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A Role of Insulin-Like Growth Factor I in Luteinizing Hormone Receptor Expression in Granulosa Cells¹

Department of Obstetrics and Gynecology School of Medicine (T.H., T.M., K.A., H.K., Y.I.), Biosignal Research Center Institute for Molecular and Cellular Regulation (K.M.), Gunma University, Maebashi, Gunma 371-8511, Japan

Address all correspondence and requests for reprints to: Dr. Takashi Minegishi, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. E-mail: tminegis{at}sb.gunma-u.ac.jp

	Abstract

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The present study was undertaken to identify the mechanisms underlying the effect of insulin-like growth factor (IGF-I) on LH receptor in rat granulosa cells. Treatment with FSH, as expected, produced a substantial increase in LH receptor messenger RNA (mRNA) level, and concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced LH receptor mRNA, with a maximal response 2.5-fold greater than that induced by FSH alone. IGF-I, either alone or in combination with FSH, did not affect intracellular cAMP levels, whereas it enhanced the effect of 8-bromo-cAMP on LH receptor mRNA production. We then investigated whether the effects of IGF-I and FSH on LH receptor mRNA levels are the results of increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into a transient expression vector, pGL-Basic, which contains luciferase as the reporter gene, and the resulting plasmids were transiently transfected into rat granulosa cells. Our studies show that the FSH-induced luciferase activity varied dependent upon the length of the 5'-flanking region sequence in the reporter gene. In addition, FSH (30 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region, but treatment with 10 ng/ml IGF-I alone did not significantly influence the activity of the LH receptor promoter or affect the increased promoter activity induced by FSH. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-I. On the other hand, the decay curves for LH receptor mRNA transcript in primary granulosa cells showed a significant increase in the half-life after the addition of IGF-I. These data suggest a possible role for changes in LH receptor mRNA stability in the IGF-I-induced regulation of LH receptor in rat granulosa cells. This interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones at the local level.

	Introduction

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THE PITUITARY gonadotropins are key hormones in the regulation of folliculogenesis. It is clear that the ability of gonadotropins to modulate ovarian function depends not only on the circulating levels of the gonadotropins, but also on the expression of appropriate receptor proteins by potential target cells in the ovary. FSH and LH act through stimulatory G protein-coupled receptors expressed on target cells and transduce their signal at least in part by the activation of adenylyl cyclase and the production of the second messenger cAMP. The expression of receptors for LH is one of the major markers of the FSH-induced differentiation of granulosa cells (1), and this process is also modified by many growth factors.

Primary cultures of rat granulosa cells obtained from immature female rats pretreated with estradiol are a frequently used model for studying cell differentiation. Using this defined model, the function of FSH to stimulate FSH and LH receptor induction has been shown to be mediated at least in part by cAMP, as exogenous cAMP or other agents that increase intracellular levels of cAMP mimic the action of FSH (2, 3, 4, 5, 6).

Recently, using the knockout mouse model it has been shown that insulin-like growth factor I (IGF-I) and FSH receptor are selectively coexpressed in growing murine follicles and that IGF-I augments granulosa cell FSH receptor expression. These data suggest that ovarian IGF-I expression serves to enhance granulosa cell FSH responsiveness by enhancing FSH receptor expression (7, 8, 9). As IGF-I enhances the proliferation of many cell types, and it has been observed that the IGF-I receptor is coexpressed with IGF-I in ovarian follicles (10), it seemed likely that IGF-I may act in an autocrine/paracrine manner to stimulate granulosa cell proliferation. Previous studies have shown that IGF-I is expressed in a subset of relatively healthy appearing follicles in the rat ovary (10, 11), suggesting that IGF-I is a marker for follicular selection.

On the other hand, knockout mouse models have indicated that IGF-I is not an obligatory participant in follicular development, at least up to the formation of antral follicles (9). The knockout ovaries contained neither corpora lutea nor corpora albicantia, indicating that ovulation never occurred in mutant females. In addition, even after a surge of exogenously administered gonadotropins, the pathway of follicular responses culminating in rupture does not function in the mutants. These observations revealed an important interdependence of the gonadotropin and IGF-I signaling pathways, which is perhaps related to events necessary for the generation of Graafian follicles and subsequent ovulation. Expression of LH receptor is one of the major markers of the FSH-induced differentiation of granulosa cells and is essential for the response to LH increase for the induction of ovulation. Therefore, the lack of ovulation after the addition of LH might be due to the insufficiency of LH receptor expression in these knockout models. In the present study we have attempted to elucidate the functional relationship between FSH and IGF-I in LH receptor expression.

	Materials and Methods

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Hormones and reagents
Rat FSH (I-8) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), gentamicin sulfate, and 8-bromo-cAMP (8-Br-cAMP) were purchased from Sigma Chemical Co. (St. Louis, MO). Rat IGF-I was purchased from GroPep Pty. Ltd. (Adelaide, Australia). DMEM, Ham’s F-12 medium, and fungizone were purchased from Life Technologies, Inc. (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Roche Molecular Biochemicals (Mannheim, Germany). [-³²P]UTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech Japan Corp. (Tokyo, Japan).

Rat granulosa cell culture
Granulosa cells were obtained from immature female Wistar rats that received an injection of 2 mg DES in 0.1 ml sesame oil once daily for 4 days. The ovaries were then excised, and granulosa cells were released by puncturing follicles with a 25-gauge needle. At all times, the animals were treated as humanely as possible, following NIH guidelines. Granulosa cells were washed and collected by brief centrifugation, and cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham’s F-12/DMEM (1:1, vol/vol) medium supplemented with 1.1 g/liter NaHCO₃, 40 mg/liter gentamicin sulfate, 1 mg/liter fungizone, and 100 mg/liter BSA on collagen-coated plates in a humidified atmosphere containing 5% CO₂-95% air at 37 C (12).

RNA isolation and analysis
Granulosa cells were cultured in 60-mm dishes containing 5 x 10⁶ viable cells in 5 ml medium, and reagents were added to the medium after 24 h of cell culture. The granulosa cells were further incubated, and the cultures were stopped at selected times as indicated using the guanidinium acid-thiocyanate-phenol-chloroform method (13). The final RNA pellet was dissolved in diethylpyrocarbonate-treated H₂O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 15 µg total RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN Biochemicals, Inc., Glen Cove, NY). Rat LH receptor complementary DNA (cDNA) was prepared as described previously and was linearized with BglII (29). Digoxigenin-labeled LH receptor complementary RNA probes corresponding to bases 440-2560 were produced by in vitro transcription with T3 RNA polymerase and a RNA labeling kit (Roche Molecular Biochemicals). A digoxigenin-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was obtained by the same method. In accordance with the standard protocol for the nucleic acid detection kit (Roche Molecular Biochemicals), Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) was then exposed to the membranes. Luminescence detection was quantified with an LKB 2202 UnitroScan laser densitometer (LKB Produkter AB, Bromma, Sweden), normalized against a corresponding relative amount of GAPDH messenger RNA (mRNA) in each sample, and expressed as relative densitometric units.

Vector preparation and transfection
Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promotor and enhancer sequences (Promega Corp., Madison, WI). The pGL3-Control contains a simian virus 40 promoter and a simian virus 40 enhancer inserted into the structure of pGL3-Basic (Promega Corp.). Two fragments of the 5'-franking region of -1389 to -1 bp and -482 to -1 bp relative to the translational initiation site were generated from genomic DNA via PCR using primers specific to the rat LH receptor sequence. For evaluating promotor activity, these fragments were ligated to a luciferase reporter vector (pGL3-Basic) and named LH-R (1389)-Luc and LH-R (482)-Luc. The luciferase assay was performed using the Dual-Luciferase Reporter System (Promega Corp.), in which transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of Renilla luciferase.

Transient transfection
Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described previously (14). Using FuGENE, a total of 0.25 µg plasmid DNA was transfected, as described previously (15), into primary granulosa cell cultures plates (2.5 x 10⁵ cells, 0.5 ml of that in a 20-mm dish). To assay regulatory elements, granulosa cells were cultured for 48 h in hormone-free conditions before transfection. Thirty hours after transfection, cells were treated with hormones for 6 h. After the incubation, cells were harvested, and luciferase activity was measured. The cells were lysed in lysis buffer supplied by the manufacturer, followed by measurement of firefly and Renilla luciferase activities on a luminometer. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities. The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments. In the luciferase assay, luciferin and Mg²⁺-ATP were added to cellular extracts, and the production of light was monitored conveniently by a luminometer. Luciferase activity was assayed as previously described (16).

Isolation of nuclei
Granulosa cells were cultured in 60-mm dishes containing 5 x 10⁶ cells in 5 ml serum-free medium. After 24 h, granulosa cells were further incubated in the presence or absence of FSH (30 ng/ml) or FSH (30 ng/ml) plus IGF-I (10 ng/ml) for 48 h before isolating the nuclei. Cells were washed three times with ice-cold Dulbecco’s PBS without calcium and magnesium, collected by scraping in Dulbecco’s PBS without calcium and magnesium, and then centrifuged for 5 mm at 1000 rpm at 4 C. The cell pellet was resuspended in 500 µl Nonidet P-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, and 0.5% Nonidet P-40]. Lysed cells were incubated on ice for 10 min and centrifuged for 5 min at 3000 rpm. The nuclear pellet was then resuspended in 500 µl Nonidet P-40 lysis buffer and centrifuged for 5 min. The final nuclear pellet was gently resuspended in 100 µl glycerol storage buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl₂, and 0.1 mM EDTA (pH 8.0)], frozen in liquid nitrogen, and stored at -80 C.

Run-on transcription assay
The nuclear run-on transcription assay was performed according to a previously described protocol (17). The relative amount of incorporation of label into specific RNAs was determined by DNA excess filter hybridization, as described previously (17), using cDNAs for rat LH receptor. Ten micrograms each of LH receptor, Bluescript, and GAPDH cDNAs were included on the DNA filter during hybridization to correct for background and to serve as internal controls. Autoradiographic bands were quantified by a fluoroimage analyzer (BAS 2000, Fuji Photo Film Co. Ltd., Tokyo, Japan).

Transcription stability analysis
Cells were preincubated with FSH alone or FSH and IGF-I for 24 h before the addition of 5 µM actinomycin D to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after the addition of the inhibitor for RNA extraction and Northern blot analysis.

Data analysis
The relative abundance of a 5.4 kb rat LH receptor mRNA in different preparations was quantified with a LKB 2202 UnitroScan laser densitometer (LKB Produkter AB), normalized against levels of GAPDH mRNA in each sample, and expressed as a percentage of the control value (100%). The data are presented as the mean ± SE of measurements from triplicate cultures for one representative experiment. Comparisons between groups were performed using one-way ANOVA. The significance of differences between the mean values in the control group and each treated group was tested using Duncan’s multiple comparison test. P < 0.05 was considered statistically significant.

	Results

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Previous studies have indicated that IGF-I is capable of enhancing FSH-stimulated LH receptor expression, but not its affinity, in a time- and dose-dependent fashion (18). In this study, to examine the effect of IGF-I on LH receptor mRNA levels, granulosa cells were cultured in the absence or presence of FSH (30 ng/ml), with or without increasing concentrations of IGF-I (1–100 ng/ml; Fig. 1). Levels of basal LH receptor mRNA were negligible and remained unchanged in treatment with IGF-I by itself. In contrast, treatment with FSH, as expected, produced a substantial increase in the LH receptor mRNA level, and concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced LH receptor mRNA, with a maximal response 2.5-fold greater than that induced by FSH alone.

View larger version (47K): [in this window] [in a new window]   Figure 1. Dose-related effect of IGF-I on FSH-induced LH receptor mRNA. A, Granulosa cells from DES-primed immature rats were cultured for 24 h, then in 30 ng/ml FSH with increasing concentrations of IGF-I for 72 h. LH receptor mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of four experiments. B, Autoradiographs of LH receptor mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH receptor mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The absorbance values obtained from this study as well as those from three other studies were standardized relative to the control value and are represented (mean ± SE; n = 4) in the bar graphs. *, Difference from the control (FSH alone) value, P < 0.01.

View larger version (36K): [in this window] [in a new window]   Figure 2. Time course of IGF-I’s effect on the FSH-induced LH receptor mRNA. A, Granulosa cells from DES-primed immature rats were cultured for 24 h (0; control = 0 h), then in 30 ng/ml FSH alone or with 10 ng/ml IGF-I. After various incubation times, total RNA was extracted, and LH receptor mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of three experiments. B, Autoradiographs of LH receptor mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH receptor mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value (FSH alone, 48 h). The absorbance values obtained from this study as well as from three other studies were standardized to the 48 h control and are represented (mean ± SE; n = 3) in the graph. *, Difference from the control (FSH alone) at each time, P < 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of IGF-I on 8-Br-cAMP-induced LH receptor mRNA. A, Granulosa cells were stimulated by 8-Br-cAMP with or without 10 ng/ml IGF-I. After 48 h, the levels of LH receptor mRNA were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of three experiments. B, Autoradiographs of LH receptor mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH receptor mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value (FSH alone, 48 h). The absorbance values obtained from this study as well as those from three other studies were standardized to the 48 h control and are represented (mean ± SE; n = 3) in the graph. *, Difference from the control value, P < 0.05.

View larger version (35K): [in this window] [in a new window]   Figure 4. Effects of FSH and IGF-I on LH-R-Luc expression in rat granulosa cells. Granulosa cells were cultured for 48 h in hormone-free conditions, then cotransfected with LH-R (1379)-Luc or LH-R (482) and pRL. After transfection 30 h later, cells were treated with FSH with or without 10 ng/ml IGF-I for 6 h, and then cells were lysed and assayed for luciferase activity. Luciferase activity was corrected for the amount of Renilla luciferase activity detected in each lysate. Each bar represents the mean ± SE of three independent experiments. *, Difference from the control value, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Figure 5. Stimulation of LH receptor gene transcription by FSH and IGF-I. A, Granulosa cells were cultured in 60-mm dishes containing 5 x 106 cells in 5 ml serum-free medium. After 24 h in culture, granulosa cells were further incubated in the presence or absence of IGF-I (10 ng/ml) for 24 h, and nuclear run-on assays were then performed as described in Materials and Methods. B, Data acquired from the nuclear run-on experiments shown in A were quantitated by a fluoroimage analyzer (BAS 2000). Data were normalized for GAPDH levels in each sample and are expressed relative to the control (cont) value. Transcriptional activities after incubation with FSH and IGF-I are expressed relative to the activity in FSH alone. The data shown are the mean ± SE of three independent experiments. *, Difference from the control value, P < 0.01.

View larger version (25K): [in this window] [in a new window]   Figure 6. A, Effect of IGF-I on LH receptor mRNA transcripts. Granulosa cells were preincubated for 24 h with FSH alone or with FSH and IGF-I. After this preincubation period, 5 µM actinomycin D was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after addition of the transcription inhibitor, and LH receptor mRNA levels were quantified by Northern blot analysis. B, mRNA levels at time zero were assigned a relative value of 100%, and mRNA levels at all other times are expressed as percentages of the time zero value.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been shown that, when levels of LH receptor mRNA are altered, all transcripts (7.5, 5.4, 3.8, 2.3, and 1.2 kb) appear to be coordinately regulated. These results suggest that the turnover of the receptor transcripts could be occurring via a common mechanisms. The observed maintenance of message levels by FSH and IGF-I may be a result of increased LH receptor gene transcription and/or message stability. Previous studies have revealed that the 5'-flanking region of the rat LH receptor gene is characteristic of that in the so-called housekeeping genes, in that this region has no apparent TATA or CAAT boxes, is rich in G and C residues, and contains multiple potential transcriptional start sites (21). Although FSH administration increased transcription of the LH receptor gene, IGF-I did not have any effect on FSH-induced transcription. Thus, we suggest that IGF-I had no effect on the interaction between FSH and LH-R-Luc in this experiment, although it may be possible that IGF-I does have an effect on the 5'-end of the LH receptor gene. Further studies will be required to clarify this point. However, data from the nuclear run-on assays demonstrate that the positive effect of IGF-I on FSH-induced LH receptor mRNA increase is not brought about by transcriptional mechanisms.

Preliminary experiments revealed that the decay of LH receptor mRNA in granulosa cells might be affected by the incubation time before actinomycin D addition. Thus, the addition of actinomycin D prolonged the half-life of LH receptor mRNA and made it impossible to estimate the stability of mRNA in this culture system after more than 48-h incubation (data not shown). Therefore, in this study, the experiment for the decay of LH receptor mRNA was performed in cells pretreated by hormones for 24 h. The data presented suggest a possible role for changes in LH receptor mRNA stability in the IGF-I-induced regulation of LH receptor in rat granulosa cells. Previous observations have suggested that a labile destabilizing factor may constitutively degrade the LH receptor mRNA (22). Our data suggest that IGF-I may act to increase LH receptor mRNA by preventing the synthesis or actions of a LH receptor mRNA-destabilizing factor in the presence of FSH. It has been also well established that the expression of specific, highly regulated mRNAs such as c-Fos, c-Myc, and ß-adrenergic receptor, are controlled at least in part at the level of mRNA degradation (23, 24). In the majority of instances of posttranscriptional mRNA regulation, the changes in stability of a particular mRNA appear to result from changes in the binding of specific proteins to defined sequences and/or structures in the target mRNA. The RNA sequences recognized by regulatory proteins often are located within discrete regions of the mRNA. In terms of LH receptor mRNA, a LH receptor mRNA-binding protein, which is a candidate for a trans-acting factor involved in the hormonal regulation of LH receptor mRNA stability in the rat ovary, has been reported (25, 26). In the rat ovary, differences in LH receptor number seen during follicular development, ovulation, and luteinization involve concomitant changes in receptor mRNA levels (27). As levels of LH receptor mRNA closely parallel receptor number, it is likely that posttranscriptional regulation plays a pivotal role in mediating physiological changes in receptor expression during the ovarian cycle.

IGF-I enhances the expression of LH receptor only in the presence of FSH, whereas IGF-I alone does not induce LH receptor mRNA. Therefore, it is possible that IGF-I has a primary effect on FSH receptor expression that secondarily potentiates FSH action and results in augmentation of LH receptor mRNA. However, follicles in the IGF-I knockout ovary are arrested at a late preantral or early antral stage of development, and the knockout ovaries contained neither corpora lutes nor corpora albicantia, indicating that ovulation never occurred in mutant females. In addition, even after a surge of exogenously administered gonadotropins, the pathway of follicular responses culminating in rupture did not function in the mutants. These observations revealed an important interdependence of the gonadotropin and IGF-I signaling pathways, which is perhaps related to events necessary for the generation of Graafian follicles and subsequent ovulation. As expression of LH receptor is one of the major markers of the FSH-induced differentiation of granulosa cells and is essential for the response to LH increase for the induction of ovulation, the pathways might be interfered with at the level of LH receptor expression. As we show in this report, IGF-I has a direct effect on LH receptor mRNA half-life in granulosa cells; therefore, it is possible that IGF-I is essential for maintaining a critical level of LH receptor expression for ovulation in the ovary.

	Acknowledgments

 
We thank the National Hormone and Pituitary Agency, NIDDK, and University of Maryland School of Medicine for the rat FSH.

	Footnotes

 
¹ This work was supported by grants from the Ministry of Education, Science, and Culture of Japan (10044235 and 10877253), Tokyo, Japan.

Received April 23, 1999.

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