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Endocrinology, doi:10.1210/en.2007-1163
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Endocrinology Vol. 149, No. 4 1524-1533
Copyright © 2008 by The Endocrine Society

Effect of Estrogen on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid in Cultured Rat Granulosa Cells

Sadatomo Ikeda, Kazuto Nakamura, Kayoko Kogure, Yuki Omori, Soichi Yamashita, Kazuko Kubota, Tetsuya Mizutani, Kaoru Miyamoto and Takashi Minegishi

Department of Gynecology and Reproductive Medicine (S.I., K.N., K.K., Y.O., S.Y., K.K., T.Min.), Gunma University Graduate School of Medicine, Gunma 371-8511, Japan; Department of Biochemistry (T.Miz., K.M.), Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan; and CREST (T.Miz., K.M.), Japan Science and Technology, Japan

Address all correspondence and requests for reprints to: Kazuto Nakamura, Department of Gynecology and Reproductive Medicine, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan. E-mail: nkazuto{at}med.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen has been considered to enhance FSH actions in the ovary, including the induction of the LH receptor (LHR). In this study, we elucidated the mechanism underlying the effect of estrogen on the induction of LHR by FSH in rat granulosa cells. Estradiol clearly enhanced the FSH-induced LHR mRNA increase in a time- and dose-dependent manner, with a maximum increase of approximately 3.5-fold at 72 h, compared with the level of LHR mRNA solely induced by FSH. We then investigated whether the effect of estrogen on LHR mRNA was due to increased transcription and/or altered mRNA stability. A luciferase assay with the plasmid containing the LHR 5'-flanking region did not show that estradiol increased the promoter activity induced by FSH. In contrast, the decay curves for LHR mRNA showed a significant increase in half-life with FSH and estradiol, suggesting that the increased stability of LHR mRNA is at least responsible for the regulation of LHR mRNA by estrogen. Recently mevalonate kinase (Mvk) was identified as a trans-factor that binds to LHR mRNA and alters LHR mRNA stability in the ovary. We found that estradiol, with FSH, decreased Mvk mRNA levels in rat granulosa cell culture, resulting in up-regulation of LHR mRNA that was inversely correlated to Mvk mRNA expression. Furthermore, the augmentation of FSH-induced LHR expression in the presence of estrogen was erased with the overexpression of Mvk by transient transfection. Taken together, these data indicate that LHR mRNA is up-regulated due to increased stability when estrogen negatively controls Mvk.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LH-HUMAN CHORIONIC gonadotropin receptor (LHR) is a G protein-coupled receptor. Consistent with the topology of other G protein-coupled receptors (1, 2), LHR consists of three distinct domains: the N-terminal region, a serpentine region, and the C-terminal tail (3, 4, 5). It has been well recognized that LHR plays a pivotal role in the reproductive functions, including steroidogenesis and ovulation in the ovary (6, 7). In addition to pituitary gonadotropins (8), various locally produced growth factors have been reported to be involved in the modulation of LHR function (7, 9, 10, 11). We previously found that activin enhanced LHR induction with FSH by stabilizing LHR mRNA (12). However, the precise mechanism of this enhancement was not fully elucidated at that time. Recently the group of Menon and colleagues (13, 14) vigorously investigated the novel trans-factor binding to LHR mRNA and found that mevalonate kinase (Mvk) was involved in the regulation of LHR mRNA expression by altering LHR mRNA stability. Mvk was initially recognized as an enzyme that phosphorylated mevalonate in the pathway of cholesterol biosynthesis (15). Wang and Menon (14) showed that Mvk existed in the rat ovary and played a role in the down-regulation of LHR, which was induced by the administration of a pharmacological dose of human chorionic gonadotropin (hCG).

It was recognized that estrogen is made mainly in the ovary and that the ovary itself is one of the targets for estrogen action in a paracrine or autocrine manner (16, 17). Moreover, estrogen has been implicated in synergizing action with FSH for granulosa cell differentiation including aromatase and LHR induction (18). Because estrogen receptor (ER)-β has been cloned (19), a number of interesting findings have emerged. ERβ was found to predominate over ER{alpha} in the ovary (20, 21). With the advantage of using target disruption of a specific gene, granulosa cell differentiation induced by FSH was seen to be attenuated or delayed in ERβ knockout female mice (22), reconfirming that estrogen is indeed crucial for many aspects of the ovarian functions. Based on this accumulated knowledge, we are motivated to elucidate whether Mvk participates in the regulation of LHR mRNA augmented by estrogen in the presence of FSH.

In this report, we studied the regulation of LHR mRNA expression by FSH and estrogen in rat primary granulosa cell culture. Northern blot analysis and luciferase assay were performed to show that estrogen enhanced the level of LHR mRNA in the presence of FSH. Meanwhile, we also measured the Mvk mRNA expression level induced by FSH and estrogen. To ascertain whether Mvk is involved in the regulation of LHR mRNA expression, we transfected Mvk plasmid to granulosa cells. Finally, we reconfirmed that Mvk was actually regulated by estrogen in the ovaries of hypophysectomized rats in vivo. We found that the induction of LHR mRNA transcripts and the prevention of LHR mRNA decay was the mechanism by which Mvk mRNA was decreased by estrogen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and reagents
Rat FSH (I-8) was obtained from the National Hormone and Peptide Program (Torrance, CA). DMEM/Ham’s nutrient mixture F-12, diethylstilbestrol (DES), 17β-estradiol, (R,R)-cis-diethyltetrahydro-2,8-chrysenediol (R,R-THC), methyl-piperidino-pyrazole (MPP), and hCG were purchased from Sigma Chemical Co. (St. Louis, MO). Gentamicin sulfate and fungizone were purchased from Invitrogen Corp. (Carlsbad, CA). The RNA labeling kit and nucleic acid detection kit were purchased from Roche Diagnostics (Mannheim, Germany). Recombinant FSH (rec-FSH) was provided by Organon Japan (Osaka, Japan).

Animals
Immature female Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) and 6-wk-old hypophysectomized female rats (Charles River Laboratories Japan, Inc., Tokyo, Japan) were maintained according to National Institutes of Health guidelines for the Care and Use of Laboratory Animals and the policies of the Gunma University Animal Care and Use Committee. Animals were housed in a temperature- and light-controlled room (12-h light, 12-h dark cycle; lights on at 0600 h) with food and water provided ad libitum.

Granulosa cell culture
The granulosa cells were obtained from immature female Wistar rats injected with 2 mg DES in 0.2 ml sesame oil daily for 4 d. The ovaries were then excised, and the granulosa cells were released by puncturing the follicles with 26-gauge needles. Granulosa cells were washed and collected by brief centrifugation, and the cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in DMEM/Ham’s nutrient mixture F-12 supplemented with 20 mg/liter gentamicin sulfate, 500 µg/liter fungizone, and 1 g/liter BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2-95% air at 37 C.

RNA isolation and Northern blot analysis
Granulosa cells were cultured in 60-mm dishes containing 5 x 106 viable cells in 5 ml of medium, and the reagents were added to the medium after 24 h of cell culture. The granulosa cells were further incubated, and the cultures were stopped at the selected times as indicated by using Isogen (Nippon Gene, Toyama, Japan). The final RNA pellet was dissolved in diethylpyrocarbonate-treated H2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For the Northern blot analysis, 15 or 20 µg of RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne; Pall Corp., Pensacola, FL). Rat LHR cDNA was prepared as described previously and linearized with BglII (12). Digoxigenin-labeled LHR cRNA probes corresponding to bases 440-2560 were produced by in vitro transcription with T3 RNA polymerase and an RNA labeling kit (Roche Diagnostics). Digoxigenin-labeled probes of rat aromatase (1555–1973 bp), rat cytochrome P450 side-chain cleavage enzyme (P450scc; 562-1162 bp), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were obtained by the same method. In accordance with the standard protocol for the nucleic acid detection kit (Roche Diagnostics, Tokyo, Japan), the membranes were then exposed on Kodak XAR film (Eastman Kodak Co., Rochester, NY). Luminescence detection was quantified using the LKB 2202 UnitroScan laser densitometer (LKB Produkter AB, Bromma, Sweden), normalized against the corresponding amount of GAPDH mRNA for each sample, and expressed in relative densitometric units.

Luciferase reporter assay
Plasmid pGL3-basic is a luciferase vector lacking the eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). The PGL3-control contains a simian virus 40 promoter and an simian virus 40 enhancer inserted into the structure of pGL3-basic (Promega). The fragment of the 5'-flanking region from the –1389 to the –1 bp relative to the translational initiation site that included the transcriptional regulating region of the rat LHR (23) was generated from the genomic DNA as described previously (11). To evaluate promoter activity, these fragments were ligated to a luciferase reporter vector (pGL3-basic) and named LH-R(1389)-Luc. Using FuGENE 6 reagent (Roche Diagnostics), a total of 1 µg of plasmid DNA was transfected into the primary granulosa cell culture plates (2.5 x 105 cells per 0.5 ml of medium in a 20 mm dish). To assay the regulatory elements, the granulosa cells were cultured for 48 h in a hormone-free condition medium before transfection. Thirty-six hours after transfection, the cells were treated with hormones for 6 h. The cells were then harvested, and the luciferase activity was measured. The luciferase assay was performed using the dual-luciferase reporter system (Promega), in which the transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of renilla luciferase.

Transcription stability analysis
Rat granulosa cells were preincubated with FSH alone or with FSH and estradiol for 60 h. The medium was replaced with fresh medium without any hormones, and 5 µM actinomycin-D was added to arrest the new RNA synthesis. Cells were harvested at 0, 2.5, 5, 7.5, and 10 h after the addition of the transcriptional inhibitor for RNA extraction and Northern blot analysis.

Reverse transcription and quantitative PCR
Isolated RNAs (2 µg of each sample) from the granulosa cell cultures were treated with DNaseI (Invitrogen Corp., Carlsbad, CA) to eliminate residual genomic DNA. Subsequently these samples were reverse transcribed, using oligo(dT)12–18 primer, deoxynucleoside triphosphate mix, and SuperScript III reverse transcriptase (Invitrogen). Additionally, the samples were incubated with RNaseH to remove RNA, and the resulting cDNAs were then diluted to 100 µl with distilled water. Each quantitative PCR consisted of 5 µl cDNA template, 12.5 µl SYBR Green real-time PCR master mix (Toyobo Co., Osaka, Japan), and 0.4 µM forward and reverse primers in a final volume of 25 µl. The primer sequences were as follows. For the detection of rat GAPDH, the forward primer was 5'-GTCATCCCAGACCTGAACGGGAAG-3' and the reverse primer was 5'-CTTGATGTCATCATACTTGGCAGG-3'. For rat LHR, the primer sequences were 5'-ATATTCAAGAGATGCACTGTGCAG-3' (forward) and 5'-AAGCAGAGTGTCAATGGGAAATAG-3' (reverse). For rat Mvk, the primer sequences were 5'-CCGCAGAGCAATGGGAAAGTG-3' (forward) and 5'-CATCGCCTTGCTCAAGAAAGCC-3' (reverse). The reactions were carried out on an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after an initial 1 min of incubation at 95 C. The fold change in the expression of each gene was calculated using the standard curve method, with the GAPDH as an internal control.

Immunoprecipitation, SDS-PAGE, and Western blotting
After the granulosa cells were incubated hormone for 96 h, the cells were washed with 2 ml of cold PBS (pH 7.4). Then they were scraped into a small volume of the same buffer containing the following protease inhibitors: phenylmethylsulfonyl fluoride (8.5 µM) and leupeptin (85 µM). From this time on, all procedures were carried out on ice or 4 C, and (unless specified otherwise) all solutions contained the protease inhibitors listed above. The cells were collected by centrifugation (3000 x g, 10 min) and soulubilized with lysis buffer containing 0.5% Nonidet P-40, 200 mM NaCl, 20 mM HEPES, 1 mM EDTA (pH 7.4). The lysate (400 µg) was incubated for 12 h at 4 C with anti-Mvk antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) prebound to 40 µl of protein A/G agarose (Santa Cruz Biotechnology). After immunoprecipitation, the protein A/G agarose was washed repeatedly with lysis buffer, and the bound material was eluted with 40 µl sample buffer [2% (wt/vol) sodium dodecyl sulfate, 10% glycerol, 20 mM EGTA, 0.5 mg/ml bromophenol blue, and 62.5 mM Tris (pH 6.8)] containing reducing agents (5% β-mercaptoethanol and 50 mM dithiothreitol). The eluant was then resolved on sodium dodecyl sulfate gels and electrophoretically transferred to polyvinylidene difluoride membrane as described previously (24). After blocking, expression of the different proteins was determined with anti-Mvk antibody and horseradish peroxidase-conjugated donkey antigoat IgG, and the proteins were finally visualized using enhanced chemiluminescence (ECL Plus; GE healthcare UK Ltd., Little Chalfont, UK).

Construction of expression plasmid for rat Mvk and overexpression in rat granulosa cells
Rats were treated with hCG to down-regulate LHR mRNA and to maximize the expression of Mvk mRNA as described in the study by Wang et al. (14). Total RNA was extracted from the ovaries, and its concentration was determined as described above. The Mvk cDNA was generated by the RT-PCR method using SuperScript III reverse transcriptase and Taq polymerase (Sigma). The sequences of the oligonucleotide primers for PCR were as follows (the underlined bases were not part of the target gene sequence and represent unique restriction enzyme digestion sites for cloning into the mammalian expression vector pcDNA3.1+ (Invitrogen)]: sense primer 5'-CGGGGTACCTGGAGAAGACCGGGAGCTA-3' and antisense primer 5'-TGCGGGCCCCAGCCCCAGATACGTGGAA-3'. After reverse transcription from isolated RNA, PCR amplification (35 cycles) was performed in a final volume of 50 µl as described by the manufacturer using the GeneAmp PCR system 2400 programmable thermal cycler (PerkinElmer Corp., Norwalk, CT). The reaction conditions were as follows: preincubation at 94 C for 4 min, denaturation at 94 C for 1 min, annealing at 55 C for 1 min, extension at 72 C for 50 sec, and final extension at 72 C for 7 min. The PCR products were resolved on a 1% agarose gel stained with ethidium bromide. The gel containing the Mvk cDNA was then excised and eluted using the QIAquick gel extraction kit (QIAGEN GmbH, Hilden, Germany). The sequence of the rat Mvk cDNA was confirmed by sequence analysis using the 3100 genetic analyzer (Applied Biosystems). The sequence was cloned into the KpnI-ApaI site of the pcDNA3.1+ vector and named pcDNA3.1-Mvk.

The constructed vector (pcDNA3.1-Mvk) or the empty vector as a control was transiently transfected into the rat granulosa cells cultured for 24 h in a hormone-free condition using FuGENE 6 reagent as described by the manufacturer. The granulosa cells were incubated for 48 h after transfection and subsequently treated with FSH and estradiol. After another 48 h incubation after the hormone treatment, the cultures were then stopped, and RNAs were extracted as described above. Isolated RNAs were analyzed by Northern blot analysis for the detection of rat LHR mRNA expression.

Hypophysectomized rat experiments
Six-week-old hypophysectomized female rats were injected subcutaneously every day with 10 IU rec-FSH alone or 10 IU rec-FSH and 1 or 2 mg DES for 4 d. Each rat was killed, and the ovaries were removed and stored in liquid nitrogen immediately. Then the ovaries were minced and homogenized in ice-cold Isogen, and RNA was isolated from them following the manufacturer’s protocol. Fifteen µg of RNA for detecting LHR mRNA and 20 µg for detecting Mvk mRNA were separated and analyzed by Northern blot analysis as described above.

Data analysis
The data represent the means ± SE from at least three independent experiments. Comparisons between groups were performed by one-way ANOVA. The significance of the differences between the mean values of the control group and each treated group was determined by Duncan’s multiple-comparison test. A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The regulation of LHR mRNA expression by FSH and estradiol in rat granulosa cells
To investigate the effect of FSH and estradiol on LHR mRNA expression, we analyzed the LHR mRNA in the primary rat granulosa cell culture. Granulosa cells were cultured for 24 h after plating on a cell culture dish, and then the cells were treated with FSH or with FSH and estradiol. Figure 1Go represents the results of LHR mRNA expression for a 72 h incubation with FSH (30 ng/ml) alone or with FSH (30 ng/ml) and estradiol (0.1–1 nM). LHR mRNA was induced by FSH and was enhanced by concurrent treatment with estradiol. LHR mRNA expression induced by simultaneous treatment with FSH and 1 nM estradiol was about 3.5-fold greater than that induced by FSH alone, and all LHR mRNA transcripts were regulated in a coordinate fashion. However, LHR mRNAs were undetected in the granulosa cells incubated with estradiol alone or with none of the hormones.


Figure 1
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  		FIG. 1. Dose-related effect of estradiol on FSH-induced LHR mRNA of rat granulosa cells. A, Granulosa cells from DES-primed immature rats were cultured for 24 h and then in 30 ng/ml FSH with increasing concentrations of estradiol for 72 h. LHR mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of three experiments. B, Levels of LHR mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LHR mRNA with FSH alone was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as from two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graphs. *, Difference from the control (FSH alone) value, P < 0.01.

 
Figure 2Go shows the time dependence of the FSH and estradiol effect on LHR mRNA accumulation. Granulosa cells were cultured for 24–96 h in the presence of FSH (30 ng/ml) with or without estradiol (1 nM). Because we detected an LHR mRNA transcript pattern identical with that observed in our previous studies (25, 26, 27), we selected a major transcript of LHR mRNA (5.4 kb) to be analyzed by Northern blot. Basal LHR mRNA levels (treatment with FSH alone) were increased until 48 h and subsequently reached a plateau. On the other hand, the concurrent treatment resulted in the augmentation of FSH-induced LHR mRNA expression for all of the time points studied and remained higher at the 96-h incubation period.


Figure 2
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  		FIG. 2. Time course of estradiol effect on FSH-induced LHR mRNA. A, Granulosa cells from DES-primed immature rats were cultured for 24 h. These cells were then further incubated with FSH (30 ng/ml) alone or with FSH plus estradiol (1 nM). After various incubation times, total RNA was extracted and LHR mRNA levels were measured using Northern blot analysis. The Northern blot is representative of three experiments. B, Levels of LHR mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LHR mRNA with FSH alone at 24 h time point was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as from two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the control (FSH alone) value, P < 0.05.

 
We then examined the effect of estrogen inhibitor on LHR mRNA in the rat granulosa cells incubated with FSH and estradiol. R,R-THC is a pure antagonist through ERβ and a partial agonist through ER{alpha} (28). Inside the ovary, ER{alpha} is expressed in thecal cells and interstitial cells, whereas ERβ is thought to be mainly located in granulosa cells (29). Thus, we used R,R-THC as an estrogen inhibitor for rat granulosa cell culture. The granulosa cells were incubated in the presence of FSH (30 ng/ml) and R,R-THC at the indicated concentrations with estradiol (1 nM). Figure 3Go shows that the enhancement of the FSH-induced LHR mRNA expression with estradiol was antagonized by the addition of R,R-THC. This antagonistic effect by R,R-THC was not toxic against granulosa cells because a high dosage of estradiol (10 nM) could override the R,R-THC. To further confirm the estrogen effect on granulosa cells through ERβ, we also used MPP as a specific ER{alpha} antagonist (28). MPP was added into the culture medium, indicating that ER{alpha} did not mediate the estrogen effect. Collectively, these results suggest that estradiol augmented LHR mRNA expression on rat granulosa cells through ERβ.


Figure 3
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  		FIG. 3. Effect of estradiol and estrogen inhibitors on FSH-induced LHR mRNA. A, Granulosa cells from DES-primed immature rats were cultured for 24 h. These cells were then further incubated with FSH and estradiol (1 nM), concurrently treated by R,R-THC and MPP at indicated concentration. After 72 h incubation, total RNA was extracted and LHR mRNA levels were measured using Northern blot analysis. The Northern blot is representative of three experiments. B, Levels of LHR mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LHR cultured with FSH and estradiol without estrogen inhibitors was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The values obtained from this experiment as well as from two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graphs. *, Difference from the control value, P < 0.01.

 
We then investigated whether the regulation of LHR mRNA by FSH and estradiol was dependent on gene transcription and/or receptor mRNA stability. In a previous experiment (23), the same region of the rat LHR gene was studied and well analyzed. Therefore, we selected the proximal 1389 bp of the LHR 5'-flanking region for this experiment. Rat granulosa cells transiently transfected with luciferase-reporting plasmids [LH-R(1389)-Luc] responded to FSH with significantly enhanced activity. However, treatment with estradiol alone did not influence the activity of the LHR promoter or affect the increased promoter activity induced by FSH (Fig. 4Go).


Figure 4
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  		FIG. 4. Effect of FSH and estradiol on LH-R-Luc expression in rat granulosa cells. Granulosa cells from DES-primed immature rats were cultured for 48 h in hormone-free condition and then cotransfected with LH-R(1389)-Luc and pRL-CMV-Rluc. Thirty-six hours after transfection, cells were treated with FSH (30 ng/ml) alone or with a combination of FSH and estradiol for 6 h, and then cells were lysed and assayed for luciferase activity. Luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. The luciferase activity of the sample treated with none of each hormone was taken as 100%. Each bar represents the mean ± SEM of the three independent experiments.

 
To assess the rate of degeneration of LHR mRNA transcripts, we preincubated the granulosa cells with FSH alone or with FSH and estradiol treatment for 60 h. After this preincubation, the medium was replaced with fresh medium without hormone, and 5 µM actinomycin-D was added to arrest the new RNA synthesis. Cells were harvested at 0, 2.5, 5, 7.5, and 10 h after the transcription inhibitor addition, and the LHR mRNA levels were quantitated by Northern blot analysis. The amount of LHR mRNA at time zero (the time of the addition of actinomycin-D) in each treatment group was assigned a value of 100%, and other values in each treatment group at different time points were expressed as a percentage of the time zero value. As shown in Fig. 5Go, the decay curves for the 5.4-kb LHR mRNA transcript in granulosa cells were significantly altered in the presence of estradiol and increased the half-life of the LHR mRNA transcript. However, Shi and Segaloff (30) demonstrated that actinomycin-D treatment was invalid for examining the mRNA stability because actinomycin-D arrested the transcription of the factor that can affect LHR mRNA stability. At present, it is not clear whether we could detect a significant difference by removing the hormone before the addition of actinomycin-D.


Figure 5
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  		FIG. 5. Effect of FSH and estradiol on LHR mRNA transcripts. A, Granulosa cells were preincubated with FSH alone or with FSH and estradiol for 60 h. Then the medium was replaced with fresh medium without hormones, and 5 µM actinomycin-D were added to arrest new RNA synthesis. Cells were harvested at 0, 2.5, 5, 7.5, and 10 h after addition of the transcription inhibitor, and LHR mRNA levels were quantitated by Northern blot analysis. B, Levels of LHR mRNA (5.4 kb) were quantified by densitometric scanning. The mRNA levels at time zero were assigned a relative value of 100%, and mRNA levels at all other times were expressed as percentages of the time zero value. The absorbance values obtained from this experiment as well as from two other experiments were standardized in relation to the control and are represented as mean ± SEM of the three independent experiments. *, Difference between values of estradiol-treated and control samples, P < 0.01.

 
It has been well acknowledged that estrogen also increases the FSH-induced expression levels of rat aromatase and cytochrome P450scc enzyme mRNA (7, 31). As shown in Fig. 6AGo, we reconfirmed the estrogen-stimulated, FSH-induced aromatase and P450scc mRNA expressions. Furthermore, the decay of both aromatase and P450scc mRNA was measured after preincubation of FSH with or without estradiol, suggesting that the stabilization of LHR mRNA by estradiol was not a general effect on gene expression regulated by estrogen (Fig. 6BGo).


Figure 6
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  		FIG. 6. Effect of estradiol on FSH-induced LHR mRNA, aromatase mRNA, and P450scc mRNA of rat granulosa cells. A, Granulosa cells from DES-primed immature rats were cultured for 24 h and further incubated in 30 ng/ml FSH with increasing concentrations of estradiol for 72 h. After Northern blot analysis was performed, levels of LHR mRNA (5.4 kb), aromatase mRNA (3.3 kb), and P450scc mRNA (2.0 kb) were quantified by densitometric scanning. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to each group treated with FSH 30ng/ml alone. The absorbance values obtained from three independent experiments were represented as the mean ± SEM in the bar graphs. *, Difference from the sample (FSH alone) value, P < 0.01. B, Granulosa cells were preincubated with FSH alone or with FSH and estradiol for 60 h. Then the medium was replaced with fresh medium without hormones, and 5 µM actinomycin-D were added to arrest new RNA synthesis. Cells were harvested at 0, 2.5, 5, 7.5, and 10 h after addition to the transcription inhibitor. Levels of aromatase mRNA (3.3 kb) and P450scc mRNA (2.0 kb) were quantified by densitometric scanning of Northern blot analysis. The mRNA levels at time zero were assigned a relative value of 100%, and mRNA levels at all other times were expressed as percentage of the time zero value. The absorbance value obtained from three independent experiments was represented as mean ± SEM in the bar graphs.

 
The interaction between LHR mRNA and Mvk mRNA
Our experiments showed that FSH-induced LHR mRNA expression of rat granulosa cells was enhanced by estradiol and increased the mRNA stability. It has been demonstrated that the regulation of mRNA turnover is influenced by the interaction of various cytoplasmic proteins (trans-factors) with regulatory regions (cis-elements) in mRNA that form a ribonucleoprotein complex (32). Nair and Menon (13) isolated Mvk from rat ovary and revealed that Mvk binds to LHR mRNA and leads it to degradation. Mvk was first cloned from rat liver as one of the enzymes involved in cholesterol biosynthesis (15) and is also thought to be the trans-acting factor of LHR mRNA in the rat ovary.

As previously noted, the FSH-induced LHR mRNA expression of rat granulosa cells was enhanced by estradiol along with the mRNA stability. We then decided to examine whether estradiol regulated the Mvk mRNA level in the rat granulosa cells. To assess the Mvk mRNA expression, total RNA was isolated from granulosa cell cultures treated with either FSH alone or FSH and estradiol for subsequent quantification of both LHR and Mvk mRNA by real-time PCR. As shown in Fig. 7AGo, FSH-induced mRNA expression of rat granulosa cells was enhanced by the addition of estradiol, whereas Mvk mRNA was suppressed by the simultaneous addition of estradiol to FSH as expected. Furthermore, the Mvk protein level was coordinately diminished by this treatment (Fig. 7BGo). These data suggest that estradiol in the presence of FSH negatively regulated Mvk, resulting in an increase of the level of LHR mRNA in rat granulosa cells.


Figure 7
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  		FIG. 7. Effect of estradiol on FSH-induced LHR mRNA, Mvk mRNA, and protein expression of rat granulosa cells. Granulosa cells from DES-primed immature rats were cultured for 24 h and then in 30 ng/ml FSH with increasing concentrations of estradiol for 72 h. A, Total RNA was isolated and cDNA was prepared from each sample. Subsequently LHR mRNA and Mvk mRNA levels were determined by quantitative-PCR as described in Materials and Methods. The amount of LHR and Mvk mRNA with FSH alone were taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The values obtained from this experiment as well as from two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graphs. *, Difference from the control value (Mvk mRNA expression with FSH alone), P < 0.05. B, The cells were solubilized as described in Materials and Methods, immunoprecipitated with anti-Mvk antibody, resolved by 7.5% reducing SDS-PAGE, transferred to a polyvinylidene difluoride membrane, probed with anti-Mvk antibody, and visualized using ECL Plus. The blot is representative of three independent experiments.

 
We then designed the following experiments to study the negative effect of Mvk expression on LHR mRNA level by the transfection of Mvk cDNA in the granulosa cell culture. Figure 8AGo shows the Mvk mRNA expression of the granulosa cells transfected with Mvk or empty vector before hormone addition, clearly indicating that Mvk mRNA was evidently overexpressed by the transfection. As shown in Fig. 8Go, B and C, the augmentation of FSH-induced LHR mRNA expression induced by estradiol was suppressed in the pcDNA3.1-Mvk-transfected group, compared with the empty vector transfected. These studies suggest that estradiol enhances FSH-induced LHR mRNA expression on rat granulosa cells along with stabilizing mRNA through the regulation of local Mvk expression.


Figure 8
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  		FIG. 8. Change of estradiol effect on FSH-induced LHR mRNA in rat granulosa cells by overexpression of mevalonate kinase. Rat granulosa cells were cultured for 24 h and then transiently transfected by pcDNA3.1-Mvk vector (Mvk) or empty vector (Emp) as described in Materials and Methods. A, Mvk mRNA expression (2.0 kb) was detected by Northern blot analysis. Each group of the transfected granulosa cells was further incubated in hormone-free condition for 48 h after transfection. Total RNAs were then prepared and Northern blotting was performed. PC, Positive control for Mvk mRNA obtained from the rat ovary. B, Transfected granulosa cells were incubated for 48 h in hormone-free condition and subsequently treated with FSH (30 ng/ml) alone or FSH plus estradiol (1 nM). After another 48 h incubation after hormones addition, cultures were then stopped and RNAs were elucidated and analyzed by Northern blot analysis for the detection of rat LHR mRNA expression. The Northern blot is representative of three experiments. C, Levels of LHR mRNA (5.4 kb) was quantified by densitometric scanning. The amount of LHR mRNA from empty vector transfected cells incubated with FSH alone was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as from two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graphs. *, Difference from the control value (LHR mRNA expression in the empty vector transfected group), P < 0.01.

 
To address the question of whether estrogen modulates the Mvk mRNA expression level in vivo, we adapted hypophysectomized rats by the injection of rec-FSH alone or both rec-FSH and DES (Fig. 9Go). DES treatment clearly demonstrated an LHR mRNA level induced by rec-FSH, whereas Mvk mRNA was inversely decreased, as in the results obtained in granulosa cell culture. This result indicates the possibility that estrogen modulates Mvk expression, whereby LHR is consequently regulated inside the ovary.


Figure 9
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  		FIG. 9. Effect of estrogen on LHR and Mvk mRNA expression in hypophysectomized rat ovaries. Six-week-old hypophysectomized female rats were injected subcutaneously every day with 10 IU rec-FSH alone or 10 IU rec-FSH and 1 or 2 mg DES for 4 d. Each rat was killed, and ovaries were removed and immediately stored in liquid nitrogen. Later total RNA was prepared for Northern blot analysis. A, Luminescence images of Northern blot of LHR mRNA, Mvk mRNA, and GAPDH mRNA. From each time point, 15 µg of total RNA for detecting LHR mRNA or 20 µg of total RNA for detecting Mvk mRNA was fractionated through denaturing agarose gels and blotted as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B, Levels of LHR mRNA (5.4 kb) and Mvk mRNA (2.0 kb) were quantified by densitometric scanning. The amount of LHR and Mvk mRNA treated by rec-FSH alone was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control (rec-FSH alone). The absorbance values obtained from this experiment as well as from two other experiments were represented of the mean ± SEM of three independent experiments in the bar graphs. *, Difference between values of DES-treated group and control group, P < 0.05.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been recognized that estradiol increases the actions of FSH for the differentiation of granulosa cell function (33), including aromatase expression (18), estradiol synthesis, FSH receptor expression, and LHR expression. In the ovary, ERβ has been demonstrated to be predominantly expressed in the ubiquitous granulosa cells of growing follicles from the primary to the ovulatory stage (20, 34). Furthermore, the recent studies by Couse et al. (22) reported that the estrogen effects mediated by ERβ are crucial for gonadotropin-induced granulosa cell differentiation as indicated by reduced aromatase activity, estradiol synthesis, and insufficient expression of LHR. However, the mechanism underlying the augmentation of LHR expression by FSH in the presence of estradiol is not yet clear. In the present study, we investigated the regulation of LHR mRNA expression in primary granulosa cell culture. Our results show that estradiol, in the presence of FSH, decreases Mvk mRNA levels in which the LHR mRNA stability is increased, resulting in the up-regulation of LHR mRNA.

As a first step of our experiment, granulosa cells were cultured for 24–96 h in the presence of FSH (30 ng/ml) with or without estradiol to confirm the effect of estrogen with FSH on LHR mRNA induction. In Figs. 1Go and 2Go, the concomitant treatment with both FSH and estradiol clearly showed that LHR mRNA level was increased in a dose- and time-dependent manner, a finding that is consistent with previous reports (18, 35). Furthermore, to ascertain whether the effect of estradiol on LHR mRNA induction is mediated through ERβ, we added R,R-THC into the culture medium. As shown in Fig. 3Go, R,R-THC clearly antagonized the estradiol effect in a dose-dependent manner. On the other hand, MPP (ER{alpha} antagonist) could not abolish the estradiol effect on FSH-induced LHR mRNA. Because it has been well acknowledged that ERβ mRNA is preferentially expressed in the granulosa cells (20, 29, 36), the present result combined with our previous report (37) leads us to conclude that ERβ mainly mediates estradiol signal transduction to increase LHR mRNA in the granulosa cells. We demonstrated that FSH and estradiol synergistically up-regulate LHR mRNA levels in the rat granulosa cell cultures. However, this estradiol action could not be produced without FSH because estradiol itself did not induce LHR mRNA (Fig. 1Go), a finding that was almost identical with the observation of Shi and Segaloff (30). A number of reports have found that estrogen augments the effect of FSH on granulosa cells, although none of them clearly show the mechanism of FSH action enhanced by estrogen. Thus, we performed a luciferase assay to elucidate the mechanism of LHR mRNA regulation by FSH and estradiol. To investigate the hormonal regulation of the 5'-flanking region of LHR, we examined the clone from –1389 to –1 bp of the 5'-flanking region. As previously indicated (38), rat LHR promoter does not contain putative TATA or CAT boxes or estrogen-responsive element (ERE), whereas specificity protein-1 Sp1 within the canonical GC box plays an important role in LHR promoter activity (39). This is consistent with the data showing that the granulosa cells transfected with a luciferase reporter construct containing this 5'-flanking region of the LHR did not additively respond to FSH and estradiol treatment, compared with treatment with FSH alone (Fig. 4Go). Thus, it is suggested that estradiol enhances the effect of FSH on LHR mRNA induction mediated through a different mechanism from that affecting LHR promoter. However, because the promoter region we cloned did not include ERE, the involvement of the ER pathway in the transcription of LHR remains questionable.

Mvk, a cytosolic enzyme in the cholesterol biosynthetic pathway, was cloned from rat liver in 1990 (15). Because the ovary is one of the major steroidogenic endocrine tissues, it is no wonder that Mvk exists and is involved in the sterol synthesis in the ovary. In fact, Mvk enzyme activity has already been demonstrated in the ovary (40). Moreover, the studies from the laboratory of Wang and Menon (14) have shown that Mvk was an LHR mRNA binding protein and that Mvk binding to LHR mRNA accelerated the LHR mRNA instability. These accumulated results prompted us to examine whether estradiol altered the Mvk levels in the granulosa cells, by which LHR mRNA was consequently modulated. As shown in Fig. 8Go, the overexpression of Mvk by transfection clearly diminished the estradiol effect on LHR mRNA expression. Additionally, Mvk mRNA and protein level was decreased in an estradiol dose-dependent manner (Fig. 7Go, A and B). From these results, we conclude that estradiol prevented Mvk from binding to LHR mRNA, resulting in the stabilizing of LHR mRNA. To confirm that estrogen regulates the FSH-induced LHR mRNA level by controlling Mvk expression in vivo, we performed Northern blot analysis by extracting RNA from the ovaries of hypophysectomized rats, which had been administered rec-FSH with or without DES treatment (Fig. 9Go). The concurrent treatment with rec-FSH and DES obviously indicated that estrogen modulated Mvk expression in the ovary. Moreover, based on the finding in an in vitro study that estradiol increased the stability of LHR mRNA by the reduction of the Mvk mRNA level, we believe that Mvk was at least involved in the mechanism by which LHR is regulated inside the ovary.

To understand the gene expression of Mvk, we cloned a 5'-untranslated region of the rat Mvk gene: –700 to –1 bp of the 5'-flanking region from the genomic library. In this region, the sequencing of the Mvk promoter showed no consensus GC, TATA, or CCAAT boxes, whereas an ERE-half-site existed. In the human Mvk 5'-flanking region, sterol regulatory element (SRE), which was a 7/8-bp match to the consensus sequences, was identified (41), whereas no SRE was detected in the rat gene. Other enzymes involved in the pathway of cholesterol biosynthesis, namely 3-hydroxy-3-methylglutaryl-coenzyme A synthase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, also contain SRE. In the ovary, ovarian steroid hormone production uses both plasma-derived cholesterol and de novo synthesized cholesterol as a precursor (42). Further experiments are required to complete our understanding of the difference in the promoter regions of Mvk between species.

Ginther et al. (43) reported that a dominant follicle contains more estradiol than other small follicles, indicating that the synergistic action of FSH and estradiol in a dominant follicle may enhance the induction of LHR more than in the secondary follicles. We speculate that, in the dominant follicle, estradiol suppresses Mvk expression to assist with the induction of LHR in coordination with other factors, namely activin, TGF-β, IGF-I, etc., before the LH surge. The LH surge activates the augmented LHRs to produce a sufficient amount of substances (e.g. cyclooxygenase-2 and tissue plasminogen activator) to complete ovulation, followed by the formation of a corpus luteum.

In this study, we have demonstrated the involvement of Mvk in the regulation of LHR mRNA stability in the rat granulosa cells. Due to the physiological aspect of Mvk in the ovary, the significance of the role of Mvk has not yet been fully elucidated. It is generally accepted that FSH is the most important factor for LHR expression because estrogen alone cannot induce LHR. Further experiments are necessary to clarify this question.


   Acknowledgments
 
We thank Dr. Yumiko Abe for her excellent technical assistance. We also thank the National Hormone and Pituitary Distribution Program of the National Institute of Diabetes and Digestive and Kidney Disease for supplying the rat FSH assay kit.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) and Initiatives for Attractive Education in Graduate Schools from MEXT.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 3, 2008

Abbreviations: DES, Diethylstilbestrol; ER, estrogen receptor; ERE, estrogen-responsive element; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; LHR, LH receptor; MPP, methyl-piperidino-pyrazole; Mvk, mevalonate kinase; P450scc, P450 side-chain cleavage; rec-FSH, recombinant FSH; R,R-THC, (R,R)-cis-diethyltetrahydro-2,8-chrysenediol; SRE, sterol regulatory element.

Received August 22, 2007.

Accepted for publication December 27, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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