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The Role of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor-Specific mRNA Binding Protein in Regulating Receptor Expression in Human Ovarian Granulosa Cells

Departments of Obstetrics/Gynecology and Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0617

Address all correspondence and requests for reprints to: Dr. K. M. J. Menon, 6428 Medical Science 1, 1301 East Catherine Street, Ann Arbor, Michigan 48109-0617.

	Abstract

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Context: In normally cycling women, LH receptor mRNA expression undergoes transient down-regulation after the LH surge. The same phenomenon is also seen during a hormonally induced ovarian cycle where the LH receptor mRNA expression is down-regulated in response to the administration of human chorionic gonadotropin (hCG). Although the granulosa cells isolated from the follicular aspirates at this stage show a decline in the expression of LH receptor mRNA, this diminished receptor expression returns to control levels upon culturing in serum-containing medium.

Objective: To understand the mechanism of hCG-induced loss of LH receptor mRNA expression, a cytosolic fraction (S100) was isolated from the granulosa cells, and its ability to bind LH receptor mRNA was assayed by performing RNA electrophoretic mobility gel shift analysis.

Results: The results showed that the mRNA binding activity of the S100 fraction was induced in the freshly isolated granulosa cells (d 1) from the follicular aspirates collected from women who had been injected with hCG to induce ovulation. The LH receptor mRNA expression in granulosa cells on d 1, as assessed by real-time PCR and Northern blot analysis, was significantly suppressed. Both the expression of LH receptor mRNA and RNA binding activity in the S100 fraction were then assessed after culturing granulosa cells for 4 d. The results showed that the LH receptor mRNA expression was significantly higher on d 4 compared with that seen on d 1. However, the RNA binding activity of the S100 fraction was significantly decreased on d 4 compared with that seen on d 1. These results show an increased association of RNA binding protein during LH receptor mRNA down-regulation.

Conclusion: The present results support the notion that LH receptor mRNA expression in the human ovaries is regulated by an RNA binding protein.

	Introduction

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IN HUMANS, THE ovarian cycle is characterized by a sequence of follicle recruitment, maturation, ovulation, and luteinization followed by luteolysis. Each of these processes occurs in response to constantly changing levels of circulating LH and FSH, as well as intraovarian secretory products including activin, IGF, and steroid hormones, notably, androgens and estrogens (1, 2). It is well established that the evolution to early antral follicles occurs in response to steadily increasing levels of FSH. Follicle maturation is accompanied by increased production of estradiol by the growing follicles that, in turn, increases the sensitivity of the follicles to FSH. The antral follicles respond to stimulation by FSH and estradiol by increasing the appearance of LH receptors (1). Finally, ovulation occurs when the oocyte reaches the metaphase of the second meiotic division and in response to LH surge.

Ovulation produces dramatic changes in the follicles. On a biochemical level, LH receptors present in the preovulatory follicles show a transient down-regulation after ovulation (3, 4, 5, 6). In our previous studies, using rat as a model, we have examined the molecular mechanisms responsible for this abrupt change in the receptor expression in response to the preovulatory LH surge. Our studies have shown that the preovulatory LH surge causes selective down-regulation of the LH/human chorionic gonadotropin (hCG) receptor expression resulting from the almost complete loss of the steady-state levels of the LH/hCG receptor mRNA (7, 8). Furthermore, we have shown that the loss of LH/hCG receptor mRNA is not due to decreased transcription but is a result of increased mRNA degradation (8). A specific LH/hCG receptor mRNA binding protein (LRBP) has been identified in the cytosolic fraction of the rat ovary that selectively binds and leads to LH/hCG receptor mRNA degradation in vitro (7, 9, 10, 11, 12, 13). More recently, we have shown that LRBP also causes an inhibition of LH receptor mRNA translation (13). In the present studies, we have extended these observations using human granulosa cells isolated from the follicular aspirates obtained from women undergoing in vitro fertilization. Our results indicate that granulosa cells show low levels of LH/hCG receptor mRNA on the day of retrieval followed by an increase after 4 d of culture. These low levels of LH/hCG receptor mRNA expression are accompanied by increased binding to an LH receptor mRNA binding protein (LRBP). When levels of LH/hCG receptor mRNA expression increased after 4 d of culture, a decline in the LH/hCG receptor mRNA binding to LRBP was observed. Our previous studies using rats have established the identity of this protein as being mevalonate kinase (10). The LH/hCG receptor mRNA binding characteristics of the protein in human granulosa cell cytosol are similar to the previously characterized rat ovarian LRBP with respect to preference for a specific mRNA nucleotide sequence and the size of the ribonucleoprotein (RNP) complex. In the present study, we show that the human granulosa cell cytosolic protein specifically interacts with human LH/hCG receptor mRNA and provides evidence for the inverse relationship between LH/hCG receptor mRNA expression and mRNA binding activity of LRBP, implicating a role for this protein as a regulator of LH/hCG receptor mRNA expression.

	Patients and Methods

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Cell isolation and culture

Ovarian follicular aspirates were obtained from women undergoing oocyte retrieval as part of the in vitro fertilization program at the University of Michigan Health System (Ann Arbor, MI). This study using discarded follicular fluids was given the exemption from continuing review by the University of Michigan Medical Institutional Review Board based on the Federal Regulations 45 CFR 46.101 (b) (4). 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 harvesting the oocytes, the remaining aspirates were pooled, divided into six to eight aliquots, and centrifuged at 500 x g for 5 min. The supernatant was removed from each aliquot, and the cell pellets were resuspended in 4 ml McCoy’s 5A medium (pH 7.4) (Invitrogen, Grand Island, NY). The suspension was layered over 3 ml Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) and centrifuged at 500 x g for 30–40 min to isolate 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 hemocytometer, and cell viability was determined by Trypan Blue exclusion, which was found to be consistently greater than 90%.

One half of the granulosa cells was processed for RNA isolation or for the preparation of the cytosolic proteins (S100 fraction) for the RNA EMSA (REMSA). The other half of the granulosa cells was plated onto 60-mm dishes at a density of 1–2 x 10⁶ viable cells with 4 ml McCoy’s 5A medium supplemented with 10% fetal bovine serum (Invitrogen), 1.65 mg/liter glutamine (Invitrogen), nonessential amino acids (Invitrogen), penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (0.25 µg/ml) (Invitrogen). Media were replaced after 24 h, and incubations were continued for an additional 3 d. These d 4 cultured cells were then processed for total RNA isolation for Northern blot analysis and real-time PCR or were processed for REMSA.

Northern blot analysis

Total RNA was extracted from granulosa cells using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions and quantitated spectrophotometrically, and the purity was determined by A260/280 ratio. Northern blot analysis was performed essentially as described before (9). Briefly, aliquots of total RNA were separated by electrophoresis in 1.2% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Blots were prehybridized for 2 h at 42 C in a solution containing salmon sperm DNA (0.5 mg/ml) and 2 x hybridization buffer [1.5 M NaCl, 0.1 M N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7), 0.1 M EDTA, 2 x Denhardt’s], diluted 1:1 with deionized formamide. The human LH receptor cDNA probe was radiolabeled with [-³² P] dCTP (MP Biomedicals, Irvine, CA) using the Invitrogen RadPrime DNA labeling kit (Carlsbad, CA). Unincorporated radioactivity was removed using Quick Spin Sephadex G-50 spin columns (Roche, Indianapolis, IN). The probe (2 x 10⁷ cpm) was hybridized to blots overnight at 42 C in fresh buffer. The blots were then washed four times with 2 x SCC containing 0.1% sodium dodecyl sulfate for 10 min each at room temperature and once at 60 C for 30 min. The washed blots were exposed to XAR film (Kodak, Rochester, NY) at –70 C in a Kodak X-Omatic cassette with intensifying screens. To monitor total RNA loading, blots were stripped and rehybridized with radiolabeled cDNA for 18S rRNA.

Real-time PCR

Aliquots (50 ng) of total RNA extracted from d 1 and 4 human granulosa cells were reverse-transcribed in a reaction volume of 20 µl using 2.5 µM random hexamer, 500 µM dNTPs, 5.5 mM MgCl₂, 8 U ribonuclease inhibitor, and 25 U Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). The 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 triplicates using predesigned primers and probes for human LH receptor from Applied Biosystems (TaqMan Assay-on-Demand Gene Expression Product). Reactions were carried out in a reaction volume of 25 µl using Applied Biosystems 7300 RealTime 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 LH receptor expression was calculated using the standard curve method with the 18S rRNA as the internal control.

Preparation of cytosolic proteins (S-100 fraction)

Day 4 granulosa cells were detached from culture dishes with PBS-EDTA and pelleted at 500 x g for 5 min. Day 1 and 4 cell pellets were 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. Homogenates were centrifuged at 105,000 x g for 90 min at 4 C, the supernatants (S100) were collected, and total protein was quantified using the BCA Protein Assay kit (Pierce, Rockford, IL).

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 sequences used for REMSA were 5'-AUCUCUCACCUAUCUCCCUGuCAA-3' for rat and 5'-AUCUCUCAGAUUGAUUCCCUGgA-3' for human. The rat LRBP contact site was mapped using hydroxyl-radical RNA footprinting and by site-directed mutagenesis as reported previously (11). The underlined sequences represent the LRBP contact sites (polypyrimidine-rich regions). The base residue identified in lowercase differs in rat and human LRBP contact sites of LH receptor mRNA. The unlabeled and labeled RNAs were prepared using the Maxiscript kit (Ambion, Inc., Austin, TX) according to the manufacturer. For radiolabeled RNA, transcription reactions were performed in the presence of 100 µCi [-³²P] UTP (800 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA) without additional unlabeled UTP. Radiolabeled RNAs were quantified by liquid scintillation counting, and unlabeled RNA was quantified by UV absorbance.

REMSA

REMSA was performed as described previously (7). Briefly, 5–25 µg of cytosolic S100 protein samples were incubated with 1–2 x 10⁵ cpm [-³²P] UTP-labeled hLBS (238–260). Binding reactions were carried out in buffer A (pH 7.5) containing 150 mM KCl and protease inhibitor mixture. Incubations were performed in the presence of 5 µg tRNA and 40 U RNasin (Promega, Madison, WI) at 30 C for 30 min. Unprotected radiolabeled RNA was then degraded by the addition of 0.5 ng ribonuclease A at 37 C for 30 min. For competition, unlabeled RNA was included in the binding reaction in mass excess as indicated in the figure legend. Samples were then incubated with heparin (final concentration, 5 mg/ml) on ice for 10 min to reduce nonspecific binding. The RNA-protein complexes were resolved by 5% native polyacrylamide (70:1) gel electrophoresis at 4 C. The gel was then dried and exposed to Kodak X-Omat AR film for visualization by autoradiography.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
As indicated in Patients and Methods, the granulosa cells isolated from the follicular aspirates were derived from women undergoing ovulation induction for in vitro fertilization. Because the patients were treated with a high dose of hCG before oocyte retrieval, we first examined whether the LH/hCG receptor expression is decreased in response to the injected hCG. The granulosa cells isolated by Ficoll gradient centrifugation were plated and cultured as described in Patients and Methods. The freshly isolated cells (d 1) and the d 4 cultured cells were subjected to total RNA extraction, and Northern blot analysis was performed by hybridization with a randomly labeled human LH/hCG receptor cDNA. The results presented in Fig. 1 show that the bands corresponding to the reported sizes (5.4 and 3.4 kb) of LH/hCG receptor mRNA transcripts appeared very faint on d 1 compared with d 4. The bottom panel shows the hybridization of 18S rRNA, which was used as loading control. These results clearly show that the LH/hCG receptor mRNA expression was low in granulosa cells on the day of retrieval. Based on our earlier studies in rodents on the regulation of LH receptor mRNA, it is likely that this decrease may have been caused by the administration of hCG before oocyte retrieval. Upon culturing in a medium devoid of hCG for 4 d, the LH/hCG receptor mRNA expression increased. The change in receptor expression was also analyzed by real-time PCR analysis, which allows more precise quantitation. The results showed that there was a 3-fold increase in the expression of the LH/hCG receptor mRNA on d 4 compared with that seen on d 1 (Fig. 2). Because the real-time PCR was more sensitive than the Northern blot analysis, low levels of expression could be demonstrated on d 1 using this technique. Thus, the real-time PCR data corroborated the Northern blot analysis data presented in Fig. 1.

View larger version (51K): [in this window] [in a new window]   FIG. 1. Northern blot analysis of LH/hCG receptor mRNA in human granulosa cells. Fifteen micrograms of total RNA extracted from d 1 (day of retrieval) and d 4 incubated granulosa cells was separated on agarose-formaldehyde gel, transferred to membrane, hybridized with the 32P-labeled 2.1-kb hLH receptor cDNA, and exposed to x-ray film. To monitor RNA loading, the blot was stripped and rehybridized with radiolabeled cDNA for 18S rRNA. The blot shown is one representative of three experiments with similar results.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Real-time PCR quantitation of LH/hCG receptor mRNA in human granulosa cells. Total RNAs from d 1 and 4 granulosa cells were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and 5-carboxyfluorescein-labeled probe as described in Patients and Methods. Mean values ± SE (n = 6) were normalized to 18S rRNA and graphed as percentage of d 1.

After establishing that the mRNA expression in the hCG-treated group was significantly lower on d 1 compared with that seen on d 4, attempts were made to determine whether a specific human LRBP was present in the cytosolic fractions isolated from the granulosa cells from the LH/hCG receptor down-regulated state (d 1). For this purpose, a 23-nucleotide ³²P-labeled RNA, representing the putative human LRBP recognition site of LH receptor mRNA as shown in Patients and Methods, was prepared and incubated with an S100 fraction isolated from granulosa cells on d 1. The formation of the RNP complex was identified using REMSA. The results presented in Fig. 3 show a prominent RNP complex in the sample incubated with S100 fraction, the intensity of which increased with protein concentration. The RNP complex formation was not seen in control reactions in the absence of S100 fraction. The RNP complex formation was abrogated in samples incubated in the presence of 10 and 25 M excess of unlabeled 23-nucleotide RNA probe (Fig. 4). This competition by unlabeled excess RNA probe showed that the interaction of the binding protein in the S100 fraction with human LH/hCG receptor mRNA was specific.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Concentration-dependent RNP complex formation. a, RNA gel mobility shift analysis was performed by incubation of 2 x 105 cpm 32P-hLBS: 238–260 in the absence (0) or presence of increasing concentrations (12.5 and 25 µg protein) of cytosolic S-100 fraction isolated from human granulosa cells on the day of retrieval, as described in Patients and Methods. The autoradiogram shown is representative of three independent experiments. b, RNP complex formed was quantitated by scanning and represented in densitometric units (mean value ± SE).

View larger version (49K): [in this window] [in a new window]   FIG. 4. RNA gel mobility shift analysis: competition with nonradiolabeled hLBS. Autoradiogram of RNA mobility shift analysis performed using 2 x 105 cpm 32P-hLBS incubated with 15 µg cytosolic S-100 fraction isolated from granulosa cells on the day of retrieval. Lane 1, 32P-hLBS alone; lane 2, hLBS + hS100; lane 3, inclusion of 10x M excess of unlabeled hLBS; lane 4, inclusion of 25x M excess of unlabeled hLBS. The autoradiogram shown is representative of three independent experiments.

View larger version (40K): [in this window] [in a new window]   FIG. 5. Autoradiogram of RNA binding activity of LH receptor mRNA binding protein (LRBP) in the cytosolic S-100 fractions of d 1 and 4 granulosa cells. a, REMSA was performed using 32P-hLBS (1.5 x 105 cpm) with no protein (lane 1) and with 20 µg S-100 protein isolated from d 1 (lane 2) and 4 (lane 3) granulosa cells as described in Patients and Methods. The autoradiogram is representative of three independent experiments. b, RNP complex formed was quantitated by scanning and represented in relative densitometric units (mean value ± SE).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

LH/hCG receptor expression undergoes significant changes during the ovarian cycle. The developing follicles acquire LH/hCG receptors by the constantly rising levels of FSH. As the preovulatory follicles begin to secrete estrogens, these estrogens synergize the action of FSH to induce LH/hCG receptors. In response to the preovulatory LH surge, the LH/hCG receptor undergoes down-regulation followed by full recovery with the formation of the corpus luteum, and the receptor expression increases further with the development of the corpus luteum. The LH/hCG receptor expression then decreases as the corpus luteum undergoes luteolysis. Our previous results, using a rodent model, suggest that the transient changes in the receptor mRNA expression are mediated by posttranscriptional mechanisms (8). We have shown that the decrease in the steady-state level of LH/hCG receptor mRNA after the preovulatory LH surge or in response to hCG administration to rats with hormonally induced follicles appears to be mediated by accelerated degradation of the mRNA rather than through a transcriptional suppression (8). This increased degradation was facilitated by the induction of LRBP in the ovary. Because the expression of this RNA binding protein was seen at higher levels in granulosa cells obtained from patients treated with hCG, it would suggest that a similar mechanism of accelerated degradation of LH/hCG receptor mRNA by this RNA binding protein might also occur in the human ovaries. The protein binding region in the rat LH/hCG receptor has been mapped to the 23 nucleotides containing a bipartite polypyrimidine-rich region in the open reading frame of the LH/hCG receptor mRNA. Sequence alignment of the rat LH/hCG receptor and the human LH/hCG receptor cDNA reveals that the bipartite sequence is similar in both mRNAs with the substitution of a U in the rat by a G in the human.

Posttranscriptional regulation of mRNA expression has now been recognized as an efficient means of controlling steady-state levels to meet the short-term needs of cells. In most cases where mRNA levels are controlled by their degradation, specific mRNA binding proteins have been shown to play a role, either by increasing or decreasing mRNA stability. The recognition site of the RNA binding protein may be localized either on the 3'- or 5'-untranslated region (15, 16, 17, 18) or in the coding region, as is the case with LH/hCG receptor (7, 10, 11). In most instances, the binding protein specifically interacts with defined structures or sequences of the mRNA and alters its stability. In the case of rat LH/hCG receptor, we have characterized the identity of the mRNA binding protein LRBP as being mevalonate kinase, an enzyme involved in cholesterol synthesis. The involvement of a metabolic enzyme as an RNA binding protein has been reported for the regulation of a variety of mRNAs (16, 19, 20). The physiological significance of the mevalonate kinase acting as an mRNA binding protein, thereby controlling its expression, is noteworthy. Mevalonate kinase is an enzyme that is regulated in the ovaries by LH or hCG (12). Thus, when the antral follicles are stimulated either in response to preovulatory LH surge or in response to hCG injection, the follicles initially respond by producing steroids, thereby depleting the endogenous cholesterol levels. This sets the stage for LH/hCG receptor down-regulation to prevent overstimulation, at which time it appears that mevalonate kinase switches its function to an LH/hCG receptor regulatory protein or maintains both functions as an enzyme in the de novo synthesis of cholesterol and as a LH receptor mRNA binding protein and triggers its degradation. The recovery from down-regulation occurs when the LH levels drop from the surge level, returning the expression of LH/hCG receptors to normal levels. Such a regulatory mechanism has the advantage of providing a fine control of the LH/hCG receptor mRNA expression by controlling its stability rather than by reprogramming the complex transcriptional assembly.

	Footnotes

 
This work was supported by National Institutes of Health Grant HD-0665.

A.K.N., H.P., and K.M.J.M. have nothing to declare.

First Published Online March 21, 2006

Abbreviations: hCG, Human chorionic gonadotropin; hLBS, human LRBP binding sequence; LRBP, LH/hCG receptor mRNA binding protein; REMSA, RNA EMSA; RNP, ribonucleoprotein.

Received December 16, 2005.

Accepted March 9, 2006.

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/6/2239    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nair, A. K. Articles by Menon, K. M. J. Search for Related Content PubMed PubMed Citation Articles by Nair, A. K. Articles by Menon, K. M. J. Related Collections Female Endocrinology