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Ribonucleic Acid Binding Protein-Mediated Regulation of Luteinizing Hormone Receptor Expression in Granulosa Cells: Relationship to Sterol Metabolism

Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: K. M. J. Menon, 6428 Medical Science I, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0617. E-mail: kmjmenon{at}umich.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Posttranscriptional mechanism plays a crucial role in regulating LH receptor (LHR) expression in the ovary. We have identified a novel trans-factor, LHR mRNA binding protein (LRBP), which binds to a polypyrimidine-rich bipartite sequence of the coding region of LHR mRNA, and its identity was established as mevalonate kinase (Mvk). Although an inverse relation between LHR mRNA expression and RNA binding activity of LRBP has been established, its intermediary role in LHR mRNA expression has not been demonstrated. The present study examined the direct role of Mvk in regulating LHR expression by using primary cultures of human granulosa cells as a model system. A marked decrease in LHR mRNA stability and an increase in Mvk expression were seen when cultured granulosa cells were treated with human chorionic gonadotropin (hCG) in vitro. This treatment also resulted in an increase in LHR mRNA binding activity in the cytosolic fractions prepared from hCG-treated cells compared with the control. Because Mvk expression is regulated by sterol response element-binding protein-1, which is sensitive to the cellular concentration of 25-hydroxycholesterol (25-OHC), cultured granulosa cells were treated with this oxysterol, and the expression of Mvk gene was examined. As expected, treatment with 25-OHC inhibited the Mvk (LRBP) expression, as well as the LHR mRNA binding activity of LRBP. To determine the role of Mvk in ligand-mediated down-regulation of LHR mRNA, cells were additionally treated with 25-OHC when treated with hCG. The results showed that the decrease in Mvk expression by oxysterol treatment abrogated ligand-induced down-regulation of LHR mRNA. These results therefore establish a direct participation of Mvk in regulating LHR expression and suggest a novel relationship between cholesterol metabolism and LHR expression in the ovary.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PREVIOUS STUDIES FROM our laboratory have shown that LH/human chorionic gonadotropin (hCG) receptor (LHR) expression in the ovary is regulated through a posttranscriptional mechanism (1). Other G protein-coupled receptors, such as ß₁- and ß₂-adrenergic receptors (2) and m1 muscarinic acetylcholine receptor (3), are also regulated through changes in their mRNA stability. Using a rodent model system, we have extensively studied the ligand-induced down-regulation of LHR mRNA and have isolated a LHR mRNA binding protein (LRBP), which binds specifically to a cytidine-rich region of LHR mRNA and accelerates its decay in vitro (4). Amino-terminal analysis and matrix-assisted laser desorption ionization mass spectrometry of the gel-purified LRBP established its identity as mevalonate kinase (Mvk) (5). Further studies showed that under varying physiological conditions, the LHR mRNA levels were inversely correlated to the binding activity of LRBP, which further suggests a role for LRBP in regulating LHR mRNA expression (4). Direct addition of purified LRBP to an in vitro LHR synthesizing system showed that LRBP caused an inhibition of LHR mRNA translation (6). It was also found that in the human granulosa cells, the expression of LHR mRNA levels had an inverse relationship with LRBP binding activity, demonstrating that a similar mechanism for the regulation of LHR mRNA expression also occurs in human ovaries (7). These results suggest that LRBP/Mvk is involved in the regulation of LHR mRNA expression in both rat and human ovaries.

Discovery of Mvk acting as a trans-factor in the regulation of LHR mRNA expression is particularly significant because it suggests that LHR mRNA expression might be linked to cholesterol metabolism in the ovarian tissue (8). Although the fact that Mvk, a cholesterol biosynthesis enzyme, could act as a trans-factor was surprising, more recent structural studies of the enzyme have revealed that Mvk belongs to the GHMP (galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase) family of kinases, a group of ATP-binding enzymes that has the structural fold known as ribosomal protein S2 domain-like fold, similar to that found in proteins interacting with rRNA (9). Thus, it is a member of a growing group of metabolic enzymes acting as RNA-binding proteins that regulate mRNA expression.

After the identity of LRBP was established as Mvk, we have shown that in the rat ovary, Mvk expression is regulated by hCG treatment in a manner consistent with its role as a regulator of LHR mRNA expression (10). Furthermore, Mvk was shown to directly interact with LHR mRNA (5). The purpose of the present study was to establish that Mvk participates in LHR mRNA destabilization during ligand-induced down-regulation of LHR mRNA. Our results show that suppression of Mvk expression in cultured human granulosa cells abrogates hCG-induced down-regulation of LHR mRNA. Thus, in this report, we provide direct evidence for the participation of Mvk in modulating LHR mRNA expression through its role as an LHR mRNA-binding protein.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Analysis of Changes in the Levels of Steady-State LHR mRNA during hCG-Induced Down-Regulation in Human Granulosa Cells
Previous studies from our laboratory demonstrated a loss of all four LHR mRNA transcripts during hCG-induced LHR down-regulation in pseudopregnant rat ovaries (4, 10, 11). Here we sought to establish whether a similar phenomenon also occurs in human granulosa cells. Unlike the rat LHR mRNA, the alternative splice variants of human LHR mRNA had not yet been fully characterized (12). Thus, before examining the regulation of LHR mRNA expression in human granulosa cells, attempts were made to measure the exact size of those mRNA transcripts in cultured human granulosa cells. Total RNA was extracted from granulosa cells and subjected to Northern blot analysis. A set of three cDNA probes was used (Fig. 1A): probe 3k, which spans the whole coding region of LHR and a large part of its 3'-untranslated region (13); probe 659 (nucleotides 3941052), which hybridizes with a portion of LHR mRNA that encodes for the last two thirds of the LHR extracellular domain (14); and probe 211 (nucleotides 8191029), which corresponds to a segment of LHR cDNA with low homology (44%) to the corresponding region of FSH receptor cDNA (15). Using each of these cDNA probes, we consistently found three bands upon Northern blot hybridization (Fig. 1B). As the Ambion’s Millennium Markers (Stratagene, La Jolla, CA) hybridized with our probe 3k, which allowed direct visualization of the markers on the film, we constructed a standard curve. Using this standard curve, we deduced the sizes of the three bands to be 7.2, 4.8, and 3.3 kb. We then determined which of these transcripts undergoes down-regulation upon treatment with hCG. The cells were treated with 10 IU/ml hCG (Sigma, St. Louis, MO), and total RNA was extracted at 0, 6, 12, and 18 h for Northern blot analysis, as described in Materials and Methods. Results presented in Fig. 2 showed a parallel decline of all three hLHR mRNA transcripts after hCG treatment, whereas there was no significant change of these transcripts in control cells. To accurately quantitate the changes in LHR mRNA levels, real-time PCR was conducted using predesigned primers and probes (TaqMan Assay-on-Demand Gene Expression Products), and employing total RNA extracted from cells treated with or without hCG for 0, 3, 6, and 12 h. The real-time PCR results showed a significant time-dependent inhibition of LHR mRNA expression in groups treated with hCG (see Fig. 4A), and these were consistent with the Northern blot results (Fig. 2). The results also showed that the time course of hCG-induced down-regulation of LHR mRNA in human granulosa cells was dependent on the concentration of hCG used (Fig. 4A). A concentration of 10 IU/ml hCG caused down-regulation after 6 h incubation whereas at 1 IU/ml hCG, the down-regulation was seen at 12 h. At a lower concentration (0.1 IU/ml), hCG caused a slight, but not significant, up-regulation of LHR mRNA. From these results, we conclude that hCG is able to induce a parallel decrease of all three LHR mRNA transcripts (7.2, 4.8, and 3.3 kb) in cultured human granulosa cells.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Analysis of LHR mRNA Transcripts in Cultured Human Granulosa Cells A, Schematic structure of the human LHR mRNA and positions of probes used. B, Cells were collected from preovulatory follicles of women undergoing ovum retrieval for in vitro fertilization and cultured as described in Materials and Methods. Total RNA (5 µg), along with the RNA ladder, was fractionated on formaldehyde-agarose gel, transferred to membrane, and hybridized to probe 3k, probe 659, or probe 211. Exposure to x-ray film lasted approximately 30–50 h. UTR, Untranslated region.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Effect of hCG on LHR mRNA Expression in Cultured Human Granulosa Cells A and B, After culture in serum free medium for 48 h, cells were treated with or without 10 IU/ml hCG and harvested at 0, 6, 12, and 18 h. A and B, Autoradiograph of Northern blot of human LHR mRNA and 18S rRNA. Total RNA (5 µg) from individual samples was fractionated on agarose gel, transferred to membrane, hybridized to probe 3k, and exposed to x-ray film. The Northern blot is representative of three independent experiments. C, Quantification of LHR mRNA levels by densitometric scans of the 3.3-kb transcript and analysis using NIH Image 1.61 software. Data were normalized for the amount of 18S rRNA in each sample and expressed relative to the value at time zero. Each point represents the average ± SE of three separate densitometric scans. Statistical differences between the time zero control value and different time intervals are indicated by the asterisk (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Regulation of LHR and Mvk Expression in Cultured Human Granulosa Cells A and B, After culture in serum free medium for 48 h, cells were treated with or without increasing concentrations of hCG and harvested at 0, 3, 6, and 12 h. Steady-state levels of LHR (A) and Mvk (B) mRNAs were measured by real-time PCR. Mean values ± SE (n = 3) were normalized to 18S rRNA and graphed as percent of control (time 0 h). Statistical differences between treated and control samples are indicated by the asterisk (P < 0.05). C, After culture in serum free medium for 48 h, cells were treated with fresh serum free medium, or 10 IU/ml hCG or 10 µg/ml 25-OHC and harvested at 12 h. Cytosolic proteins were extracted and subjected to SDS-PAGE on a 12% gel, transferred to membrane, and incubated with antibodies (1:1000 dilution) against Mvk or ß-tubulin. The Western blot is representative of two independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effect of hCG on the Stability of LHR mRNA in Cultured Human Granulosa Cells After culture in serum free medium for 48 h, cells were preincubated for 6 h with or without hCG. After this preincubation period, 2 µg/ml actinomycin D was added to arrest new RNA synthesis. Cells were harvested at 0, 4, and 8 h after addition of the transcription inhibitor, and LHR mRNA levels were quantified by real-time PCR. Mean values ± SE (n = 3) were normalized to 18S rRNA and graphed as percent of the value at time zero.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Autoradiogram of RNA Binding Activity of Mvk in the Cytosolic S-100 Fraction of Cultured Human Granulosa Cells After culture in serum free medium for 48 h, cells were treated with fresh serum free medium, or 10 IU/ml hCG or 10 µg/ml 25-OHC and harvested at 12 h. REMSA was performed using 32P-labeled hLBS (1.5 x 105 cpm) with no protein (lane 1), or with S-100 proteins isolated from control cells (lanes 2 and 5), hCG-treated cells (lanes 3 and 6), and 25-OHC-treated cells (lanes 4 and 7), as described in Materials and Methods. S-100 protein (20 µg) was used for REMSA in lanes 2–4, and 40 µg of S-100 protein was used for lanes 5–7. The autoradiogram shown is representative of two independent experiments. RNP, Ribonucleoprotein.

The granulosa cells, after 48 h culture in serum free medium, were treated with vehicle or 10 µg/ml 25-OHC. Unless indicated otherwise, 25-OHC was always added together with 100 µg/ml aminoglutethimide, which prevents the metabolism of cholesterol to steroid hormones. After 0-, 12-, and 24-h intervals, total RNA was extracted for real-time PCR. The results showed that both Mvk and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) mRNA were significantly suppressed by treatment with 25-OHC (Fig. 6, A and B), whereas the same treatment did not decrease the mRNA expression of LHR and a control enzyme, cytochrome P450_scc (CYP11a1) (Fig. 6, C and D), which indicates that the effect of 25-OHC is gene specific. The S-100 fractions were then prepared from cells treated with 10 µg/ml 25-OHC for 12 h and were subjected to both Western blot analysis and REMSA. Western blot results presented in Fig. 4C, lane 4, showed that treatment with 25-OHC suppressed Mvk protein levels, which is consistent with the reduction in Mvk mRNA levels (Fig. 6A). Treatment with 25-OHC also caused a reduction in LRBP activity due to decreased levels of Mvk in the oxysterol-treated group (Fig. 5, lanes 4 and 7). Taken together, these results conclusively show that in human granulosa cells, 25-OHC suppresses Mvk expression and consequently reduces its binding to LHR mRNA. Furthermore, these experiments substantiate the role of Mvk as an LHR mRNA binding protein.

View larger version (25K): [in this window] [in a new window]   Fig. 6. 25-OHC Suppresses Specific mRNA Expression in Cultured Human Granulosa Cells After culture in serum free medium for 48 h, cells were treated with or without 10 µg/ml 25-OHC and harvested at 0, 12, and 24 h. Steady-state levels of Mvk, HMGR, CYP11a1, and LHR mRNAs (A–D) were measured by real-time PCR. Mean values ± SE (n = 3) were normalized to 18S rRNA and graphed as percent of control (time 0 h). Statistical differences between treated and control samples are indicated by the asterisk (P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 7. 25-OHC Affects Gene Expression in hCG Down-Regulated Human Granulosa Cells A–D, After culture in serum free medium for 48 h, cells were treated with serum free medium, or 10 IU/ml hCG, or 10 IU/ml hCG plus 10 µg/ml 25-OHC, and harvested at 0 and 12 h. Steady-state levels of Mvk, HMGR, CYP11a1, and LHR mRNAs were measured by real-time PCR. Mean values ± SE (n = 3) were normalized to 18S rRNA and graphed as percent of control (time 0 h). Statistical differences between treated and control samples are indicated by the asterisk (P < 0.05). E, After culture in serum free medium for 48 h, cells were treated with serum free medium, or 10 IU/ml hCG or 10 IU/ml hCG plus 10 µg/ml 25-OHC, and harvested at 12 h. mRNA levels of SREBP-1 and SREBP-2 were measured by real-time PCR. Mean values ± SE (n = 3) were normalized to 18S rRNA and graphed as percent of control (time 12 h). Statistical differences between treated and control samples are indicated by the asterisk (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Our previous studies in the rodent system showed a rapid decline in the steady-state levels of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) during hCG-induced down-regulation (1, 4, 10, 11). Like the rat LHR gene, the human LHR gene also gives rise to multiple mRNA species in gonadal tissues. Three alternatively spliced variants of the hLHR have been reported (15, 19, 20, 21), but they are not fully characterized with regard to size and abundance (12). In the present studies, the total RNA from granulosa cells was hybridized with three different LHR cDNA probes (Fig. 1). As Probe 3k hybridized directly with our RNA markers, we were able to calculate the exact size of the three bands (7.2, 4.8, and 3.3 kb) based on a reliable standard curve. Because Probe 211 (nucleotides 819-1029) corresponds to the last one third of the LHR extracellular domain, we deduce that each of the three mRNA transcripts (7.2, 4.8, and 3.3 kb) contains either a complete or truncated hLHR coding sequence.

Human granulosa cells treated with 10 IU/ml hCG gave results consistent with our observations in the rodent system. All three LHR mRNA transcripts were coordinately down-regulated after 6 h hCG treatment. At 12 h, the steady-state level of the 3.3-kb transcript declined approximately 40–60% in comparison with the control (Figs. 2 and 4A). At this time interval, the Mvk mRNA expression increased 3-fold (Fig. 4B). LHR mRNA binding activity in the S-100 fractions also showed an increase (Fig. 5, lanes 3 and 6). To verify that the decrease in LHR mRNA is due to accelerated mRNA degradation, we measured mRNA levels after transcription arrest in control and hCG-pretreated cells. The exact LHR mRNA half-lives were not calculated, mainly because the granulosa cells obtained from different subjects demonstrated different sensitivities to actinomycin D. Therefore, it was not possible to calculate accurate values. However, our experiments consistently showed an increased LHR mRNA decay in hCG-treated cells, supporting the notion of a posttranscriptional regulation of LHR mRNA expression (Fig. 3).

These cultured human granulosa cells provided a useful model system to investigate the effect of cellular sterol on Mvk/LRBP during hCG-induced LHR mRNA down-regulation. Our results clearly show that 25-OHC inhibited the expression of Mvk along with another sterol-responsive element (SRE)-containing gene, HMGR, which was used as a representative marker for the induction of SRE-containing genes (Fig. 6). It is not surprising that 25-OHC causes a marked reduction in Mvk protein levels because previous studies have shown that cholesterol loading inhibits the expression of SRE-containing genes as well as causing degradation of HMGR through ubiquitylation (22, 23). Most interestingly, at the same time interval (12 h), LHR mRNA binding activity of LRBP in the S-100 fraction also showed a decline (Fig. 5, lanes 4 and 7). These results therefore directly show that Mvk serves as a LHR mRNA-binding protein in human granulosa cells, confirming the conclusions deduced from the in vivo studies (4, 6, 7). In cells treated with both hCG and 25-OHC for 12 h, the presence of 25-OHC caused a suppression of Mvk mRNA expression despite the presence of hCG, whereas treatment with hCG alone led to a nearly 150% increase in Mvk expression (Fig. 7). Along with the suppression of Mvk expression, the hCG-induced LHR mRNA down-regulation was abrogated. This experiment provides strong evidence for the crucial role of Mvk in mediating hCG-induced down-regulation: inhibition of Mvk expression results in the abolishment of hCG-induced LHR mRNA down-regulation.

Based on these results, we propose that hCG-induced cholesterol depletion, as a consequence of steroidogenesis in ovarian cells, facilitates positive regulation of Mvk expression (10), leading to accelerated LHR mRNA decay. Conversely, increased cellular sterol has a negative regulatory effect on Mvk, which abrogates the hCG-induced down-regulation of LHR mRNA. As an SRE-containing gene, Mvk expression may be mainly regulated through SREBPs. Generally, when cellular sterol levels are low, active SREBPs bind to the promoters of SRE-containing genes and activate their transcription. When sterol levels are sufficient, SREBPs form a complex with Scap and Insig and are retained on the endoplasmic reticulum membrane (18), and transcription of SRE-containing genes is suppressed. We show that hCG treatment led to a 3- to 4-fold increase in SREBP mRNA abundance (Fig. 7E), which is consistent with the observations of Lopez et al. (24), who showed that hCG induced the expression of SREBPs in pseudopregnant rat ovaries. However, 25-OHC did not affect the SREBP mRNA expression because it acts to suppress the transport of the precursor form of SREBP from the endoplasmic reticulum to the Golgi for proteolytic processing (18).

In summary, we have shown a crucial role of Mvk in accelerating LHR mRNA decay, by the formation of a ribonucleoprotein complex. Our results suggest a novel mechanism by which a member of the G protein receptor family can be regulated by the cellular sterol content.

	MATERIALS AND METHODS

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Cell Isolation and Culture
Ovarian follicular aspirates were obtained from women aged 22–38 yr undergoing oocyte retrieval at the IVF Michigan Laboratory. This study, using discarded follicular fluid, was approved by the local ethics committee. Ovulation was induced by sequential treatment with recombinant human FSH (Gonal F), followed by hCG. The follicles were aspirated 36 h after hCG administration. After the oocytes were harvested, the remaining aspirates were pooled, divided into six to eight aliquots, and centrifuged at 500 x g for 5 min. The supernatants were removed, and the cell pellets were resuspended in 4 ml McCoy’s 5A medium (pH 7.4) (Invitrogen, Carlsbad, CA), as described previously (7). The suspension was layered over 3 ml Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ) and centrifuged at 400 x g for 40 min to separate the granulosa cells from the red blood cells associated with the initial cell pellet. The granulosa cells, free of red blood cells, were removed from the interface and washed twice. Cells were counted with a hemacytometer, and cell viability was determined by Trypan Blue staining.

Cells were plated into 35-mm dishes at a density of 0.5 x 10⁶ viable cells with 2 ml McCoy’s 5A medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 µg/ml gentamycin, and 10 U/ml nyastatin. Cells were cultured at 37 C in an atmosphere of 5% CO₂ humidified air. After an initial period of 48 h, serum-supplemented medium was replaced by serum free medium for an additional 48-h period. Subsequently, this medium was replaced with serum free medium containing the treatments described in Results.

Isolation of Total RNA
Total RNA was extracted from cultured human granulosa cells using the commercial product TRIzol Reagent (Invitrogen) with a modified TRI reagent procedure (25) and stored at –80 C.

Probe Construction
A 3-kb cDNA fragment, which encodes the full-length hLH receptor (13), was subcloned into pSG5 plasmid at an EcoRI restriction site, as described previously (7). Probe 659 (394–1052 bases) and probe 211 (819–1029 bases) were prepared by RT-PCR, using total RNA extracted from human granulosa cells as template. Sequences of these probes were verified by dideoxy chain termination sequencing (26).

Northern Blot Analysis
Aliquots of total RNA (5 µg per lane) were separated by electrophoresis in 1.2% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Blots were subjected to UV cross-linking and prehybridized at 42 C for 2 h in a solution containing 0.5 mg/ml salmon sperm DNA and 2x hybridization buffer [1.5 M NaCl-0.1 M TES (pH 7.1)-0.1 M EDTA-2x Denhardt’s solution] diluted 1:1 with deionized formamide. Probes were radiolabeled with [-³²P]dCTP (PerkinElmer, Boston, MA) and hybridized to blots overnight at 42 C in fresh hybridization buffer. Hybridized blots were washed five times with 2x saline sodium citrate containing 0.1% sodium dodecyl sulfate and then exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) at –80 C.

Real-Time PCR
Aliquots (50 ng) of total RNA extracted from human granulosa cells were reverse transcribed in a reaction volume of 20 µl using 2.5 µM random hexamer, 500 µM deoxynucleotide triphosphates, 5.5 mM MgCl₂, 8 U ribonuclease inhibitor, and 25 U Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). Reactions were carried out in a PTC-100 (MJ Research, Watertown, MA) thermal controller (25 C for 10 min, 48 C for 30 min, and 95 C for 5 min). The resulting cDNAs were diluted with water. The real-time PCR quantitation was then performed using 5 µl of the diluted cDNAs in triplicate using predesigned primers and probes for human LHR, Mvk, HMGR, CYP11a1, and SREBP, from Applied Biosystems (TaqMan Assay-on-Demand Gene Expression Product). Reactions were carried out in a volume of 25 µl using Applied Biosystems 7300 Real-Time PCR system for 40 cycles (95 C for 15 sec, 60 C for 1 min) after initial incubation for 10 min at 95 C. The fold change in gene expression was calculated using the standard curve method with 18S rRNA as the internal control (7).

Preparation of Cytosolic Proteins
Granulosa cells were detached from culture dishes with PBS-EDTA and pelleted at 500 x g for 5 min (7). The pellet was homogenized at 4 C in buffer A (10 mM HEPES, pH 7.9; 0.5 mM MgCl₂; 50 µM EDTA; 5 mM dithiothreitol; and 10% glycerol) containing 50 mM KCl and EDTA-free protease inhibitor mixture (11). Homogenates were centrifuged at 105,000 x g for 90 min at 4 C, and the supernatants (S-100) were quantified using the BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL).

Western Blot Analysis
Cytosolic protein samples (50 µg) were denatured by boiling for 5 min in the presence of loading buffer and were subjected to SDS-PAGE on a 12% gel. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (0.2-µm pore) and blocked with 5% milk overnight at 4 C. The membranes were then incubated for 1 h with antibody (1:1000 dilution) at room temperature. [The antibody was raised against the first 15 N-terminal amino acids of Mvk (MLSEVLLVSAPGKVI).] The protein loading was standardized on the basis of ß-tubulin using a commercial antibody (Sigma). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature, and signals were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).

Preparation of Radiolabeled RNAs for EMSA
The cDNAs used to generate the human LRBP binding sequence (hLBs; 238–260) were chemically synthesized and contained the T7 RNA polymerase promoter sequence at the 5'-end. The probe sequence used for REMSA was 5'-AUCUCUCAGAUUGAUUCCCUGGA-3'. The underlined sequences represent the LRBP contact sites (polypyrimidine-rich regions). The labeled RNAs were prepared using the Maxiscript kit (Ambion, Inc., Austin, TX). Transcription reactions were performed in the presence of 100 µCi [-³²P] UTP (800 Ci/mmol; PerkinElmer) without additional unlabeled UTP. Radiolabeled RNAs were quantified by liquid scintillation counting.

REMSA
REMSA was performed as described previously (7, 10). In brief, cytosolic S-100 protein samples were incubated with 1 x 10⁵ cpm radiolabeled gel-purified hLBS (238–260). Binding reactions were carried out in homogenization buffer A for 10 min at 30 C, in the presence of 5 µg tRNA and 40 U of RNasin (Promega Corp., Madison, WI). Unprotected radiolabeled RNA was degraded by the addition of 2 U of RNase T1, and the RNA-protein complexes were then resolved by 5% native polyacrylamide (70:1) gel electrophoresis at 4 C. The gel was dried and exposed to Kodak x-ray film.

Statistical Analysis
Statistical analysis was carried out using the unpaired t test, with Sigma Stat software (version 2.0; SPSS Inc., Chicago, IL). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SEM of at least three experiments.

	ACKNOWLEDGMENTS

 
We thank Helle Peegel and Dr. Pradeep Kayampilly for their critical reading of the manuscript and valuable comments. We thank Dr. Iqbal Khan (IVF Michigan Laboratory, Rochester Hills, MI) for providing ovarian follicular aspirates.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grant R37 HD 06656.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 5, 2007

Abbreviations: CYP11a1, Also known as cytochrome P450scc, cholesterol side chain cleavage P450; hCG, human chorionic gonadotropin; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; LBS, LRBP-binding sequence; LHR, LH/hCG receptor; LRBP, LHR mRNA binding protein; Mvk, mevalonate kinase; 25-OHC, 25-hydroxycholesterol; REMSA, RNA EMSA; SRE, sterol-responsive element; SREBP, SRE-binding protein.

Received for publication February 23, 2007. Accepted for publication May 28, 2007.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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