BIOLOGY OF REPRODUCTION 49, 416-422 (1993)
Control of Rat Granulosa Cell Mitosis by Phorbol Ester-, Cyclic AMP-,
and Estradiol-171-Dependent Pathways
JJ. PELUSO,'
2
A. PAPPALARDO, 2 and B.A. WHITE 3
3
Departments of Obstetrics and Gynecology, 2 and Anatomy, University of Connecticut Health Center
Farmington, Connecticut 06032
ABSTRACT
The present studies examined the effect of 8-bromo-cAMP (8-Br-cAMP), phorbol ester (TPA), and estradiol-170 (E,) on the
capacity of rat granulosa cells (GC) to undergo mitosis. In the first series of experiments, GC were either maintained within
immature rat ovaries in perifusion culture or isolated and placed in tissue culture. GC were cultured for 24 h with 1) control
3
3
medium, 2) 8-Br-cAMP, 3) TPA, or 4) 8-Br-cAMP plus TPA in the presence of H-thymidine ( H-T). In perifusion culture, 8-Br5
3
cAMP stimulated both H-T incorporation (p < 0.05) and E, secretion (p < 0.05) while TPA increased H-T (p < 0.05) without
3
and suppressed E,
incorporation
H-T
enhanced
and
TPA
to
8-Br-cAMP
exposure
altering E, secretion (p > 0.05). Simultaneous
as compared to 8-Br-cAMP treatment alone (p < 0.05). In tissue culture, 8-Br-cAMP did not increase H-T incorporation or cell
3
number. TPA increased both H-T incorporation (p < 0.05) and cell number (p < 0.05), while 8-Br-cAMP suppressed both of
these TPA-induced responses. In the presence of testosterone, 1) TPA's mitogenic action was also observed, 2) basal E, secretion
ranged between 30 and 35 pg/ml, 3) neither 8-Br-cAMP nor TPA stimulated E, secretion over basal levels, and 4) Rp-cAMP, a
cAMP antagonist, blocked TPA-induced cell proliferation. E, at 250 pg/ml also blocked TPA's mitogenic action. In a second series
of experiments, GC were collected from eCG-treated rats. Although E, levels ranged from 250 to 350 pg/ml, TPA induced mitosis
that was inhibited by 8-Br-cAMP. These data demonstrate that cAMP- and TPA-dependent pathways interact to determine whether
the GC will undergo mitosis. Specifically, low endogenous levels of cAMP synergize with TPA-dependent pathways to promote
mitosis, while high levels of 8-Br-cAMP inhibit proliferation. An E,-dependent mechanism also exists that attenuates TPA's mitogenic action in GC isolated from immature rats but not eCG-treated rats. Since 8-Br-cAMP blocks mitosis in the absence of
androgen, it is unlikely that cAMP mediates its action by increasing E, secretion. It appears then that the anti-mitogenic actions
of cAMP and E, are mediated through redundant, independent mechanisms that prevent GC mitosis. Further, the anti-mitogenic
properties of E, but not cAMP diminish as a result of gonadotropin-induced GC maturation.
one secretion from GC collected from eCG-primed immature rats [6, 12, 13]. Since in other systems TPA stimulates
cell proliferation [8, 9], it is possible that activation of PKC
is part of the cellular mechanism through which gonadotropins and/or growth factors promote GC proliferation.
To test this hypothesis, the effect of TPA and/or 8-bromocAMP (8-Br-cAMP) on GC 3 H-thymidine (3 H-T) incorporation and E2 secretion was determined. These studies were
initially conducted using an ovarian perifusion culture system, since in this system GC can be stimulated to proliferate. In order to further characterize the cellular responses to TPA, subsequent studies utilized standard tissue
culture procedures. Finally, the effect of TPA and 8-Br-cAMP
on mitosis and E2 secretion from GC isolated from eCGtreated immature rats was determined and compared to effects in GC isolated from immature ovaries.
INTRODUCTION
Granulosa cells (GC) undergo mitosis and/or secrete estradiol-17P (E2 ), these functions being essential for follicular growth [1]. These diverse physiological processes are
controlled by a complex interaction between gonadotropins and intraovarian growth factors [2]. Both gonadotropins and growth factors regulate GC function by binding to
receptors that in turn activate specific second messenger
systems. Gonadotropins stimulate adenylate cyclase, and this
leads to an increase in intracellular cAMP and subsequent
activation of protein kinase A (PKA) [3]. Since both gonadotropins and cAMP analogs induce GC E2 secretion in vitro,
it is generally believed that gonadotropins mediate their
steroidogenic action through a cAMP/PKA-dependent pathway [4, 5].
Gonadotropins as well as various growth factors increase
the intracellular levels of both 1,2-diacylglycerol (DAG) and
inositol triphosphate (IP 3) [6, 7]. DAG acts as second messenger by directly stimulating protein kinase C (PKC) [8, 9].
PKC can also be directly stimulated by the phorbol ester,
12-0-tetradecanoyl phorbol 13-acetate (TPA) [8, 91. TPA can
influence steroidogenesis, inhibiting progesterone secretion from rat luteal cells [10, 11] and enhancing progester-
MATERIALS AND METHODS
Preparationof Culture Medium
Medium 199 (Sigma Chemical Company, St. Louis, MO)
was supplemented with 2.2 g NaHCO 2 /L, 2.0 g BSA (fraction V)/L, 1.0 g glucose/L, 50 mg streptomycin sulfate/L,
100 000 IU k-penicillin-G/L, and 2.38 g Hepes/L. The pH
was adjusted to 7.4 and the medium filtered through a 0.2r.m filter. Sigma was also the supplier of 8-bromoadenosine 3'-5' cyclic monophosophate (8-Br-cAMP), 12-0-tetrad-
Accepted April 12, 1993.
Received January 4, 1993
'Correspondence. FAX: (203) 679-1286.
416
CONTROL OF GRANULOSA CELL MITOSIS
radecanoyl phorbol 13-acetate (TPA), testosterone, and E2.
A stock solution of 8-Br-cAMP was made fresh daily at a
concentration of 10 mg/ml of PBS. Dilutions were made
from the stock solution in Medium 199 for a final concentration of 0.1 mg/ml. TPA was made at stock solution concentration of 1 mg/10 ml dimethylsulfoxide (DMSO) and
then frozen in aliquots. Dilutions of TPA were made daily
from frozen stock solutions. TPA was first diluted in PBS
and then with Medium 199. E2 and testosterone were dissolved in ethanol at a concentration of 1 mg/ml and then
diluted in Medium 199 to the desired final concentration.
Rp-cAMP (Biolog Life Science, La Jolla, CA) was dissolved
in Medium 199 and used at a final concentration of 0.1 IiM.
Prior to culture, 3 H-thymidine (New England Nuclear, Cambridge MA; specific activity 45-50 Ci/mmol) was added at
a concentration of 2.5 Ci/ml for the perifusion experiments or 10 [jCi/ml for the tissue culture experiments.
Animals
Immature female Wistar rats were obtained from Charles
River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L: 12D; lights-on at 0700 h). On the day of the experiment, the immature animals (25-30 days of age) were
cervically dislocated between 0930 and 1000 h. Only animals without fluid in their uteri were used, ensuring that
the animals were in the juvenile stage. Both ovaries were
removed and placed in oxygenated medium supplemented
with 400 IU heparin/L. After 5-10 min, the ovaries were
weighed and placed in perifusion culture. To isolate GC for
tissue culture, immature ovaries were placed in PBS at 4C
and pricked with 25-gauge needles to express the GCs.
"Mature" GC were obtained from immature rats that were
injected i.p. with 20 IU of eCG. Forty-eight hours later, GC
were collected as described previously and placed in tissue
culture.
Experimental Design
For the perifusion experiments, ovaries were placed in
perifusion culture as previously described [14]. Briefly, individual ovaries were placed in a perifusion culture chamber that was in a 370C water bath. Each chamber was connected to a holding flask containing 25 ml of medium that
was recirculated at 6 ml/h. The ovaries were exposed to
either 0.0, 0.6, or 1.2 ng/ml (2 nM) of TPA in the presence
(0.1 mg/ml, 0.2 mM) or absence of 8-Br-cAMP. The medium in the holding flask was continuously gassed with 5%
CO2 in oxygen throughout the culture. Perifusate was collected after 24 h, frozen, and stored at -20°C until assayed
for E2. The ovaries were removed from the culture chambers and placed into 1 ml of cold PBS. The GC were then
expressed and the amount of 3H-T incorporated was determined. Regardless of treatment, an average of 7.6 ± 0.7
x 105 cells were harvested from each ovary.
417
For the tissue culture experiments, GC were collected
from the ovaries of 5 to 6 immature rats, pooled together,
and centrifuged for 10 min at 500 x g at 6°C. The cell pellet
was then resuspended in Medium 199 and the number of
cells/ml determined via a hemocytometer. Then 5-10 x
104 GC were placed in 1 ml of medium with or without
TPA and/or 8-Br-cAMP in either a 12 x 75-mm polystyrene
tube (Falcon Plastic model #2054, Oxnard, CA) or a 35-mm
culture dish (Falcon Plastic model #1008). Culture tubes
were placed in a shaking water bath at 37°C and gassed
continuously with 5% CO 2 and 95% 02. Culture dishes were
placed in a 37 0C incubator in a 5% CO2/air atmosphere.
Regardless of whether the cells were cultured in tubes or
dishes, the responses to TPA and 8-Br-cAMP were similar.
Because equivalent results were obtained, the culture procedure will not be reported in the Results section of this
paper but will be specified in the legend to each figure.
After 24 h, the cells were collected, placed in a 1.5-ml microfuge tube, and centrifuged at 13 000 x g for 1 min. In
some experiments the supernatant was frozen and stored
at -20°C until assayed for E2 or cAMP content. The cell
pellet was used to determine the rate of 3H-T incorporation
and/or cell number.
[3H] Thymidine Incorporation and Cell Proliferation
Assays
After centrifugation at 13 000 x g for 1 min, the cell pellet was resuspended in 200 1 of cold PBS. A 175-pl aliquot
was placed in a 96-well plate and the cells were harvested
onto filtermats and washed via a Skatron Titerteck cell harvester (Skatron Inc., Steriling, VA) [15,16]. The filters were
dried at 37°C, placed into 7 ml of liquid scintillation fluid
(ACS, Amersham Arlington Heights, IL), and counted in a
scintillation counter. The number of cells/ml was determined from the remaining 25-ulA aliquot using a hemocytometer. Incorporation of 3H-T was expressed as cpm/10 4
cells. This procedure accurately measures the amount of
3
H-thymidine incorporated into DNA, since the glass-fiber
filters bind only intact DNA and not DNA fragments or unincorporated 3 H-thymidine [17].
Estradiol-17f3 Assay
The assay was a direct solid-phase radioimmunoassay
(Diagonistic Products Corp, Los Angeles, CA). The antiserum to estradiol cross-reacts 100% with E2 and <0.001%
with estrone and several other steroids. The assay sensitivity was 5 pg/ml and the intra- and interassay coefficients
of variation were 8% and 14%.
Cyclic AMP Assay
Cyclic AMP content within the medium was measured by
a double-antibody radioimmunoassay. To increase the assay
sensitivity, media samples were acetylated according to the
manufacturer's instructions (Amersham Corp, Arlington
418
PELUSO ET AL.
**
3 Control
400
|
T1
8-Br-eAMP
4
I-
a.
300
*
L
T
200
I
*
T
'o
I100
a.
0
0
11
F0
ir
1.2
0.6
TPA
(ng/ml)
O Control
an
w1
I *
t
I-
60 -
8-Br-cAMP
15
40 -
T
T
C
._
do
201
O'
C
0
0.6
TPA
1.2
(ng/ml)
3
FIG. 1. Granulosa cell H-thymidine incorporation (upper panel) and E2
secretion (lower panel) from immature ovaries perifused for 24 h with 8Br-cAMP and/or TPA. Values represent mean ± standard error (n = 4-8).
* Indicates a value that is significantly different from control value (p <
0.05). ** Indicates a value that is significantly different from values obtained both with 8-Br-cAMP and with TPA treatments (p < 0.05).
Heights, IL). All samples were run in the same assay with
an intraassay coefficient of variation of 8% and a sensitivity
of 2 fmol/ml.
StatisticalAnalysis
Within each experiment all treatments were replicated
4-8 times; each experiment was repeated at least once. The
data were examined by either two-way or one-way analysis
of variance followed by Student-Newman-Keuls Multiple
Range test with only p values <0.05 considered to be significant.
3
FIG. 2. The effect of 8-Br-cAMP and TPA on GC H-thymidine incorporation (upper panel) and cell number (lower panel). GC were isolated
from immature ovaries maintained in suspension culture for 24 h with 8Br-cAMP and/or TPA. Values represent mean + standard error n = 4-8).
* Indicates a value that is significantly different from values for all other
groups (p < 0.05). ** Indicates a value that is significantly greater than
control or 8-Br-cAMP values but less than the TPA value (p < 0.05).
RESULTS
GC of immature ovaries perifused with control medium
for 24 h incorporated 3H-T into DNA at a relatively low rate.
Both 8-Br-cAMP (0.1 mg/ml) and TPA (1.2 ng/ml) increased 3 H-T incorporation over control levels (p < 0.05).
Simultaneous exposure to TPA and 8-Br-cAMP increased 3HT incorporation over control (p < 0.05) and 8-Br-cAMP (p
< 0.05) groups (Fig. 1). Basal E2 secretion was enhanced
over control values by 8-Br-cAMP (p < 0.05). Although TPA
did not affect basal E2 secretion, it did attenuate 8-Br-cAMPstimulated E2 secretion (Fig. 1).
In contrast to its effects on perifused ovaries, 8-Br-cAMP
did not enhance 3 H-T incorporation or increase the number of isolated GC present after 24 h of tissue culture (Fig.
2). Further, 3H-T incorporation was not increased by 8-BrcAMP when tested at doses ranging from 0.05 to 1 mg/ml
419
CONTROL OF GRANULOSA CELL MITOSIS
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Time in Culture (h)
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.
FIG. 3. The effect of time in culture on TPA-induced 3H-thymidine incorporation (upper panel) and extracellular cAMP levels. GC were isolated
from immature ovaries maintained in suspension culture for up to 12 h
with either control medium or TPA. Four to eight replicated cultures from
both the control and TPA groups were collected at each time point. * Indicates a value that is significantly different from values for all other groups
(p < 0.05). Extracellular cAMP levels are shown in the lower panel with
the mean (-SE) for the control values represented by the open bar and
TPA treatment value represented by the shaded bars.
(data not shown). Conversely, TPA increased 3H-T incorporation (p < 0.05) and cell number (p < 0.05) (Fig. 2).
When isolated GC were exposed to 8-Br-cAMP and TPA, 3HT incorporation was increased over that in control (p <
0.05) and 8-Br-cAMP (p < 0.05) groups but was less than
that in the TPA treatment group (p < 0.05). In addition,
the number of cells harvested after combined 8-Br-cAMP
and TPA treatment was similar to control values (Fig. 2).
Incorporation of 3 H-T was constant throughout the first
8 h of culture in both control and TPA groups but increased
by 12 h in the TPA group (p < 0.05) (Fig. 3). TPA treatment
also altered extracellular levels of cAMP. Previous studies
30
I
-04
25
.
Control
A
.
l-br-cAMP
Rp
I-Dr-cAMP + Rp
FIG. 4. The effect of Rp-cAMP on TPA-induced cell proliferation (upper
panel), TPA-induced estradiol-17B secretion (middle panel), and 8-Br-cAMPinduced estradiol-17P secretion (lower panel). GC were isolated from
immature ovaries cultured in 35-mm culture dishes in a 5% C0 2/air atmosphere for 24 h with the indicated agents in the presence of 0.5 i.M
testosterone. * Indicates a value that is significantly different from control
value (p < 0.05).
have shown that extracellular cAMP levels increase and reach
a maximal level by 2 h and then remain constant over the
next 10 h of culture [18]. Extracellular cAMP levels in the
control group did not change from 2 to 12 h of culture,
averaging 4.37 + 0.08 fmol/ml. TPA reduced extracellular
cAMP levels by 15-20% (p < 0.05) during the first 8 h,
with extracellular cAMP levels increasing to control levels
by 12 h of culture (Fig. 3).
As shown in Figure 4, TPA stimulated GC proliferation
in the presence of testosterone. This mitogenic effect was
420
PELUSO ET AL.
Without Estradiol-17B
!
0
Estradiol-17B (250 pg/ml)
I
After 24 h of culture, estradiol-7l
ranged
between 250 and 350 pg/ml in all treatments
*
*
12-
T
9
V
C
C,
-4
10-
mm
0
w
7
:
is
E
2
i"
.M
u
Vz
5
6-
4
3
Control
TPA
FIG. 5. The effect of 8-Br-cAMP and TPA on GC number. GC were isolated from eCG-treated ovaries maintained in suspension culture for 24 h
with 8-Br-cAMP and/or TPA in the presence of 0.5 F.M testosterone. Values
represent mean
standard error (n = 4). * Indicates a value that is significantly different from values for all other groups (p < 0.05).
blocked by the cAMP antagonist, Rp-cAMP. Rp-cAMP also reduced basal E2 secretion, but E2 secretion was restored to
basal levels with the addition of 8-Br-cAMP (Fig. 4). Similarly, a 250-pg/ml dose of E2 in the absence of testosterone
attenuated TPA-induced mitosis of GC isolated from immature rats (Fig. 5).
As previously demonstrated for GC isolated from immature ovaries, TPA in the presence of testosterone stimulated mitosis, and 8-Br-cAMP blocked this mitogenic action in "mature" GC isolated from eCG-treated immature
rats (Fig. 6). In contrast to findings for immature GC, TPA's
mitogenic action was observed in the "mature" GC in the
presence of endogenous E2 levels between 250 and 350 pg/
ml (Fig. 6).
DISCUSSION
Although gonadotropins promote GC mitosis in vivo, the
intracellular pathways that mediate this response are unknown. Gonadotropins ultimately activate PKA, and this leads
to cellular differentiation (i.e., enhanced E2 secretion) by
48 h [3]. In addition to enhancing intracellular cAMP levels,
gonadotropins increase intracellular IP3 and DAG levels with
DAG stimulating PKC activity [6, 7]. The present data provide evidence that both PKA- and PKC-dependent pathways
are involved in controlling GC proliferation. The perifusion
studies demonstrate that 8-Br-cAMP stimulates 3H-T incorporation. Similar studies of intact hamster or murine follicles also indicate that 8-Br-cAMP activates cellular processes that lead to mitosis [19, 20]. However, the mitogenic
I
&,
-
- -
Control
T
.,,
,,
b
8-Br-cAMP
Tr
.
......
TPA
v - - - - - -
cAMP + TPA
FIG. 6. The effect of TPA and estradiol-17P on GC cell proliferation.
GC were isolated from immature ovaries cultured in 35-mm culture dishes
in a 5% C0 2 /air atmosphere for 24 h with the indicated agents in the absence of testosterone. * Indicates a value that is significantly different from
control value (p < 0.05).
effects of cAMP are not observed when isolated GC are placed
in tissue culture. This finding suggests that the mitogenic
response to exogenous cAMP is not mediated by a direct
effect on the GC but is most likely the result of an interaction with the thecal cells. The thecal cells are responsive
to cAMP and are the source of transforming growth factor
,3 as well as other growth factors [21, 22]. Since transforming growth factor 13(TGFP3) in the presence of low levels
of FSH stimulates rat GC proliferation in vitro [23], cAMP
may stimulate the thecal cells to secrete this or other thecaderived growth factors with this secretion in turn promoting GC proliferation.
In spite of the fact that exogenous 8-Br-cAMP does not
directly stimulate GC mitosis, low endogenous levels of cAMP
play an essential role in regulating GC cell cycle traverse.
This conclusion is based on the findings that Rp-cAMP, a
cAMP antagonist, blocks TPA-induced mitosis. This is not
the result of a general toxic effect, since the Rp-cAMP reduction of basal E2 secretion can be overridden by additional 8-Br-cAMP (present study) or FSH [24]. In other cell
types, cAMP promotes "competence" or the capability to
enter the S-phase of the cell cycle as well as stimulating
spindle formation [25, 26]. Thus, low intracellular levels of
cAMP may act at two points within the cell cycle to stimulate
GC mitosis.
While low endogenous levels of cAMP synergize with TPAdependent pathways to promote GC mitosis, sustained exposure to 8-Br-cAMP impedes entry into the cell cycle
(present study, [27, 28]). On the basis of data from other
cell systems, it appears that once the cell has become "corn-
421
CONTROL OF GRANULOSA CELL MITOSIS
petent," endogenous cAMP levels must be lowered in order
for the cell to enter the S-phase of the cell cycle [29]. In
immature GC, TPA initially suppresses by 15-20% intracellular and extracellular FSH-induced cAMP levels, with
cAMP levels returning to control values by 14 h of culture
[18]. In the present study, TPA decreased extracellular cAMP
levels by 15% immediately prior to an increase in 3 H-T incorporation. This slight drop in extracellular cAMP levels
could reflect important changes in intracellular cAMP levels
that allow some of the GC to progress through the cell cycle.
The precise site or sites within the cell cycle that are
blocked by sustained levels of intracellular cAMP are not
known. It is likely that 8-Br-cAMP prevents GC from entering the S-phase of the cell cycle, since 8-Br-cAMP reduces
mitogen-induced 3 H-T incorporation by approximately 30%
(present study, [5]). Treatment with 8-Br-cAMP also inhibits
3H-T incorporation
in transformed GC and dramatically reduces their proliferation in nude mice [30]. In Balb/c-3T3
cells, cjun and c-myc expression are inhibited by increased cAMP levels [31]. These proto-oncogenes are expressed early in the GoG, transition, and their suppression
could be at least partially responsible for preventing Balb/
c-3T3 cells from entering the S-phase of the cell cycle. A
similar molecular mechanism could account for cAMP's antimitogenic properties in GC, since both of these protooncogenes are expressed prior to an increase in 3H-T incorporation [16, 32]. However, 8-Br-cAMP may also inhibit
mitosis at other stages in the cell cycle, since TPA-induced
3H-T incorporation is
suppressed by 30% but GC proliferation is completely inhibited.
The growth-inhibiting action of 8-Br-cAMP is observed
in the presence or absence of testosterone, suggesting that
this anti-mitogenic effect is independent of 8-Br-cAMP's ability
to stimulate and/or maintain estrogen synthesis. E2 at doses
similar to the amount secreted by "mature" GC isolated from
eCG-treated rats neither stimulates nor inhibits the proliferation of GC of immature rats. However, E2 blocks TPA
from inducing mitosis of GC isolated from immature ovaries. This effect of E2 was not observed when "mature" GCs
from eCG-treated rats were studied. Further, simultaneous
addition of E2 does not block insulin-induced mitosis of
"mature" GC of eCG-treated rats (insulin plus E2 increased
cell numbers by 16 + 3% compared to controls [p < 0.05];
similarly, insulin alone stimulated a 20 + 2% increase in
cell number compared to controls [p < 0.05]; n = 10; Peluso, unpublished data). It appears then that the ability of
GC to respond to a mitotic stimulus (TPA) can be attenuated by either 8-Br-cAMP and/or E2. The reasons for the
loss of E2 regulation of TPA-induced mitosis of "mature"
GCs are unknown but could be related to E2 receptor content. Richards [33] has shown that 48 h after eCG treatment,
the number of GC estrogen receptors is dramatically reduced. In addition, TPA decreases E2 receptor mRNA and
protein levels by 50% within 3 h in the human mammary
carcinoma cell line (MCF-7) [34]. The combined effects of
eCG pretreatment and TPA could account for both the loss
of E2 receptors and the absence of E2's anti-mitogenic effects in the "mature" GC isolated from eCG-treated rats.
The differential response to the combined action of TPA
and E2 could represent an important physiologic mechanism that determines which follicles continue to grow and
which become atretic during the estrous cycle. It has been
known for a long time that E2 has both growth-promoting
and inhibitory properties. E2's growth-promoting actions are
mainly observed within GC of preantral follicles [35]. E2 does
not directly stimulate GC proliferation but acts on the thecal cells [36], presumably to stimulate the production of
TGF[, which in turn induces rat GC mitosis [37]. In contrast, the inhibitory effects of E2 are most frequently noted
for mid-sized antral follicles [35, 38, 39]. On the basis of the
data from the present studies, it is proposed that in those
antral follicles that are destined to grow and subsequently
ovulate, GC lose E2 receptors and begin to secrete E2. Since
the GC of these follicles have few E2 receptors, they will
continue to undergo mitosis in response to appropriate
stimuli even in the presence of high E2 levels. Conversely,
when immature GC, which possess higher numbers of E2
receptors, are exposed to increasing intraovarian levels of
E2, they are unable to proliferate in response to mitogenic
stimuli and therefore become atretic. In this manner E2 secreted from the "dominant" follicles could act to induce
atresia in the less mature follicles, thus limiting the number
of follicles that develop and ovulate during each cycle. This
concept is in agreement with the observation that continuous E2 treatment reduces 3H-T incorporation and induces
atresia of mid-sized but not large antral follicles [38, 39].
ACKNOWLEDGMENTS
We gratefully acknowledge Ms. Susan Kavel for running the cAMP assays and Mr.
Richard Krasuski for his help in conducting the perifusion experiments.
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