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Dihydrotestosterone Inhibits Granulosa Cell Proliferation by Decreasing the Cyclin D2 mRNA Expression and Cell Cycle Arrest at G1 Phase

Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. K. M. J. Menon, 6428 Medical Science I, 1150 West Medical Center Drive, University of Michigan, Ann Arbor, Michigan 48109. E-mail: .

	Abstract

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Hyperandrogenism is known to perturb ovarian physiology resulting in anovulatory conditions. In the ovary, androgens produced by theca-interstitial cells are converted to estrogens in granulosa cells under the influence of FSH and LH. In some of the target organs, including the ovary, androgens are also converted into their 5 reduced metabolites. In the present study, we examined the molecular mechanism by which dihydrotestosterone (DHT), a 5 reduced metabolite of testosterone, mediates the inhibition of granulosa cell proliferation, using a rat model. Immature female rats were primed with estradiol, followed by DHT administration for 2 d and granulosa cells were cultured in the presence or absence of forskolin. Granulosa cells from the DHT-treated rats showed reduced [³H]thymidine incorporation into DNA and reduced cell number in response to forskolin stimulation, compared with control. The decreased responsiveness of DHT-treated granulosa cells to forskolin was not due to increased apoptosis because the expression of cleaved caspase 3 remained the same in both control and DHT-exposed granulosa cells stimulated with forskolin. Forskolin treatment stimulated the expression of cyclin D2 mRNA in control granulosa cells, whereas DHT treatment abolished this response. In vitro DHT treatment of granulosa cells for 48 h resulted in a cell cycle arrest with 70% of cells at G1 phase and 26% at S phase, and control cells exhibited a distribution of 42% and 55% at G1 and S phase, respectively. In conclusion, the present study shows that DHT inhibits the granulosa cell proliferation through a decrease in cyclin D2 mRNA expression, which leads to cell cycle arrest at the G1 phase.

	Introduction

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PROLIFERATION AND DIFFERENTIATION of granulosa cells are regulated by sequential and combined actions of FSH and LH. Androgens produced by the ovarian theca-interstitial cells play a critical regulatory role in follicle development because they serve as the precursors of estrogen synthesis in granulosa cells (1, 2, 3). Estrogens, in turn, enhance the responsiveness of ovarian follicles to gonadotropin stimulation (4) and increase granulosa cell proliferation (5). However, excess androgen production by theca-interstitial cells impairs follicular function. In the human, hyperandrogenism has been identified as a crucial factor in the pathogenesis of anovulation (6). Androgens produced by the ovary are known to inhibit the effect of estrogen on follicular growth (7) and increase atresia among the follicles in rat ovaries (8). FSH has been shown to induce the LH receptors in granulosa cells (9), whereas androgens inhibit FSH induction of LH receptors in granulosa cells (10, 11).

In many target organs, including the ovary, androgens have been shown to undergo conversion to 5 reduced metabolites by 5 reductase (12, 13, 14, 15). These earlier reports were further confirmed by the demonstration of 5 reductase mRNA expression in rat ovarian tissues by Lephart et al. (16). They have also reported an inverse relationship between 5 reductase and aromatase mRNA expression in the ovarian tissues. Dihydrotestosterone (DHT), a 5 reduced metabolite of testosterone, has been reported to act as a competitive inhibitor of aromatase activity in the ovary (17). Administration of DHT has been shown to arrest the growth, ovulation, and function of ovarian follicles in intact, immature rats primed with pregnant mare’s serum gonadotropin (18). Recent studies by Jakimiuk et al. (19) have shown that women with polycystic ovarian syndrome express elevated level of 5 reductase mRNA in the granulosa cells. Thus, it is conceivable that the deleterious effects of androgens in the ovary may be mediated through the conversion to 5 reduced metabolites. The present studies were undertaken to examine the molecular basis of DHT mediated inhibition of granulosa cell proliferation. Using rat granulosa cells, we have examined the effect of DHT on the progression of cell cycle at the G1/S interphase. Our results show that DHT inhibits the cyclin D2 mRNA expression in granulosa cells and arrests the cell cycle at G1 phase.

	Materials and Methods

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Materials
DHT (5-androstan-17ß-ol-3-one) and 17ß estradiol (1,3,5 [10]-estratriene-3, 17ß-diol) were purchased from Sigma (St. Louis, MO). Forskolin was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [³²P]Deoxy-CTP (3000 Ci/mmol) and [methyl-³H]thymidine (77 Ci/mmol) were obtained from ICN Biomedicals, Inc. (Irvine, CA). Cleaved caspase 3 antibody and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) cell extraction buffer were products of Cell Signaling Technology (New England Biolabs, Inc., Beverly, MA), and ß tubulin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antirabbit IgG horseradish peroxidase conjugate and enhanced chemiluminescence Western blotting detection reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). The RTS RadPrime DNA labeling kit, DMEM F-12 (phenol red free), and TRIzol reagent were products of Life Technologies, Inc. (Gaithersburg, MD).

Animals and treatments
Twenty-two-day-old Sprague Dawley rats (Charles River, Wilmington, MA) were used as a model system. Animals were housed in a temperature-controlled room with proper dark-light cycles under the care of University of Michigan Unit of Laboratory Animal Medicine. They were primed with estradiol (1.5 mg/d) for 3 d to stimulate the development of large preantral follicles (20). In some experiments DHT was administered in vivo. For in vivo treatment, DHT (1.5 mg/d) was injected sc on d 3 of estradiol priming and then on d 4, whereas for in vitro treatment, granulosa cells from 3-d estradiol-primed rats were harvested and cultured in a medium containing 90 ng/ml DHT. After treatment as indicated for each experiment, the animals were killed by CO₂ asphyxiation, and ovaries were collected.

Granulosa cell isolation and culture
Ovaries were cleared from the surrounding fat. Granulosa cells were collected from the ovaries by the method of Campbell (21). In brief, the ovaries were punctured with 27-gauge needles, and cells were collected into phenol red free DMEM-F12 containing 0.2% BSA, 10 mM HEPES, and 6.8 mM EGTA. Cells were incubated for 15 min at 37 C under 95% O₂:5% CO₂ and centrifuged for 5 min at 250 x g. The pellets were suspended in a solution containing 0.5 M sucrose, 0.2% bovine serum albumin and 1.8 mM EGTA in DMEM-F12 and incubated for 5 min. Following the incubation, the suspension was diluted with 3 vol DMEM-F12 and centrifuged 5 min at 250 x g. Granulosa cells were then treated sequentially with trypsin (20 µg/ml) for 1 min, 300 µg/ml soybean trypsin inhibitor for 6 min, and DNase I (100 µg/ml) for 6 min at 37 C to remove dead cells. The cells were then rinsed twice with serum-free media and suspended in DMEM-F12, and cell number was determined. Cell viability was examined by trypan blue exclusion technique, and cells were cultured in serum-free DMEM-F12 supplemented with 20 mM HEPES (pH 7.4), 4 mM glutamine, 100 IU penicillin, and 100 µg/ml streptomycin. Before plating, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed twice with DMEM-F12.

[³H]Thymidine incorporation studies
Thymidine incorporation studies were performed in 12-well plates. Granulosa cells from control or DHT-treated rats were plated at a density of 5 x 10⁵ cells/well. After overnight attachment cells were incubated with fresh media containing [³H]thymidine (1.5 µCi/well) in the presence or absence of forskolin (10 µM) for 12, 24, 36, and 48 h. After incubation, cells were washed three times with ice-cold PBS and incubated with 500 µl ice-cold trichloroacetic acid (10%) to remove the acid-soluble [³H]thymidine pool. Cells were washed again with ice-cold PBS and dissolved in 500 µl 0.2 N NaOH containing 0.1% SDS. Following incubation for 1 h at 37 C, these were transferred to scintillation vials and neutralized with 0.2 N HCl, and radioactivity was measured using a scintillation counter.

Cell proliferation assay
Equal numbers of cells (from control or DHT-treated rats) were cultured in 12-well plates, and after overnight attachment the medium was replaced with fresh medium. One group of cells (control and DHT treated) received forskolin (10 µM), and the other group (control and DHT) received vehicle. They were incubated for 12, 24, 36, and 48 h. Following the incubation, cells were harvested using 500 µl EDTA solution. Trypsin was neutralized with fresh medium containing 300 µg/ml trypsin inhibitor. Cells were transferred to microcentrifuge tubes. The wells containing attached cells were once again washed with 500 µl media and pooled with the first wash. The cells were then pelleted by centrifugation at 3000 rpm for 5 min and resuspended in fresh media. Four replicates of each experiment were done, and cell number was determined in four aliquots from each replicate using a hemocytometer.

Western blot analysis for cleaved caspase 3
Granulosa cells from 3-d estradiol-primed rats were harvested and plated in 60-mm culture plates at a density of 2.5 x 10⁶/plate. After overnight attachment the media was replaced with fresh media containing DHT (90 ng/ml) and forskolin (10 µM), whereas the control group received vehicle and forskolin and incubated for 24 h. At the end of the incubation, cells were washed with PBS (three times) and harvested with PBS-EDTA. The cells were pelleted by centrifugation (10,000 rpm for 5 min at 4 C) and lysed using Chaps cell extraction buffer [50 mM piperazine-N,N’-bis(2-ethane sulfonic acid) (pH 6.5), 2 mM EDTA, 0.1% Chaps, 20 mg/ml leupeptin, 10 mg/ml pepstatin A, 10 mg/ml aprotinin, 5 mM dithiothreitol, and 1 mM PMSF] and centrifuged at 14,000 rpm for 5 min at 4 C to remove the cell debris. The lysates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Membrane was incubated with cleaved caspase 3-specific rabbit polyclonal antibody followed by secondary horseradish peroxidase-conjugated rabbit IgG. Detection was performed with enhanced chemiluminescence Western blotting detection system. The blot was then stripped and reprobed with ß-tubulin antibody to measure the protein loading. The signals were measured using an Arcus II scanner (Agfa, Wilmington, MA) and quantitated using the NIH Image 1.61 program.

Northern blot analysis for cyclin D2
Cells from the control or DHT treatment group were plated in 100-mm culture dishes at a density of 6 x 10⁶/plate. After treatment as indicated for each experiment, the cells were incubated in the presence or absence of forskolin (10 µM) for 2 h. At the end of incubation, the medium was decanted and washed once with PBS. The cells were harvested and total RNA was extracted using TRIzol reagent using the manufacturer’s protocol (Life Technologies, Inc.). The cyclin D2 cDNA (1.1 kb) probe was radiolabeled using [³²P]deoxy-CTP and RTS Rad Prime DNA labeling system (Life Technologies, Inc.) and was hybridized to blots overnight at 42 C using 2 x 10⁷ cpm-labeled probe. The hybridized blots were washed and exposed at -70 C to XAR film (Kodak, Rochester, NY). The films were developed and the signals were measured using the Arcus II scanner (Agfa) and quantitated using the NIH Image 1.61 program. After the blots were stripped, they were rehybridized with a radiolabeled cDNA probe corresponding to 18S rRNA to monitor total RNA loading.

Cell cycle analysis
Granulosa cells from estradiol-primed rats were cultured in 60-mm plates (3 x 10⁶ cells/plate) containing 90 ng/ml DHT or vehicle. After 48 h, cells were washed once with PBS and harvested using PBS-EDTA, pelleted by centrifugation (250 x g for 5 min) and lysed in a hypotonic buffer containing 0.1% sodium citrate, 0.1% Triton X-100, 100 µg/ml Rnase, and 50 µg/ml propidium iodide. The pellet was agitated gently and incubated for 20 min at room temperature. Analysis was carried out by a fluorescence-activated Elite ESP flow cytometer (Beckman Coulter, Inc., San Jose, CA).

Statistical analysis
The statistical analysis was initially carried out using ANOVA. If ANOVA indicated significant differences within the data set, pair-wise comparisons were performed using an unpaired t test. Each experiment was repeated three times. The results shown represent a single experiment.

	Results

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Effect of DHT on forskolin-stimulated [³H]thymidine incorporation
The effect of DHT on granulosa cell proliferation in response to forskolin was examined by [³H]thymidine incorporation assay. Forskolin, a diterpene, is known to activate adenylate cyclase and increase the adenosine cAMP pool in the granulosa cells and thereby mimics the effect of FSH (22, 23). The results shown in Fig. 1 indicate a time-dependent increase in [³H]thymidine incorporation in granulosa cells in all treatment groups. This effect was maximum in forskolin-stimulated control cells up to 48 h (Fig. 1). Although the granulosa cells obtained from DHT-treated animals showed an enhancement in DNA synthesis (maximum at the end of 48 h) in response to forskolin, it was significantly lower than that of control cells under same treatment conditions at all time points tested. Cells treated with DHT showed the lowest level of [³H]thymidine incorporation throughout the incubation period in the absence of forskolin. These results show that DHT exerts an inhibitory effect on DNA synthesis.

View larger version (27K): [in this window] [in a new window]   Figure 1. Time course of the effect of forskolin on [3H]thymidine incorporation into DNA. Female rats (22 d old) were primed with estradiol (1.5 mg/d) for 3 d followed by DHT (1.5 mg/d) on d 3 and d 4. Granulosa cells were harvested and plated at a density of 5 x 105 / well in 12-well plates. After overnight attachment, the cultures were incubated with [3H]thymidine (1.5 µCi/well) in the presence or absence of forskolin (10 µM) for the indicated time. Radioactivity incorporated into the DNA was measured at 12, 24, 36, and 48 h. Each experiment was done in triplicate (a, P < 0.001; b, P < 0.005; c, P < 0.05 when forskolin-stimulated control and DHT treatment groups are compared). FSK, Forskolin.

View larger version (49K): [in this window] [in a new window]   Figure 2. Time course of the effect of forskolin on granulosa cell proliferation. Twenty-two-day-old female rats were primed with estradiol for 3 d (1.5 mg/d) followed by DHT (1.5 mg/d) on d 3 and d 4. Granulosa cells were harvested and equal numbers were plated in 12-well plates. After overnight attachment, the cells were incubated in the presence or absence of forskolin (10 µM) and harvested using PBS-EDTA at 12, 24, 36, and 48 h of incubation. Each experiment was performed in replicates of four, and cell number was quantitated in four aliquots from each replicate using a hemocytometer (a, P < 0.001; b, P < 0.002 when forskolin-stimulated control and DHT treatment groups are compared). FSK, Forskolin.

View larger version (40K): [in this window] [in a new window]   Figure 3. Effect of DHT on cleaved caspase 3 expression in granulosa cells in response to forskolin. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml) for 24 h with forskolin (10 µM). After the incubation, cells were washed with PBS and lysate was prepared using Chaps cell extraction buffer. Total protein concentration was estimated and equal amounts of total protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with specific cleaved caspase 3 antibody (top panel). The blot was then stripped and reprobed with ß-tubulin antibody to normalize the protein loading (middle panel). Bottom panel shows the densitometric scan of cleaved caspase 3 normalized for ß-tubulin. FSK, Forskolin.

View larger version (40K): [in this window] [in a new window]   Figure 4. In vivo effect of DHT on cyclin D2 mRNA expression in granulosa cells in response to forskolin. Twenty-two-day-old female rats were primed with estradiol for 3 d (1.5 mg/d) and then treated with DHT (1.5 mg/d) on d 3 and d 4. Granulosa cells were harvested and cultured. After overnight attachment, the cultures were incubated in the presence or absence of forskolin (10 µM) for 2 h. Total RNA was extracted and Northern blot analysis was performed using a radiolabeled cyclin D2 cDNA probe (top panel). Radiolabeled cDNA for 18S rRNA was used to monitor RNA loading (middle panel). Bottom panel shows the densitometric scan of cyclin D2 mRNA normalized for 18S rRNA. The figure is a representative of three separate experiments.

View larger version (44K): [in this window] [in a new window]   Figure 5. In vitro effect of DHT on cyclin D2 mRNA expression in granulosa cells in response to forskolin. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in presence of DHT (90 ng/ml) for 24 h. The cultures were then incubated in the presence or absence of forskolin (10 µM) for 2 h. Total RNA was extracted and Northern blot analysis was performed using [32P] cyclin D2 cDNA probe (top panel). [32P] cDNA for 18S rRNA was used to monitor RNA loading (middle panel). Bottom panel shows the densitometric scan of cyclin D2 mRNA normalized for 18S rRNA. The figure is a representative of three separate experiments.

	Discussion

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Previous studies have shown that granulosa cell proliferation is regulated by FSH, as demonstrated by its stimulation of [³H]thymidine incorporation into DNA (5, 31). FSH has also been shown to increase cAMP production in undifferentiated rat granulosa cells (32). Forskolin mimics the actions of FSH by activating adenylyl cyclase in the granulosa cell (23, 33). In our efforts to understand the molecular basis for the inhibitory effect of DHT in granulosa cell proliferation, we examined the effect of DHT on forskolin-stimulated DNA synthesis in granulosa cells in vitro. Our results clearly show that granulosa cells from DHT-treated rats exhibited a reduction in [³H]thymidine incorporation in the presence of forskolin, compared with control. This impaired DNA synthesis in the DHT-treated rats might account for the reduced cell number seen, compared with the control group. Together these data demonstrate an inhibition in granulosa cell proliferation in response to DHT treatment. Because cells in primary culture may undergo apoptosis, we examined the possibility that the decreased [³H]thymidine incorporation and cell number in DHT-treated granulosa cells might be due to increased cell death. To test this, we examined the cleaved caspase 3 expression in granulosa cells of control and DHT-treated granulosa cells in response to forskolin. Caspase 3, a cysteine protease is one of the key executioners of apoptosis, and is produced in response to agents that cause cell death by cleavage from a zymogen precursor (34). The presence of cleaved caspase 3 has been previously demonstrated in the cytosolic compartment of oocytes and granulosa cells of rats (35), and its expression has been used as a marker of apoptosis (36). Western blot analysis of cleaved caspase 3 in control and DHT-treated granulosa cells in response to forskolin revealed no differences. These data rule out the possibility that the decrease in [³H]thymidine incorporation and cell number seen in cells exposed to DHT might be due to cell death.

Because forskolin-mediated cell proliferation was inhibited by DHT, the mechanism underlying the inhibitory effect was examined. Mitogenesis in eukaryotic cells is primarily regulated at the G1 phase of the cell cycle. During the G1 phase, the cells respond to extracellular signals by either advancing toward another division or withdrawing from the cycle to G₀ (37, 38). D-type cyclins are the positive regulators of G1 progression in the cell cycle (25, 39). Once cells are stimulated by mitogenic signals, one or more D-type cyclins (D1, D2, and D3) are induced and activate their catalytic partners, cyclin-dependent kinases 4 and 6, which in turn, through a cascade of reactions culminates in cell division (38). Sicinski et al. (24) demonstrated that in transgenic mice lacking functional cyclin D2 (cyclin D2^-/-), granulosa cell proliferation in response to FSH and forskolin was abolished. Although cyclins D and E have been implicated in cell cycle progression at the G1 phase, studies by Robker and Richards (26) showed that cyclin D2 mRNA is specifically localized to granulosa cells, whereas cyclin D1 and D3 are restricted to theca cells. They also reported that in normal rats, FSH increases the expression of cyclin D2 mRNA expression in the rat granulosa cells.

In light of these reports, we examined whether DHT mediated inhibition of granulosa cell proliferation in response to forskolin is due to inhibition of cyclins expressed during G1/S transition. Granulosa cells from DHT-treated rats showed reduced cyclin D2 mRNA expression in response to forskolin, compared with the control. This in vivo inhibitory effect of DHT on cyclin D2 mRNA expression was seen within 24 h under in vitro conditions. Because DHT treatment of granulosa cells reduced the expression of cyclin D2 mRNA, it is conceivable that the inhibition of proliferation by DHT might be due to inhibition of cyclin D2 production.

Our results are consistent with previous reports in which DHT has been shown to arrest cell cycle in androgen receptor-transfected prostatic cells (PC-3) (40) and in prostate cancer cell line LNCaP (41). The critical role of cyclin D2 in granulosa cell proliferation is strengthened by the findings of Drummond and Findlay (42), in which they reported a retarded proliferation of granulosa cells because of blocked cyclin D2. The present observations show that cyclin D2 plays an important role in cell proliferation in granulosa cells in response to mitogenic stimuli. Because cyclin D2 is expressed during the G1 phase, a reduced expression in the DHT-treated group might block the cell cycle progression at the G1 phase. This conclusion is supported by the flow cytometric analysis data, which showed that more than 70% of granulosa cells exposed to DHT were unable to progress from the G1 to the S phase, exhibiting a block at the G1/S transition. The blockade of cell cycle progression produced by DHT could eventually lead the granulosa cells to undergo apoptosis. For instance, it has been demonstrated that DHT blocks the cell cycle progression of androgen receptor-transfected human PC-3 prostate carcinoma cells resulting in growth inhibition culminating in apoptosis (40). Collectively, these observations support the hypothesis that DHT-mediated inhibition of granulosa cell proliferation is mediated through cell cycle arrest at the G1 phase as a result of reduced cyclin D2 mRNA expression. The upstream events that are responsible for the decrease in cyclin D2 mRNA expression by DHT are currently under investigation.

	Acknowledgments

 

	Footnotes

 
This work was supported by NIH Grant HD-38424.

Abbreviations: Chaps, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; DHT, dihydrotestosterone.

Received December 10, 2001.

Accepted for publication April 16, 2002.

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