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a Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan
b Department of Biochemistry, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data from several laboratories, including our own, have shown that FSH, a major regulator of FSHR induction, increases FSHR mRNA levels both in vivo and in vitro [1–4]. Many of the effects of FSH on granulosa cells are mediated by the cAMP second-messenger system [5]. A model system that has been widely used to study this phenomenon consists of primary cultures of rat granulosa cells obtained from immature female rats pretreated with estradiol. The use of this defined system has showed that the ability of FSH to stimulate the induction of FSHR is mediated, at least in part, by cAMP, since exogenous cAMP or other agents that increase intracellular levels of cAMP mimic the actions of FSH [4].
Follicle selection is known to be affected by gonadotropins. In cycling animals, small follicles are recruited into the growth pool each cycle by the FSH surge. The development of larger follicles is stimulated by and dependent on FSH. However, the initial growth of granulosa cells in small follicles is independent of FSH and is observed in hypophysectomized animals [6,7]. Recent evidence suggests that activin may play a major role as a local regulator in ovarian follicles that both produce and respond to activin [8–10]. The expression of activin subunit messenger RNA (mRNA) in granulosa cells is limited in growing follicles, but not in atretic follicles [11]. Of particular importance is the stimulatory influence of activin on FSHRs as a mechanism whereby preantral follicles may become responsive to FSH. We have described how FSH and cAMP analogs suppress the level of activin in the medium produced by these granulosa cells [12]. Moreover, follistatin, the activin-binding protein, is produced from granulosa cells under the control of FSH; follistatin suppresses activin's effect on FSHR expression [13,14]. These data suggest that while activin is indispensable for FSHR expression in the earlier stage of follicle development, once the FSH effect is mediated by FSHR, the maintenance of the FSHR level is not dependent on activin.
Previous studies have shown that insulin-like growth factor I (IGF-I) is expressed in a subset of relatively healthy-appearing follicles in the rat ovary [15,16], suggesting that IGF-I is a marker for follicular selection. Recently, it was shown by means of a knockout mouse model that IGF-I and FSHR are selectively coexpressed in growing murine follicles and that IGF-I augments granulosa cell FSHR expression [17]. These data suggest that ovarian IGF-I expression serves to enhance granulosa cell FSH responsiveness by enhancing FSHR expression [6,17,18]. Amplification of FSHR expression by IGF-I is positively reinforced by FSH-induced augmentation of IGF-I receptor expression, which has been demonstrated in vivo at the mRNA level [16] and in vitro at the IGF-I binding level [19]. Thus, local IGF-I expression creates an intrafollicular positive feedback loop in which IGF-I enhances FSH action and FSH enhances IGF-I action through mutually complementary receptor up-regulation. The present studies were undertaken to elucidate the molecular mechanisms underlying the induction of FSH mRNA by IGF-I in the presence of FSH.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activin A was kindly donated by Dr. Eto (Ajinomoto Co., Inc., Central Research Laboratories, Kawasaki, Japan). Rat FSH (I-8) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), gentamicin sulfate, and 8-bromo-adenosine 3,5-cyclic monophosphate (8-Br-cAMP) were purchased from Sigma Chemical Company (St. Louis, MO). Rat IGF-I was purchased from GroPep Pty Ltd. (Adelaide, SA, Australia). Dulbecco's modified Eagle's 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).
Rat Granulosa Cell Culture
Granulosa cells were obtained from immature female Wistar rats, which received an injection of 2 mg DES in 0.2 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 National Institutes of Health (NIH) guidelines. This study was approved by the Gunma University School Institutional Review Board. 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) supplemented with 1.1 g/L NaHCO3, 40 mg/L gentamicin sulfate, 1 mg/L fungizone, and 100 mg/L BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2, 95% air at 37°C [20].
RNA Isolation and Analysis
Granulosa cells were cultured in 60-mm dishes containing 5 x 106 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 indicated in the guanidinium acid-thiocyanate-phenol-chloroform method [21]. The final RNA pellet was dissolved in diethyl pyrocarbonate-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 was separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN, Glen Cove, NY). 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. Luminescence detection was quantified with an LKB 2202 UnitroScan Laser Densitometer (LKB Produkter AB, Bromma, Sweden), normalized against a corresponding relative amount of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA in each sample, and expressed as relative densitometric units.
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 24 h before the nuclei were isolated. Cells were washed three times with ice-cold Dulbecco's PBS without calcium and magnesium (PBS[-]), collected by scraping in PBS(-), and then centrifuged for 5 min 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, 0.5% Nonidet P-40 [Sigma]). 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, 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 [22]. The relative amount of incorporation of label into specific RNAs was determined by DNA excess filter hybridization, as described previously [22], using cDNAs for rat FSHR. Ten micrograms each of FSHR, Bluescript, and ß-actin cDNA was included on the DNA filter during hybridization in order to correct for background and to serve as internal controls. Autoradiographic bands were quantified by a fluoro-image analyzer (BAS 2000, Fuji Co Ltd., Japan).
Vector Preparation and Transfection
The luciferase assay was performed by using the Dual-Luciferase Reporter System (Promega, Madison, WI), in which the transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of renilla luciferase. The rat FSHR promotor from -1862 to -1 base pairs (bp) relative to the transcriptional start site was cloned from genomic DNA via polymerase chain reaction (PCR) using a DNA walking kit (Clontech Laboratories, Palo Alto, CA; Fig. 1). Briefly, adaptor-ligated rat genomic DNA fragments were amplified using an adaptor-specific primer and a primer specific to the FSHR gene. A second PCR reaction was done by using a nested adaptor-specific primer and a nested primer specific to FSHR. The distinct DNA band on an agarose gel corresponding to the FSHR gene was isolated and subcloned into T-vector (Promega) and sequenced by the dye primer or terminator cycle sequencing method using automated DNA sequencer (373 A, Applied Biosystems, Foster City, CA). The DNA sequence of the clone was compared to the published sequence of a cloned rat FSHR from -1105 to -1 [23]. Sequence comparison indicated individual base pair substitution between our and the reported clone at the following positions: -1103 (A to G), -1102 (A to T), -1101 (G to A), -1093 (A to G), -1092 (C to A), -1091 (G to C), -1090 (T to A), -887 (T to C), -187 (A to G).
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Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promotor and enhancer sequences (Promega). The pGL3-Control contains an SV40 promoter and an SV40 enhancer inserted into the structure of pGL3-Basic (Promega). For evaluating promotor activity, -1862 to -1 bp of the 5' flanking sequence of the rat FSHR promoter was ligated to a luciferase reporter vector (pGL3-Luc) and named FSHR-Luc. The cells were lysed in lysis buffer supplied by manufacturer before measurement of the firefly and the renilla luciferase activities on 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.
Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described previously [16]. Using FuGENE (Boehringer Mannheim), a total of 1 µg of plasmid DNA was transfected, as described previously [17], into primary granulosa cell culture plates (2.5 x 105 cells/0.5 ml of medium 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. 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 [6].
Data Analysis
The relative abundance of a 2.4-kilobase (kb) signal for rat FSHR mRNA in different preparations was quantified with an LKB 2202 UnitroScan Laser Densitometer, 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 three independent experiments. Comparisons between groups were performed by one-way ANOVA. The significance of differences between the mean values in the control group and each treated group was tested with Duncan's multiple-comparison test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study the time dependence of the IGF-I effect on FSHR mRNA accumulation, granulosa cells were cultured for 24–96 h in the presence of FSH (30 ng/ml) with and without IGF-I at a concentration of 10 ng/ml (Fig. 3). Basal FSHR mRNA remained low throughout the 96-h incubation period and was not significantly affected by treatment with IGF-I alone. The concurrent treatment with IGF-I resulted in significant augmentation of the FSH-induced FSHR mRNA from 72 to 96 h, with a peak at the 72-h time point. As can also be seen for this time course, the effect of IGF-I on the mRNA levels was not significant during the increase of receptor mRNA, whereas after the maximal levels of expression of receptor, the effect of IGF-I was significant.
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It is known that the action of FSH is mediated by cAMP and that a considerable amount of cAMP is accumulated in the granulosa cells upon stimulation by FSH. We observed that FSH increased intracellular cAMP levels; however, IGF-I, either alone or in combination with FSH, did not have an effect on the intracellular cAMP levels (data not shown). As shown in Figure 4, treatment with 8-Br-cAMP produced a significant increase in FSHR mRNA, and concurrent treatment with IGF-I (10 ng/ml) produced a significant increase in 8-Br-cAMP action.
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We next examined whether IGF-I regulation of FSHR mRNA is dependent on gene transcription and/or receptor mRNA stability. The following sets of experiments were designed to address the potential contribution made by changes in FSHR gene transcription to the regulation of FSHR mRNA. To determine whether the FSHR 5'-flanking region plays a role in directing FSHR mRNA expression, the proximal end and 1862 bp of the FSHR 5'-flanking region was inserted into a transient expression vector, pGL3-Basic, which contains luciferase as the reporter gene, and the resulting plasmid (FSHR-Luc) was transiently transfected into rat granulosa cells. Gene transfer studies were performed using FuGENE transfection, and luciferase enzyme activity was 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 5'-flanking region, we analyzed the effect of FSH on 1862 bp of FSHR promoter in rat granulosa cells. Treatment with 10 ng/ml FSH significantly enhanced the activity of the 1862 bp of the FSHR 5'-flanking region in a dose-dependent manner (Fig. 5A). FSH (30 ng/ml) significantly enhanced the activity of the 1862 bp of the FSHR 5'-flanking region, but treatment with 10 ng/ml IGF-I alone did not significantly influence the activity of the FSHR promotor or affect the increased promotor activity induced by FSH (Fig. 5B).
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To test whether IGF-I has an effect on transcription of the FSHR gene in the presence of FSH, we performed nuclear run-on assays on granulosa cells. As shown in Figure 6, while 30 ng/ml FSH significantly enhanced the activity of FSHR transcription, no difference in FSHR gene transcription was observed in granulosa cells treated with FSH vs. those treated with FSH plus IGF-I. These data, therefore, suggest that the synergistic effects of IGF-I on FSHR induction are not mediated by an increase in FSHR gene transcription.
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In order to assess the rates of degradation of FSHR mRNA transcripts, granulosa cells were preincubated without treatment and with FSH, IGF-I, or FSH plus IGF-I for 24 h. After this preincubation period, 5 µM actinomycin-D was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, and 9 h after addition of the transcription inhibitor, and FSHR mRNA levels were quantitated by Northern blot analysis. The amount of FSHR mRNA at time 0 (the time of addition of actinomycin-D) in each group was assigned a value of 100%, and other values in each group at different time-points were expressed as a percentage of the Time 0 value. As shown in Figure 7, in the absence of FSH, the decay curves for the 2.4-kb FSHR mRNA transcript in primary granulosa cells were not significantly different in the absence and presence of IGF-I. However, in the presence of FSH, IGF-I significantly increased stability of FSHR mRNA.
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Since activin alone increases FSHR mRNA levels through its own receptor in this culture and does not increase intracellular cAMP accumulation, we examined the effect of IGF-I on activin-induced FSHR mRNA. To study the time dependence of the IGF-I effect on activin-induced FSHR mRNA, granulosa cells were cultured for the duration indicated in the presence of activin with and without IGF-I at a concentration of 10 ng/ml. IGF-I did not affect the time course of the activin-induced FSHR increase (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The observed maintenance of message levels of FSHR by FSH and IGF-I may be the result of increased FSHR gene transcription and/or message stability. Determination of the transcriptional mechanisms that regulate FSHR expression in the gonads will provide important insights into both cell-specific transcriptional events that are important for gonadal function and mechanisms that control the response of the gonads to FSH through modulation of receptor levels. As described in a paper by Goetz et al. [23], an E box is required for full promoter function of the rat FSHR gene. The promoters of the rat, human, sheep, and mouse FSHR genes all contain an E box consensus sequence, CANNTG, which is known to bind members of the basic helix-loop-helix (bHLH) family of proteins that consist mostly of transcriptional regulators involved in control of growth and differentiation. To investigate the hormonal regulation of the 5'-flanking region, we cloned 1862 bp of the FSHR 5'-flanking region and analyzed the effect of FSH on 1862 bp of FSHR promoter in rat granulosa cells. FSH enhanced the activity of the 1862 bp of the FSHR 5'-flanking region in a dose-dependent manner, and FSH (30 ng/ml) significantly enhanced the activity of the 1862 bp of the FSHR 5'-flanking region, but the treatment with 10 ng/ml IGF-I alone did not significantly influence the activity of the FSHR promotor or affect the increased promotor activity induced by FSH.
Further characterization of both the FSHR gene and its promoter region is required to complete our understanding of the transcriptional mechanisms activating FSHR in granulosa cells. Although FSH significantly stimulates the rate of transcription of the FSHR gene, the results of the nuclear run-on experiments also presented herein demonstrated that IGF-I did not stimulate the rate of FSH-induced transcription of the FSHR.
The experiment for the decay of FSHR mRNA was performed in cells pretreated by hormone for 24 h. The data presented suggest a possible role for changes in FSHR mRNA stability in the IGF-I induced regulation of FSHR in rat granulosa cells. Also, it has been well established that the expression of specific, highly regulated mRNAs like c-fos, c-myc, and ß-adrenergic receptor are controlled, at least in part, at the level of mRNA degradation [24,25]. In the majority of instances of posttranscriptional regulation of mRNA, 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 are often located within a discrete region of the mRNA. In terms of LHR mRNA, an LHR mRNA binding protein, which is a candidate for a trans-acting factor involved in the hormonal regulation of LHR mRNA stability in rat ovary, has been reported [26,27]. Since levels of FSHR mRNA closely parallel the receptor number, it is likely that post-transcriptional regulation has a pivotal role in mediating physiological changes in receptor expression seen during the ovarian cycle. Since IGF-I clearly prolonged FSHR mRNA stability according to the result of time-course and half-life experiments, IGF-I may relate to the production of certain proteins that stabilize the FSHR mRNA in granulosa cells.
Questions remain concerning the process of ovarian follicular selection. Why, for example, do only a few follicles develop in response to gonadotropins when all are exposed to equal circulating levels? The exposure to FSH does not result in an equal effect among follicles, since selective growth factor expression significantly amplifies FSHR gene expression and presumably FSH action in a subset of follicles. In a previous study, it was reported that activin supported granulosa cell survival and cell proliferation, and maintained the functional FSHR in the absence of FSH throughout long-term culture [28]. In our cultured cells, activin alone induced FSHR mRNA and protein expression, whereas the IGF-I effect on FSHR mRNA was dependent on the presence of FSH. The IGF-I signal is mediated by the presence of FSH, and it is possible that FSHR was present on the cell surface in the absence of IGF-I. Therefore, activin has the unique role of introducing FSHR in the early stage of folliculogenesis since activin itself in the absence of FSH can induce FSHR. However, while activin still has a coordinated stimulatory effect with FSH on FSHR expression, FSH suppressed activin production and increased production of follistatin, which is able to bind to activin and neutralize the effect of activin [14,29]. Thus, the effect of FSH on granulosa cells may result in diminishing the effect of activin in the follicles. In addition, there was no additive effect of activin and IGF-I on FSHR, as shown in this experiment. On the other hand, amplification by IGF-I of FSHR expression is positively reinforced by FSH-induced augmentation of IGF-I receptor expression, which has been demonstrated in vivo at the mRNA level [16] and in vitro at the IGF-I binding level [19]. Thus, local IGF-I expression creates an intrafollicular positive feedback loop in which IGF-I enhances FSH action and FSH enhances IGF-I action through mutually complementary receptor up-regulation. IGF-I is not essential for induction of granulosa cell FSHR gene expression de novo, since FSHR mRNA is still present, albeit at low levels, in the IGF-I knockout ovary [18]. It is possible that activin has a primary effect on granulosa cell FSHR expression, and that IGF-I secondarily enhances FSHR expression to maintain the response to FSH.
A previous study showed that IGF-I and FSHR gene expression were selectively localized in granulosa cells in a subset of healthy growing follicles in the murine ovary [17]. In addition, a reduction in FSHR mRNA and aromatase expression in IGF-I knockout follicles was detected in IGF-I knockout mice. The reduced level of granulosa cell FSHR expression in IGF-I knockout ovaries may explain the infertility of the IGF-I knockout female [18]. Follicles in the IGF-I knockout ovary are arrested at a late preantral or early antral stage of development. Thus it appears that a critical level of FSHR expression, normally ensured by local IGF-I action, is essential for gonadotropin responsiveness and follicular development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the Ministry of Education, Science and Culture of Japan (10044235, 10877253), Tokyo, Japan. 
2 Correspondence. FAX: 81-27-220-8443;tminegis{at}sb.gunma-u.ac.jp
Accepted: October 6, 1999.
Received: July 29, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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