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Endocrinology Vol. 142, No. 6 2230-2237
Copyright © 2001 by The Endocrine Society

ARTICLES

Gonadotropins Decrease Estrogen Receptor-ß Messenger Ribonucleic Acid Stability in Rat Granulosa Cells1

Caixia Guo, Lisa Savage, Kevin D. Sarge and Ok-Kyong Park-Sarge2

Department of Physiology (C.G., L.S., O.-K.P.-S.) and Department of Biochemistry (C.G., K.D.S.), University of Kentucky, Lexington, Kentucky 40536-0084

Address all correspondence and requests for reprints to: Dr. Ok-Kyong Park-Sarge, Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0084. E-mail: okps{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that the preovulatory LH surge down-regulates estrogen receptor-ß (ERß) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ERß mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ERß gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERß gene transcription. In contrast, when ERß mRNA levels were estimated in granulosa cells that were cultured in the presence of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ERß mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agents that mimic LH actions in granulosa cells, also showed similar effects. Thus, these results suggest that LH decreases ERß mRNA expression in the granulosa cells of preovulatory follicles, primarily by destabilizing the preexisting ERß mRNA. We next determined the decay rate of the ERß mRNA in granulosa cells that were cultured in the presence of DRB and additional hCG, FSK, or TPA for various time periods, by estimating ERß mRNA levels, using semiquantitative RT-PCR assays and subsequent linear regression analyses. The half-life of the ERß mRNA in the presence of vehicle was 17.87 ± 1.2 h (n = 4). hCG dramatically decreased the half-life of the ERß mRNA (4.85 ± 0.49 h, n = 4). Similarly, both FSK and TPA decreased the half-life of the ERß mRNA to 3.57 ± 0.31 h and 4.02 ± 0.13 h, respectively. We extended these findings by examining whether the LH-induced down-regulation of the ERß mRNA is cycloheximide-sensitive. When granulosa cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor, the inhibitory effects of hCG, FSK, and TPA on ERß mRNA levels were abolished. Similar results were obtained in the presence or absence of DRB, indicating that the hormone-induced destabilization of the ERß mRNA is coupled with translation processes. Taken together, our results demonstrate that LH decreases ERß mRNA expression, predominantly at the posttranscriptional level, in a cycloheximide-sensitive manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN exerts diverse physiological effects in whole-body physiology, including oogenesis, reproduction, and maintenance of the healthy bone and blood vessels (1). Many, if not all, of these effects are mediated by the nuclear estrogen receptors (ER{alpha} and ERß) that display cell- and promoter context-dependent transcriptional activities (1, 2). In the rat ovary, estrogen stimulates the proliferation of granulosa cells of preantral follicles (3); increases granulosa cell LH receptor levels (4, 5); enhances gap junction formation among granulosa cells (6), maintaining granulosa cell health (7, 8); and modulates granulosa (9) and luteal (10) steroidogenic capacity. These estrogen actions are presumed to be mediated by estrogen-binding activities observed in granulosa cells (11, 12), as well as luteal cells (13). Although both ER{alpha} and ERß are expressed in the ovary, their expression is cell-specific. ER{alpha} is expressed predominantly in theca cells (14), stromal cells (14, 15), and luteal cells (16) and, to much less degree, in granulosa cells (14, 15, 16). ERß is expressed predominantly in granulosa cells (12, 14, 16, 17, 18, 19). Conversely, in granulosa cells that seem to be the main target for estrogen actions, ERß is the predominant ER subtype, as we (17, 18) and others (12, 14, 16, 19) have demonstrated. Indeed, changes in ERß messenger RNA (mRNA) (17) and protein (12, 19) levels in granulosa cells, during follicular development, mirror the previously observed changes in estrogen binding activities (11, 12). Consistent with the potential modulatory role that ERß plays in aforementioned granulosa cell functions, ERß knockout female mice show compromised folliculogenesis and reduced fertility (20).

We have previously demonstrated that the preovulatory LH surge decreases ERß mRNA levels selectively in the granulosa cells of preovulatory follicles that express LH receptors (17) and, in turn, decrease ERß protein levels (12, 19). Similar observations have been made in other species, including monkeys (21). LH-induced decrease in ERß mRNA levels may be attributable to decreased synthesis and/or increased degradation of transcripts, because steady-state mRNA levels represent the net changes at the level of transcription and posttranscription (22). The preovulatory LH surge has been shown to induce the transcription of a variety of genes (23), including the progesterone receptor (PR) gene (19, 24, 25) in granulosa cells. The same preovulatory LH surge has also been shown to decrease the stability of several genes, including the LH receptor gene (26, 27). In this study, we have determined whether the down-regulation of ERß mRNA after the LH surge is attributable to changes at the level of transcription and/or mRNA stability. Our results demonstrate that the LH-induced down-regulation of ERß mRNA level predominantly reflects increased degradation of ERß mRNA in a cycloheximide-sensitive manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM/Hams-F12 and antibiotics for tissue culture were from Life Technologies, Inc. (Gaithersburg, MD). 5,6-Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), human CG (hCG), 2-O-tetradecanol-phorbol-13-acetate (TPA), forskolin (FSK), and all general reagents were from Sigma (St. Louis, MO). Radioisotopes were purchased from NEN Life Science Products (Boston, MA), and oligonucleotides were synthesized by Integrated DNA Technologies, INC (Coralville, IA).

Granulosa cell isolation and culture
All animal experiments were conducted with the approval of the University of Kentucky Institutional Animal Care and Use Committee. Immature female rats (21–23 days old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed in a photoperiod of 14-h light,10-h darkness, with light on at 0500 h. At 22 or 23 days of age, rats were injected sc with 10–15 IU PMSG (Sigma); and 40–44 h afterwards, granulosa cells were isolated using follicular puncture, essentially as described (24, 25, 28). Cells were plated in 4F medium [15 mM HEPES (pH 7.4), 50% DMEM, and 50% Ham’s F12 with bovine transferrin (5 µg/ml), human insulin (2 µg/ml), hydrocortisone (40ng/ml) and antibiotics] supplemented with 5% FBS (Life Technologies, Inc.) at a density of approximately 1 x 106 cells, in 60-mm dishes, and cultured in the humidified atmosphere of 5% CO2 at 37 C. Two hours later, cells were pretreated with DRB (65–200 µM) or vehicle [dimethylsulfoxide (DMSO)] for 30–60 min before addition of hormone [hCG (1 IU/ml), FSK (5 x 10-5 M), TPA (10-7 M), or vehicle (DMSO)] to ensure the blockade of transcription. The cells were treated for the indicated time periods. For cycloheximide experiments, cycloheximide (10 µg/ml) or vehicle was added to cells, 1 h before hormone treatments, to ensure the blockade of translation before and during hormone challenges [hCG (1 IU/ml), FSK (5 x 10-5 M), TPA (10-7 M), or vehicle (DMSO)] for 3 h. Upon termination of hormone treatments, cells were harvested for RNA extraction.

RT-PCR
Total RNA from granulosa cells was purified by homogenization in a guanidium thiocyanate solution and ultracentrifugation through a cesium-chloride gradient. RT-PCR analyses for ERß and PR transcripts were performed as described (17, 25), with modifications. Each PCR reaction was carefully monitored to run under the condition that PCR products reflect the amount of input RNA in a linear range. Total RNA (2 µg) was reverse-transcribed at 37 C in a 20-µl vol using random hexamer (250 ng) and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (10 U; New England Biolabs, Inc., Boston, MA). Complementary DNA (cDNA) samples (5 µl) were used for subsequent PCR amplification of ERß, PR cDNA using oligonucleotide primer pairs specific for the rat ERß mRNA (5'-AAAGCCAAGAGAAACGGTGGGC-3' and 5'-GCCAATCATGTGCACCAGTTCC-3', 203 bp) (29), PR mRNA (5'-CCCACAGGAGTTTGTCAAGCTC-3' and 5'-TAACTTCAGACATCATTTCCGG-3', 328 bp) (25), and for an internal control S16 mRNA (5'-TCCAAGGGTCCGCTGCAGTC-3' and 5'-CGTTCACCTTGATGAGCCCATT-3', 100 bp). A 25-µl mix, containing the primers (0.1 µM each), {alpha}-32P-deoxycycidine triphosphate (1 µCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) in 1x PCR buffer [10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin] were added to each cDNA sample and overlaid with light mineral oil. Amplification was carried out for 15, 20, 25, and 30 cycles, using an annealing temperature of 64 C, on a thermocycler (Perkin-Elmer Cetus, Norwalk, CT) in each RT-PCR assay. The samples were separated on an 8% nondenaturing polyacrylamide gel and exposed to a phosphoimager screen. The intensity of each band that was in the linear range of amplification was analyzed using a Phosphoimager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA). ERß and PR signals were normalized to those of the ribosomal protein S16 internal control.

Isolation of nuclei
The isolation of nuclei was performed according to a previously described protocol (30), with modification. Granulosa cells were treated with hCG (1 IU/ml) or vehicle for 3 h. Cells were washed three times with ice-cold Dulbecco’s PBS without calcium and magnesium [PBS(-)], collected by scraping in PBS(-), then centrifuged for 5 min at 500 x g at 4 C. The cell pellet was resuspended in 4 ml Nonidet P-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40]. Lysed cells were incubated on ice for 10 min and centrifuged for 5 min at 3500 rpm. The nuclear pellet was then resuspended in 1 ml Nonidet P-40 lysis buffer and centrifuged for 5 min. The final nuclear pellet was gently resuspended in 150 µl glycerol storage buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA (pH 8.0)], snap-frozen, and stored at -80 C until use.

Nuclear run-off assay
The nuclear run-off assay was performed as previously described, with modification (30). In brief, in vitro transcription was performed with nuclei (1–2 x 107) incubated for 30 min at 30 C with 100 µCi of {alpha}-32P-uridine 5'-triphosphate, ATP, cytidine 5'-triphosphate, GTP (0.5 mM), Tris-HCl (5 mM), MgCl2 (2.5 mM), KCl (150 mM), and RNasin ribonuclease inhibitor (20 U). After ribonuclease-free deoxyribonuclease-I (15 U) (Promega Corp., Madison, WI) and proteinase-K (0.3 mg/ml) treatment, the labeled transcripts were purified by phenol, phenol/chloroform (1:1) extraction, and precipitated twice with 100% ethanol in the presence of 2.5 M ammonium acetate. The radiolabeled RNAs were dissolved in 500 µl hybridization solution [50% formamide, 5 x SSC, 50 mM NaPO4 (pH 6.5), 1 x Denhardt’s solution, 20 µg/ml yeast transfer RNA, 20 µg/ml poly A]. Five micrograms each of ERß, vector PSP72, CHO-B [a cDNA of ribosomal protein S2 (31)] cDNAs were fixed onto nitrocellulose membranes as described by the manufacturer, using their Bio-Dot SF slot-blot apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). The filters were prehybridized at 42 C for at least 4 h before hybridization for 36 h. The hybridization reaction typically contained 6 x 106 cpm/ml labeled RNA. After hybridization, the filters were washed in 2 x SSC at 65 C twice, 1 h each time, then in 2 x SSC containing 10 µg/ml ribonuclease A at 37 C for 30 min. The final wash was carried out in 0.1 x SSC plus 0.1% SDS at 65 C, and the membranes were exposed to a phosphoimager screen.

Data analysis
The relative ERß mRNA level was expressed as a percentage of the control value. The half-life was calculated using t1/2 = 0.693/k (22), where k is the slope derived from the linear equation lnC = lnC0 - kt, where C is the relative level of ERß mRNA in granulosa cells after hormone treatment. Data were presented as mean ± SEM of measurements from at least three independent experiments. Statistical analysis of data was performed using Systat for Windows (Indiana University, Bloomington, IN) and ANOVA followed by Dunnett test. P values less than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously shown that LH rapidly decreases steady-state ERß mRNA levels in the granulosa cells of preovulatory follicles in vivo, as well as in granulosa cells cultured in vitro (17, 18). To gain insight into the mechanisms by which LH regulates ERß gene expression in granulosa cells, we first examined whether LH regulates the ERß gene at the level of transcription. The rate of ERß gene transcription in granulosa cells was determined in nuclear run-off assays. Nascent 32P-labeled RNA transcripts were prepared from nuclei of granulosa cells treated with hCG (1 IU/ml) or vehicle, for 3 h, and hybridized to ERß cDNA, CHO-B cDNA as an internal control, and nonspecific vector sequences. Hybridization signals of ERß cDNA were normalized to those of CHO-B cDNA, because a similar approach was successfully used for measuring the effect of estrogen on the transcription of the PRL gene (32). Shown in Fig. 1Go are a representative autoradiogram (upper panel) of three independent experiments and their quantitative results (mean ± SEM, as a percent of vehicle-treated control) (lower panel). Under our experimental condition, hCG minimally decreased in vivo transcription of the ERß mRNA, compared with vehicle control (93.8 ± 8% over control, n = 3), suggesting that ERß gene transcription in granulosa cells is minimally altered by LH.



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  		Figure 1. hCG minimally affects ERß gene expression at the transcriptional level. Granulosa cells from PMSG (10–15 IU, 40–44 h)-treated immature rats were cultured in the presence of hCG (1 IU/ml), FSK (50 µM), TPA (100 nM), or vehicle (DMSO) for 3 h. Nuclei of these cells were isolated and used for the in vitro synthesis of 32P-labeled nascent RNA for hybridization with ERß cDNA, CHO-B cDNA, or vector PSP72 (5 µg each). A representative autoradiogram (upper panel) and the quantitated data (mean ± SEM of vehicle control) of three independent experiments are shown.

 
As an independent approach, we also assessed the effect of an inhibitor of RNA synthesis, DRB (65 µM), on the ability of hCG to decrease ERß mRNA levels in granulosa cells. This concentration of DRB has been shown to effectively (90–95%) inhibit hnRNA/mRNA transcription while maintaining cell viability (33, 34). Thus, we reasoned that the changes in mRNA levels in the presence of DRB should reflect the changes at the level of mRNA stability. To minimize the input from newly synthesized ERß transcripts, DRB (65 µM) was included in the media 30–60 min before hormonal treatments. Granulosa cells were cultured in the presence of hCG (1 IU/ml), FSK (50 µM), TPA (100 nM), or vehicle for 3 h, and ERß mRNA levels were assessed by semiquantitative RT-PCR assays. As described in Materials and Methods, each PCR reaction was carefully monitored to run under the condition that PCR products reflect the amount of input RNA in a linear range. To minimize variation between assays, PCR amplification was also carried out for 15, 20, 25, and 30 cycles for individual samples, and the intensity of the band from the linear range of amplification in each RT-PCR assay (15–20 cycles for S16, 20–25 cycles for ERß, 25-30 cycles for PR) was used for normalization of ERß or PR signals over S16 internal control. In the absence of hormones, DRB itself inhibited ERß mRNA levels to approximately 70% of the value experienced in the absence of DRB (Fig. 2AGo, n = 3), suggesting that DRB may have effectively blocked basal transcription of the ERß gene in granulosa cells. Under this condition, the efficiency of hCG to significantly decrease ERß mRNA expression (Fig. 2AGo, n = 3) was comparable with that in the absence of DRB. FSK and TPA, both of which mimic LH actions (17, 23), also decreased ERß mRNA levels regardless of the presence or absence of DRB. The same samples were examined for the effect of DRB on LH-induced PR mRNA expression in granulosa cells, because the PR gene is regulated at the level of transcription by LH and FSK (24, 25). Results (Fig. 2BGo, n = 3) show that DRB effectively blocked the stimulatory effect of LH and FSK on PR mRNA levels. Interestingly, the stimulatory effect of TPA on PR mRNA expression in these granulosa cells was rather weak. Taken together, these results demonstrate that the effect of LH on ERß mRNA levels in granulosa cells is primarily attributable to posttranscriptional changes of ERß gene expression.



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  		Figure 2. hCG effectively decreases ERß mRNA levels in the presence of DRB, a transcription inhibitor. Granulosa cells were prepared as described in Fig. 1Go. Media was supplemented with vehicle (DMSO) or DRB (65 µM) 30–60 min before, and during, treatment with hCG (1 IU/ml), FSK (50 µM), TPA (100 nM), or vehicle for 3 h. ERß (A) and PR (B) mRNA levels were assessed by semiquantitative RT-PCR assays (see Materials and Methods for details) and normalized over the S16 internal control. The quantitated data (mean ± SEM) of three independent experiments are shown. *, Difference from the vehicle values (DMSO and DMSO plus DRB) at P < 0.005 by Systat for Windows (SPSS, Inc.) and ANOVA followed by Dunnett test, respectively.

 
We next determined the decay rate of the ERß mRNA in granulosa cells cultured in vitro. As mentioned above, DRB (65 µM) was included in the media 30–60 min before, and during, hormonal treatments. Granulosa cells were first cultured in the presence of hCG (1 IU/ml), FSK (50 µM), TPA (100 nM), or vehicle for 0–24 h for ERß mRNA measurement. Results (Fig. 3AGo, n = 4) show that there is a rapid decline in ERß mRNA levels below the 30% of the 0-h level during the first 12-h period of hormonal treatments. Interestingly, a much slower decline in ERß mRNA levels was seen in the vehicle-treated group. Indeed, only after the 24-h period of hormonal treatment, ERß mRNA levels declined to approximately 45% of the 0-h level. To estimate ERß mRNA half-life in a reliable manner, we cultured granulosa cells in the presence of DRB and treated them with hormones for 0–7 h for hormone-treated groups and for 0–24 h for the vehicle-treated control group (Fig. 3BGo, n = 4). All the hormonal agents (hCG, FSK, and TPA) that were used to mimic LH actions decreased ERß mRNA levels at a faster rate. Although no significant changes in ERß mRNA levels were observed within the 1st hour of hormone treatments, there was a significant drop in ERß mRNA levels during the subsequent 1–7 h of hormone treatments. The maximal change in ERß mRNA levels was achieved during the 1–3 h of hormone treatments since ERß mRNA levels already dropped to 49.2 ± 0.4% (hCG), 56.3 ± 6.8% (FSK), and 54.1 ± 3.6% (TPA) of the 0-h value (100%), at 3 h after hormone treatments. In contrast, ERß mRNA levels in the vehicle-treated group gradually declined, over the course of 24 h, with the estimated half-life of 17.87 ± 1.2 h. hCG (4.85 ± 0.49 h) or FSK (3.57 ± 0.31 h) or TPA (4.02 ± 0.13 h) significantly (P < 0.001) reduced ERß mRNA half-life.



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  		Figure 3. hCG decreases the stability of ERß mRNA in granulosa cells. Granulosa cells were cultured and treated with DRB as described in Fig. 2Go. After being cultured in the presence of hCG, FSK, TPA, or vehicle for the indicated times, granulosa cells were harvested, and ERß mRNA levels were assessed by semiquantitative RT-PCR assays. The ERß mRNA level at 0 h was assigned a relative value of 100%, and ERß mRNA levels at all other times are expressed as percentages of this value. Each data point represents the mean ± SEM of four independent experiments. A, Representative autoradiogram and quantitation data of RT-PCR assays for granulosa cells treated with hormones or vehicle for 0–24 h; B, representative autoradiogram of RT-PCR assays for granulosa cells treated with hormones for 0–7 h and with vehicle for 0–24 h, the linear regression plots of the quantitated ERß mRNA level on an ln (loge) scale, and the calculated half-life of the ERß mRNA in each experimental condition; *, statistical difference at P < 0.001 by Systat for Windows (SPSS, Inc.) and ANOVA followed by Dunnett test.

 
We then determined whether LH-induced down-regulation of ERß mRNA expression in granulosa cells requires ongoing protein synthesis, by examining the effect of cycloheximide, a protein synthesis inhibitor. To maximize the effect of cycloheximide, granulosa cells were cultured in the presence of cycloheximide (10 µg/ml) or vehicle for 1 h before addition of hCG (1 IU/ml), FSK (50 µM), TPA (100 nM), or vehicle. Granulosa cells were then cultured for an additional 3 h, and ERß mRNA levels were estimated by semiquantitative RT-PCR assays. As demonstrated above, hCG, FSK, and TPA all significantly decreased ERß mRNA expression, compared with vehicle treatment. Cycloheximide fully blocked this hormone-induced decrease in ERß mRNA levels (Fig. 4AGo, n = 3). Interestingly, the inhibitory effect of cycloheximide on the hormone-induced decrease in ERß mRNA levels was also observed in granulosa cells cultured in the presence of DRB (Fig. 4BGo, n = 3).



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  		Figure 4. Ongoing protein synthesis is required for LH to decrease the stability of ERß mRNA in rat granulosa cells. Granulosa cells were cultured in the presence of cycloheximide (10 µg/ml) or vehicle (ethanol) for 1 h before addition of hCG, FSK, TPA, or vehicle, to maximize the blockade of protein synthesis. Addition of vehicle (A) or DRB (DMSO, B) was achieved as described in Fig. 2Go. The amounts of ERß transcripts were estimated after 3 h of hormone treatments by RT-PCR. A representative autoradiogram (upper panel) and the quantitated data (mean ± SEM, lower panel) of three independent experiments were shown. *, Statistical significance at P < 0.01 by Systat for Windows (SPSS, Inc.) and ANOVA followed by Dunnett test.

 
In summary, our results demonstrated that LH down-regulates ERß mRNA expression, primarily at the posttranscription level, in a cycloheximide-sensitive manner.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The predominant ERß (12, 14, 16, 17, 18, 19) and scarce ER{alpha} (14, 15, 16, 17, 18) expression defines ovarian granulosa cells as one of a few estrogen target cells with an ERß homodimeric intracellular environment. Thus, ERß is likely responsible for the previously observed estrogen effects on rat granulosa cell proliferation (3), LH receptor expression (4, 5), gap junction formation (6), health maintenance (7, 8), and steroidogenic capacity (9, 10). Despite the lack of our understanding the exact molecular mechanisms by which estrogen modulates granulosa cell function, the estrogen-regulated granulosa cell function(s) must be controlled by ERß levels, as shown, by a positive relationship between the estrogen-responsiveness and ER content of a particular cell type (11, 19, 35). Thus, the previous reports demonstrating that the preovulatory LH surge down-regulates ERß mRNA expression selectively in the granulosa cells of preovulatory follicles (17, 18) and that estrogen binding activities also decrease in the granulosa cells of preovulatory follicles in response to hCG (12, 19) underscore the importance of the regulatory mechanisms controlling ERß mRNA content in granulosa cells. One such mechanism is provided by our results demonstrating that LH down-regulates ERß mRNA expression primarily at the posttranscription level in a cycloheximide-sensitive manner.

The most reliable and generally accepted method for direct measurement of transcription is the nuclear run-off assay that uses isolated nuclei and quantifies the elongations in vitro of nascent mRNA chains already initiated in vivo (36). When we used this approach, with the CHO-B gene as an internal control that has successfully been used to measure the transcription of the rat PRL gene (32), only a minor effect of hCG was observed on the transcription rate of the ERß gene. Consistent with this data, DRB, a transcription inhibitor-blocking transcription in general (33, 34), decreased basal ERß mRNA levels in the absence of hCG but did not significantly alter the ability of hCG to decrease ERß mRNA levels in cultured granulosa cells. Similarly, ERß mRNA levels decreased in the presence of FSK or TPA, which was used to mimic LH action in these cells, in a DRB-insensitive manner. The small difference in the ability of these hormonal agents to decrease ERß mRNA expression exists between vehicle- and DRB-treated cells, suggesting the possibility of a minor contribution from transcriptional control. It is possible that a heterogeneous population of granulosa cells, prepared from PMSG-primed immature rats, responds to hormones differently. Indeed, the activator of protein kinase A has been shown to stimulate the {alpha}-inhibin gene in the granulosa cells of small and antral follicles but inhibit the same gene in the granulosa cells of preovulatory follicles (37).

Though hCG marginally affects the transcription of the ERß gene, it clearly decreases the ERß mRNA levels in the presence of DRB. The concentration of DRB used for our experiments effectively minimized the input from on-going transcription. The inhibitory effect of DRB itself on ERß mRNA levels in our granulosa cell preparations (~70% of vehicle-treated cells) was consistent through the doses from 65–200 µM (the data obtained in the presence of 200 µM DRB not shown), the doses that have been shown fully blocking hnRNA/mRNA transcription in other system (33, 34). In addition, the stimulatory effect of LH and FSK on PR mRNA expression that is known to occur at the level of transcription (19, 24, 25) was also effectively blocked by DRB, as assessed from the same samples used for ERß mRNA measurements. Under these conditions, steady-state ERß mRNA levels, over the culture period, should represent the decay rate of the preexisting ERß mRNA, because this type of approach has successfully been used to monitor the stability of several other genes, including immunoglobulin heavy- and light-chain mRNA (34), epidermal growth factor receptor mRNA (38), and FSHß mRNA (39). The decay rate of the ERß mRNA estimated this way was much faster when granulosa cells were cultured in the presence of hCG, as opposed to vehicle, regardless of the presence or absence of DRB, strongly suggesting that the decline in steady-state ERß mRNA levels in the granulosa cells of preovulatory follicles after the LH surge is largely attributable to the increased degradation of the preexisting ERß mRNA. Interestingly, the stability of the closely related ER{alpha} mRNA is also regulated by hormones such as estrogen (40, 41, 42, 43, 44, 45) and a pharmacological agent, TPA (46).

Curiously, ERß mRNA levels decreased only to approximately 30% of the 0-h period level, regardless of whether the cells were treated with vehicle or hormones. This may be attributable to the technical sensitivity of RT-PCR assays, in which the extremely low ERß mRNA level may not be within the linear range of amplification. Alternatively, this may be attributable to the DRB’s general effect, leading to a decrease in protein synthesis necessary for ERß degradation (34). It is also possible that ERß mRNA in granulosa cells undergoing hormone-induced luteinization may respond to hormonal agents differently. When granulosa cells were cultured in the presence of vehicle for a longer culture period (>36 h), which is known to induce spontaneous luteinization, they maintained ERß mRNA levels at 30–40% of the 0-h level. Consistent with this result, ERß mRNA levels seem to increase significantly with spontaneous luteinization in vitro in human granulosa-luteal cells (47).

The stability of many mRNA species is mediated by the molecular interaction between the AU-rich cis-element (AURE) in the mRNA and cytosol trans-acting protein(s) (22). It is possible that an AUUUA motif, the most common AURE, conserved in the 3'-untranslated region (UTR) of the rat (29) and mouse (48) ERß mRNA, may mediate the LH-induced destabilization of the ERß mRNA. Alternatively, other regulatory element(s) within the coding sequence of mRNA and secondary RNA structure may be involved. Consistent with this possibility, human ER{alpha} mRNA seems to use a cooperation among several subfragments in its 3'-UTR, not AURE, to mediate mRNA destabilization (49, 50). Similarly, the open reading frame of LH receptor mRNA is responsible for the rapid LH receptor mRNA turnover in luteinizing granulosa cells induced by the preovulatory LH surge, although their 3'-UTR contains multiple copies of AUUUA motifs (27, 51). It remains to be determined whether predominant ERß (mRNA and protein) and scarce ER{alpha} (mRNA and protein) expression in the granulosa cells of preantral and antral follicles (12, 14, 15, 16, 17, 18, 19) is attributable to the stability of ER products. On one hand, it may take longer for ERß mRNA (~18 h of half-life, this study), compared with ER{alpha} mRNA (~3 h of half-life, 49, 50), to decay in granulosa cells. On the other hand, ERß protein may be more resistant to protease digestion in granulosa cells than is ER{alpha} protein, as shown in in vitro assays (52).

Although the molecular mechanisms underlying the ability of LH to destabilize the ERß mRNA await further studies, our data demonstrating the inhibitory effect of cycloheximide on LH-induced destabilization of the ERß mRNA indicate the requirement of ongoing protein synthesis during this process. Interestingly, the preovulatory LH surge also destabilizes the LH-receptor mRNA in a similar manner (27). The inhibitory effect of cycloheximide on the ability of LH to destabilize ERß mRNA may be attributable to the blockade of translation elongation of ERß transcripts themselves, as demonstrated, as autoregulatory mechanisms of ß-tubulin and histone mRNA stability (22). Alternatively, the synthesis of a new protein(s) may be required for LH to destabilize the ERß mRNA. Requirement of a new protein, vigilin, has been demonstrated for the estrogen-induced stabilization of the vitellogenin mRNA in Xenopus liver (53). The molecular interaction between LH-induced new protein(s) and the ERß mRNA may alter tertiary folding of the ERß mRNA that can be easily targeted by ribonucleases.

In summary, our results demonstrating the ability of LH to decrease ERß gene expression primarily at the level of mRNA stability provide a finely tuned action of the preovulatory surge to suppress the estrogen-mediated regulation of granulosa cell function. Further studies to identify and characterize the cis-element within the ERß mRNA and granulosa cell trans-acting factors that are involved in the LH-induced down-regulation of ERß mRNA should elucidate the biochemical and molecular mechanisms that govern the autocrine/paracrine action of estrogen in granulosa cells.


   Acknowledgments
 
We wish to thank Dr. Chemyong Ko for help with granulosa cell isolation and Dr. Carolyn Komar for help with statistical analyses.


   Footnotes
 
1 This work was supported by NIH Grants HD-30719 and HD-36879 (to O.-K.P.-.S.). Back

2 Receipient of NIH Research Career Development Award HD-01135. Back

Received September 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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