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Endocrinology 143(8):2930 –2935
Copyright © 2002 by The Endocrine Society
Dihydrotestosterone Inhibits Granulosa Cell
Proliferation by Decreasing the Cyclin D2 mRNA
Expression and Cell Cycle Arrest at G1 Phase
P. K. PRADEEP, XIAOLING LI, HELLE PEEGEL,
AND
K. M. J. MENON
Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
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
P
ROLIFERATION 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 (1–3). Estrogens, in turn,
enhance the responsiveness of ovarian follicles to gonadotropin stimulation (4) and increase granulosa cell proliferation (5). However, excess androgen production by thecainterstitial 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 (12–15). 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, ovuAbbreviations: Chaps, 3-[(3-Cholamidopropyl)dimethylammonio]1-propanesulfonate; DHT, dihydrotestosterone.
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. (Endocrinology 143: 2930 –2935,
2002)
lation, 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
Materials
DHT (5␣-androstan-17␤-ol-3-one) and 17␤ estradiol (1,3,5 [10]-estratriene-3, 17␤-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 ␤ 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).
2930
Pradeep et al. • DHT Reduces Cyclin D2 mRNA Expression in Granulosa Cells
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 ⫻ 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 DMEMF12 and centrifuged 5 min at 250 ⫻ g. Granulosa cells were then treated
sequentially with trypsin (20 ␮g/ml) for 1 min, 300 ␮g/ml soybean
trypsin inhibitor for 6 min, and DNase I (100 ␮g/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 ␮g/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 ⫻ 105 cells/well. After overnight attachment cells were incubated
with fresh media containing [3H]thymidine (1.5 ␮Ci/well) in the presence or absence of forskolin (10 ␮m) for 12, 24, 36, and 48 h. After
incubation, cells were washed three times with ice-cold PBS and incubated with 500 ␮l 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 ␮l 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.
Endocrinology, August 2002, 143(8):2930 –2935 2931
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 ␤-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 ⫻ 106/plate. After treatment as
indicated for each experiment, the cells were incubated in the presence
or absence of forskolin (10 ␮m) 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 ⫻ 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 ⫻ 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 ⫻ g for 5 min) and lysed in a hypotonic
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 ␮m), 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 ␮l EDTA solution. Trypsin was neutralized with fresh medium containing 300
␮g/ml trypsin inhibitor. Cells were transferred to microcentrifuge tubes.
The wells containing attached cells were once again washed with 500 ␮l
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 ⫻ 106/plate. After
overnight attachment the media was replaced with fresh media containing DHT (90 ng/ml) and forskolin (10 ␮m), whereas the control
FIG. 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 ⫻ 105 / well in 12-well
plates. After overnight attachment, the cultures were incubated with
[3H]thymidine (1.5 ␮Ci/well) in the presence or absence of forskolin
(10 ␮M) 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 forskolinstimulated control and DHT treatment groups are compared). FSK,
Forskolin.
2932
Endocrinology, August 2002, 143(8):2930 –2935
FIG. 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 ␮M) 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.
buffer containing 0.1% sodium citrate, 0.1% Triton X-100, 100 ␮g/ml
Rnase, and 50 ␮g/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
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 forskolinstimulated 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
Pradeep et al. • DHT Reduces Cyclin D2 mRNA Expression in Granulosa Cells
FIG. 3. Effect of DHT on cleaved caspase 3 expression in granulosa
cells in response to forskolin. Granulosa cells from 3-d estradiolprimed immature rats (1.5 mg/d) were cultured in the presence or
absence of DHT (90 ng/ml) for 24 h with forskolin (10 ␮M). 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 SDSPAGE 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 ␤-tubulin antibody to normalize the protein loading (middle panel). Bottom panel shows the
densitometric scan of cleaved caspase 3 normalized for ␤-tubulin.
FSK, Forskolin.
TABLE 1. Distribution of control and DHT-treated granulosa
cells in the G1, S, and G2/M phase of the cell cycle
Phase of cell cycle
Control cells (%)
DHT-treated cells (%)
G1
S
G2/M
42.0
55.0
3.0
70.0
26.0
3.5
Immature female rats (22 d old) were treated sc with estradiol (1.5
mg/d) for 3 d. Granulosa cells were harvested and plated at a density
of 3 ⫻ 106 cells in 60-mm culture plates. After overnight attachment,
cells were incubated with or without DHT (90 ng/ml) for 48 h. The cells
were harvested using PBS-EDTA and lysed in hypotonic medium
containing propidium iodide as described in Materials and Methods.
Cell cycle analysis was performed using flow cytometry. Percentage
of cells in each phase is presented.
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.
DHT-mediated inhibition of granulosa cell proliferation
The DHT-mediated inhibition of [3H]thymidine incorporation in granulosa cells in response to forskolin was further
confirmed by determining the cell number. Consistent with
Pradeep et al. • DHT Reduces Cyclin D2 mRNA Expression in Granulosa Cells
the [3H]thymidine incorporation assay, forskolin-stimulated
control cells showed a significantly higher number of cells at
all time intervals tested (Fig. 2), compared with the cells from
DHT-treated rats.
Effect of DHT on cleaved caspase 3 expression
To examine whether the changes in [3H]thymidine incorporation and cell number in forskolin-stimulated control and
DHT-treated cells is due to apoptosis, the expression of
cleaved caspase 3 in control and DHT-treated granulosa cells
in response to forskolin (Fig. 3) was analyzed. After 24 h of
incubation with forskolin, both control and DHT-treated
granulosa cells showed identical expression of cleaved
caspase 3. This result shows that the decrease in [3H]thymidine incorporation and cell number in DHT-treated granulosa cells in response to forskolin was not due to apoptosis.
DHT reduces cyclin D2 mRNA expression in response
to forskolin
Cyclin D2 is a critical molecule in hormone-dependent
proliferation of granulosa cells (24). Alteration in cyclin D2
mRNA expression in granulosa cells in response to forskolin
was examined in control and DHT-treated animals. The effect of in vivo DHT treatment in cyclin D2 mRNA expression
is shown in Fig. 4. The results show that forskolin increased
Endocrinology, August 2002, 143(8):2930 –2935 2933
cyclin D2 mRNA expression by 100% in cells from control
rats, whereas this response was blunted with DHT treatment.
We also tested cyclin D2 mRNA expression in granulosa cells
treated with DHT in vitro. Forskolin enhanced the cyclin D2
mRNA expression 3-fold over control, and DHT treatment
abolished this increase in cyclin D2 mRNA expression in
response to forskolin under in vitro conditions as well (Fig.
5). These results show that DHT blocks the responsiveness of
granulosa cells to forskolin with respect to cyclin D2 mRNA
expression under both in vivo and in vitro conditions.
DHT arrests the cell cycle at G1/S interphase of the
cell cycle
Because cyclin D2 is an important molecule in the G1/S
transition of the cell cycle (25, 26), we examined the possibility of a cell cycle arrest at the G1/S interphase under DHT
treatment. Granulosa cells from control and in vitro DHT
treatments were analyzed for cell cycle phase distribution
based on DNA content using flow cytometry. In the control
group, 42% of cells were in G1 phase and 55% in S phase,
whereas DHT treatment caused a majority of cells (70%) to
arrest at G1 phase, compared with S phase (26%) (Table. 1).
This result shows that DHT treatment of granulosa cells
causes cell cycle arrest at the G1/S interphase.
Discussion
Although it is well established that in rodents and humans
overproduction of androgens results in anovulatory conditions, the underlying mechanism is less well understood. It
FIG. 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 ␮M) 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.
FIG. 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 ␮M) 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.
2934
Endocrinology, August 2002, 143(8):2930 –2935
has been recognized for some time that the androgen-secreting cells of the ovaries are the theca-interstitial cells. The
biosynthetic pathway for androgen in the theca-interstitial
cells is similar to that in other steroid-producing tissues (2).
In addition to aromatization of testosterone to estradiol, testosterone is also converted to 5␣ androstan-17␤-ol-3-one
(DHT), 5␣-androstan-3␣, 17␤-diol, and 5␣ androstan-3␤,
17␤-diol (27). Androgens and their 5␣ reduced metabolites
like DHT have been shown to perturb the ovarian physiology
in rats (17, 18, 28 –30). One of the targets of this disruptive
action is the granulosa cell in the ovarian follicle.
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 DHTtreated 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
Pradeep et al. • DHT Reduces Cyclin D2 mRNA Expression in Granulosa Cells
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
Received December 10, 2001. Accepted April 16, 2002.
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: kmjmenon@
umich.edu.
This work was supported by NIH Grant HD-38424.
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