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Female Reproductive Tract |
Department of Obstetrics and Gynecology,3 School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan
Department of Biochemistry,4 Fukui Medical University, Matsuoka, Fukui 910-1193, Japan
CREST, JST (Japan Science and Technology Corporation),5 Saitama 322-0012, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activin, follicle-stimulating hormone receptor, follistatin, granulosa cells, growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent evidence suggests that activin may play a major role as a local regulator in ovarian follicles that both produce and respond to activin [4–6]. The expression of activin subunit mRNA in granulosa cells is limited in growing follicles but not in atretic follicles [7]. The particular importance is the stimulatory influence of activin on FSH-R as a mechanism whereby preantral follicles may become responsive to FSH [8]. However, we have described how FSH and cAMP analogs suppress the level of activin in the medium produced by these granulosa cells [9]. Additionally, follistatin, the activin-binding protein, is produced from granulosa cells under the control of FSH; follistatin suppresses activin's effect on FSH-R expression [9–11]. These data suggest that whereas activin is indispensable for FSH-R expression during the earlier stage of follicle development, once the FSH effect is mediated by FSH-R, the maintenance of the FSH-R level is not dependent on activin.
Among the growth factors, transforming growth factor ß1 (TGFß1), which belongs to the family of multifunctional cytokines, is expressed in the ovary [12]. This suggests that TGFß may serve as a local regulator of granulosa cell development, particularly as a regulator of FSH-R expression [13, 14]. The present report provides further evidence of a modulatory action of TGFß on FSH-R expression [13, 14].
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 estrogen. Using this system, we have examined the mechanism of action of TGFß to determine the functional difference between activin and TGFß in follicular development.
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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. Y. Eto (Ajinomoto Co., Inc., Central Research Laboratories, Kawasaki, Japan). Rat FSH (I-8) and human follistatin were obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol and gentamicin sulfate were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). The TGFß was purchased from Pepro Tech EC Ltd. (St. Janes' Square, London, U.K.). Dulbecco modified Eagle (DME) medium, Ham 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 that received an injection of 2 mg of diethylstilbestrol in 0.2 ml of 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 guidelines. The present study was approved by the Gunma University School Institutional Review Board. Granulosa cells were washed and collected by brief centrifugation (300 g, 10 min), and cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham F-12/DME (1:1 vol/vol) medium supplemented with 1.1 g/L of NaHCO3, 40 mg/L of gentamicin sulfate, 1 mg/L of fungizone, and 100 mg/L of BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2 and 95% air at 37°C [8].
Receptor-Binding Assay
Granulosa cells were cultured in Immulon-2 Removawell (Dynatech Laboratories, Inc., Chantilly, VA). Each well contained 1 x 105 viable cells (determined by trypan blue exclusion) in 0.1 ml of medium. After 24 h of incubation, hormone was added to the medium. The cells were placed on ice and quickly washed three times with 0.2 ml of cold medium after 72 h of incubation.
Next, the granulosa cells were incubated in a 1:1 (vol/vol) mixture of DME/Ham F-12 medium containing 0.1% BSA (pH 7.4) at 37°C with 5 x 104 cpm [125I]FSH (0.5 ng, 100 000 cpm/ng). The FSH was iodinated according to the chloramine-T method. The incubation medium was removed after 2 h of incubation, and the cells were washed twice with 0.2 ml of medium. Each well was then torn off from the Removawell strip, and the amount of radioactivity remaining in the well (cell-bound hormone) was quantified by a 
-counter. Nonspecific binding was determined by adding excess unlabeled FSH (1.25 IU/well).
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 without and with reagents, and the cultures were stopped at the selected time as indicated in the guanidinium acid-thiocyanate-phenol-chloroform method [15]. 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 of total RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne; ICN, Glen Cove, NY), and RNA was fixed by ultraviolet cross-linking. The membranes were hybridized with digoxigenin-labeled cRNA probe for 16 h at 68°C, and membranes were rinsed twice for 5 min with blocking reagent and treated for 30 min with 5 ml of antidigoxigenin Fab fragment conjugated to alkaline phosphatase diluted 1:10 000 in blocking reagent and filtered through a 0.22-µm filter before use. Each membrane was incubated for 10 min in 0.26 mM 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)-penyl-1,2-dioxetane (AMPPD) in fresh substrate buffer. The membrane was then sealed in a hybridization bag and exposed to Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). 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.
Preparation of cRNA Probes
Rat FSH-R cDNA was subcloned into the EcoRI site of the Bluescript KS(+) (Stratagene, La Jolla, CA) vector and linearized with HindIII [8]. Digoxigenin-labeled FSH-R cRNA probes corresponding to bases 239–2368 were produced by in vitro transcription with T7 RNA polymerase and the RNA-labeling kit. Rat follistatin cDNA (kindly donated by Dr. Shunichi Shimasaki, Whittier Institute, La Jolla, CA) was subcloned into the PstI and XbaI site of the Bluescript SK(+) (Stratagene) vector and linearized with PstI [16]. Digoxigenin-labelled follistatin cRNA probe corresponding to bases 1–1018 was produced by in vitro transcription with T3 RNA polymerase and the RNA-labeling kit. Rat GAPDH cDNA was subcloned into pGEM-T easy vector and linearized with SphI. Digoxigenin-labelled rat GAPDH cRNA probe corresponding to bases 519–970 was produced by in vitro transcription with SP6 RNA polymerase and the RNA-labeling kit.
Vector Preparation and Transfection
Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). The pGL3-Control contains an SV40 promoter and an SV40 enhancer inserted into the structure of pGL3-Basic (Promega). For evaluating promoter activity, bases -1862 to -1 of the 5'-flanking sequence of the rat FSH-R promoter were ligated to a luciferase reporter vector (pGL3-Luc) and named FSH-R-Luc. Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described previously [17]. Using FuGENE (Roche Molecular Biochemicals, Indianapolis, IN), a total of 1 µg of plasmid DNA was transfected as described previously [18] into primary granulosa cell culture plates (2.5 x 105 cells per 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. The cells were lysed in lysis buffer supplied by the manufacturer, followed by measurement of the firefly and the renilla luciferase activities on a luminometer. The relative firefly luciferase activities were normalized 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 [18].
Transcription Stability Analysis
Cells were preincubated with and without TGFß 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 addition of the inhibitor for RNA extraction and Northern blot analysis.
Data Analysis
The relative abundance of a 2.4-kilobase (kb) signal for rat FSH-R mRNA and of a 2.6-kb signal for rat follistatin mRNA in different preparations was quantified with the 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 ± SEM 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 the Duncan multiple-comparison test. A P value of less than 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because the time-course experiment of FSH-R mRNA resulted in maximum levels being reached at 48 h, we investigated the dose-dependent effects of TGFß under the same conditions. The FSH-R mRNA levels increased in a dose-dependent manner compared to the control in the presence of increasing concentrations (0.1–30 ng/ml) of TGFß (Fig. 2).
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Treatment with FSH produced a substantial increase in the FSH-R mRNA level, and concurrent treatment with increasing concentrations of TGFß brought about dose-dependent increases in FSH-induced FSH-R mRNA (Fig. 3).
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As shown in Figure 4, 72-h treatment with TGFß (10 ng/ml) increased FSH-R levels, and TGFß treatment in the presence of 100 ng of follistatin did not affect the FSH-R level.
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We next examined whether TGFß regulation of FSH-R mRNA is dependent on gene transcription and/or receptor mRNA stability. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the effect of FSH on 1862 base pairs (bp) of FSH-R promoter in rat granulosa cells. The treatment with FSH (30 ng/ml) significantly enhanced the activity of the 1862 bp of the FSH-R 5'-flanking region, but the treatment with 10 ng/ml of TGFß alone did not significantly influence the activity of the FSH-R or affect the increased promoter activity induced by FSH (Fig. 5).
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To assess the rates of degradation of FSH-R mRNA transcripts, we preincubated granulosa cells with and without TGFß treatment 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, 9, and 12 h after addition of the transcription inhibitor, and FSH-R mRNA levels were quantitated by Northern blot analysis. The amount of FSH-R 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 6, the decay curves for the 2.4-kb FSH-R mRNA transcript in primary granulosa cells in the presence of TGFß significantly increased the stability of FSH-R mRNA.
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Because our previous data showed that activin treatment induced the expression of follistatin mRNA in granulosa cells [11], we examined the effect of TGFß on follistatin mRNA. The level of follistatin mRNA in the presence of 10 ng/ml of TGFß increased in a time-dependent manner; it resulted in a maximum at 12–24 h and gradually decreased after 48 h (Fig. 7). Follistatin mRNA accumulation was stimulated in a dose-dependent manner by TGFß, with a maximum increase of 1.7-fold at a dose of 10 ng/ml of TGFß (Fig. 8).
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To compare the effect of follistatin on activin- and TGFß-induced expression of FSH-R mRNA in granulosa cells, we examined the effect of follistatin on activin and TGFß action with respect to FSH-R mRNA induction. Activin (50 ng/ml) induced FSH-R mRNA significantly, and the addition of 100 ng/ml of follistatin antagonized this effect. On the other hand, the addition of follistatin had no effect on TGFß-induced FSH mRNA (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous experiments suggested that TGFß potentiates the action of FSH at sites distal to cAMP generation in the rat granulosa cells [22]. Therefore, the observed increase of message levels of FSH-R by TGFß may be the result of increased FSH-R gene transcription and/or message stability. Determination of the transcriptional mechanisms that regulate FSH-R expression in the gonads will provide important insights regarding both cell-specific transcriptional events that are important for gonadal function and mechanisms that control the response of the gonads to FSH through the modulation of receptor levels. To investigate the hormonal regulation of the 5'-flanking region, we have cloned 1862 bp of the FSH-R 5'-flanking region and analyzed the effect of FSH and TGFß in rat granulosa cells. We found that FSH enhanced the activity of the 1862 bp of the FSH-R 5'-flanking region in a dose-dependent manner. At a dose of 30 ng/ml, FSH significantly enhanced the activity, whereas at a dose of 10 ng/ml, TGFß alone did not significantly influence the activity of the FSH-R promoter or affect the increased promoter activity induced by FSH. Further characterization of both the FSH-R gene and its promoter region is required to complete our understanding of the transcriptional mechanisms activating FSH-R in granulosa cells.
We examined the decay of FSH-R mRNA in cells pretreated with TGFß for 24 h. The data presented suggest a possible role for changes in FSH-R mRNA stability in the TGFß-induced regulation of FSH-R in rat granulosa cells. Also, it has been 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 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 LH-receptor (LH-R) mRNA, an LH-R mRNA-binding protein, which is a candidate for a trans-acting factor involved in the hormonal regulation of LH-R mRNA stability in rat ovary, has been reported [25, 26]. Posttranscriptional regulation likely has a pivotal role in mediating the physiological changes in receptor expression seen during the ovarian cycle. Because TGFß clearly prolonged FSH-R mRNA stability according to the results of time-course and half-life experiments, TGFß may relate to the production of certain proteins that stabilize the FSH-R mRNA in granulosa cells.
Earlier studies showed that FSH stimulated preantral follicular growth [27] and that a decrease of circulating FSH levels retarded folliculogenesis in immature rats [28]. Granulosa cells respond to FSH, in part, by elaborating peptide autocrine/paracrine factors, such as inhibin, activin, and TGFß [29].
Activin and TGFß both stimulate the expression of follistatin mRNA in cultured granulosa cells. In addition, activin has a strong, positive effect on the expression of FSH-R mRNA, as described before, and TGFß also induces and prolongs the elevated level of FSH-R mRNA compared with the time course of activin action. Because activin and TGFß have a common signal transduction system, which is comprised of serine/threonine kinase receptor type I and type II, and of Smad proteins [30], someone may expect a similar effect of these two reagents. However, the effect of TGFß on the FSH-R mRNA was not neutralized by the addition of follistatin under the condition in which the effect of activin was completely neutralized. Because the effect of TGFß on FSH-R mRNA lasted longer compared with that of activin, this result might be caused by a lack of sensitivity against follistatin, which might be produced by the presence of activin and TGFß.
The identification and characterization of follistatin-related protein (FSRP) suggests that the follistatin gene family may actually contain two subfamilies. The FSRP inhibits activin-mediated gene transcription in heterologous assays. Additionally, FSRP is much less active than follistatin in the rat pituitary bioassay. When overexpressed in transgenic mice, FSRP may lead to interruption of follicular development and fertility in females, but it appears to have only a modest effect on males [31]. These results indicated that FSRP may interfere with the development of normal follicles. Although further studies of FSRP will be required to know the actions of FSRP, some of the FSRP might interact with the TGFß action in the ovary.
As we have shown in a previous report, FSH increases the production of follistatin and reduces the production of activin, resulting in the suppression of activin bioactivity [9–11]. The effect of activin on the granulosa cell starts by increasing the sensitivity to FSH, which causes an increase of follistatin and suppression of the production of activin. Local feedback terminates activin action after only a short period of time. This reduction of activin activity induced by FSH might be important for the normal growth of each follicle, and we speculate that the suspended effect of activin might interfere with the physiological action of gonadotropin. For example, inhibin 
knockout mice produce gonadal tumors, and a report of increased activin and FSH production in those mice has appeared [32]. The authors of the report suggested that the uncontrolled production of activin eventually caused the tumor growth in the mouse gonads.
Activin and TGFß stimulate the expression of FSH-R, either alone or in the presence of FSH. Both enhance the effect of FSH on the growing follicle and might be important factors in selection of the leading follicle. However, the action of TGFß is completely different from that of activin in terms of autofeedback regulation by production of follistatin. Because GDF-9, activin, and TGFß are included in the TGF superfamily and the members of this family might have an important, stage-specific function in follicular development, further studies of the regulation of the TGFß superfamily in granulosa cells are required.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 81 27 220 8443; tminegis{at}showa.gunma-u.ac.jp
Received: 20 December 2002.
First decision: 20 January 2003.
Accepted: 21 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1994 50:940-948[Abstract]2-macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element. Mol Endocrinol 1996 10:171-184[Abstract]-Inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 1992 26:313-319This article has been cited by other articles:
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  D. Rosairo, I. Kuyznierewicz, J. Findlay, and A. Drummond Transforming growth factor-{beta}: its role in ovarian follicle development Reproduction, December 1, 2008; 136(6): 799 - 809. [Abstract] [Full Text] [PDF]
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 C. Andreu-Vieyra, R. Chen, and M. M. Matzuk Conditional Deletion of the Retinoblastoma (Rb) Gene in Ovarian Granulosa Cells Leads to Premature Ovarian Failure Mol. Endocrinol., September 1, 2008; 22(9): 2141 - 2161. [Abstract] [Full Text] [PDF]
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 H.-F. Kwok, W.-K. So, Y. Wang, and W. Ge Zebrafish Gonadotropins and Their Receptors: I. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Receptors-- Evidence for Their Distinct Functions in Follicle Development Biol Reprod, June 1, 2005; 72(6): 1370 - 1381. [Abstract] [Full Text] [PDF]
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D. C. Woods and A.L. Johnson Regulation of Follicle-Stimulating Hormone-Receptor Messenger RNA in Hen Granulosa Cells Relative to Follicle Selection Biol Reprod, March 1, 2005; 72(3): 643 - 650. [Abstract] [Full Text] [PDF]
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