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Control of the Expression of Luteinizing Hormone Receptor by Local Factors in Rat Granulosa Cells
Archives of Biochemistry and Biophysics Vol. 367, No. 2, July 15, pp. 185–192, 1999 Article ID abbi.1999.1241, available online at http://www.idealibrary.com on
Control of the Expression of Luteinizing Hormone Receptor by Local Factors in Rat Granulosa Cells Megumi Tsuchiya,* Takashi Minegishi,* ,1 Hiroshi Kishi,* Mari Tano,* ,2 Takashi Kameda,* Takashi Hirakawa,* Yoshito Ibuki,* Tetsuya Mizutani,† and Kaoru Miyamoto† *Department of Obstetrics and Gynecology, School of Medicine, and †Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8511, Japan Received December 14, 1998, and in revised form April 6, 1999
To identify the mechanisms underlying the hormone-dependent induction and maintenance of luteinizing hormone receptor (LH-R) in rat granulosa cells, the effect of follicle-stimulating hormone (FSH) and local factors on the LH-R mRNA levels were studied. LH-R mRNA levels of the cells incubated with FSH decreased rapidly after medium removal, and readdition of FSH with the fresh medium did not restore these levels. On the other hand, 8-bromoadenosine 3,5cyclic monophosphate significantly enhanced the expression of LH-R mRNA after medium removal, while the level of LH-R mRNA was lower than that of the cells replaced by original medium including FSH. In addition, the incubation with 8-Br-cAMP produced dose-dependent responses for LH-R mRNAs and enhanced the activity of 1379 bp of the LH-R 5*-flanking region, while the level of LH-R mRNA decreased 3 days after medium removal. Further studies were undertaken to assess the role of factors in maintaining the LH receptor once induced by FSH. Since FSH and cAMP increase follistatin production in granulosa cells, we examined the effect of follistatin on LH-R induction in the presence of activin and FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dose-dependent manner. However, insulinlike growth factor-I (IGF-I) induced LH-R mRNA in the presence of FSH even after medium change. IGF-I might be one of the important factors that act in the medium to maintain LH-R levels in granulosa cells. © 1999 Academic Press Key Words: LH receptor; FSH receptor; activin; follistatin; IGF-I.
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To whom correspondence should be addressed. Fax: 272-20-8443. E-mail: [email protected]. 2 Supported by Fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists.
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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. Folliclestimulating hormone (FSH) 3 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 FSHinduced differentiation of granulosa cells (1) and this process is also modified by many growth factors. Primary cultures of rat granulosa cells obtained from hypophysectomized or 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-R and LH-R induction has been shown to be mediated, at least in part, by cAMP, since exogenous cAMP or other agents that increase intracellular levels of cAMP mimic the action of FSH (2– 6). Therefore, we first analyzed the effect of cAMP on the LH-R mRNA levels and on the 59-flanking region of LH-R using transient expression. In this system, the continual presence of FSH is required to maintain the LH-R at elevated steady levels after induction, associated with changes in LH-R mRNA levels (7, 8). Presently, it is unknown whether these FSH-induced signals directly enhance granulosa cell differentiation and/or whether they instigate the 3 Abbreviations used: FSH, follicle-stimulating hormone; LH, luteinizing hormone; FSH-R, FSH receptor; LH-R, LH receptor; IGF-I, insulinlike growth factor-I; DES, diethylstilbestrol; 8-Br-cAMP, 8-bromoadenosine 3,5-cyclic monophosphate; DMEM, Dulbecco’s modified Eagle’s medium.
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FIG. 1. Maintenance of LH-R mRNA levels by FSH and 8-Br-cAMP. (A) Granulosa cells from DES-primed immature rats were incubated with and without (1) 30 ng/ml FSH (added 24 h after cell plating) for 24 h (2). The cells incubated with FSH for 24 h were incubated with fresh FSH (30 ng/ml) (3–5) or 8-Br-cAMP (1 mM) (6 – 8) after medium exchange for the indicated times (3 and 5, 24 h; 4 and 7, 48 h; 5 and 8, 72 h). The medium from the cells, incubated with FSH for 24 h, was transferred to the cells which were also incubated with FSH for 24 h. These cells were further incubated for the indicated times (9, 24 h; 10, 48 h; 11, 72 h). LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with FSH was taken as 100%. 1, 24 h after cell plating; 2, incubated with FSH for 24 h; 3–5, incubated with fresh FSH after medium change; 6 – 8, incubated with 8-Br-cAMP after medium change; 9 –11, incubated with transfered medium from the cells incubated with FSH for 24 h. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
production of local mediators, which then stimulate differentiation. Previously, our studies using granulosa cell culture have shown that follistatin synthesis is stimulated by FSH (9), while activin production is suppressed by FSH (10), leading to the suggestion that FSH attenuates the effect of activin in the granulosa cell. This is due to follistatin being an activin-binding protein (11) and it neutralizes the diverse activin bioactivities in various systems by stoichiometrically forming inactive complexes with activins (12). In this experiment, we examined the effect of follistatin on LH-R expression induced by activin and FSH. Insulinlike growth factor-I (IGF-I) is one of the growth factors involved in the FSH-induced differentiation of granulosa cells and more importantly FSH increases the synthesis of IGF-I (13, 14). Since we have found that the exchange of medium incubated with or without FSH disturbed the expression of LH-R in rat granulosa cells, it might suggest that the factors produced in this culture medium are essential for the function of FSH to stimulate LH-R expression. The present studies were therefore undertaken to elucidate the molecular mechanisms underlying the hormonal induction of LH-R mRNA in granulosa cells. MATERIALS AND METHODS Hormones and reagents. Activin A was kindly donated by Dr. Eto (Ajinomoto Co., Inc., Central Research Laboratories, Kawasaki, Ja-
pan). Rat FSH (I-8), hCG (CR-119), and human follistatin were obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), gentamicin sulfate, 8-bromo-adenosine 3,5-cyclic monophosphate (8-Br-cAMP) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). Rat IGF-I was purchased from GroPep Pty Ltd. (Adelaide, Australia). Dulbecco’s modified Eagle medium (DMEM), Ham’s F-12 medium, and fungizone were purchased from GIBCO Laboratories (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Boehringer-Mannheim (Mannheim, Germany). Animals. Immature female Wistar rats (21 days old) were obtained from Imai Experimental Animal Farm (Saitama Pref., Japan). Since estrogen treatment in vivo results in the formation of multiple preantral follicles and provides a large number of relatively homogeneous cells at the same stage of development, rats were pretreated with 2 mg DES in 0.1 ml sesame oil, sc, once daily for 4 days. The rats were killed at 26 days and their ovaries were aseptically excised. All procedures followed NIH guidelines. Rat granulosa cell culture. Granulosa cells were obtained by puncturing ovaries from DES-treated immature rats. The granulosa cells were then cultured in Ham’s F-12:DMEM (1:1, v/v) supplemented with 1.1 g/liter NaHCO 3, 40 mg/liter gentamicin sulfate, 1 mg/liter fungizone, and 100 mg/liter bovine serum albumin in a humidified atmosphere containing 5% CO 2, 95% air at 37 C. RNA isolation and analysis. Rat LH-R cDNA was prepared as described previously and linearized with BglII (15). Digoxigeninlabeled LH/hCG receptor cRNA probes corresponding to bases 440 – 2560 were produced by in vitro transcription with T3 RNA polymerase and an RNA labeling kit (Boehringer-Mannheim). A digoxigeninlabeled GAPDH cRNA probe was obtained by the same method. Granulosa cells were cultured in 60-mm dishes containing 5 3 10 6 viable cells in 5 ml of 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 the selected time as
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FIG. 2. Dose-dependent effect of 8-Br-cAMP on LH-R mRNA. (A) Granulosa cells from DES-primed immature rats were cultured for 24 h (0, control t 5 0 h), and 8-Br-cAMP was added at the indicated concentrations. The cells were incubated for 24 h (left) or further incubated for 72 h (right) after a medium change with cAMP-free medium. LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with 0.2 mM cAMP was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
indicated in the guanidinium acid–thiocyanate–phenol– chloroform method (16). The final RNA pellet was dissolved in diethyl pyrocabonate-treated H 2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 15 mg total RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN). In accordance with the standard protocol for the nucleic acid detection kit (Boehringer-Mannheim), Kodak X-Omat film (Eastman Kodak, Rochester, NY) was then exposed to the membranes. The relative abundance of 2.4 kb for rat FSH-R mRNA and 5.4 kb for rat LH-R mRNA in different preparations was quantified with a LKB 2202 UnitroScan Laser Densitometer (LKB Produkter AB, Bromma, Sweden) and normalized against levels of GAPDH mRNA in each sample and expressed as relative to the control value (100%). For all experiments, a representative Northern blot of three replicates is presented. The data are presented as means 6 SE of measurements from triplicate cultures for one representative experiment. The significance of differences between the mean values in the control group and each treated group were tested with Duncan’s multiple comparison test. A value of P , 0.05 was considered statistically significant. Vector preparation and transfection. Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). pGL3-Control contains a SV40 promoter and a SV40 enhancer inserted into structure of pGL3-Basic (Promega Corp). The pRL-SV40 vector contains the SV40 early enhancer/promoter region, which provides strong, constitutive expression of Rluc in a variety of cell types. The pRL vector provide constitutive expression of Renilla luciferase. The rat LH-R promoter from 21389 to 21 bp relative to the transcriptional start site was generated from genomic DNA via PCR using primers specific to the rat LH/hCG receptor sequence. For evaluating promoter activity, 21379 to 21 bp of the 59 flanking sequence of the rat LH-R promoter was ligated to a luciferase reporter vector (pGL3-Luc) and named LH-R-Luc. Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described (17). Using FuGENE, a total of 10 mg of plasmid DNA was transfected, as previously described (18), into primary granulosa cell cultures plate (5 3 10 5 cells in 2 ml in a 35-mm dish). To assay regulatory elements, granulosa cells were cultured 48 h under hormone-free conditions
before transfection. Thirty hours after transfection, cells were treated by hormones for 6 h and processed. After the incubation, cells were harvested and luciferase activity was measured. In the luciferase assay, luciferin and Mg 21 ATP were added to cellular extracts, and the production of light was monitored conveniently by a luminometer. Luciferase activity was assayed as previously described (19).
FIG. 3. Effect of 8-Br-cAMP on the expression of LH-R-Luc in rat granulosa cells. Granulosa cells were cultured for 48 h under hormone-free conditions and cotransfected with LH-R-Luc and pRL. After transfection 30 h later, cells were treated by 8-Br-cAMP for 6 h and were processed. Luciferase activity was corrected for the amount of Renilla luciferase activity detected in each lysate. Each bar represents the mean 6 SE of three independent experiments.
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FIG. 4. Effect of follistatin on activin-induced LH-R mRNA levels. (A) Total RNA was prepared from cells incubated for 72 h in the presence or absence of FSH and activin with increasing concentrations of follistatin, and LH-R mRNA and GAPDH mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA with FSH 30 ng/ml was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE. Values with asterisks differed significantly from values in cells treated by FSH plus activin: *P , 0.05 and **P , 0.01.
RESULTS
Previous studies have shown that a continual presence of FSH is required to maintain LH-R at elevated steady-state levels after induction (3). It has been shown that the function of FSH to stimulate LH-R induction is mediated, at least in part, by cAMP since exogenously added cAMP or other agents that increase intracellular levels of cAMP mimic the actions of FSH (3– 6). Moreover, we have detected comparable effect of 2 mM of 8-Br-cAMP to 30 ng/ml FSH on the expression of LH-R mRNA. Figure 1 showed that the LH-R mRNA increased with the addition of FSH (30 ng/ml) for 24 h and this LH-R mRNA decreased rapidly after a change of medium and even after the readdition of FSH. Alternatively, LH-R mRNA remained elevated with the addition of the medium, which was incubated with cells for 24 h in the presence of FSH. In addition, after the removal of FSH, the level of LH-R mRNA during incubation increased with the presence of 2 mM 8-BrcAMP. Since we detected the fact that the addition of FSH after a 24 h preincubation with FSH could not increase intracellular cAMP levels (20), data from the readdition of FSH might be due to the functional downregulation of FSH-R. To evaluate the effect of cAMP on LH-R mRNA levels, the cAMP effects on LH-R mRNA levels were ex-
amined before and after removal of cAMP. LH-R mRNA was also dose-dependently induced with a maximum response at 2 mM at 24 h incubation. Furthermore, LH-R mRNA levels decreased rapidly after removal of 8-Br-cAMP, and by the third day after removal, LH-R mRNA was not detectable in the cells (Fig. 2). For many genes that are transcriptionally regulated by cAMP, the 59 flanking promoter regions are necessary and sufficiently carry out this activity. To determine whether the LH/hCG-R 59-flanking region can direct expression, the proximal and 1379 bp of the LH/hCG-R 59-flanking region was inserted into a transient expression vector, pGL3-Basic, which contains luciferase as the reporter gene. The resulting plasmid (LH-R-Luc) was transiently transfected into rat granulosa cells. Gene transfer studies were performed by FuGENE transfection and luciferase enzyme activity measured in light units as an indication of promoter activity. Cells were cotransfected with pRL as an internal control for transfection efficiency. To investigate the hormonal regulation of the 59-flanking region, we analyzed the effect of cAMP on 1379 bp of the LH-R promoter region in the rat granulosa cells. Treatment with 8-Br-cAMP (0.5– 4 mM) significantly enhanced activity of 1379 bp of the LH-R 59-flanking region in a
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FIG. 5. Maintenance of LH-R mRNA by the presence of IGF-I. (A) Granulosa cells were incubated with 30 ng/ml FSH (added 24 h after cell plating) for 24 h. The cells then continued to be incubated for a total of 72 h with either no medium change or exchange (wash) at 24 h and with FSH alone or FSH and IGF-I (30 ng/ml). LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. LH-R mRNA levels were not significantly affected by treatment with IGF-I alone. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with FSH was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
dose dependent manner (Fig. 3). These data clearly demonstrate that FSH stimulates the induction of the LH-R in granulosa cells of DES-primed immature rats, at least in part, due to increasing the rate of transcription of the LH-R gene. Since our data showed that the presence of medium incubated with FSH enhanced the effect of FSH on LH-R expression, further studies were undertaken to identify the local factors induced by FSH exposure. In our previous experiment, the treatment of cAMP or FSH treatment suppressed the production of activin and increased follistatin production in granulosa cells (10, 21). We examined the effect of follistatin on activin action for LH-R induction in the presence of FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dosedependent manner (Fig. 4). Therefore, follistatin, whose production is induced by FSH and activin, suppresses the effect of activin on LH-R mRNA expression. Since IGF-I enhances the proliferation of many cell types and since the IGF-I receptor is coexpressed with IGF-I in ovarian follicles (22) it seemed likely that IGF-I may act in an autocrine/paracrine manner to stimulate granulosa cell differentiation. In the previous experiment (23), the relatively low concentrations of exogenously added IGF-I are capable of synergizing with FSH in the induction of LH-R expression in gran-
ulosa cell. Figure 5 shows that FSH itself could not induce LH-R after medium change, and FSH in the presence of IGF-I increased LH-R mRNA. DISCUSSION
The present studies were undertaken to determine the mechanisms underlying the induction and maintenance of LH-R in the differentiating granulosa cell. Consistent with previous studies (4, 24 –28), our data show that FSH is able to dramatically induce LH-R mRNA in granulosa cells from DES-primed immature rats from undetectable basal levels. The data obtained by the other group suggest that levels of LH-R mRNA decrease rapidly if FSH is removed from the cultured cells and that the levels of mRNA remain elevated despite the removal of medium if FSH is present (7). However, in the present experiment, the LH-R mRNA level of the cells incubated with FSH decreased rapidly after medium removal, and the readdition of FSH with the fresh medium did not restore these levels. These discrepancies might be due to the differences in the experimental conditions such as the coating of the surface of the culture plates. For example, the previous reports claimed that a time-related increase in the level of FSH-R mRNA was observed after increasing the lengths of culture in the absence of hormone treat-
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FIG. 6. (A) (1) Activin induces FSH-R. (2) FSH (mediated by cAMP) induces LH-R. (3) FSH also stimulates the expression of follistatin, activin-binding protein, and inhibits the production of activin. (4) FSH stimulates the expression of IGF-I and IGF-I-R. (B) (5) Follistatin binds to activin and suppresses the effect of activin on LH-R expression. (6) IGF-I maintains the expression of LH-R in the presence of FSH. FSH actions result in the suppression of activin’s effects and enhance IGF-I’s effect.
ment, compared to the levels found in cells at the start of culture (29). In contrast, the granulosa cells on collagen-coated dishes in our experiments indicated that FSH-R mRNA levels did not change during 4 days of culture in the absence of hormone treatment (30). Therefore, the level of gonadotropin receptor expression in the untreated cells may depend on the characteristics of the culture system. The ability of cAMP to increase LH-R mRNA to a greater extent than FSH at the second addition may be explained by the FSH receptor desensitization induced by FSH pretreatment. In the previous experiment, we showed that 24 h presence of FSH causes downregulation of FSH-R function of increasing intracellular cAMP levels in response to the next FSH stimulation. It took about 48 h to recover the ability to increase cAMP levels in response to the second addition of FSH after the first exposure to FSH in this culture model (20). Since addition of 8-Br-cAMP significantly enhanced the expression of LH-R mRNA after medium removal, the data showed that this marked decrease in LH-R mRNA levels after removal of medium including FSH is due, at least in part, to a significant decrease in cAMP production. In addition, the present results showed that 8-Br-cAMP more potently induced LH-R mRNA after medium removal and that cAMP alone is not sufficient to maintain the levels of LH-R mRNA induced by the first FSH.
The incubation with 8-Br-cAMP for 24 h produced a dose-dependent response in LH-R mRNA in granulosa cells of DES-primed immature rats, and the results of the nuclear run-on experiments clearly demonstrated that 8-Br-cAMP significantly stimulates the rate of transcription of the LH-R gene (7). Therefore, the effect of 8-Br-cAMP on the LH-R mRNA at 24 h might be due to an increased rate of transcription. As shown in previous studies (4, 24 –28), our data demonstrated that 8-Br-cAMP induces LH-R mRNA which is initially at undetectable basal levels in granulosa cells. In our previous experiments, we have shown that FSH (1–100 ng/ml) significantly enhanced the activity of the LH-R 59-flanking region in a dose-dependent manner (in press). Therefore, the increased LH-R mRNA gene transcription elicited by 8-Br-cAMP suggests that FSH mediates this effect through cAMP, i.e., the protein kinase A pathway. The results from transient transfection of LH-R promotor luciferase constructs into granulosa cells, which yield a stimulation of luciferase activity in response to 8-Br-cAMP, support the results of the previous nuclear run-on transcription assays (9). Our studies show that DNA sequences present within 1379 bp of the translation start site greatly enhance the expression of a luciferase reporter gene in granulosa cells. The possibility that sequences located upstream of 21379 bp would further enhance this expression, however, remains open. More detailed studies of
EXPRESSION OF LEUTINIZING HORMONE RECEPTOR IN GRANULOSA CELLS
this region by mutational analysis and other approaches aimed at identifying sequences that may be specifically recognized by cAMP-regulated nuclear factors will allow more precise characterization of this element. At present it’s unknown whether FSH-induced signals directly enhance LH-R expression or whether they instigate the production of local mediators such as activin and IGF-I, which then stimulate LH-R synthesis in the presence of FSH. According to present data, some local factors might be secreted into the medium by the cAMP dependent mechanisms. Although activin stimulates granulosa cell proliferation, follicular development in vitro (31, 32), and LH-R expression in the presence of FSH, FSH increased follistatin production and suppressed activin production in this culture system in a dose-dependent manner (10). Since follistatin is an activin-binding protein that has been shown to antagonize the function of activin in a number of assays, including the present data (11, 33), and cAMP signals increase the production of follistatin, the activin–follistatin model seems to be unrelated to the increase of LH-R mRNA level (Fig. 6). Previous studies showed that IGF-I is selectively expressed in a subset of relatively healthy-appearing follicles in the rat ovary (22, 34), leading to the suggestion that IGF-I is a marker for follicular selection. Since IGF-I enhances the proliferation of many cell types and since it has been previously shown that the IGF-I receptor is coexpressed with IGF-I in ovarian follicles (22), it seemed likely that IGF-I may act in an autocrine/paracrine manner to stimulate granulosa cell proliferation. For example, IGF-I amplifies FSHinduced aromatase expression and LH-R induction (23, 35). In addition, amplification by IGF-I of FSH receptor expression is positively reinforced by FSH-induced augmentation of IGF-I receptor expression, which has been demonstrated in vivo at the mRNA level (22) and in vitro at the IGF-I-binding level (36). This local IGF-I expression creates an intrafollicular positive feedback loop in which IGF-I enhances FSH action and FSH enhances IGF-I action through mutual complementary receptor upregulation (37). In addition, the effect of IGF-I on the induction of FSH receptor enhances FSH action of cAMP production, which results secondarily in augmentation of the LH-R gene. However, as shown in this experiment, the cAMP dose does not completely compensate for the effect of medium in the presence of FSH to maintain mRNA levels of LH-R. Therefore, it is also suspected that IGF-I has a primary effect on LH-R production in the presence of FSH. Further studies are clearly required to elucidate the mechanism of the IGF-I effect on the gonadotropin receptor.
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ACKNOWLEDGMENTS We thank the National Hormone and Pituitary Agency, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, and the University of Maryland School of Medicine for the rat FSH. This work was supported by grants from the Ministry of Education, Science and Culture of Japan (10044235, 09470353, and 10877253), Tokyo, Japan.
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Control of the Expression of Luteinizing Hormone Receptor by Local Factors in Rat Granulosa Cells Megumi Tsuchiya,* Takashi Minegishi,* ,1 Hiroshi Kishi,* Mari Tano,* ,2 Takashi Kameda,* Takashi Hirakawa,* Yoshito Ibuki,* Tetsuya Mizutani,† and Kaoru Miyamoto† *Department of Obstetrics and Gynecology, School of Medicine, and †Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8511, Japan Received December 14, 1998, and in revised form April 6, 1999
To identify the mechanisms underlying the hormone-dependent induction and maintenance of luteinizing hormone receptor (LH-R) in rat granulosa cells, the effect of follicle-stimulating hormone (FSH) and local factors on the LH-R mRNA levels were studied. LH-R mRNA levels of the cells incubated with FSH decreased rapidly after medium removal, and readdition of FSH with the fresh medium did not restore these levels. On the other hand, 8-bromoadenosine 3,5cyclic monophosphate significantly enhanced the expression of LH-R mRNA after medium removal, while the level of LH-R mRNA was lower than that of the cells replaced by original medium including FSH. In addition, the incubation with 8-Br-cAMP produced dose-dependent responses for LH-R mRNAs and enhanced the activity of 1379 bp of the LH-R 5*-flanking region, while the level of LH-R mRNA decreased 3 days after medium removal. Further studies were undertaken to assess the role of factors in maintaining the LH receptor once induced by FSH. Since FSH and cAMP increase follistatin production in granulosa cells, we examined the effect of follistatin on LH-R induction in the presence of activin and FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dose-dependent manner. However, insulinlike growth factor-I (IGF-I) induced LH-R mRNA in the presence of FSH even after medium change. IGF-I might be one of the important factors that act in the medium to maintain LH-R levels in granulosa cells. © 1999 Academic Press Key Words: LH receptor; FSH receptor; activin; follistatin; IGF-I.
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To whom correspondence should be addressed. Fax: 272-20-8443. E-mail: [email protected]. 2 Supported by Fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists.
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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. Folliclestimulating hormone (FSH) 3 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 FSHinduced differentiation of granulosa cells (1) and this process is also modified by many growth factors. Primary cultures of rat granulosa cells obtained from hypophysectomized or 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-R and LH-R induction has been shown to be mediated, at least in part, by cAMP, since exogenous cAMP or other agents that increase intracellular levels of cAMP mimic the action of FSH (2– 6). Therefore, we first analyzed the effect of cAMP on the LH-R mRNA levels and on the 59-flanking region of LH-R using transient expression. In this system, the continual presence of FSH is required to maintain the LH-R at elevated steady levels after induction, associated with changes in LH-R mRNA levels (7, 8). Presently, it is unknown whether these FSH-induced signals directly enhance granulosa cell differentiation and/or whether they instigate the 3 Abbreviations used: FSH, follicle-stimulating hormone; LH, luteinizing hormone; FSH-R, FSH receptor; LH-R, LH receptor; IGF-I, insulinlike growth factor-I; DES, diethylstilbestrol; 8-Br-cAMP, 8-bromoadenosine 3,5-cyclic monophosphate; DMEM, Dulbecco’s modified Eagle’s medium.
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FIG. 1. Maintenance of LH-R mRNA levels by FSH and 8-Br-cAMP. (A) Granulosa cells from DES-primed immature rats were incubated with and without (1) 30 ng/ml FSH (added 24 h after cell plating) for 24 h (2). The cells incubated with FSH for 24 h were incubated with fresh FSH (30 ng/ml) (3–5) or 8-Br-cAMP (1 mM) (6 – 8) after medium exchange for the indicated times (3 and 5, 24 h; 4 and 7, 48 h; 5 and 8, 72 h). The medium from the cells, incubated with FSH for 24 h, was transferred to the cells which were also incubated with FSH for 24 h. These cells were further incubated for the indicated times (9, 24 h; 10, 48 h; 11, 72 h). LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with FSH was taken as 100%. 1, 24 h after cell plating; 2, incubated with FSH for 24 h; 3–5, incubated with fresh FSH after medium change; 6 – 8, incubated with 8-Br-cAMP after medium change; 9 –11, incubated with transfered medium from the cells incubated with FSH for 24 h. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
production of local mediators, which then stimulate differentiation. Previously, our studies using granulosa cell culture have shown that follistatin synthesis is stimulated by FSH (9), while activin production is suppressed by FSH (10), leading to the suggestion that FSH attenuates the effect of activin in the granulosa cell. This is due to follistatin being an activin-binding protein (11) and it neutralizes the diverse activin bioactivities in various systems by stoichiometrically forming inactive complexes with activins (12). In this experiment, we examined the effect of follistatin on LH-R expression induced by activin and FSH. Insulinlike growth factor-I (IGF-I) is one of the growth factors involved in the FSH-induced differentiation of granulosa cells and more importantly FSH increases the synthesis of IGF-I (13, 14). Since we have found that the exchange of medium incubated with or without FSH disturbed the expression of LH-R in rat granulosa cells, it might suggest that the factors produced in this culture medium are essential for the function of FSH to stimulate LH-R expression. The present studies were therefore undertaken to elucidate the molecular mechanisms underlying the hormonal induction of LH-R mRNA in granulosa cells. MATERIALS AND METHODS Hormones and reagents. Activin A was kindly donated by Dr. Eto (Ajinomoto Co., Inc., Central Research Laboratories, Kawasaki, Ja-
pan). Rat FSH (I-8), hCG (CR-119), and human follistatin were obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), gentamicin sulfate, 8-bromo-adenosine 3,5-cyclic monophosphate (8-Br-cAMP) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). Rat IGF-I was purchased from GroPep Pty Ltd. (Adelaide, Australia). Dulbecco’s modified Eagle medium (DMEM), Ham’s F-12 medium, and fungizone were purchased from GIBCO Laboratories (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Boehringer-Mannheim (Mannheim, Germany). Animals. Immature female Wistar rats (21 days old) were obtained from Imai Experimental Animal Farm (Saitama Pref., Japan). Since estrogen treatment in vivo results in the formation of multiple preantral follicles and provides a large number of relatively homogeneous cells at the same stage of development, rats were pretreated with 2 mg DES in 0.1 ml sesame oil, sc, once daily for 4 days. The rats were killed at 26 days and their ovaries were aseptically excised. All procedures followed NIH guidelines. Rat granulosa cell culture. Granulosa cells were obtained by puncturing ovaries from DES-treated immature rats. The granulosa cells were then cultured in Ham’s F-12:DMEM (1:1, v/v) supplemented with 1.1 g/liter NaHCO 3, 40 mg/liter gentamicin sulfate, 1 mg/liter fungizone, and 100 mg/liter bovine serum albumin in a humidified atmosphere containing 5% CO 2, 95% air at 37 C. RNA isolation and analysis. Rat LH-R cDNA was prepared as described previously and linearized with BglII (15). Digoxigeninlabeled LH/hCG receptor cRNA probes corresponding to bases 440 – 2560 were produced by in vitro transcription with T3 RNA polymerase and an RNA labeling kit (Boehringer-Mannheim). A digoxigeninlabeled GAPDH cRNA probe was obtained by the same method. Granulosa cells were cultured in 60-mm dishes containing 5 3 10 6 viable cells in 5 ml of 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 the selected time as
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FIG. 2. Dose-dependent effect of 8-Br-cAMP on LH-R mRNA. (A) Granulosa cells from DES-primed immature rats were cultured for 24 h (0, control t 5 0 h), and 8-Br-cAMP was added at the indicated concentrations. The cells were incubated for 24 h (left) or further incubated for 72 h (right) after a medium change with cAMP-free medium. LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with 0.2 mM cAMP was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
indicated in the guanidinium acid–thiocyanate–phenol– chloroform method (16). The final RNA pellet was dissolved in diethyl pyrocabonate-treated H 2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 15 mg total RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN). In accordance with the standard protocol for the nucleic acid detection kit (Boehringer-Mannheim), Kodak X-Omat film (Eastman Kodak, Rochester, NY) was then exposed to the membranes. The relative abundance of 2.4 kb for rat FSH-R mRNA and 5.4 kb for rat LH-R mRNA in different preparations was quantified with a LKB 2202 UnitroScan Laser Densitometer (LKB Produkter AB, Bromma, Sweden) and normalized against levels of GAPDH mRNA in each sample and expressed as relative to the control value (100%). For all experiments, a representative Northern blot of three replicates is presented. The data are presented as means 6 SE of measurements from triplicate cultures for one representative experiment. The significance of differences between the mean values in the control group and each treated group were tested with Duncan’s multiple comparison test. A value of P , 0.05 was considered statistically significant. Vector preparation and transfection. Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). pGL3-Control contains a SV40 promoter and a SV40 enhancer inserted into structure of pGL3-Basic (Promega Corp). The pRL-SV40 vector contains the SV40 early enhancer/promoter region, which provides strong, constitutive expression of Rluc in a variety of cell types. The pRL vector provide constitutive expression of Renilla luciferase. The rat LH-R promoter from 21389 to 21 bp relative to the transcriptional start site was generated from genomic DNA via PCR using primers specific to the rat LH/hCG receptor sequence. For evaluating promoter activity, 21379 to 21 bp of the 59 flanking sequence of the rat LH-R promoter was ligated to a luciferase reporter vector (pGL3-Luc) and named LH-R-Luc. Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described (17). Using FuGENE, a total of 10 mg of plasmid DNA was transfected, as previously described (18), into primary granulosa cell cultures plate (5 3 10 5 cells in 2 ml in a 35-mm dish). To assay regulatory elements, granulosa cells were cultured 48 h under hormone-free conditions
before transfection. Thirty hours after transfection, cells were treated by hormones for 6 h and processed. After the incubation, cells were harvested and luciferase activity was measured. In the luciferase assay, luciferin and Mg 21 ATP were added to cellular extracts, and the production of light was monitored conveniently by a luminometer. Luciferase activity was assayed as previously described (19).
FIG. 3. Effect of 8-Br-cAMP on the expression of LH-R-Luc in rat granulosa cells. Granulosa cells were cultured for 48 h under hormone-free conditions and cotransfected with LH-R-Luc and pRL. After transfection 30 h later, cells were treated by 8-Br-cAMP for 6 h and were processed. Luciferase activity was corrected for the amount of Renilla luciferase activity detected in each lysate. Each bar represents the mean 6 SE of three independent experiments.
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FIG. 4. Effect of follistatin on activin-induced LH-R mRNA levels. (A) Total RNA was prepared from cells incubated for 72 h in the presence or absence of FSH and activin with increasing concentrations of follistatin, and LH-R mRNA and GAPDH mRNA levels were measured using Northern blot analysis as described under Materials and Methods. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA with FSH 30 ng/ml was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE. Values with asterisks differed significantly from values in cells treated by FSH plus activin: *P , 0.05 and **P , 0.01.
RESULTS
Previous studies have shown that a continual presence of FSH is required to maintain LH-R at elevated steady-state levels after induction (3). It has been shown that the function of FSH to stimulate LH-R induction is mediated, at least in part, by cAMP since exogenously added cAMP or other agents that increase intracellular levels of cAMP mimic the actions of FSH (3– 6). Moreover, we have detected comparable effect of 2 mM of 8-Br-cAMP to 30 ng/ml FSH on the expression of LH-R mRNA. Figure 1 showed that the LH-R mRNA increased with the addition of FSH (30 ng/ml) for 24 h and this LH-R mRNA decreased rapidly after a change of medium and even after the readdition of FSH. Alternatively, LH-R mRNA remained elevated with the addition of the medium, which was incubated with cells for 24 h in the presence of FSH. In addition, after the removal of FSH, the level of LH-R mRNA during incubation increased with the presence of 2 mM 8-BrcAMP. Since we detected the fact that the addition of FSH after a 24 h preincubation with FSH could not increase intracellular cAMP levels (20), data from the readdition of FSH might be due to the functional downregulation of FSH-R. To evaluate the effect of cAMP on LH-R mRNA levels, the cAMP effects on LH-R mRNA levels were ex-
amined before and after removal of cAMP. LH-R mRNA was also dose-dependently induced with a maximum response at 2 mM at 24 h incubation. Furthermore, LH-R mRNA levels decreased rapidly after removal of 8-Br-cAMP, and by the third day after removal, LH-R mRNA was not detectable in the cells (Fig. 2). For many genes that are transcriptionally regulated by cAMP, the 59 flanking promoter regions are necessary and sufficiently carry out this activity. To determine whether the LH/hCG-R 59-flanking region can direct expression, the proximal and 1379 bp of the LH/hCG-R 59-flanking region was inserted into a transient expression vector, pGL3-Basic, which contains luciferase as the reporter gene. The resulting plasmid (LH-R-Luc) was transiently transfected into rat granulosa cells. Gene transfer studies were performed by FuGENE transfection and luciferase enzyme activity measured in light units as an indication of promoter activity. Cells were cotransfected with pRL as an internal control for transfection efficiency. To investigate the hormonal regulation of the 59-flanking region, we analyzed the effect of cAMP on 1379 bp of the LH-R promoter region in the rat granulosa cells. Treatment with 8-Br-cAMP (0.5– 4 mM) significantly enhanced activity of 1379 bp of the LH-R 59-flanking region in a
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FIG. 5. Maintenance of LH-R mRNA by the presence of IGF-I. (A) Granulosa cells were incubated with 30 ng/ml FSH (added 24 h after cell plating) for 24 h. The cells then continued to be incubated for a total of 72 h with either no medium change or exchange (wash) at 24 h and with FSH alone or FSH and IGF-I (30 ng/ml). LH-R mRNA levels were measured using Northern blot analysis as described under Materials and Methods. LH-R mRNA levels were not significantly affected by treatment with IGF-I alone. (B) Autoradiographs of LH-R mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH-R mRNA at 24 h with FSH was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The Northern blot is representative of three replicate experiments and the data are presented as means 6 SE.
dose dependent manner (Fig. 3). These data clearly demonstrate that FSH stimulates the induction of the LH-R in granulosa cells of DES-primed immature rats, at least in part, due to increasing the rate of transcription of the LH-R gene. Since our data showed that the presence of medium incubated with FSH enhanced the effect of FSH on LH-R expression, further studies were undertaken to identify the local factors induced by FSH exposure. In our previous experiment, the treatment of cAMP or FSH treatment suppressed the production of activin and increased follistatin production in granulosa cells (10, 21). We examined the effect of follistatin on activin action for LH-R induction in the presence of FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dosedependent manner (Fig. 4). Therefore, follistatin, whose production is induced by FSH and activin, suppresses the effect of activin on LH-R mRNA expression. Since IGF-I enhances the proliferation of many cell types and since the IGF-I receptor is coexpressed with IGF-I in ovarian follicles (22) it seemed likely that IGF-I may act in an autocrine/paracrine manner to stimulate granulosa cell differentiation. In the previous experiment (23), the relatively low concentrations of exogenously added IGF-I are capable of synergizing with FSH in the induction of LH-R expression in gran-
ulosa cell. Figure 5 shows that FSH itself could not induce LH-R after medium change, and FSH in the presence of IGF-I increased LH-R mRNA. DISCUSSION
The present studies were undertaken to determine the mechanisms underlying the induction and maintenance of LH-R in the differentiating granulosa cell. Consistent with previous studies (4, 24 –28), our data show that FSH is able to dramatically induce LH-R mRNA in granulosa cells from DES-primed immature rats from undetectable basal levels. The data obtained by the other group suggest that levels of LH-R mRNA decrease rapidly if FSH is removed from the cultured cells and that the levels of mRNA remain elevated despite the removal of medium if FSH is present (7). However, in the present experiment, the LH-R mRNA level of the cells incubated with FSH decreased rapidly after medium removal, and the readdition of FSH with the fresh medium did not restore these levels. These discrepancies might be due to the differences in the experimental conditions such as the coating of the surface of the culture plates. For example, the previous reports claimed that a time-related increase in the level of FSH-R mRNA was observed after increasing the lengths of culture in the absence of hormone treat-
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FIG. 6. (A) (1) Activin induces FSH-R. (2) FSH (mediated by cAMP) induces LH-R. (3) FSH also stimulates the expression of follistatin, activin-binding protein, and inhibits the production of activin. (4) FSH stimulates the expression of IGF-I and IGF-I-R. (B) (5) Follistatin binds to activin and suppresses the effect of activin on LH-R expression. (6) IGF-I maintains the expression of LH-R in the presence of FSH. FSH actions result in the suppression of activin’s effects and enhance IGF-I’s effect.
ment, compared to the levels found in cells at the start of culture (29). In contrast, the granulosa cells on collagen-coated dishes in our experiments indicated that FSH-R mRNA levels did not change during 4 days of culture in the absence of hormone treatment (30). Therefore, the level of gonadotropin receptor expression in the untreated cells may depend on the characteristics of the culture system. The ability of cAMP to increase LH-R mRNA to a greater extent than FSH at the second addition may be explained by the FSH receptor desensitization induced by FSH pretreatment. In the previous experiment, we showed that 24 h presence of FSH causes downregulation of FSH-R function of increasing intracellular cAMP levels in response to the next FSH stimulation. It took about 48 h to recover the ability to increase cAMP levels in response to the second addition of FSH after the first exposure to FSH in this culture model (20). Since addition of 8-Br-cAMP significantly enhanced the expression of LH-R mRNA after medium removal, the data showed that this marked decrease in LH-R mRNA levels after removal of medium including FSH is due, at least in part, to a significant decrease in cAMP production. In addition, the present results showed that 8-Br-cAMP more potently induced LH-R mRNA after medium removal and that cAMP alone is not sufficient to maintain the levels of LH-R mRNA induced by the first FSH.
The incubation with 8-Br-cAMP for 24 h produced a dose-dependent response in LH-R mRNA in granulosa cells of DES-primed immature rats, and the results of the nuclear run-on experiments clearly demonstrated that 8-Br-cAMP significantly stimulates the rate of transcription of the LH-R gene (7). Therefore, the effect of 8-Br-cAMP on the LH-R mRNA at 24 h might be due to an increased rate of transcription. As shown in previous studies (4, 24 –28), our data demonstrated that 8-Br-cAMP induces LH-R mRNA which is initially at undetectable basal levels in granulosa cells. In our previous experiments, we have shown that FSH (1–100 ng/ml) significantly enhanced the activity of the LH-R 59-flanking region in a dose-dependent manner (in press). Therefore, the increased LH-R mRNA gene transcription elicited by 8-Br-cAMP suggests that FSH mediates this effect through cAMP, i.e., the protein kinase A pathway. The results from transient transfection of LH-R promotor luciferase constructs into granulosa cells, which yield a stimulation of luciferase activity in response to 8-Br-cAMP, support the results of the previous nuclear run-on transcription assays (9). Our studies show that DNA sequences present within 1379 bp of the translation start site greatly enhance the expression of a luciferase reporter gene in granulosa cells. The possibility that sequences located upstream of 21379 bp would further enhance this expression, however, remains open. More detailed studies of
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this region by mutational analysis and other approaches aimed at identifying sequences that may be specifically recognized by cAMP-regulated nuclear factors will allow more precise characterization of this element. At present it’s unknown whether FSH-induced signals directly enhance LH-R expression or whether they instigate the production of local mediators such as activin and IGF-I, which then stimulate LH-R synthesis in the presence of FSH. According to present data, some local factors might be secreted into the medium by the cAMP dependent mechanisms. Although activin stimulates granulosa cell proliferation, follicular development in vitro (31, 32), and LH-R expression in the presence of FSH, FSH increased follistatin production and suppressed activin production in this culture system in a dose-dependent manner (10). Since follistatin is an activin-binding protein that has been shown to antagonize the function of activin in a number of assays, including the present data (11, 33), and cAMP signals increase the production of follistatin, the activin–follistatin model seems to be unrelated to the increase of LH-R mRNA level (Fig. 6). Previous studies showed that IGF-I is selectively expressed in a subset of relatively healthy-appearing follicles in the rat ovary (22, 34), leading to the suggestion that IGF-I is a marker for follicular selection. Since IGF-I enhances the proliferation of many cell types and since it has been previously shown that the IGF-I receptor is coexpressed with IGF-I in ovarian follicles (22), it seemed likely that IGF-I may act in an autocrine/paracrine manner to stimulate granulosa cell proliferation. For example, IGF-I amplifies FSHinduced aromatase expression and LH-R induction (23, 35). In addition, amplification by IGF-I of FSH receptor expression is positively reinforced by FSH-induced augmentation of IGF-I receptor expression, which has been demonstrated in vivo at the mRNA level (22) and in vitro at the IGF-I-binding level (36). This local IGF-I expression creates an intrafollicular positive feedback loop in which IGF-I enhances FSH action and FSH enhances IGF-I action through mutual complementary receptor upregulation (37). In addition, the effect of IGF-I on the induction of FSH receptor enhances FSH action of cAMP production, which results secondarily in augmentation of the LH-R gene. However, as shown in this experiment, the cAMP dose does not completely compensate for the effect of medium in the presence of FSH to maintain mRNA levels of LH-R. Therefore, it is also suspected that IGF-I has a primary effect on LH-R production in the presence of FSH. Further studies are clearly required to elucidate the mechanism of the IGF-I effect on the gonadotropin receptor.
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ACKNOWLEDGMENTS We thank the National Hormone and Pituitary Agency, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, and the University of Maryland School of Medicine for the rat FSH. This work was supported by grants from the Ministry of Education, Science and Culture of Japan (10044235, 09470353, and 10877253), Tokyo, Japan.
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