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A novel mechanism for the modulation of luteinizing hormone receptor mRNA expression in the rat ovary
Molecular and Cellular Endocrinology 233 (2005) 65–72
A novel mechanism for the modulation of luteinizing hormone receptor mRNA expression in the rat ovary夽 H. Peegel, R. Towns, A. Nair, K.M.J. Menon ∗ Departments of Obstetrics and Gynecology and Biological Chemistry, 6428 Medical Science I, 1150 West Medical Center Drive, University of Michigan Medical School, Ann Arbor, MI 48109, USA Received 18 October 2004; accepted 16 December 2004
Abstract Luteinizing hormone receptor (LHR) is a G-protein-coupled receptor that exerts its effects mainly through increased cAMP synthesis. Our previous studies have shown that a ovarian cytosolic protein, designated as LHR mRNA binding protein (LRBP) is an important regulator of the steady state levels of LHR expression. To test whether LHR mRNA expression is modulated by cAMP through LRBP activity, we used rolipram, a type IV phosphodiesterase inhibitor that is known to promote intracellular cAMP accumulation. On day 4 of pseudopregnancy, rats were treated with rolipram (1.25 mg/injection) to raise intracellular levels of cAMP. In order to maintain higher cAMP levels, up to four injections of rolipram were given, with the last injection 4 h before collecting the ovaries. Measurement of cAMP levels showed an increase (p ≤ 0.05) at 8, 12, and 24 h after rolipram injections at total dosages of 2.5, 3.75 and 5.0 mg/rat, respectively. Northern blot analysis of LHR mRNA showed that rolipram treatment also markedly reduced ovarian LHR mRNA levels by up to 75%. LHR mRNA binding activity of LRBP, assayed by RNA electrophoretic mobility shift analysis, using S-100 fractions from control or rolipram-treated ovaries showed increased LHR mRNA binding activity in the S-100 fractions from rolipram treated groups. These data indicate that chronic elevation of ovarian cAMP leads to a decreased expression of LHR mRNA with a concomitant increase in LHR mRNA binding activity of LRBP. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: LH receptor; Luteinizing hormone receptor mRNA; Phosphodiesterase inhibitor; Cyclic AMP; LH receptor mRNA binding protein; Rat ovary
1. Introduction The interaction of luteinizing hormone (LH) with its receptor leads to activation of adenylate cyclase through stimulatory guanine nucleotide regulatory protein (Gs protein) to increase cyclic AMP levels (Marsh, 1975; Menon and Gunaga, 1974). LH has also been shown to activate phospholipase C resulting in phosphoinositide breakdown to inositol tris phosphate and diacylglycerol (Davis et al., 1984; Gundermann et al., 1992). While the significance of reactions stimulated by cyclic AMP on steroidogenesis and ovulation has been well documented, the role of the products of phosphoinositide breakdown on the LH-mediated regulation of specific ovarian function is less well understood.
夽 This work is supported by NIH Grant HD06656. ∗
Corresponding author. Tel.: +1 734 764 8142; fax: +1 734 936 8617. E-mail address: [email protected] (K.M.J. Menon).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.12.009
LH receptor is a member of the rhodopsin/2 adrenergic receptor-like family A of G protein coupled receptors (Ascoli et al., 2002). The expression of LH/hCG receptor (LHR) in the growing follicle is stimulated by FSH (Zeleznik et al., 1974). In response to the preovulatory LH surge or in response to pharmacological doses of hCG, LHR has been shown to undergo transient down regulation as evidenced by the loss of cell surface receptor expression (Hoffman et al., 1991; Lapolt et al., 1990). During this process, the steady state levels of LHR mRNA show a dramatic decline starting at about 4 h, reaching non-detectable level at 24 h, followed by a full recovery of the mRNA which occurs by 48 h (Hoffman et al., 1991; Lapolt et al., 1990; Lu et al., 1993; Peegel et al., 1994). Further studies on the mechanism of the loss of the steady levels of LHR mRNA revealed an increase in LHR mRNA degradation with no change in transcription (Lu et al., 1993). It is now clear that regulated degradation of mRNA is a general strategy used by many cells as a mechanism to con-
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trol the expression of steady state levels of different mRNAs (Ross, 1995). A main feature of this mechanism is the binding of the mRNA by a specific mRNA binding protein. The specific sequence recognized by the mRNA binding protein resides either in the open reading frame or in the 5 or 3 untranslated region of the mRNA transcripts (Burd and Dreyfuss, 1994; Fan and Steitz, 1998; Klausner et al., 1993; Peng et al., 1998; Ross, 1995). In the case of LHR mRNA, we have identified a trans-acting protein that interacts with the pyrimidine-rich region of the coding region of the mRNA (Kash and Menon, 1998, 1999; Nair et al., 2002; Nair and Menon, 2004). Furthermore, using RNA electrophoretic gel mobility shift assay (REMSA), we have shown that the LHR mRNA binding protein, designated as LRBP, is up-regulated in the ovary in response to treatment with hCG when the LHR mRNA is down-regulated (Kash and Menon, 1998). The purpose of the present study was to determine whether the down-regulation of LHR mRNA and the up-regulation of LRBP activity are related to the ovarian levels of cyclic AMP under in vivo conditions. In the present studies we have employed rolipram, an inhibitor of type IV phosphodiesterase, the most ubiquitous form of phosphodiesterase expressed in the ovary (Conti, 2000), to increase the intracellular levels of cyclic AMP. Using this inhibitor we have examined if chronic elevation of cyclic AMP in the pseudopregnant corpus luteum mimics the effect of LH/hCG to down regulate LHR mRNA expression and up regulate the LHR mRNA binding activity of LRBP in the ovarian cytosolic fractions.
received two treatments, 12 h group received three injections and the 24 h group received four treatments. Control rats received equal volumes of vehicle at corresponding intervals as the treatment group. Three animals were used for each treatment, control or rolipram, at each time point. The animals were sacrificed by CO2 asphyxiation, ovaries were removed and further processed. The experimental protocols were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
2. Materials and methods
2.5. RNA extraction and Northern blot analysis
2.1. Reagents
Total RNA was extracted from tissues using the procedure of Chomczynski and Saachi (Klausner et al., 1993). Briefly, tissues were homogenized in a solution of guanidine isothiocyanate acidified with 2 M sodium acetate, pH 4.0 and extracted with water-saturated phenol and chloroformisoamyl alcohol (49:1). The RNA in the aqueous phase was precipitated overnight at −20 ◦ C using three volumes of ethanol. RNA was quantitated spectrophotometrically and the purity was determined by the ratio of A260/280. Northern blot analysis was essentially the same as that described by Sambrook et al. (Kash and Menon, 1998). Aliquots of total RNA were separated by electrophoresis in 1.2% agarose formaldehyde gels and transferred to nitrocellulose membrane. Blots were prehybridized for 2 h at 42 ◦ C in a solution containing 0.5 mg/ml salmon sperm DNA and 2 × hybridization buffer [1.5 M NaCl–0.1 M TES (pH 7.1)–0.1 M EDTA–2 × Denhardt’s], diluted 1:1 with deionized formamide. The probe (2 × 107 cpm) was hybridized to blots overnight at 42 ◦ C in fresh buffer. Hybridized blots were washed four times with 2 × SCC containing 0.1% sodium dodecyl sulfate (SDS) at room temperature 10 min each and once at 60 ◦ C (30 min). The washed blots were exposed to XAR film (Kodak, Rochester, NY) at −70 ◦ C in a Kodak XOmatic cassette with intensifying screens. The intensity of
Pregnant mare serum gonadotropin (PMSG) was purchased from Calbiochem (La Jolla, CA). Human chorionic gonadotropin (hCG), rolipram and heparin were obtained from Sigma (St. Louis, MO). [␣-32 P] deoxy CTP (3000 Ci/mmol) was purchased from MP Biomedicals Inc. (Irvine, CA). EDTA-free protease inhibitor mixture tablets and RNase T1 were from Roche (Indianapolis, IN). [␣-32 P] UTP (800 Ci/mol) was obtained from Perkin Elmer Life Sciences (Boston, MA). MAXI script kit was from Ambion (Austin, TX). RNasin was obtained from Promega (Madison, WI). BCA Protein Assay reagents were from Pierce (Rockford, IL). All other chemicals used were of analytical grade. 2.2. Animals Pseudopregnancy was induced in immature female rats (Sprague-Dawley, Harlan, Indianapolis, IN) by subcutaneous injection of 50 IU of pregnant mare serum gonadotropin (PMSG) followed by 25 IU of human chorionic gonadotropin (hCG) 56 h later. The day of hCG injection was taken as day 0. On day 4, the animals were treated with subcutaneous injections of rolipram (1.25 mg) at 4 h intervals. The 8 h group
2.3. cAMP Assay cAMP was measured by following the protocol in the cAMP radioimmunoassay (RIA) Kit from Biomedical Technologies Inc. (Stoughton, MA). Briefly, tissues were extracted by homogenizing with 6% trichloroacetic acid (TCA), clarified by centrifugation and the supernatants extracted with water-saturated ether. Trace amounts of [3 H] cAMP were added to monitor recovery. After removing ether under a stream of nitrogen, samples were reconstituted in working buffer for RIA. 2.4. Labelling of probes for Northern blot analysis LH/hCG receptor, 18S rRNA and SCC probes were radiolabelled with [␣-32 P] dCTP using Invitrogen RadPrime DNA labelling Kit (Carlsbad, CA). Unincorporated radioactivity was removed using Quick Spin Sephadex G-50 spin columns (Roche, Indianapolis, IN).
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the bands was measured using the Arcus II scanner (Agfa) and quantitated using the NIH Image 1.61 program. After the blots were stripped, they were rehybridized with radiolabelled 18S rRNA to monitor total RNA loading. 2.6. RNA electrophoretic mobility shift analysis RNA electrophoretic mobility shift analysis (REMSA) was performed as described previously (Kash and Menon, 1998). Briefly, 10 g of cytosolic S-100 protein samples were incubated with 1 × 105 cpm of [␣-32 P] UTP labelled RNA, the LRBP binding sequence (LBS-188–228), in homogenization buffer A (10 mM HEPES, pH, 7.9, 0.5 mM MgCl2 , 50 M EDTA, 5 mM DTT and 10% glycerol containing 50 mM KCl and protease inhibitor mixture) in the presence of 5 g of tRNA and 40 units of RNasin at 30 ◦ C for 20 min. Unprotected radiolabelled RNA was then degraded by the addition of 20 units of RNase T1 at 37 ◦ C for 30 min. Samples were then incubated with heparin at a final concentration of 5 mg/ml for 10 min on ice to decrease 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 and visualized by autoradiography. 2.7. Morphological integrity of ovaries after rolipram treatment Ovaries from day 4 pseudopregnant rats injected with vehicle or with rolipram were embedded in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, IN) on dry ice and stored at −80 ◦ C. Frozen sections of 10 m were cut, mounted and stained with hematoxylin–eosin. Morphological integrity was further tested by measuring LHR mRNA by Northern blot analysis 24–48 h after the treatments. 2.8. Statistical analysis The statistical analysis of the cAMP tissue levels was performed by standard one-way analysis of variance, followed by Tukey’s test for multiple pair-wise comparisons. Statistical significance was accepted at p < 0.05.
3. Results 3.1. Effect of chronic inhibition of type IV PDE on intraovarian cAMP concentrations The chronic administration of rolipram, an inhibitor of the type IV phosphodiesterase (PDE), was expected to promote the accumulation of intracellular cAMP concentrations and thus bypass LH receptor-mediated enhancement of cyclic AMP production. The effect of rolipram to chronically inhibit type IV PDE and promote the accumulation of intraovarian cAMP was therefore examined. The results of these experi-
Fig. 1. Effect of rolipram treatment on intraovarian cAMP concentrations. Ovaries were collected from vehicle-injected control (C) and rolipramtreated (R) rats at 8, 12 and 24 h following the first rolipram or vehicle injection. The ovaries were homogenized and cAMP was extracted and subsequently measured by RIA. The values shown were normalized on the basis of the protein concentration in the homogenates. Columns stemming from the bars represent the standard error of the means of three samples from each treatment group.
ments are described in Fig. 1. The concentrations of cAMP in the ovaries of rolipram-treated rats were higher (p < .001) at 8, 12 and 24 h time-intervals selected. Actual mean values ± standard errors, expressed in pmol cAMP/mg protein were 8.6 ± 0.29 for the pseudopregnant controls treated with vehicle (C) versus 9.9 ± 0.07 for pseudopregnant rats treated with rolipram (R) at the 8 h time interval; 8.4 ± 0.08 for C versus 10.4 ± 0.05 for R at 12 h and 8.1 ± 0.06 For C versus 12.2 ± 0.07 for R at the 24 h time interval. Since no exogenous agonists were given to stimulate cAMP production, the extent of stimulation of cAMP accumulation by treatment with rolipram alone was lower than what would be expected with hCG treatment. However, the cAMP levels in rolipramtreated animals were significantly higher than the controls. 3.2. Effect of rolipram treatment on ovarian levels of LH receptor mRNA The effect of chronic elevation in cAMP levels produced by rolipram treatment on the levels of LHR mRNA were then determined. Fig. 2 illustrates the expression of LHR mRNA in the ovaries of rolipram-treated and control rats at 8, 12 and 24 h following rolipram injection. The top panel presents the autoradiograph of the expression of the LHR mRNA and shows a sharp decrease in the expression of the receptor mRNA in the ovaries of rolipram-treated rats compared to controls at all time intervals examined. The bottom panel depicts the densitometric scans of the 6.7 kb transcript of the LHR mRNA normalized to the density of the bands for the 18S rRNA (middle panel). The results show that the
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Fig. 2. Down regulation of the LH receptor mRNA following rolipram treatment. The top panel is the autoradiograph of the Northern blot of the LH receptor mRNA from the ovaries of vehicle-injected (C) or rolipram-injected (R) rats at 8, 12 and 24 h after the first injection of vehicle or the PDE inhibitor. Total RNA was isolated from the ovaries and 25 g RNA were loaded on 1.2% agarose-formaldehyde gels and subsequently transferred to a nitrocellulose membrane following electrophoresis. The densitometric scans of the bands of the 6.7 kb transcript of LHR (upper panel) were standardized to the density of the autoradiographic signal for the 18S rRNA (middle panel). The lower panel depicts the magnitude of the inhibitory effect of rolipram treatment in arbitrary densitometric units. Bars represent the mean ± S.E.M. of six scannings of the autoradiograph. The blot is a representative of three experiments.
ovaries of the rats treated with the PDE inhibitor exhibited a marked, consistent decrease of up to 75% in the expression of the LHR mRNA. The sharp decrease in LHR mRNA suggests that the increase in the intracellular accumulation of cyclic AMP produced in response to rolipram treatment causes the down-regulation of LHR mRNA. 3.3. Determination of selectivity of type IV phosphodiesterase inhibition on LH receptor mRNA down-regulation The specificity of the effect of rolipram in decreasing the expression of LHR mRNA was then examined. To test the
Fig. 3. Expression of p450scc mRNA in the ovaries following rolipram treatment. The top panel shows the autoradiograph of the Northern blot of the mRNA of p450scc from the ovaries of control, vehicle injected rats (C) compared to the ovaries from rats treated with rolipram (R). The ovaries were collected 12 and 24 h after the first administration of vehicle or the PDE inhibitor. Total RNA was isolated from the ovaries and 25 g were run in each lane on a 1.2% agarose-formaldehyde gel and transferred to a nitrocellulose membrane following electrophoresis. The lower panel depicts the densitometric scanning of the autoradiograph in arbitrary densitometric units, normalized to 18S rRNA. Bars represent the mean ± S.E.M. of three scannings of the autoradiograph. The blot is a representative of three experiments.
specificity, rats were treated with rolipram or vehicle as described previously and the ovaries were collected at 12 and 24 h for the assay of the expression of the mRNA of p450scc by Northern hybridization. The autoradiography and densitometric scanning of Fig. 3 illustrate the results of these studies. The images show that inhibition of the type IV PDE did not lead to a decrease in the expression of p450scc but instead resulted in an increase in the expression of this key steroidogenic enzyme in the rolipram-treated animals compared to vehicle-treated control rats. This increase is consistent with an increase in P450scc mRNA levels seen following hCG injection (Lu et al., 1993). These results indicate that the effect of cAMP to down-regulate LHR mRNA is specific.
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3.4. Assessment of potential toxicity of rolipram administration The possibility that the decline in LHR mRNA expression in response to rolipram treatment is perhaps due to a potential toxic effect of the PDE inhibitor on the ovarian tissues was then examined. This was explored by examining the morphology of the ovaries of rolipram-treated rats compared to control rats. Fig. 4 shows microscopic images of representative areas of ovarian tissue sections of rolipram-
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treated (panel A), and control rats treated with vehicle (panel B). The sections were stained with hematoxylin/eosin and the images clearly show that the ovaries of rolipram-treated rats exhibited normal microscopic structures, similar to the healthy morphology observed in the ovaries of the control rats. The physiological integrity of the ovaries of rolipramtreated rats was also assessed by testing the recovery of LHR mRNA expression to pre-treatment levels between 24 and 48 h following the administration of the PDE inhibitor using Northern blot analysis (data not shown). The results showed
Fig. 4. Microscopic appearance of ovaries of rolipram-treated rats. The images show representative ovarian frozen sections from control vehicle-injected rat (panel B) and rolipram-treated rat (panel A). The sections were cut to a thickness of 10 m and were stained with hematoxylin/eosin, g: granulosa cells; ti: theca-interstitial cells; cl: corpus luteum.
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that the decreases in LHR mRNA expression seen in response to rolipram treatment were transient and returned to pretreatment levels comparable to those found in the control rats. 3.5. Evaluation of LHR mRNA binding activity of LRBP following inhibition of type IV PDE The results showing a decreased expression of the LH receptor mRNA in the rolipram-treated rats underscored the effects of a chronic increase in ovarian cAMP on LHR mRNA expression. Previous reports from our laboratory have shown that the decreased expression of LH receptor mRNA following the administration of hCG is associated with an increase in the RNA binding activity of LH receptor mRNA binding protein (LRBP) (Kash and Menon, 1998). Therefore, we conducted additional studies to examine if rolipram treatment could also mimic the effect of LH or hCG in stimulating the activity of LRBP which is a modulator of hCG-induced LHR mRNA down-regulation. This possibility was investigated by treating rats with rolipram or vehicle and determining the activity of the LRBP by RNA electrophoretic mobility shift as-
Fig. 5. Activity of the LH receptor mRNA binding protein in ovaries of control and rolipram-treated rats. The autoradiograph is a representative RNA electrophoretic mobility shift assay (REMSA) conducted with 1 × 105 cpm of radiolabelled RNA, the LRBP binding sequence (LBS-188–228), and incubated with no protein (NP): lane 1, which is the (−) control, or with 10 g of cytosolic S-100 fractions of ovaries from vehicle-injected control animals (C); lane 2, which is the (+) control, or from rolipram-treated rats (R); lane 3, 8 h after the administration of the PDE inhibitor. The samples were then treated with RNase T1 to eliminate unprotected RNA and were then resolved by electrophoresis on a 5% native polyacrylamide gel. The samples were subsequently visualized by autoradiography. The blot is a representative of three experiments.
say (REMSA). The REMSA of LRBP activity was conducted with the cytosolic (S-100) fractions of the ovaries incubated with the radiolabelled LHR mRNA sequence (nucleotides 188–228) which contains the target sequence for the binding of LRBP. The 47 kDa bands of LRBP-RNA complexes were then visualized by autoradiography. The results of these experiments are presented in Fig. 5. The autoradiograph of the REMSA is a representative image of the effect of rolipram administration on RNA binding activity of LRBP in the ovaries of rolipram- and vehicle-treated rats 8 h after treatment. These results show that the mRNA binding activity of LRBP was enhanced by treatment with rolipram. The assessment of LRBP activity indicated that rolipram caused an increase in the RNA binding activity. The increase in LRBP activity at 8 h after the first rolipram dosage is also in agreement with the decrease in LHR mRNA expression observed at the same time interval (Fig. 2) and with the increased concentrations of intraovarian cAMP depicted in Fig. 1 in the rolipram-treated group.
4. Discussion Our laboratory has previously reported that ligandinduced down regulation of LHR mRNA occurs through a post-transcriptional mechanism and this process is accompanied by an increase in the binding activity of a cytosolic protein termed LH receptor mRNA binding protein (LRBP) to the LHR mRNA (Hoffman et al., 1991; Kash and Menon, 1998, 1999; Lu et al., 1993; Nair et al., 2002; Nair and Menon, 2004). Furthermore, we have shown an inverse correlation between LHR mRNA expression and LRBP binding activity during the course of follicle maturation, ovulation and the life span of the corpus luteum (Nair et al., 2002). A major pathway by which the activation of the LH receptor, a G protein coupled receptor, leads to the stimulation of ovarian function is mediated through the cAMP-PKA pathway (Marsh, 1975; Menon and Gunaga, 1974), although LH may also exert some of its actions through the Akt/PKB cascade (Salvador et al., 2002). The present studies were designed to determine whether the down-regulation of the LH receptor mRNA expression is mediated by cAMP through modulation of LRBP activity. The overall approach to bypass the activation of the LH receptor by its ligands involved the repeated administration of rolipram, a type IV PDE inhibitor, in order to promote the intra-ovarian accumulation of cAMP. The selection of a type IV PDE inhibitor was based on a body of evidence showing that this is the major phosphodiesterase present in the granulosa cells in the ovary (Conti et al., 2002; Jin et al., 1999; Thomas et al., 2002; Tsafriri et al., 1996). Additionally, it has been shown that inhibition of type IV PDE can enhance LHinduction of oocyte maturation in cultured follicles (Tsafriri et al., 1996). Our results showing a moderate but chronic and consistent enhancement of intraovarian concentrations of cAMP in response to treatment with rolipram indeed sug-
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gest that Type IV phosphodiesterase plays a role in regulating the intracellular cAMP in the ovary. The sharp decreases in the expression of the LHR mRNA in response to treatment with rolipram clearly show that the down-regulation of LHR mRNA following hCG injection is mediated through increased cyclic AMP production (Hoffman et al., 1991; Kash and Menon, 1998, 1999; Nair et al., 2002). The repeated dosage regimen utilized for the administration of rolipram is at the upper range of the dosages reported in the literature (Krause and Kuhne, 1998; Larson et al., 1996). The repeated approach was considered necessary in view of the short half-life reported for rolipram in rats (Krause and Kuhne, 1998) and the need to ensure a chronic inhibition of the type IV PDE in the experiments. The consequence of this dosage regimen also made it important to assess whether there were any toxic compounding effects involved in the experiments. This possibility we assessed by examining the ovarian morphology. The morphology of the ovaries shown in the hematoxylin/eosin images revealed normal, healthy corpora lutea, granulosa cells and theca cells in the ovaries of the rolipram-treated rats compared to the controls. Additionally, the studies conducted to determine the recovery of expression of the LH receptor mRNA indicated that the LHR mRNA level exhibited a return to normal, pre-treatment ranges after 24–48 h, indicating a transient inhibitory effect of rolipram without producing an adverse toxic effect (Peegel et al., 1994). The effect of rolipram was specific for LHR mRNA since the expression of the mRNA for the key steroidogenic enzyme p450scc did not show any decrease in response to rolipram but interestingly showed an increase at the 12 h time interval. This increase is also significant since it would be expected as a normal response to the increased concentrations of cAMP, in the transduction of the LH signal for ovarian steroid synthesis. This result also underscores the preservation of normal ovarian physiology in the ovaries of the rats during the administration of rolipram and indicates a selective effect of the chronic Type IV PDE inhibition in the down-regulation of the mRNA for the LH receptor. Previous reports from our laboratory showed an increase in LHR mRNA binding activity of an ovarian cytosolic protein specific for the LH receptor mRNA (Kash and Menon, 1999). These reports also suggest a major role for this protein in promoting the degradation of the LHR mRNA as a mechanism for the modulation of the expression of the LH receptor (Nair et al., 2002). The experiments conducted in the present study show that the administration of the PDE inhibitor also results in an increase in LHR mRNA binding activity of LRBP. The REMSA assay showed an increased LRBP binding activity in a representative sample taken at the 8 h time-interval. At the 8 h time interval, rolipram treatment showed an increase in cAMP accumulation as well as a drastic reduction in the LH receptor mRNA. Thus, as previously reported (Nair et al., 2002), the decline in LHR mRNA expression and the induction of the LHR mRNA binding protein activity caused by hCG administration can be mimicked by increasing the
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intracellular cAMP level produced in response to treatment with phosphodiesterase inhibitor (Nair et al., 2002). Furthermore, hCG-induced changes in LHR mRNA expression can be mimicked by increasing the intracellular cAMP concentration in vivo suggesting that cAMP plays an intermediary role in the down-regulation of LHR mRNA levels in the ovary. Acknowledgment The authors wish to thank Nicole Acevedo for helpful assistance with photography of the ovarian histologic sections. References Ascoli, M., Fanelli, F., Segaloff, D., 2002. The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocrinol. Rev. 23, 141– 174. Burd, C., Dreyfuss, G., 1994. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621. Conti, M., 2000. Phosphodiesterases and cyclic nucleotide signaling in endocrine cells. Mol. Endocrinol. 14, 1317–1327. Conti, M., Andersen, C.B., Richard, F., Mehats, C., Chun, S.Y., Horner, K., Jin, C., Tsafriri, A., 2002. Role of cyclic nucleotide signaling in oocyte maturation. Mol. Cell Endocrinol. 187, 153–159. Davis, J., West, L., Farese, R., 1984. Effects of luteinizing hormone on phosphoionostitde metabolism in rat granuloda cells. J. Biol. Chem. 259, 15028–15034. Fan, X., Steitz, J., 1998. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases in in vivo stability of ARE-containing mRNAs. EMBO J. 17, 3448–3460. Gundermann, T., Birnbaumer, M., Birnbaumer, L., 1992. Evidence of dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoiositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J. Biol. Chem. 267, 4479–4488. Hoffman, Y., Peegel, H., Sprock, M., Zhang, Q., Menon, K.M.J., 1991. Evidence that human chorionic gonadotropin/luteinzing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocriniology 128, 388–393. Jin, S., Richard, F., Kuo, W., D’Ercole, A., Conti, M., 1999. Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4Ddeficient mice. Proc. Natl. Acad. Sci. U.S.A.. Kash, J., Menon, K.M.J., 1998. Identification of a hormonally regulated luteinizing hormone/human chorionc gonadotropin receptor mRNA binding protein. J. Biol. Chem. 273, 10658–10664. Kash, J., Menon, K.M.J., 1999. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame. Biochemistry 38, 16889–16897. Klausner, R., Rouault, T., Harford, J., 1993. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72, 19–28. Krause, W., Kuhne, G., 1998. Pharmacokinetics of rolipram in the rhesus and cynomolgus monkeys, the rat and the rabbit. Studies on species differences. Xenobiotica 18, 561–571. Lapolt, P., Oikawa, M., Jia, X., Dargan, C., Hsueh, A., 1990. Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 126, 3277–3279. Larson, J., Ino, M., Geiger, L., Simeone, C., 1996. The toxicity of repeated exposures to rolipram, a type IV phosphodiesterase inhibitor, in rats. Pharmacol. Toxicol. 78, 44–49. Lu, D., Peegel, H., Mosier, S., Menon, K.M.J., 1993. Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs posttranscriptionally. Endocrinology 132, 235–240.
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Ross, J., 1995. mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450. Salvador, L., Maizels, E., Hales, D., Miyamoto, E., Yamamoto, H., Hunzicker-Dunn, M., 2002. Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 143, 2986–2994. Thomas, R., Armstrong, D., Gilchrist, R., 2002. Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev. Biol. 244, 215–225. Tsafriri, A., Chun, S., Zhang, R., Hsueh, A., Conti, M., 1996. Oocye maturation involves comparatmentalization and opposing changes of cAMP levels in follicular somatic and term cells: Studies using selective phosphodiesterase inhibitors. Dev. Biol. 178, 393– 402. Zeleznik, A., Midgley Jr., A., Reichert Jr., L., 1974. Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormone in vivo. Endorcinology 95, 818–825.
A novel mechanism for the modulation of luteinizing hormone receptor mRNA expression in the rat ovary夽 H. Peegel, R. Towns, A. Nair, K.M.J. Menon ∗ Departments of Obstetrics and Gynecology and Biological Chemistry, 6428 Medical Science I, 1150 West Medical Center Drive, University of Michigan Medical School, Ann Arbor, MI 48109, USA Received 18 October 2004; accepted 16 December 2004
Abstract Luteinizing hormone receptor (LHR) is a G-protein-coupled receptor that exerts its effects mainly through increased cAMP synthesis. Our previous studies have shown that a ovarian cytosolic protein, designated as LHR mRNA binding protein (LRBP) is an important regulator of the steady state levels of LHR expression. To test whether LHR mRNA expression is modulated by cAMP through LRBP activity, we used rolipram, a type IV phosphodiesterase inhibitor that is known to promote intracellular cAMP accumulation. On day 4 of pseudopregnancy, rats were treated with rolipram (1.25 mg/injection) to raise intracellular levels of cAMP. In order to maintain higher cAMP levels, up to four injections of rolipram were given, with the last injection 4 h before collecting the ovaries. Measurement of cAMP levels showed an increase (p ≤ 0.05) at 8, 12, and 24 h after rolipram injections at total dosages of 2.5, 3.75 and 5.0 mg/rat, respectively. Northern blot analysis of LHR mRNA showed that rolipram treatment also markedly reduced ovarian LHR mRNA levels by up to 75%. LHR mRNA binding activity of LRBP, assayed by RNA electrophoretic mobility shift analysis, using S-100 fractions from control or rolipram-treated ovaries showed increased LHR mRNA binding activity in the S-100 fractions from rolipram treated groups. These data indicate that chronic elevation of ovarian cAMP leads to a decreased expression of LHR mRNA with a concomitant increase in LHR mRNA binding activity of LRBP. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: LH receptor; Luteinizing hormone receptor mRNA; Phosphodiesterase inhibitor; Cyclic AMP; LH receptor mRNA binding protein; Rat ovary
1. Introduction The interaction of luteinizing hormone (LH) with its receptor leads to activation of adenylate cyclase through stimulatory guanine nucleotide regulatory protein (Gs protein) to increase cyclic AMP levels (Marsh, 1975; Menon and Gunaga, 1974). LH has also been shown to activate phospholipase C resulting in phosphoinositide breakdown to inositol tris phosphate and diacylglycerol (Davis et al., 1984; Gundermann et al., 1992). While the significance of reactions stimulated by cyclic AMP on steroidogenesis and ovulation has been well documented, the role of the products of phosphoinositide breakdown on the LH-mediated regulation of specific ovarian function is less well understood.
夽 This work is supported by NIH Grant HD06656. ∗
Corresponding author. Tel.: +1 734 764 8142; fax: +1 734 936 8617. E-mail address: [email protected] (K.M.J. Menon).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.12.009
LH receptor is a member of the rhodopsin/2 adrenergic receptor-like family A of G protein coupled receptors (Ascoli et al., 2002). The expression of LH/hCG receptor (LHR) in the growing follicle is stimulated by FSH (Zeleznik et al., 1974). In response to the preovulatory LH surge or in response to pharmacological doses of hCG, LHR has been shown to undergo transient down regulation as evidenced by the loss of cell surface receptor expression (Hoffman et al., 1991; Lapolt et al., 1990). During this process, the steady state levels of LHR mRNA show a dramatic decline starting at about 4 h, reaching non-detectable level at 24 h, followed by a full recovery of the mRNA which occurs by 48 h (Hoffman et al., 1991; Lapolt et al., 1990; Lu et al., 1993; Peegel et al., 1994). Further studies on the mechanism of the loss of the steady levels of LHR mRNA revealed an increase in LHR mRNA degradation with no change in transcription (Lu et al., 1993). It is now clear that regulated degradation of mRNA is a general strategy used by many cells as a mechanism to con-
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trol the expression of steady state levels of different mRNAs (Ross, 1995). A main feature of this mechanism is the binding of the mRNA by a specific mRNA binding protein. The specific sequence recognized by the mRNA binding protein resides either in the open reading frame or in the 5 or 3 untranslated region of the mRNA transcripts (Burd and Dreyfuss, 1994; Fan and Steitz, 1998; Klausner et al., 1993; Peng et al., 1998; Ross, 1995). In the case of LHR mRNA, we have identified a trans-acting protein that interacts with the pyrimidine-rich region of the coding region of the mRNA (Kash and Menon, 1998, 1999; Nair et al., 2002; Nair and Menon, 2004). Furthermore, using RNA electrophoretic gel mobility shift assay (REMSA), we have shown that the LHR mRNA binding protein, designated as LRBP, is up-regulated in the ovary in response to treatment with hCG when the LHR mRNA is down-regulated (Kash and Menon, 1998). The purpose of the present study was to determine whether the down-regulation of LHR mRNA and the up-regulation of LRBP activity are related to the ovarian levels of cyclic AMP under in vivo conditions. In the present studies we have employed rolipram, an inhibitor of type IV phosphodiesterase, the most ubiquitous form of phosphodiesterase expressed in the ovary (Conti, 2000), to increase the intracellular levels of cyclic AMP. Using this inhibitor we have examined if chronic elevation of cyclic AMP in the pseudopregnant corpus luteum mimics the effect of LH/hCG to down regulate LHR mRNA expression and up regulate the LHR mRNA binding activity of LRBP in the ovarian cytosolic fractions.
received two treatments, 12 h group received three injections and the 24 h group received four treatments. Control rats received equal volumes of vehicle at corresponding intervals as the treatment group. Three animals were used for each treatment, control or rolipram, at each time point. The animals were sacrificed by CO2 asphyxiation, ovaries were removed and further processed. The experimental protocols were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
2. Materials and methods
2.5. RNA extraction and Northern blot analysis
2.1. Reagents
Total RNA was extracted from tissues using the procedure of Chomczynski and Saachi (Klausner et al., 1993). Briefly, tissues were homogenized in a solution of guanidine isothiocyanate acidified with 2 M sodium acetate, pH 4.0 and extracted with water-saturated phenol and chloroformisoamyl alcohol (49:1). The RNA in the aqueous phase was precipitated overnight at −20 ◦ C using three volumes of ethanol. RNA was quantitated spectrophotometrically and the purity was determined by the ratio of A260/280. Northern blot analysis was essentially the same as that described by Sambrook et al. (Kash and Menon, 1998). Aliquots of total RNA were separated by electrophoresis in 1.2% agarose formaldehyde gels and transferred to nitrocellulose membrane. Blots were prehybridized for 2 h at 42 ◦ C in a solution containing 0.5 mg/ml salmon sperm DNA and 2 × hybridization buffer [1.5 M NaCl–0.1 M TES (pH 7.1)–0.1 M EDTA–2 × Denhardt’s], diluted 1:1 with deionized formamide. The probe (2 × 107 cpm) was hybridized to blots overnight at 42 ◦ C in fresh buffer. Hybridized blots were washed four times with 2 × SCC containing 0.1% sodium dodecyl sulfate (SDS) at room temperature 10 min each and once at 60 ◦ C (30 min). The washed blots were exposed to XAR film (Kodak, Rochester, NY) at −70 ◦ C in a Kodak XOmatic cassette with intensifying screens. The intensity of
Pregnant mare serum gonadotropin (PMSG) was purchased from Calbiochem (La Jolla, CA). Human chorionic gonadotropin (hCG), rolipram and heparin were obtained from Sigma (St. Louis, MO). [␣-32 P] deoxy CTP (3000 Ci/mmol) was purchased from MP Biomedicals Inc. (Irvine, CA). EDTA-free protease inhibitor mixture tablets and RNase T1 were from Roche (Indianapolis, IN). [␣-32 P] UTP (800 Ci/mol) was obtained from Perkin Elmer Life Sciences (Boston, MA). MAXI script kit was from Ambion (Austin, TX). RNasin was obtained from Promega (Madison, WI). BCA Protein Assay reagents were from Pierce (Rockford, IL). All other chemicals used were of analytical grade. 2.2. Animals Pseudopregnancy was induced in immature female rats (Sprague-Dawley, Harlan, Indianapolis, IN) by subcutaneous injection of 50 IU of pregnant mare serum gonadotropin (PMSG) followed by 25 IU of human chorionic gonadotropin (hCG) 56 h later. The day of hCG injection was taken as day 0. On day 4, the animals were treated with subcutaneous injections of rolipram (1.25 mg) at 4 h intervals. The 8 h group
2.3. cAMP Assay cAMP was measured by following the protocol in the cAMP radioimmunoassay (RIA) Kit from Biomedical Technologies Inc. (Stoughton, MA). Briefly, tissues were extracted by homogenizing with 6% trichloroacetic acid (TCA), clarified by centrifugation and the supernatants extracted with water-saturated ether. Trace amounts of [3 H] cAMP were added to monitor recovery. After removing ether under a stream of nitrogen, samples were reconstituted in working buffer for RIA. 2.4. Labelling of probes for Northern blot analysis LH/hCG receptor, 18S rRNA and SCC probes were radiolabelled with [␣-32 P] dCTP using Invitrogen RadPrime DNA labelling Kit (Carlsbad, CA). Unincorporated radioactivity was removed using Quick Spin Sephadex G-50 spin columns (Roche, Indianapolis, IN).
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the bands was measured using the Arcus II scanner (Agfa) and quantitated using the NIH Image 1.61 program. After the blots were stripped, they were rehybridized with radiolabelled 18S rRNA to monitor total RNA loading. 2.6. RNA electrophoretic mobility shift analysis RNA electrophoretic mobility shift analysis (REMSA) was performed as described previously (Kash and Menon, 1998). Briefly, 10 g of cytosolic S-100 protein samples were incubated with 1 × 105 cpm of [␣-32 P] UTP labelled RNA, the LRBP binding sequence (LBS-188–228), in homogenization buffer A (10 mM HEPES, pH, 7.9, 0.5 mM MgCl2 , 50 M EDTA, 5 mM DTT and 10% glycerol containing 50 mM KCl and protease inhibitor mixture) in the presence of 5 g of tRNA and 40 units of RNasin at 30 ◦ C for 20 min. Unprotected radiolabelled RNA was then degraded by the addition of 20 units of RNase T1 at 37 ◦ C for 30 min. Samples were then incubated with heparin at a final concentration of 5 mg/ml for 10 min on ice to decrease 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 and visualized by autoradiography. 2.7. Morphological integrity of ovaries after rolipram treatment Ovaries from day 4 pseudopregnant rats injected with vehicle or with rolipram were embedded in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, IN) on dry ice and stored at −80 ◦ C. Frozen sections of 10 m were cut, mounted and stained with hematoxylin–eosin. Morphological integrity was further tested by measuring LHR mRNA by Northern blot analysis 24–48 h after the treatments. 2.8. Statistical analysis The statistical analysis of the cAMP tissue levels was performed by standard one-way analysis of variance, followed by Tukey’s test for multiple pair-wise comparisons. Statistical significance was accepted at p < 0.05.
3. Results 3.1. Effect of chronic inhibition of type IV PDE on intraovarian cAMP concentrations The chronic administration of rolipram, an inhibitor of the type IV phosphodiesterase (PDE), was expected to promote the accumulation of intracellular cAMP concentrations and thus bypass LH receptor-mediated enhancement of cyclic AMP production. The effect of rolipram to chronically inhibit type IV PDE and promote the accumulation of intraovarian cAMP was therefore examined. The results of these experi-
Fig. 1. Effect of rolipram treatment on intraovarian cAMP concentrations. Ovaries were collected from vehicle-injected control (C) and rolipramtreated (R) rats at 8, 12 and 24 h following the first rolipram or vehicle injection. The ovaries were homogenized and cAMP was extracted and subsequently measured by RIA. The values shown were normalized on the basis of the protein concentration in the homogenates. Columns stemming from the bars represent the standard error of the means of three samples from each treatment group.
ments are described in Fig. 1. The concentrations of cAMP in the ovaries of rolipram-treated rats were higher (p < .001) at 8, 12 and 24 h time-intervals selected. Actual mean values ± standard errors, expressed in pmol cAMP/mg protein were 8.6 ± 0.29 for the pseudopregnant controls treated with vehicle (C) versus 9.9 ± 0.07 for pseudopregnant rats treated with rolipram (R) at the 8 h time interval; 8.4 ± 0.08 for C versus 10.4 ± 0.05 for R at 12 h and 8.1 ± 0.06 For C versus 12.2 ± 0.07 for R at the 24 h time interval. Since no exogenous agonists were given to stimulate cAMP production, the extent of stimulation of cAMP accumulation by treatment with rolipram alone was lower than what would be expected with hCG treatment. However, the cAMP levels in rolipramtreated animals were significantly higher than the controls. 3.2. Effect of rolipram treatment on ovarian levels of LH receptor mRNA The effect of chronic elevation in cAMP levels produced by rolipram treatment on the levels of LHR mRNA were then determined. Fig. 2 illustrates the expression of LHR mRNA in the ovaries of rolipram-treated and control rats at 8, 12 and 24 h following rolipram injection. The top panel presents the autoradiograph of the expression of the LHR mRNA and shows a sharp decrease in the expression of the receptor mRNA in the ovaries of rolipram-treated rats compared to controls at all time intervals examined. The bottom panel depicts the densitometric scans of the 6.7 kb transcript of the LHR mRNA normalized to the density of the bands for the 18S rRNA (middle panel). The results show that the
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Fig. 2. Down regulation of the LH receptor mRNA following rolipram treatment. The top panel is the autoradiograph of the Northern blot of the LH receptor mRNA from the ovaries of vehicle-injected (C) or rolipram-injected (R) rats at 8, 12 and 24 h after the first injection of vehicle or the PDE inhibitor. Total RNA was isolated from the ovaries and 25 g RNA were loaded on 1.2% agarose-formaldehyde gels and subsequently transferred to a nitrocellulose membrane following electrophoresis. The densitometric scans of the bands of the 6.7 kb transcript of LHR (upper panel) were standardized to the density of the autoradiographic signal for the 18S rRNA (middle panel). The lower panel depicts the magnitude of the inhibitory effect of rolipram treatment in arbitrary densitometric units. Bars represent the mean ± S.E.M. of six scannings of the autoradiograph. The blot is a representative of three experiments.
ovaries of the rats treated with the PDE inhibitor exhibited a marked, consistent decrease of up to 75% in the expression of the LHR mRNA. The sharp decrease in LHR mRNA suggests that the increase in the intracellular accumulation of cyclic AMP produced in response to rolipram treatment causes the down-regulation of LHR mRNA. 3.3. Determination of selectivity of type IV phosphodiesterase inhibition on LH receptor mRNA down-regulation The specificity of the effect of rolipram in decreasing the expression of LHR mRNA was then examined. To test the
Fig. 3. Expression of p450scc mRNA in the ovaries following rolipram treatment. The top panel shows the autoradiograph of the Northern blot of the mRNA of p450scc from the ovaries of control, vehicle injected rats (C) compared to the ovaries from rats treated with rolipram (R). The ovaries were collected 12 and 24 h after the first administration of vehicle or the PDE inhibitor. Total RNA was isolated from the ovaries and 25 g were run in each lane on a 1.2% agarose-formaldehyde gel and transferred to a nitrocellulose membrane following electrophoresis. The lower panel depicts the densitometric scanning of the autoradiograph in arbitrary densitometric units, normalized to 18S rRNA. Bars represent the mean ± S.E.M. of three scannings of the autoradiograph. The blot is a representative of three experiments.
specificity, rats were treated with rolipram or vehicle as described previously and the ovaries were collected at 12 and 24 h for the assay of the expression of the mRNA of p450scc by Northern hybridization. The autoradiography and densitometric scanning of Fig. 3 illustrate the results of these studies. The images show that inhibition of the type IV PDE did not lead to a decrease in the expression of p450scc but instead resulted in an increase in the expression of this key steroidogenic enzyme in the rolipram-treated animals compared to vehicle-treated control rats. This increase is consistent with an increase in P450scc mRNA levels seen following hCG injection (Lu et al., 1993). These results indicate that the effect of cAMP to down-regulate LHR mRNA is specific.
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3.4. Assessment of potential toxicity of rolipram administration The possibility that the decline in LHR mRNA expression in response to rolipram treatment is perhaps due to a potential toxic effect of the PDE inhibitor on the ovarian tissues was then examined. This was explored by examining the morphology of the ovaries of rolipram-treated rats compared to control rats. Fig. 4 shows microscopic images of representative areas of ovarian tissue sections of rolipram-
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treated (panel A), and control rats treated with vehicle (panel B). The sections were stained with hematoxylin/eosin and the images clearly show that the ovaries of rolipram-treated rats exhibited normal microscopic structures, similar to the healthy morphology observed in the ovaries of the control rats. The physiological integrity of the ovaries of rolipramtreated rats was also assessed by testing the recovery of LHR mRNA expression to pre-treatment levels between 24 and 48 h following the administration of the PDE inhibitor using Northern blot analysis (data not shown). The results showed
Fig. 4. Microscopic appearance of ovaries of rolipram-treated rats. The images show representative ovarian frozen sections from control vehicle-injected rat (panel B) and rolipram-treated rat (panel A). The sections were cut to a thickness of 10 m and were stained with hematoxylin/eosin, g: granulosa cells; ti: theca-interstitial cells; cl: corpus luteum.
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that the decreases in LHR mRNA expression seen in response to rolipram treatment were transient and returned to pretreatment levels comparable to those found in the control rats. 3.5. Evaluation of LHR mRNA binding activity of LRBP following inhibition of type IV PDE The results showing a decreased expression of the LH receptor mRNA in the rolipram-treated rats underscored the effects of a chronic increase in ovarian cAMP on LHR mRNA expression. Previous reports from our laboratory have shown that the decreased expression of LH receptor mRNA following the administration of hCG is associated with an increase in the RNA binding activity of LH receptor mRNA binding protein (LRBP) (Kash and Menon, 1998). Therefore, we conducted additional studies to examine if rolipram treatment could also mimic the effect of LH or hCG in stimulating the activity of LRBP which is a modulator of hCG-induced LHR mRNA down-regulation. This possibility was investigated by treating rats with rolipram or vehicle and determining the activity of the LRBP by RNA electrophoretic mobility shift as-
Fig. 5. Activity of the LH receptor mRNA binding protein in ovaries of control and rolipram-treated rats. The autoradiograph is a representative RNA electrophoretic mobility shift assay (REMSA) conducted with 1 × 105 cpm of radiolabelled RNA, the LRBP binding sequence (LBS-188–228), and incubated with no protein (NP): lane 1, which is the (−) control, or with 10 g of cytosolic S-100 fractions of ovaries from vehicle-injected control animals (C); lane 2, which is the (+) control, or from rolipram-treated rats (R); lane 3, 8 h after the administration of the PDE inhibitor. The samples were then treated with RNase T1 to eliminate unprotected RNA and were then resolved by electrophoresis on a 5% native polyacrylamide gel. The samples were subsequently visualized by autoradiography. The blot is a representative of three experiments.
say (REMSA). The REMSA of LRBP activity was conducted with the cytosolic (S-100) fractions of the ovaries incubated with the radiolabelled LHR mRNA sequence (nucleotides 188–228) which contains the target sequence for the binding of LRBP. The 47 kDa bands of LRBP-RNA complexes were then visualized by autoradiography. The results of these experiments are presented in Fig. 5. The autoradiograph of the REMSA is a representative image of the effect of rolipram administration on RNA binding activity of LRBP in the ovaries of rolipram- and vehicle-treated rats 8 h after treatment. These results show that the mRNA binding activity of LRBP was enhanced by treatment with rolipram. The assessment of LRBP activity indicated that rolipram caused an increase in the RNA binding activity. The increase in LRBP activity at 8 h after the first rolipram dosage is also in agreement with the decrease in LHR mRNA expression observed at the same time interval (Fig. 2) and with the increased concentrations of intraovarian cAMP depicted in Fig. 1 in the rolipram-treated group.
4. Discussion Our laboratory has previously reported that ligandinduced down regulation of LHR mRNA occurs through a post-transcriptional mechanism and this process is accompanied by an increase in the binding activity of a cytosolic protein termed LH receptor mRNA binding protein (LRBP) to the LHR mRNA (Hoffman et al., 1991; Kash and Menon, 1998, 1999; Lu et al., 1993; Nair et al., 2002; Nair and Menon, 2004). Furthermore, we have shown an inverse correlation between LHR mRNA expression and LRBP binding activity during the course of follicle maturation, ovulation and the life span of the corpus luteum (Nair et al., 2002). A major pathway by which the activation of the LH receptor, a G protein coupled receptor, leads to the stimulation of ovarian function is mediated through the cAMP-PKA pathway (Marsh, 1975; Menon and Gunaga, 1974), although LH may also exert some of its actions through the Akt/PKB cascade (Salvador et al., 2002). The present studies were designed to determine whether the down-regulation of the LH receptor mRNA expression is mediated by cAMP through modulation of LRBP activity. The overall approach to bypass the activation of the LH receptor by its ligands involved the repeated administration of rolipram, a type IV PDE inhibitor, in order to promote the intra-ovarian accumulation of cAMP. The selection of a type IV PDE inhibitor was based on a body of evidence showing that this is the major phosphodiesterase present in the granulosa cells in the ovary (Conti et al., 2002; Jin et al., 1999; Thomas et al., 2002; Tsafriri et al., 1996). Additionally, it has been shown that inhibition of type IV PDE can enhance LHinduction of oocyte maturation in cultured follicles (Tsafriri et al., 1996). Our results showing a moderate but chronic and consistent enhancement of intraovarian concentrations of cAMP in response to treatment with rolipram indeed sug-
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gest that Type IV phosphodiesterase plays a role in regulating the intracellular cAMP in the ovary. The sharp decreases in the expression of the LHR mRNA in response to treatment with rolipram clearly show that the down-regulation of LHR mRNA following hCG injection is mediated through increased cyclic AMP production (Hoffman et al., 1991; Kash and Menon, 1998, 1999; Nair et al., 2002). The repeated dosage regimen utilized for the administration of rolipram is at the upper range of the dosages reported in the literature (Krause and Kuhne, 1998; Larson et al., 1996). The repeated approach was considered necessary in view of the short half-life reported for rolipram in rats (Krause and Kuhne, 1998) and the need to ensure a chronic inhibition of the type IV PDE in the experiments. The consequence of this dosage regimen also made it important to assess whether there were any toxic compounding effects involved in the experiments. This possibility we assessed by examining the ovarian morphology. The morphology of the ovaries shown in the hematoxylin/eosin images revealed normal, healthy corpora lutea, granulosa cells and theca cells in the ovaries of the rolipram-treated rats compared to the controls. Additionally, the studies conducted to determine the recovery of expression of the LH receptor mRNA indicated that the LHR mRNA level exhibited a return to normal, pre-treatment ranges after 24–48 h, indicating a transient inhibitory effect of rolipram without producing an adverse toxic effect (Peegel et al., 1994). The effect of rolipram was specific for LHR mRNA since the expression of the mRNA for the key steroidogenic enzyme p450scc did not show any decrease in response to rolipram but interestingly showed an increase at the 12 h time interval. This increase is also significant since it would be expected as a normal response to the increased concentrations of cAMP, in the transduction of the LH signal for ovarian steroid synthesis. This result also underscores the preservation of normal ovarian physiology in the ovaries of the rats during the administration of rolipram and indicates a selective effect of the chronic Type IV PDE inhibition in the down-regulation of the mRNA for the LH receptor. Previous reports from our laboratory showed an increase in LHR mRNA binding activity of an ovarian cytosolic protein specific for the LH receptor mRNA (Kash and Menon, 1999). These reports also suggest a major role for this protein in promoting the degradation of the LHR mRNA as a mechanism for the modulation of the expression of the LH receptor (Nair et al., 2002). The experiments conducted in the present study show that the administration of the PDE inhibitor also results in an increase in LHR mRNA binding activity of LRBP. The REMSA assay showed an increased LRBP binding activity in a representative sample taken at the 8 h time-interval. At the 8 h time interval, rolipram treatment showed an increase in cAMP accumulation as well as a drastic reduction in the LH receptor mRNA. Thus, as previously reported (Nair et al., 2002), the decline in LHR mRNA expression and the induction of the LHR mRNA binding protein activity caused by hCG administration can be mimicked by increasing the
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intracellular cAMP level produced in response to treatment with phosphodiesterase inhibitor (Nair et al., 2002). Furthermore, hCG-induced changes in LHR mRNA expression can be mimicked by increasing the intracellular cAMP concentration in vivo suggesting that cAMP plays an intermediary role in the down-regulation of LHR mRNA levels in the ovary. Acknowledgment The authors wish to thank Nicole Acevedo for helpful assistance with photography of the ovarian histologic sections. References Ascoli, M., Fanelli, F., Segaloff, D., 2002. The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocrinol. Rev. 23, 141– 174. Burd, C., Dreyfuss, G., 1994. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621. Conti, M., 2000. Phosphodiesterases and cyclic nucleotide signaling in endocrine cells. Mol. Endocrinol. 14, 1317–1327. Conti, M., Andersen, C.B., Richard, F., Mehats, C., Chun, S.Y., Horner, K., Jin, C., Tsafriri, A., 2002. Role of cyclic nucleotide signaling in oocyte maturation. Mol. Cell Endocrinol. 187, 153–159. Davis, J., West, L., Farese, R., 1984. Effects of luteinizing hormone on phosphoionostitde metabolism in rat granuloda cells. J. Biol. Chem. 259, 15028–15034. Fan, X., Steitz, J., 1998. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases in in vivo stability of ARE-containing mRNAs. EMBO J. 17, 3448–3460. Gundermann, T., Birnbaumer, M., Birnbaumer, L., 1992. Evidence of dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoiositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J. Biol. Chem. 267, 4479–4488. Hoffman, Y., Peegel, H., Sprock, M., Zhang, Q., Menon, K.M.J., 1991. Evidence that human chorionic gonadotropin/luteinzing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocriniology 128, 388–393. Jin, S., Richard, F., Kuo, W., D’Ercole, A., Conti, M., 1999. Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4Ddeficient mice. Proc. Natl. Acad. Sci. U.S.A.. Kash, J., Menon, K.M.J., 1998. Identification of a hormonally regulated luteinizing hormone/human chorionc gonadotropin receptor mRNA binding protein. J. Biol. Chem. 273, 10658–10664. Kash, J., Menon, K.M.J., 1999. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame. Biochemistry 38, 16889–16897. Klausner, R., Rouault, T., Harford, J., 1993. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72, 19–28. Krause, W., Kuhne, G., 1998. Pharmacokinetics of rolipram in the rhesus and cynomolgus monkeys, the rat and the rabbit. Studies on species differences. Xenobiotica 18, 561–571. Lapolt, P., Oikawa, M., Jia, X., Dargan, C., Hsueh, A., 1990. Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 126, 3277–3279. Larson, J., Ino, M., Geiger, L., Simeone, C., 1996. The toxicity of repeated exposures to rolipram, a type IV phosphodiesterase inhibitor, in rats. Pharmacol. Toxicol. 78, 44–49. Lu, D., Peegel, H., Mosier, S., Menon, K.M.J., 1993. Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs posttranscriptionally. Endocrinology 132, 235–240.
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Ross, J., 1995. mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450. Salvador, L., Maizels, E., Hales, D., Miyamoto, E., Yamamoto, H., Hunzicker-Dunn, M., 2002. Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 143, 2986–2994. Thomas, R., Armstrong, D., Gilchrist, R., 2002. Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev. Biol. 244, 215–225. Tsafriri, A., Chun, S., Zhang, R., Hsueh, A., Conti, M., 1996. Oocye maturation involves comparatmentalization and opposing changes of cAMP levels in follicular somatic and term cells: Studies using selective phosphodiesterase inhibitors. Dev. Biol. 178, 393– 402. Zeleznik, A., Midgley Jr., A., Reichert Jr., L., 1974. Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormone in vivo. Endorcinology 95, 818–825.