whether estradiol up-regulates ER mRNA by increasing the stability of the message. Endometrial tissue was collected from ovariectomized ewes 18 h after the ewes were injected with 50 µg estradiol. Previous work indicated rapid accumulation of ER mRNA at this time. Estradiol increased uterine weights (to 157 ± 15%) as well as steady-state concentrations of ER (to 309 ± 37%), progesterone receptor (PR; to 165 ± 19%), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; to 374 ± 32%) mRNAs in endometrium, compared to control levels of 100%. The effects of estradiol on ER mRNA stability in endometrium were measured in explants cultured with the transcription inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole, as well as by labeling RNA in vivo with 4-thiouridine. Both assays indicated that estradiol enhanced ER mRNA stability (half-life increased from 9 h to 
24 h). The estradiol effect was specific, because the stabilities of PR, GAPDH, and c-fos mRNAs were unaffected by treatment. Thus, estradiol up-regulates steady-state concentrations of ER mRNA in endometrium by a novel posttranscriptional mechanism.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) and progesterone receptor (PR) [1, 2], are up-regulated by the preovulatory surge of estrogen in all species examined [3–6]. The sheep is a good animal model for studying these phenomena because it is similar to other farm and primate species in its profiles of plasma estrogen and progesterone concentrations, as well as its modulation of ER and PR gene expression during the estrous cycle [7]. In addition, the large yield of endometrium harvested from each ewe allows analyses of several estrogen-responsive mRNA species in individual animals, even when sampling across a time course. The ovariectomized ewe is our model for studying the effects of the proestrous surge of estrogen on the regulation of ER and PR gene expression in endometrium. Injection of a single 50-µg dose of estradiol produces physiological levels of hormone in the plasma and endometrium [8, 9]. In addition, it increases steady-state concentrations of ER and PR gene products (both mRNAs and proteins) in most uterine cell types at 24 h postinjection [8, 10]. Nuclear runoff assays showed that estradiol increases the transcription rate of the PR gene [8], as demonstrated in other model systems [11, 12]. In contrast, estradiol increased endometrial ER mRNA concentrations 500% within 24 h in the absence of any increase in the transcription rate of the ER gene [8]. This indicates that estradiol enhances steady-state concentrations of ER mRNA by a highly effective posttranscriptional mechanism.
Posttranscriptional regulation of mRNA stability is rapidly gaining recognition as a powerful and widespread mechanism for controlling gene expression. Modulation of mRNA stability is a quick and efficient mechanism for altering steady-state concentrations of mRNAs [13]. The best-characterized example of hormone-modulated message stability is the 30-fold stabilization of vitellogenin mRNA in frog and chicken liver by estradiol [14]. In mammalian tissues, the short half-lives of both ER mRNA and ER protein make ER gene expression exquisitely sensitive to posttranscriptional regulation [15, 16].
The purpose of this study was to directly determine the effects of estradiol on ER mRNA stability in endometrium. Tissue was collected during the time of rapid ER mRNA accumulation from ovariectomized ewes treated with estradiol [8]. Stability of ER mRNA was measured in endometrium from estradiol-treated and control ewes by two diverse methods, one in vitro and one in vivo. Messenger RNAs for ER, PR, glyceraldehyde phosphate dehydrogenase (GAPDH; a long-lived mRNA), and c-fos (a short-lived mRNA) were analyzed simultaneously to characterize estradiol effects on expression of a variety of estrogen-responsive genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crossbred Rambouillet ewes (Ovis aries) were ovariectomized during the breeding season after exhibiting estrous cycles of normal duration (16–18 days). In experiment I, ewes (n = 6 ewes per treatment group) were ovariectomized 4 wk prior to the beginning of treatments and were injected i.m. with 50 µg estradiol or vehicle (0.5 ml of charcoal-stripped corn oil; Eastman Kodak, Rochester, NY). At hysterectomy, 18 h later, whole uteri and dissected endometrium were weighed, and endometrium was cultured (Fig. 1). Three additional estradiol-treated ewes provided endometrium for a pilot study of transcription inhibitor effectiveness and liver for production of dual-labeled (3H- and thio-labeled) RNA in explant cultures.
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In experiment II, ewes were ovariectomized and fitted with indwelling uterine catheters [17] 2 wk prior to the initiation of treatments. Ewes were treated as in experiment I (n = 3 ewes per treatment group). In addition, 4 h prior to hemihysterectomy, 3 ml of 2.5 mM 4-thiouridine in PBS containing 1 mg/ml ovine serum albumin was infused into each uterine horn via the uterine catheters (Fig. 1). One uterine horn was collected by hemihysterectomy at 18 h post-estradiol (Time 0 h), and the rest of the uterus was collected 6 h later. Each surgical procedure lasted 20 min, with ewes awakening 5 min after each surgery. Endometrium was snap-frozen and stored at -80°C prior to RNA preparation and analysis. All animal procedures were approved by the Texas A&M University Laboratory Animal Care and Use Committee. Chemicals were of molecular biology grade and, unless otherwise indicated, were purchased from Sigma Chemical Co. (St. Louis, MO).
Explant Cultures
Finely minced endometrium (500 mg) from each ewe was cultured in 10 ml of phenol red-free Ham's F12-Dulbecco's Modified Eagle's medium at 37°C on a rocker platform under O2:N2:CO2 (50:45:5) [18]. To evaluate inhibition of transcription by 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) and actinomycin D, replicate cultures of endometrial explants from three ewes were cultured with DRB (75 µg/ml) or its vehicle (0.1% dimethylsulfoxide), or with actinomycin D (5 µg/ml) or its vehicle (0.25% ethanol). After 30 min of culture, medium was replaced with medium containing 5 mCi of [5,6-3H]uridine added (40 Ci/mmol; New England Nuclear; Boston, MA) for pulse-labeling of RNA in addition to the inhibitor treatments. Three hours later, medium was changed to "chase" medium, which contained 2.5 mM unlabeled uridine and cytidine in place of the [3H]uridine [19]. Explants were harvested 6 h after the addition of [3H]uridine, snap-frozen in liquid nitrogen, and stored at -80°C. Ten-microgram samples of total cellular RNA (TriPure reagent; Boehringer Mannheim, Indianapolis, IN) from each endometrial sample were used to purify poly(A)+ RNA [20] in duplicate. Briefly, poly(A)+ RNA bound to 25 µl of oligo(dT) cellulose in 1 ml buffer containing 0.5 M LiCl in 1.5-ml microfuge tubes. After washing five times in 1 ml 0.15 M LiCl buffer, poly(A)+ RNA was pooled from two elutions in 200 µl buffer lacking LiCl. Incorporation of [3H]uridine into poly(A)+ RNA was determined by scintillation counting 10 µl of poly(A)+ RNA preparations.
For analysis of mRNA stability in vitro, endometrial explants from each ewe in experiment I were cultured with DRB as described above. Estradiol (10-9 M) or vehicle (1 µl ethanol) was added to the medium of explant cultures from estradiol-treated and control ewes, respectively. At 0, 3, 6, 9, and 12 h of culture, samples of explants (100 mg) were harvested, snap-frozen in liquid nitrogen, and stored at -80°C until RNA analysis.
For use as trace RNA to calculate efficiencies of purification of thio-labeled RNA by affinity chromatography, RNA was dual-labeled with [3H]uridine and 4-thiouridine in explant cultures of sheep liver (0.4 g). Explants were cultured for 6 h in medium containing 0.5 mCi/ml [3H]uridine and 2.5 mM 4-thiouridine.
Preparation and Analysis of mRNAs
Total cellular RNA was purified from each endometrial explant sample with TriPure Reagent (Boehringer Mannheim). The RNA preparations were analyzed on replicate slot blots for hybridization to ovine ER, PR, GAPDH, and c-fos cRNA probes cloned in our lab. The cDNA sequences have GenBank accession numbers U30299, U30300, U94718, and U94719, respectively [8, 9]. The human 18S rRNA template was purchased from Ambion (Austin, TX). The ER cRNA probe was synthesized with T7 RNA polymerase (Maxiscript kit; Ambion) from plasmid poER8 linearized with EcoRI, as described previously [8]. PR, GAPDH and c-fos cRNA probes were generated similarly from their respective plasmids (poPR77A, poGAPDH-48, and poC-fos 6–8) after linearization with BamHI. Because PR and c-fos mRNAs are relatively low in abundance, the specific activities of PR and c-fos cRNA probes were increased by eliminating unlabeled UTP and doubling [32P]UTP in those in vitro transcription reactions. Unincorporated radionucleotides were removed on spin columns (Bio101, La Jolla, CA).
Use of slot blots for quantitation was justified by Northern blots that demonstrated that these homologous probes hybridized to specific RNA bands of appropriate sizes [8, 9]. Samples of 20 µg RNA, estimated by A260, were denatured and immobilized in slots [21] on nitrocellulose (Schleicher & Schuell, Keene, NH) in 4-µg aliquots (except for blots to be probed with c-fos cRNA, which were loaded with 8 µg). Replicate blots were hybridized with antisense cRNAs (described above). The very high stringency hybridization (55°C) and washing conditions (three times in 0.1-strength SSC [single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate] and 0.1% SDS for 20 min at 68°C; 1 µg/ml RNase A digestion for 10 min at 25°C; and 0.1-strength SSC and 0.1% SDS for 15 min at 55°C) were consistent with Northern blot analyses [8]. In experiment I, blots were exposed to Fuji (Tokyo, Japan) x-ray film for 30 min to 4 days, depending on the probe, to record hybridization signals in the gray (linear) range of film sensitivity. Hybridization signals on autoradiographs were quantified by densitometry using Adobe Photoshop (Mountain View, CA) scanning and Intelligent Quantifier (Bio Image, Ann Arbor, MI) quantitation software programs. In experiment II, hybridization signals were counted directly from blots with an InstantImager (Packard, Meriden, CT). Steady-state levels of mRNAs are reported from two independent analyses of endometrial samples from each ewe.
Purification of RNA Labeled with 4-Thiouridine
Total cellular RNA was prepared from 1-g samples of endometrium collected at hemihysterectomy and complete hysterectomy 6 h later. In duplicate, thio-labeled RNA was isolated at 4°C from 500 µg of total cellular RNA by affinity chromatography on Affigel 501 (Bio-Rad, Hercules, CA) [22]. Briefly, endometrial RNA, combined with 40 000 cpm of 3H- and thio-labeled liver RNA trace (70 µg), bound 0.5 ml of the organomercurial matrix in 1 ml of binding buffer (50 mM sodium acetate, pH 5.5, 0.05% SDS, 0.15 M NaCl, and 4 mM EDTA) for 3 h with gentle agitation. Samples were loaded in 1-ml columns and washed with 15 ml each of binding buffer and binding buffer containing 0.5 M NaCl. Thio-labeled RNA was eluted in binding buffer containing 10 mM dithiothreitol while 250-µl fractions were collected. Counts of 3H in 20-µl aliquots of fractions identified the peak fraction and were used to calculate the purification efficiency of each column. Slot blots were loaded with equal amounts of column-purified RNA, based on duplicate A260 measurements, and analyzed for ER and GAPDH mRNAs and 18S rRNA as described above.
Data Analyses
Quantitative data were analyzed by least-squares ANOVA using the General Linear Models procedures of the Statistical Analysis System [23]. Results from estradiol-treated and control groups were compared by orthogonal contrasts. Data presented are least-squares means and SEs for treatment groups. In the evaluation of transcription inhibitor effectiveness, values were normalized to control 3H-poly(A)+ RNA counts from vehicle-treated cultures to show percentage of transcription in control (untreated) cultures. In ER, PR, GAPDH, and c-fos mRNA analyses, hybridization signals were normalized to 18S rRNA hybridization signals to account for unequal RNA loading between samples. In steady-state comparisons, values were normalized to the average value of the control group to show estradiol-induced changes. In mRNA stability data, mRNA concentrations were normalized to the average Time 0 value (set at 100%) to illustrate the percentage mRNA remaining. Message concentrations in experiment II were also normalized to the efficiency of the affinity-chromatography procedure. Messenger RNA degradation was assumed to reflect first-order kinetics [24], so mRNA half-lives were estimated from linear plots of mRNA concentrations on a semi-log scale against time on a linear scale (not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estradiol treatment resulted in obvious changes in gross characteristics of the uteri and endometrium. It increased the size, redness, and tone of the uteri compared to these characteristics in vehicle-treated control ewes. Uterine weights of the ewes in experiment I were 57 ± 15% greater (p < 0.05) in estradiol-treated ewes compared to controls (41.5 ± 4.3 vs. 26.5 ± 4.3 g). Although estradiol appeared to increase the weight of dissected endometrium by 37%, the increase was not significant (6.1 ± 0.9 g vs. 4.5 ± 0.9 g). We conclude that the brief physiological estradiol treatment evoked obvious changes in gross uterine characteristics.
Estradiol Enhanced Steady-State Concentrations of Endometrial ER, PR, and GAPDH mRNAs
In addition to eliciting the gross changes described above, estradiol treatment altered the expression of genes in endometrium. In the ewes in experiment I, estradiol increased steady-state concentrations of ER and GAPDH mRNAs to 309 ± 37% and 374 ± 32%, respectively, of control values (p < 0.001, Fig. 2). PR mRNA levels also rose in response to estradiol, but to a lesser extent (to 165 ± 19% of control values, p < 0.05). Steady-state concentrations of c-fos mRNA also increased with estradiol treatment (to 193 ± 37% of control values, p < 0.08). Thus, a single injection of estradiol strongly up-regulated steady-state concentrations of ER and GAPDH mRNA, while PR and c-fos mRNA concentrations increased more moderately in endometrium.
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Effectiveness of Transcription Inhibitors in Endometrial Explant Cultures
The most common way to measure mRNA stability is to block transcription and follow the decay of the mRNA population of interest over time. To find an effective inhibitor of transcription in endometrial explant cultures, we tested 75 µg/ml DRB, which acts by inhibiting transcription elongation factor TFIIH, and 5 µg/ml actinomycin D, which intercalates the DNA template [25]. Incorporation of [3H]uridine into poly(A)+ RNA was compared between inhibitor-treated and control cultures. Surprisingly, actinomycin D, at a dose five times greater than one effective in inhibiting transcription in cultured cells [26], showed poor inhibition of transcription in the endometrial explant cultures (40%). However, DRB inhibited the majority of transcription (90%). Because of its successful inhibition of transcription in this test, DRB (75 µg/ml) was used in the endometrial explant cultures for the in vitro mRNA stability measurements.
Estradiol Specifically Increased ER mRNA Stability In Vitro
To determine the effects of estradiol on stability of ER mRNA, explant cultures from each ewe in experiment I were sampled at 3-h intervals over a 12-h time course following inhibition of transcription by DRB treatment. Total cellular RNA was prepared from the endometrial explant samples and analyzed for ER mRNA, GAPDH mRNA, and 18S rRNA concentrations on replicate slot blots. Figure 3 shows degradation of ER, PR, GAPDH, and c-fos mRNA concentrations over time relative to their initial concentrations (100%). ER mRNA declined rapidly in endometrial explant cultures from control animals, exhibiting a half-life of 9 h. Estradiol treatment enhanced the stability of ER mRNA at least 3-fold, to a half-life 24 h (p < 0.01).
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In contrast to effects on ER mRNA, estradiol did not affect degradation rates of GAPDH, PR, or c-fos mRNAs (Fig. 3). PR and GAPDH mRNA concentrations were relatively stable: both had mRNA half-lives of approximately 24 h. Surprisingly, c-fos mRNA appeared to be stable in the culture system. This anomalous stability of c-fos mRNA is reported to be an artifact of DRB treatment [26]. Because of it, we tested estradiol effects on ER mRNA stability in endometrium again, using a different approach: pulse-labeling with 4-thiouridine of endometrial RNA in vivo.
Estradiol Specifically Increased ER mRNA Stability In Vivo
In experiment II (Fig. 1), the 4-thiouridine treatment in utero was effective in labeling a large population of endometrial RNA. The thio-labeled RNA was reproducibly isolated by affinity chromatography, with consistent efficiencies of purification of the dual-labeled (3H- and thio-) liver RNA (62.3 ± 1.0%) added as trace. The high yield of endometrium from each ewe allowed for duplicate purification of thio-RNA by affinity chromatography from each endometrial sample. The yields of the thio-RNA preparations were sufficient for replicate slot-blot analyses to follow the fates of pulse-labeled ER and GAPDH mRNAs.
Relative to the initial time point, concentrations of pulse-labeled ER mRNA remaining 6 h later were dependent upon estradiol treatment (p < 0.02, Fig. 4). Without it, ER mRNA at 6 h decreased to 59 ± 11% of initial levels (half-life = 9 h). Estradiol treatment stabilized ER mRNA so that its concentrations were unchanged between the initial and 6-h sampling times. Estradiol treatment did not affect the stability of GAPDH mRNA, the concentrations of which were not different between the initial and 6-h time points in the pulse-labeled pools of RNA in either treatment group. Thus, the results of the in vivo pulse-labeling assays confirmed those from the in vitro transcription inhibitor assays: estradiol treatment specifically stabilized ER mRNA in endometrium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the molecular level, estradiol increased steady-state concentrations of ER and PR mRNA in endometrium, consistent with results from a complete time course study from 0 to 48 h posttreatment [8]. The magnitudes of the effects are comparable to increases in endometrial ER and PR gene expression during proestrus in ovary-intact female mammals [3, 4, 30]. Estradiol up-regulated steady-state concentrations of GAPDH mRNA in endometrium as strongly as it did ER mRNA. This is consistent with induction of the GAPDH gene by estrogen in the uterus and pituitary of the rat [31, 32]. The magnitude of estradiol up-regulation of c-fos mRNA concentrations appeared small, but this was probably due to collection of tissue at 18 h postinjection. Others have reported maximal increases in c-fos mRNA concentrations at 1–2 h after estrogen treatment [33]. However, the estradiol effect resembled up-regulation of the c-fos gene in endometrium subsequent to the preovulatory surge of estrogen in naturally cycling ewes [29].
To understand how estradiol increases steady-state ER mRNA concentrations while the transcriptional activity of the ER gene is unchanged [8], we investigated whether estradiol stabilized ER mRNA. Two RNA stability assays were used: one employed a transcription inhibitor drug in explant culture and the other pulse-labeled a pool of RNAs in vivo. The former technique allows extensive tissue sampling across time, while the latter avoids possible artifacts of explant culture and inhibitor drugs. The two mRNA stability assays generated strikingly similar results and indicated that endometrial ER mRNA was at least 3-fold more stable in estradiol-treated ewes. The magnitude of the estradiol effect is consistent with the 3-fold increase in steady-state concentrations of ER mRNA in endometrium. Additionally, the stabilities of PR, GAPDH, and c-fos mRNAs were not affected by estradiol, demonstrating the specificity of this estradiol action.
The mRNAs for ER and PR are similar to those of oncogenes in stability and structure: ER mRNA has a very short half-life in breast cancer cell lines [15], and both human ER and PR mRNAs carry 13 AUUUA sequences in their extensive 3' untranslated regions. Transfer of the 3' untranslated region of the human ER mRNA to heterologous messages decreased their stability [34]. The AUUUA motif has been identified as an instability element in oncogene mRNAs, including that of c-fos [26]. However, the degradation pathways of ER and c-fos mRNAs appear to be distinct because DRB treatment, which ablates the primary degradation pathway of c-fos mRNA, did not affect ER mRNA stability in vitro.
Our results indicate that estrogen acts via a posttranscriptional mechanism in mammalian tissue. Others have proposed similar mechanisms for estradiol-mediated up-regulation of ER mRNA in human breast cancer cells [35] and thyrotropin-releasing hormone receptor mRNA in rat pituitary cells [36]. It is likely that estradiol stabilizes mammalian mRNAs as it does vitellogenin mRNA in the liver of egg-laying animals—requiring specific sequences of the mRNA and the presence of ER [14]. This may be an ancient mechanism of gene regulation that cooperates with transcriptional effects of estrogen to tightly control gene expression. It appears that mammals utilize mRNA stabilization to up-regulate ER gene expression in endometrium, which prepares it for production of steroid-dependent secretions that are critical to survival of embryos in the earliest days of pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 409 862 3399; ning{at}cvm.tamu.edu
3 Current address: Department of Animal and Veterinary Science, 216 Agricultural Science Building, University of Idaho, Moscow, ID 83844-2330.
Accepted: September 1, 1998.
Received: April 15, 1998.
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and ß. Endocrinology 1997; 138:863–870.This article has been cited by other articles:
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