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Department of Cell Biology Baylor College of Medicine Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(11), follicular development is arrested at
the preantral stage. These models illustrate that although the
early/slow stages of granulosa cell proliferation and follicle growth
occur in the absence of gonadotropins and estradiol, these hormones are
required for normal growth, including the final rapid stages of
development that form preovulatory follicles. In response to the surge of LH that triggers ovulation, granulosa cells of preovulatory follicles cease dividing (1, 6) and initiate a program of terminal differentiation (12, 13). Whereas proliferation occurs over days, the cessation of granulosa cell division is rapid and within 7 h of exposure to LH, these cells are irreversibly committed to become luteal cells (14). These dramatic events indicate that hormones such as FSH, estradiol, and LH control both the rate of granulosa cell proliferation, as well as exit from the cell cycle during differentiation. With the recent explosion of information concerning cell cycle-regulatory molecules, the mechanisms involved in controlling successive stages of granulosa cell proliferation and terminal differentiation can begin to be elucidated.
Cellular proliferation is controlled by molecules positively or negatively affecting the kinase cascades that regulate transitions through the checkpoints of the cell cycle. Briefly summarized (for reviews, see Refs. 15, 16, 17, 18, 19, 20, 21), entry into the cell cycle is positively regulated by D-cyclins, D1, D2, or D3 (22, 23). The D-cyclins bind cyclin-dependent kinase (cdk) 4 or cdk6, which allows phosphorylation by cdk-activating kinase (CAK), thereby forming an active complex. The cyclin-cdk complex phosphorylates cellular substrates, including the retinoblastoma protein RB and related proteins, leading to activation of the E2F family of transcription factors. This results in the activation of numerous genes involved in DNA synthesis and ultimately enables cells to begin the transition from G1 to S phase. Further progression depends on activation of cyclin E-cdk2 complexes and the sequential activation of additional cdks. In contrast to cyclins, cdk inhibitors (or CKIs) are negative regulators that arrest the cell cycle in G1 by binding and inhibiting the activity of cyclin-cdk complexes. The Cip/Kip family of CKIs, p21Cip1 (24, 25, 26, 27, 28), p27Kip1 (29, 30), and p57Kip2 (31, 32), bind cyclinD-cdk4/6 as well as cyclinE-cdk2 complexes and prevent their activation.
These regulatory molecules, D cyclins and Cip/Kips, although widely expressed in overlapping as well as tissue-specific patterns (28, 32, 33, 34), can exert dramatic effects on cell cycle progression when their relative amounts are altered by external signals. For instance, when overexpressed in fibroblasts, cyclin D2 stimulated a shortened G1 phase and reduced cell generation time, and reduced cellular dependence on serum for growth (35). When proliferating macrophages were treated with cAMP, it caused p27 expression to increase and triggered cell cycle arrest (36). Expression of p27 was also increased when glial cell precursors were induced to differentiate into astrocytes (37). Of particular interest, the targeted deletion of cyclin D2 and p27 genes indicated that they are essential for normal ovarian function. In mice null for cyclin D2 (cyclin D2-/-), granulosa cell proliferation is impaired, the ovarian follicles remain small, and ovulation fails to occur (38). In mice null for p27 (p27-/-), follicular growth is not compromised but granulosa cells do not luteinize properly in response to LH leading to female infertility (39, 40, 41).
These advances in our understanding of hormonal control of ovarian function and of the roles of cell cycle regulatory molecules have proceeded independently. Therefore, we sought to determine when cyclin D2 and p27 are expressed in the ovary and whether they are regulated by hormones at specific stages of follicular growth and luteinization. Since cyclin D2 is a rate-limiting activator of cell cycle progression and essential for granulosa cell proliferation, we have investigated whether the hormones, FSH and estradiol, known to trigger rapid granulosa cell proliferation during the formation of preovulatory follicles, also regulate cyclin D2. Since p27 is known to be involved in exit from the cell cycle and, in particular, in the differentiation of granulosa cells to luteal cells, we analyzed its regulation by the surge of LH. Our results demonstrate that gonadotropins and steroids, acting via cAMP and estrogen receptor-mediated mechanisms, impinge upon the cell cycle regulatory machinery to control the function and fate of granulosa cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and cyclin-cdk complexes (42).
PCNA is expressed in proliferating cells (43) and has been shown in the
rat ovary to be a sensitive marker of granulosa cell proliferation
(44). In mice treated with PMSG to stimulate the growth of preovulatory
follicles, cyclin D2 mRNA was localized specifically to the granulosa
cells (Fig. 1
). The abundance of PCNA in
these cells indicates that they are proliferating. This pattern of
expression was specific to cyclin D2. In situ hybridization
using antisense probes for cyclin D1 and cyclin D3 showed their mRNA
predominantly in the theca and interstitial cells. Therefore, the
proliferating granulosa cells in the ovary specifically express cyclin
D2.
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) in ovaries from mice treated with PMSG
followed by hCG to trigger ovulation and luteinization. PCNA was absent
in the luteal cells that are no longer dividing and have terminally
differentiated. Interestingly, mRNA for p21Cip1, another
member of the Cip/Kip family of cdk inhibitors, was also selectively
localized to luteal cells (Fig. 2), indicating that these cdk
inhibitors are specifically expressed in these terminally
differentiated cells.
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). Ovaries from mice at day 5 (D5) and
10 (D10) of age contain growing follicles that consist of only one or
two layers of granulosa cells. Although serum FSH increases between day
5 and 10, the follicles of these prepubertal mice do not mature and do
not synthesize appreciable amounts of estradiol (45). Despite the small
size of these follicles and the slow proliferative rate (1, 2, 46),
cyclin D2 mRNA is expressed in the granulosa cells.
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). FSH has long been known to be important for
follicular growth (4). In mice null for the FSHß subunit
(FSHß-/-), follicular growth progresses until follicles
are comprised of several layers of granulosa cells, but arrests before
antral formation indicating that only early stages of follicular growth
are gonadotropin independent (10). Cyclin D2 is also expressed in
the granulosa cells of the small follicles in the
FSHß-/- mice (Fig. 3). In situ hybridization
performed on ovarian sections from these same mice using a p27
antisense probe showed that p27 mRNA is expressed at low levels and not
localized to any particular cell type (data not shown). Collectively,
these results demonstrate that cyclin D2 is expressed very early during
the course of follicular growth, i.e. in all follicles that
have initiated growth from the primordial stage. Second, the expression
of cyclin D2 mRNA in granulosa cells of small follicles is gonadotropin
independent and can occur in the absence of FSH, estradiol, and GDF-9.
Third, in these early stages of proliferation and follicular growth,
granulosa cells lack appreciable levels of p27. In the mice analyzed above, the follicles are in a gonadotropin-independent phase of growth, and granulosa cell proliferation is slow (1, 2). In contrast, the most rapid phase of granulosa cell proliferation occurs when small follicles become responsive to gonadotropins and begin to synthesize estradiol (1, 6). To determine whether these hormones regulate the expression of cyclin D2 or p27, we used the hypophysectomized rat model, which enables precise hormonal treatments, provides sufficient material for quantitative studies, and allows for comparison with the mouse. Most importantly, it is a physiological model in which the effects of estradiol, FSH, and LH on ovarian cell proliferation and function have been well characterized (4, 5, 7).
Cyclin D2 and p27Kip1 Are Hormonally
Regulated during Follicular Development
In the hypophysectomized (H) rat, follicular growth is initiated
but not sustained due to lack of gonadotropins and steroids (4).
Treatment of H rats with estradiol for 3 days (HE) promotes the
proliferation of granulosa cells (6) and growth of large preantral
follicles (4, 5, 6). Subsequent treatment of HE rats with FSH for 2 days
(HEF) stimulates further proliferation and promotes the growth of large
antral follicles that exhibit characteristics of normal preovulatory
follicles (4, 5, 6). Finally, an ovulatory dose of hCG triggers ovulation
and luteinization. The expression of cyclin D2 and p27 mRNA in the
ovaries of these rats was analyzed by in situ hybridization
(Fig. 4A). In H rats, cyclin D2 mRNA is
expressed at low levels and is localized to the granulosa cells of the
small growing follicles, a pattern similar to that observed in the
follicles of FSHß-/- or prepubertal mice (Fig. 3). In
ovaries of HEF rats, cyclin D2 mRNA expression was increased and
specifically localized to the rapidly proliferating granulosa cells of
large preovulatory follicles. In response to hCG, cyclin D2 mRNA was
rapidly down-regulated within 4 h (HEF hCG4 h) in the large
follicles that are committed to ovulate and luteinize and remained low
in the terminally differentiated corpora lutea (HEF hCG48 h). In these
same ovaries, cyclin D2 expression was not down-regulated in the small
follicles; these lack LH receptor and therefore are not exposed to
elevated levels of intracellular cAMP (3, 4, 49, 50, 51).
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). p27 mRNA was expressed at low levels
throughout the ovary in H rats, but appeared to be preferentially
localized to granulosa cells of the preovulatory follicles in HEF rats.
Treatment with hCG resulted in decreased expression of p27 within
4 h but this effect was transient. Highest levels of p27 mRNA were
observed 48 h after hCG specifically in the terminally
differentiated luteal cells.
To provide a more detailed and quantitative analysis of the effects of
estradiol, FSH, and LH on cyclin D2 and p27 expression, RNA and protein
were obtained from granulosa cells and corpora lutea isolated from the
ovaries of hormone-treated H rats. Northern analysis (Fig. 5A) using the cyclin D2 cDNA as probe
showed that cyclin D2 transcripts of approximately 6 kb and 2.5 kb (34, 52) were present at low levels in granulosa cells of H rats. Cyclin D2
mRNA levels were greatly increased by estradiol treatment alone (HE)
and subsequent treatment with FSH (HEF) sustained the elevated
expression of the 6-kb transcript and increased the 2.5-kb transcript.
Consistent with the in situ hybridization results, cyclin D2
mRNA decreased rapidly within 2 h following treatment with hCG and
further by 8 h. Cyclin D2 mRNA levels remained low in corpora
lutea isolated 24 and 48 h after hCG. Western analysis of cellular
extracts showed that cyclin D2 protein is regulated in a similar
manner: major increases occurred with E alone, were sustained by
subsequent treatment with FSH, and were down-regulated in response
to hCG (Fig. 5A, lower panel).
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transcript of approximately 2.4 kb (53) was present in granulosa cells
of H and HE rats and increased in response to FSH. Consistent with
results from in situ hybridization, p27 mRNA levels were
transiently decreased in HEF rats 2 and 8 h after treatment with
hCG, but increased in corpora lutea isolated at 24 and 48 h. (The
additional band at 4 kb hybridizing to the p27 cDNA appears to be
nonspecific and is not hormonally regulated.) Western analysis of
cellular extracts showed that changes in levels of p27 protein were
quite similar to those of its mRNA: p27 protein was present in
granulosa cells of small and preovulatory follicles of H rats,
exhibited a transient decrease in response to ovulatory hCG, but then
increased to highest levels in corpora lutea. However, unlike its mRNA,
p27 protein was lower in granulosa cells of HE rats compared with H
rats and did not exhibit a marked increase in granulosa cells of HEF
rats, indicating that the half-life of p27 protein and mRNA in response
to estradiol and FSH may differ.
To dissect the effects of FSH vs. estradiol, and to
determine whether FSH can independently regulate the expression of
cyclin D2 expression, H rats were treated for short periods with either
FSH or estradiol, granulosa cells were isolated at selected timepoints,
and cellular protein was prepared. To stimulate a rapid response
in vivo (i.e. at 2 and 12 h) rats were
injected iv with 5.0 µg FSH, a dose previously shown to mimic the FSH
surge by markedly elevating intracellular levels of cAMP (50).
Additional rats were injected subcutaneously with FSH (1.0 µg) twice
daily, a regimen that closely mimics basal levels of FSH and maintains
granulosa cell function (5), and granulosa cells were isolated at
24 h. Estradiol treatments consisted of 1.5 mg estradiol injected
ip (for 2 h) or subcutaneously (for 12 or 24 h). Western
analysis of granulosa cell protein showed that each hormone alone
regulates cyclin D2 protein levels and that each hormone causes a
distinct temporal pattern of expression (Fig. 5C). Specifically,
exposure of granulosa cells of H rats to elevated (surge)
concentrations of FSH resulted in a marked 7.2-fold induction of cyclin
D2 protein within 2 h compared to untreated H animals.
Interestingly, the induction of cyclin D2 by elevated FSH was
transient; protein levels returned to control levels (or below) after
12 h of FSH. In rats given the lower dose of FSH to mimic basal
hormone secretion, cyclin D2 protein was elevated 2.6-fold by 24 h
compared to untreated H controls. In contrast, the response to
estradiol occurred more gradually, 2.8-fold increase by 2 h, but
was then sustained at an elevated level, 9.7-fold at 24 h. When
the same blot was reprobed using a p27 antibody, the levels of this
protein were not acutely regulated by either FSH or estradiol. The fold
induction in response to estradiol was never greater than 1.5-fold.
Cyclin D2 Is Regulated by FSH in Vivo
To further analyze the oscillating temporal expression pattern of
cyclin D2 in response to FSH, H rats were injected iv with 5.0 µg FSH
and granulosa cells were isolated at 2, 4, 8, and 12 h. Additional
rats were injected subcutaneously with 1.0 µg FSH and granulosa cells
were isolated at 24 and 48 h. Western analyses of granulosa cell
extracts were analyzed using ECL (Fig. 6A, upper) and densitometric
quantification (lower). As shown in Fig. 5C above and in
additional experiments, the levels of cyclin D2 protein respond to
acute as well as tonic treatment with FSH. High levels of FSH given iv
stimulated a rapid increase (6.6 ± 1.7 fold; n = 3) in
cyclin D2 protein at 2 h. This response was transient, and levels
of cyclin D2 returned to near control levels at 4, 8, and 12 h
(1.4 ± 0.5; n = 3) after FSH treatment. In response to the
tonic dose of FSH given subcutaneously, cyclin D2 protein increased
(2.8 ± 0.3; n = 3) at 24 h and 48 h. Analysis of
p27 protein levels in these same extracts (Fig. 6B) showed that the
increase in cyclin D2 in response to FSH is specific. When compared
with untreated controls, the levels of p27 protein remained relatively
constant at 2 h (1.2 ± 0.1; n = 3), 4 h, 8 h,
12 h (1.5 ± 0.1; n = 3), or 24 h (1.4 ± 0.5;
n = 3) and were even decreased 50% at 48 h.
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showed that FSH increased cyclin D2 mRNA expression within 1 and 2
h (9.0 ± 2.4, n = 3), but that expression was decreased to
1.6-fold by 6 and 10 h (not shown). After 24 h of FSH, cyclin
D2 is again elevated to high levels (10.2-fold) compared with cells
cultured 24 h in the absence of hormone (-). The increases in
cyclin D2 mRNA are induced specifically by FSH since the addition of
10% FBS, which contains numerous growth factors, did not affect cyclin
D2 expression at 2 or 6 h.
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showed that forskolin (Fo) increased cyclin D2 mRNA expression within 1
and 2 h compared to untreated cells (-). Estradiol had only a
minimal effect after 1 h, but increased cyclin D2 mRNA expression
within 2 h. This increase was specific since the estrogen receptor
antagonist ICI 164,384 blocked the estradiol-mediated increase in
cyclin D2 (E+ICI).
LH Regulates Expression of Cyclin D2,
p27Kip1, and
p21Cip1
Follicular growth is terminated by the LH surge. As a consequence
of elevated cAMP, granulosa cells exit the cell cycle (Fig. 2 and Ref.
6), exhibit altered patterns of expression of cyclin D2 and p27 (Fig. 4), and undergo terminal differentiation (3, 12, 13, 14). To determine more
precisely the time course of changes in cyclin D2 and p27 protein
levels after the LH surge, granulosa cells and corpora lutea were
isolated from hormone-primed H rats 2, 4, 8, 12, and 24 h after an
ovulatory dose of hCG. Western analysis (Fig. 8) showed that the elevated levels of
cyclin D2 protein present in preovulatory granulosa cells of HEF rats
were decreased within 2 h, even lower at 4 h, and remained
low in corpora lutea (24 and 48 h). In contrast, p27, although
initially decreased by hCG after 2, 4, and 8 h, is elevated to
high levels during the course of luteinization, between 12 and 24
h after hCG.
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) but is increased rapidly in response
to hCG, within 2–4 h. Interestingly, at these early times, p21 mRNA
was localized to the granulosa cells surrounding the antrum and oocyte.
As luteinization proceeded, p21 was localized to granulosa cells
throughout the follicle (hCG 12 h) and continued to be expressed
in the luteal cells 24 h after hCG. Therefore, hCG induced rapid
changes in the expression of p21, as well as cyclin D2 and p27, during
the terminal differentiation of granulosa cells to luteal cells.
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). In
both normal and cyclin D2-/- immature mice, PCNA staining
of granulosa cells was observed in the small follicles that are in the
slow/gonadotropin-independent phase of growth (Fig. 10A). When normal
mice were treated with PMSG, many more PCNA-immunopositive cells were
present and associated with the rapidly dividing granulosa cells of
large preovulatory follicles. However, when the cyclin
D2-/- mice were treated with PMSG, proliferation did not
occur, and few of the granulosa cells expressed PCNA.
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) showed that aromatase was induced by
PMSG in cyclin D2-/- mice, indicating that the FSH
response system in granulosa cells of these mice is intact. Likewise,
in cyclin D2-/- mice, hCG (LH) decreased aromatase mRNA
and rapidly induced PGS-2 and PR mRNA, indicating that these responses
of preovulatory granulosa cells to LH are not impaired. The lower
levels of aromatase, PGS-2, and PR mRNA detected by RT-PCR in cyclin
D2-/- mice most likely reflect the reduced number of
granulosa cells in these ovaries. Lastly, hCG stimulated the formation
of small corpora lutea that do not exhibit PCNA staining (Fig. 10C),
indicating that the mechanisms mediating complete exit from the cell
cycle are intact. However, despite the ability of cyclin
D2-/- granulosa cells to differentiate in response to LH,
the oocytes are not ovulated and remain trapped in the corpora
lutea. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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During the earliest stages of follicle growth, granulosa cell proliferation occurs by mechanisms that are independent of FSH and estradiol as well as of cyclin D2. Specifically, we have shown that cyclin D2 mRNA is present in granulosa cells of small follicles in which follicular growth is restricted by the absence of hormones or growth factors; FSHß-/- mice, gdf-9-/- mice, prepubertal mice, and hypophysectomized rats. Conversely, some, albeit limited, proliferation of granulosa cells occurs in cyclin D2-/- mice. These observations indicate that in small follicles, cyclin D2 is one, but not the exclusive, factor regulating granulosa cell entry into the cell cycle and that its expression occurs independently of gonadotropins and steroids. The slow rate of growth is not related to elevated amounts of p27 or p21; expression of both was low in these small follicles. Therefore, this protracted phase of granulosa cell proliferation appears to involve a unique set of cell cycle regulators (or inhibitors)—a specific hormone/growth factor (GDF-9 or one yet to be identified) or the controlled lack of responsiveness to stimulatory molecules such as FSH and estradiol.
Once the follicles begin to mature, respond to the gonadotropins, and produce estradiol, the rate of granulosa cell proliferation is exponentially increased (1). Based on data from the H rat, this period of rapid proliferation is due, at least in part, to the combined effects of estradiol and FSH/cAMP (6). Our data indicate that FSH/cAMP and estradiol increase cyclin D2 expression by specific receptor-mediated mechanisms in a fashion that mimics the effects of these hormones on the labeling index/proliferation of granulosa cells in H rats (6). Previous studies have estimated that the doubling time of granulosa cells during this time is reduced to 24 h (1). Consistent with this report, the results presented herein show that in vivo cyclin D2 expression not only increased rapidly in response to a surge concentration of FSH, but also increased between 12 and 24 h in response to tonic, basal levels of FSH. Likewise, when cultured granulosa cells were exposed to FSH in vitro, cyclin D2 expression showed a transient increase at 2 h followed by a secondary increase at 24 h. These observations combined with the markedly impaired proliferation in FSH-stimulated cyclin D2-/- mice indicate that cyclin D2 is an essential downstream mediator of hormone- stimulated proliferation at this period of exponential cell growth. The ability of estradiol and FSH to increase cyclin D2 is specific. Neither hormone increases the other D-cyclins or p27. Therefore, our data suggest that FSH and estradiol stimulate granulosa cell proliferation by increasing the levels of cyclin D2 relative to those of p27, thereby triggering a burst of proliferation that results in the development of large preovulatory follicles.
The individual effects of estradiol and FSH/cAMP indicate that each hormone may regulate distinct points in the cell cycle and may utilize parallel mechanisms. For example, the temporal increase in expression of cyclin D2 expression within 24 h in response to FSH, as well as estradiol, is consistent with the doubling time of granulosa cells during the period of exponential growth (1), indicating that cyclin D2 is directly or indirectly involved in timing cell cycle transit. An oscillatory pattern of cyclin D2 expression has been observed in other cell types in response to stimulatory factors (18, 52). However, the apparent oscillatory pattern observed in response to FSH may also involve an inherent periodicity in the activation of the A-kinase pathway (61) in granulosa cells, thereby integrating many facets of granulosa cell function. Additionally, FSH (in the presence of insulin) has been shown to induce c-myc and c-fos (62), and cAMP has been shown to regulate the levels of cyclin B (63), indicating that FSH may act at multiple steps during the cell cycle. Estradiol, in contrast to FSH, not only stimulates a greater increase in cyclin D2 mRNA and protein, but may also decrease p27 protein levels. The apparent down-regulation of the cell cycle inhibitor p27 concurrent with enhanced induction of cyclin D2 may be responsible for the greater mitogenic activity of estradiol compared with FSH in the H rat (5, 6). Furthermore, in other cell types, estradiol has been observed to induce cyclin E (64), whose activation of cdk2 is essential for cells to enter S phase. Therefore estradiol may also induce cyclin E in granulosa cells providing another explanation for its greater effect on granulosa cell proliferation compared with FSH.
In addition to FSH and estradiol, other growth factors are able to stimulate granulosa cell proliferation implicating a role in follicular growth. For example, activin has been observed to stimulate DNA synthesis (65) and maintain proliferation (66) in cultured granulosa cells, and insulin-like growth factor I (IGF-I) expression in situ has been correlated with granulosa cell DNA synthesis (67). However, targeted deletion of the activin ßB subunit in mice (68, 69) did not result in an ovarian phenotype, indicating that activins alone are not the essential mediators of granulosa cell proliferation in vivo. In mice null for IGF-I, although the females are unable to ovulate, the ovarian follicles develop to the large antral stage, indicating IGF-I may be involved primarily in granulosa cell differentiation and responsiveness to ovulatory hormones as opposed to granulosa cell proliferation, per se (70).
The importance of gonadotropin stimulation for granulosa cell
proliferation during the formation of preovulatory follicles is
highlighted by observations in mice deficient in the inhibin subunit
. In these mice, the appearance of ovarian tumors is coincident with
puberty (71), when the first group of growing, gonadotropin-responsive
follicles reaches the phase of exponential proliferation. However, if
the inhibin null mice are cross-bred with hypogonadal mice that
lack FSH/LH, no ovarian tumors form (72), suggesting that when
gonadotropin/steroid action is unopposed, such as in the absence of
inhibin, granulosa cell proliferation is uncontrolled. Consistent with
this idea, chronic overexpression of LHß results in elevated levels
of estradiol and occasional ovarian tumors (73). Therefore, granulosa
cell proliferation is tightly regulated, and it appears that
appropriate ratios of estradiol, FSH/LH, and transforming growth
factor-ß family members (activin, inhibin, GDF-9), as well as growth
factors such as IGF-I, determine the progression of proliferative and
antiproliferative events within these cells. Interestingly, the human
granulosa cell tumors that have been studied overexpress cyclin D2 but
not cyclin D1 or D3 (38), supporting the evidence that expression of
cyclin D2 causes rapid granulosa cell proliferation and thereby, may
contribute to enhanced oncogenic potential.
Most dramatic is the ability of the LH surge to rapidly and completely halt cell cycle progression as well as initiate the process of luteinization, potentially utilizing distinct mechanisms for each. Our results indicate that the LH surge (high cAMP) induces exit from the cell cycle not only by elevating the levels of cell cycle inhibitors, such as p27 and p21, but by completely down-regulating the activator, cyclin D2. Cyclin D2 mRNA and protein are undetectable within 2–4 h after the LH surge. The temporal differences in the expression of cyclin D2, p27, and p21 may be indicative of distinct roles for each of these regulatory molecules in the control of cell cycle progression and cellular differentiation. Specifically, granulosa cell exit from the cell cycle occurs rapidly after the LH surge and is likely due to the drastic down-regulation of cyclin D2, since its absence would prevent or markedly reduce progression through G1 phase. The early induction of p21 in response to hCG may indicate it plays a role in exit from the cell cycle as well. Since p27 is not elevated until 12–24 h after hCG, it may not affect the immediate exit of granulosa cells from the cell cycle but rather contributes to cell differentiation and maintenance of cell cycle arrest. These observations are consistent with those presented by the phenotype of p27-/- mice (39, 40, 41) as well as evidence that cAMP increases p27 in glial cells (36). Furthermore, p27 and p21 inhibit many cdks and may be most important for blocking the activity of cyclin E (and cyclin A)-cdk2 complexes (15, 18); therefore, the LH-mediated induction of high levels of these CKIs would inhibit cell cycle progression at multiple points. Collectively, these (and additional) mechanisms permit the LH surge (high cAMP) to rapidly reprogram granulosa cells to enter a nonproliferative luteal state. In contrast, FSH and low levels of cAMP promote granulosa cell proliferation by increasing expression of cyclin D2. Such different effects in response to changes in cAMP levels underscore the diverse roles that the A-kinase pathway plays in regulating ovarian cell function.
Lastly, our results offer insight into the relationship between cell number and follicular function. Specifically, we have shown that although follicles in the cyclin D2-/- mice have a reduced number of granulosa cells and fail to ovulate, the hormonal activation of the PGS-2 and PR genes that are essential for ovulation (74, 75, 76) occurs normally in these follicles. These results implicate cell number as a critical factor in ovulation, presumably due to the production by granulosa cells of a critical (as yet unknown) factor controlling an event in the ovulatory process. In contrast, cell number is not a factor for differentiation and the formation of the corpus luteum. In response to the LH surge, granulosa cells of cyclin D2-/- mice exit the cell cycle (loss of PCNA staining) and undergo terminal differentiation including hypertrophy and expression of the luteal-specific gene, P450 side-chain cleavage (14, 38).
In summary, the control of granulosa cell proliferation is complex, essential for normal follicular growth, and critical for ovulation. It is dependent on the cell-specific expression of regulators of G1 progression: the cdk activator cyclin D2 and the cdk inhibitor p27Kip1. After the initial gonadotropin-independent phase of growth, the regulation of these molecules by FSH, LH, and estradiol controls the exponential phase of granulosa cell proliferation as well as the exit of granulosa cells from the cell cycle and their terminal differentiation to luteal cells. Critical to the entry into and exit from the period of rapid proliferation are the differential responses of the granulosa cells to estradiol, as well as to the low vs. high levels of cAMP stimulated by FSH and LH, respectively. Thus, the coordinated interactions of estradiol and cAMP are able to tightly control the highly proliferative and nonproliferative events in granulosa cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In Situ Hybridization
In situ hybridization was done as described by
Wilkensen (77). Briefly, [35S]UTP-labeled antisense and
sense probes of mouse cyclin D1, cyclin D2, and cyclin D3 cDNAs
(provided by Dr. Piotr Sicinski and Dr. Robert Weinberg, Whitehead
Institute, MIT, Boston, MA) and mouse p27Kip1 and
p21Cip1 cDNAs (provided by Dr. Stephen Elledge, Department
of Biochemistry, Baylor College of Medicine) were made using the
Riboprobe In Vitro Transcription Systems kit (Promega,
Madison, WI). Ovaries were fixed in 4% paraformaldehyde, embedded in
paraffin, and sectioned at 6 µm onto silane-coated slides (Histology
Control Systems, Glen Head, NY). After rehydration, sections were
pretreated with 20 µg/ml proteinase K and 0.1 M
triethanolamine/acetic anhydride before coating with the labeled probe
and then incubated at 55 C overnight. Slides were washed at high
stringency including two washes of 50% formamide, 2x saline sodium
citrate, 100 mM ß-mercaptoethanol for 30 min each at 65
C, then treated with 20 µg/ml RNase A and dehydrated. After drying,
slides were dipped in photographic NTB-2 emulsion (Kodak, Rochester,
NY) and exposed at 4 C for 2–5 days. The slides were developed with
D-19 developer and fixer (Kodak) and counter-stained with hematoxylin
for 30 sec. Light field photography shows the tissue histology while
dark field illumination allows visualization of the mRNA probe.
Northern Analysis
Granulosa cells were isolated from ovaries by needle puncturing
(78). Northern analysis was done according to the method of Fitzpatrick
and Richards (79). Briefly, total cellular RNA was obtained by
homogenizing granulosa cells in buffer (140 mM NaCl, 5
mM KCl, 3 mM MgCl2, 25 mM Tris-HCl)
containing 1% NP-40 followed by phenol extraction and ethanol
precipitation. The RNA (20 µg) was separated on formaldehyde-agarose
gels and transferred to 0.2-µm Biotrans nylon membranes (ICN
Biomedicals Inc, Aurora, OH). Probes consisted of isolated cDNA inserts
labeled by random hexanucleotide priming (79) using
[32P]dCTP (ICN Radiochemicals, Los Angeles, CA) and were
incubated with the membrane at 42 C overnight, according to the
specifications of ICN. Membranes were washed for high stringency with
0.5x saline sodium citrate, 0.1% SDS at 55 C for 1 h and exposed
to X-OMAT film (Kodak).
Western Analysis
Soluble cellular protein was obtained by suspending isolated
granulosa cells in whole cell extract buffer: 10 mM
NaHPO4, pH 7.4, 1 mM Na2EDTA, 1
mM dithiothreitol, 400 mM KCl, 10% glycerol, 5
µg/ml aprotinin, 5 µg/ml leupeptin, 1 µM pepstatin, 1
mM phenylmethylsulfonyl fluoride, 5 µM NaF,
0.5 µg/ml ocadeic acid; followed by two freeze/thaw cycles and
centrifugation at 4 C. Fifty micrograms of soluble protein were run on
12% acrylamide/SDS gels and transferred to Immobilon-P nylon membranes
(Millipore Co, Bedford, MA). Membranes were blocked with 5% nonfat
milk followed by incubation with the recommended dilution of primary
antibody in 5% milk for 1 h at room temperature. Antibodies used
were mouse monoclonal anticyclin D2 (DCS-3.1, Neomarkers Inc, Fremont,
CA) at 2 µg/ml and rabbit polyclonal anti-p27 (C-19, Santa Cruz
Biotechnology Inc, Santa Cruz, CA) at 1 µg/ml. Blots were then washed
extensively in 10 mM Tris-buffered saline containing 0.5%
Tween-20 (TBS-T) and incubated with 1:10,000 dilution of antimouse IgG
(or antirabbit IgG) peroxidase-linked antibody (Amersham Life Sciences,
Inc., Arlington Heights, IL) in 5% milk for 1 h at room
temperature. After washing in TBS-T, ECL was performed using
Supersignal chemiluminescent detection reagents (Pierce, Rockford, IL).
Western blots were quantitated by densitometry (Molecular Dynamics,
Sunnyvale, CA) of autoradiographic films. For quantification of Western
blots shown in Fig. 8, proteins were transferred to Protran
nitrocellulose membrane (Schleicher & Schuell, Keene, NH), blocked with
5% milk, and incubated with primary antibody as above but detected
with 125I-labeled protein A (ICN) and exposed to film.
Bands detected by autoradiography were cut from the membrane, dissolved
in scintillation fluid, and counted in a Beckman LS-6500 multipurpose
scintillation counter set for 
-detection.
Cell Culture
For in vitro experiments, granulosa cells were
isolated from either immature (day 22 of age) rats (Holtzman, Madison
WI) or immature rats primed with 1.5 mg/0.2 ml 17ß-estradiol (Sigma)
once daily for 3 days, as indicated. The cells were treated with 20
µg/ml trypsin and 160 µg/ml DNaseI as described previously (78).
The cells were then plated on bovine serum-coated dishes in serum-free
DMEM:F12 medium and cultured overnight at 37 C, 95% air/5%
CO2 (79). Hormone treatments included 100 ng/ml FSH (NIH
oFSH-16, National Hormone and Pituitary Agency, Rockville, MD), 10%
FBS (Hyclone Labs., Logan, UT), 10 µM forskolin
(Calbiochem, La Jolla, CA), 10 nM estradiol (Sigma), and
100 nM ICI 164,384 (Zeneca Pharmaceuticals, Macclesfield,
UK).
Immunocytochemistry
Proliferating Cell Nuclear Antigen (PCNA) was detected by
immunolocalization as described previously (44) with modifications.
Briefly, rehydrated paraffin sections were washed in PBS, blocked with
1% equine serum for 10 min at 37 C followed by overnight incubation at
room temperature with 1:150 dilution monoclonal anti-PCNA antibody
(Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). Slides were
washed with PBS and then incubated with 15 µl/ml mouse IgG
peroxidase-linked antibody (Amersham Life Sciences) at 37 C for 30 min.
After washing with PBS, slides were incubated in 50 ml
NaPO4 buffer containing 25 mg 3,3'-diaminobenzidine
substrate (Amersham) for 10 min, rinsed with H2O, and
counter-stained with eosin.
RT-PCR
RT-PCR was performed as previously described (80). Total
RNA (100 ng for aromatase; 350 ng for PGS-2 and PR) was reverse
transcribed using 500 ng poly dT (Pharmacia Biotech Inc, Piscataway,
NJ) and 0.25 U avian myeloblastosis virus (AMV) reverse transcriptase
(Promega) at 42 C for 75 min. After the addition of 500 ng of each
specific primer, 2 µCi [32P]dCTP (ICN), 2.5 U
Taq Polymerase (Promega) and Thermocycle Buffer (Promega),
the 100 µl reaction completed 20 cycles of PCR at 94 C for 1 min, 55
C (aromatase) or 65 C (PGS-2, PR) for 2 min, and 72 C for 3 min.
Primers for the ribosomal protein L19 were included in each
experiment as an internal control (60). Products were separated on a
5% polyacrylamide gel, which was then dried and exposed to film. PCR
primers are: aromatase for 5'-TGCACAGGCTCGAGTATTTCC-3'; aromatase
rev 5'-ATTTCCACAATGGGGCTGTCC-3' (80); PGS-2 for
5'-TGTACAAGCAGTGGCAAAGG-3'; PGS-2 rev 5'-GCTGTGGATCTTGCACATTG-3';
PR for 5'-CCCACAGGAGTTTGTCAAGCT-3'; PR rev 5'-TAACTTCAGACATATTCCGG-3'
(60); and L19 for 5'-CTGAAGGTCAAAGGGAATGTG-3'; L19 rev
5'-GGACAGAGTCTTGATGATCTC-3' (60), which result in products of 271 bp,
430 bp, 328 bp, and 194 bp, respectively.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Supported by NIH Grant HD-16272.
Received for publication September 23, 1997. Revision received March 17, 1998. Accepted for publication March 19, 1998.
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J. J. Buzzard, N. G. Wreford, and J. R. Morrison Thyroid Hormone, Retinoic Acid, and Testosterone Suppress Proliferation and Induce Markers of Differentiation in Cultured Rat Sertoli Cells Endocrinology, September 1, 2003; 144(9): 3722 - 3731. [Abstract] [Full Text] [PDF]
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 S. Perlman, B. van den Hazel, J. Christiansen, S. Gram-Nielsen, C. B. Jeppesen, K. V. Andersen, T. Halkier, S. Okkels, and H. T. Schambye Glycosylation of an N-Terminal Extension Prolongs the Half-Life and Increases the in Vivo Activity of Follicle Stimulating Hormone J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3227 - 3235. [Abstract] [Full Text] [PDF]
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S. Jirawatnotai, D. S. Moons, C. O. Stocco, R. Franks, D. B. Hales, G. Gibori, and H. Kiyokawa The Cyclin-dependent Kinase Inhibitors p27Kip1 and p21Cip1 Cooperate to Restrict Proliferative Life Span in Differentiating Ovarian Cells J. Biol. Chem., May 2, 2003; 278(19): 17021 - 17027. [Abstract] [Full Text] [PDF]
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C. L. Chaffin, R. S. Brogan, R. L. Stouffer, and C. A. VandeVoort Dynamics of Myc/Max/Mad Expression during Luteinization of Primate Granulosa Cells in Vitro: Association with Periovulatory Proliferation Endocrinology, April 1, 2003; 144(4): 1249 - 1256. [Abstract] [Full Text] [PDF]
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J. Cottom, L. M. Salvador, E. T. Maizels, S. Reierstad, Y. Park, D. W. Carr, M. A. Davare, J. W. Hell, S. S. Palmer, P. Dent, et al. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase J. Biol. Chem., February 21, 2003; 278(9): 7167 - 7179. [Abstract] [Full Text] [PDF]
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N. A. Grieshaber, C. Ko, S. S. Grieshaber, I. Ji, and T. H. Ji Follicle-Stimulating Hormone-Responsive Cytoskeletal Genes in Rat Granulosa Cells: Class I {beta}-Tubulin, Tropomyosin-4, and Kinesin Heavy Chain Endocrinology, January 1, 2003; 144(1): 29 - 39. [Abstract] [Full Text] [PDF]
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 M. A. Ciemerych, A. M. Kenney, E. Sicinska, I. Kalaszczynska, R. T. Bronson, D. H. Rowitch, H. Gardner, and P. Sicinski Development of mice expressing a single D-type cyclin Genes & Dev., December 15, 2002; 16(24): 3277 - 3289. [Abstract] [Full Text] [PDF]
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P. K. Pradeep, X. Li, H. Peegel, and K. M. J. Menon Dihydrotestosterone Inhibits Granulosa Cell Proliferation by Decreasing the Cyclin D2 mRNA Expression and Cell Cycle Arrest at G1 Phase Endocrinology, August 1, 2002; 143(8): 2930 - 2935. [Abstract] [Full Text] [PDF]
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N. Danilovich, D. Javeshghani, W. Xing, and M. R. Sairam Endocrine Alterations and Signaling Changes Associated with Declining Ovarian Function and Advanced Biological Aging in Follicle-Stimulating Hormone Receptor Haploinsufficient Mice Biol Reprod, August 1, 2002; 67(2): 370 - 378. [Abstract] [Full Text] [PDF]
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 J. E. Dumont, S. Dremier, I. Pirson, and C. Maenhaut Cross signaling, cell specificity, and physiology Am J Physiol Cell Physiol, July 1, 2002; 283(1): C2 - C28. [Abstract] [Full Text] [PDF]
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G. E. Owens, R. A. Keri, and J. H. Nilson Ovulatory Surges of Human CG Prevent Hormone-Induced Granulosa Cell Tumor Formation Leading to the Identification of Tumor-Associated Changes in the Transcriptome Mol. Endocrinol., June 1, 2002; 16(6): 1230 - 1242. [Abstract] [Full Text] [PDF]
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S. Chu, S. Rushdi, E.T. Zumpe, P. Mamers, D.L. Healy, T. Jobling, H.G. Burger, and P.J. Fuller FSH-regulated gene expression profiles in ovarian tumours and normal ovaries Mol. Hum. Reprod., May 1, 2002; 8(5): 426 - 433. [Abstract] [Full Text] [PDF]
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J. S. Richards, S. C. Sharma, A. E. Falender, and Y. H. Lo Expression of FKHR, FKHRL1, and AFX Genes in the Rodent Ovary: Evidence for Regulation by IGF-I, Estrogen, and the Gonadotropins Mol. Endocrinol., March 1, 2002; 16(3): 580 - 599. [Abstract] [Full Text] [PDF]
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M. Hsieh, M. A. Johnson, N. M. Greenberg, and J. S. Richards Regulated Expression of Wnts and Frizzleds at Specific Stages of Follicular Development in the Rodent Ovary Endocrinology, March 1, 2002; 143(3): 898 - 908. [Abstract] [Full Text] [PDF]
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D. S. Moons, S. Jirawatnotai, T. Tsutsui, R. Franks, A. F. Parlow, D. B. Hales, G. Gibori, A. T. Fazleabas, and H. Kiyokawa Intact Follicular Maturation and Defective Luteal Function in Mice Deficient for Cyclin- Dependent Kinase-4 Endocrinology, February 1, 2002; 143(2): 647 - 654. [Abstract] [Full Text] [PDF]
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D. Choi, S. Yoon, E. Lee, S. Hwang, S. Song, J. Kim, B.-K. Yoon, and J.-H. Lee Characterization of Cyclin D2 Expression in Human Endometrium Reproductive Sciences, January 1, 2002; 9(1): 41 - 46. [Abstract] [PDF]
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 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
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L. M. Salvador, Y. Park, J. Cottom, E. T. Maizels, J. C. R. Jones, R. V. Schillace, D. W. Carr, P. Cheung, C. D. Allis, J. L. Jameson, et al. Follicle-stimulating Hormone Stimulates Protein Kinase A-mediated Histone H3 Phosphorylation and Acetylation Leading to Select Gene Activation in Ovarian Granulosa Cells J. Biol. Chem., October 19, 2001; 276(43): 40146 - 40155. [Abstract] [Full Text] [PDF]
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T. El-Hefnawy and A. J. Zeleznik Synergism Between FSH and Activin in the Regulation of Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D2 Expression in Rat Granulosa Cells Endocrinology, October 1, 2001; 142(10): 4357 - 4362. [Abstract] [Full Text] [PDF]
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C. L. Chaffin, K. M. Schwinof, and R. L. Stouffer Gonadotropin and Steroid Control of Granulosa Cell Proliferation During the Periovulatory Interval in Rhesus Monkeys Biol Reprod, September 1, 2001; 65(3): 755 - 762. [Abstract] [Full Text] [PDF]
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K. H. Burns, C. Yan, T. R. Kumar, and M. M. Matzuk Analysis of Ovarian Gene Expression in Follicle-Stimulating Hormone {beta} Knockout Mice Endocrinology, July 1, 2001; 142(7): 2742 - 2751. [Abstract] [Full Text] [PDF]
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S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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J. S. Richards Perspective: The Ovarian Follicle--A Perspective in 2001 Endocrinology, June 1, 2001; 142(6): 2184 - 2193. [Full Text] [PDF]
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K. F. Roby Alterations in Follicle Development, Steroidogenesis, and Gonadotropin Receptor Binding in a Model of Ovulatory Blockade Endocrinology, June 1, 2001; 142(6): 2328 - 2335. [Abstract] [Full Text] [PDF]
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R. S. Muraoka, A. E.G. Lenferink, J. Simpson, D. M. Brantley, L. R. Roebuck, F. M. Yakes, and C. L. Arteaga Cyclin-Dependent Kinase Inhibitor P27Kip1 Is Required for Mouse Mammary Gland Morphogenesis and Function J. Cell Biol., May 28, 2001; 153(5): 917 - 932. [Abstract] [Full Text] [PDF]
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D. Waltregny, I. Leav, S. Signoretti, P. Soung, D. Lin, F. Merk, J. Y. Adams, N. Bhattacharya, N. Cirenei, and M. Loda Androgen-Driven Prostate Epithelial Cell Proliferation and Differentiation in Vivo Involve the Regulation of p27 Mol. Endocrinol., May 1, 2001; 15(5): 765 - 782. [Abstract] [Full Text]
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J. S. Richards New Signaling Pathways for Hormones and Cyclic Adenosine 3',5'-Monophosphate Action in Endocrine Cells Mol. Endocrinol., February 1, 2001; 15(2): 209 - 218. [Abstract] [Full Text]
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J. S. Richards Graafian Follicle Function and Luteinization in Nonprimates Reproductive Sciences, January 1, 2001; 8(1_suppl): S21 - S23. [Abstract] [PDF]
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S. K. Roy and L. Albee Requirement for Follicle-Stimulating Hormone Action in the Formation of Primordial Follicles during Perinatal Ovarian Development in the Hamster Endocrinology, December 1, 2000; 141(12): 4449 - 4456. [Abstract] [Full Text] [PDF]
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T. L. Beumer, H. L. Roepers-Gajadien, I. S. Gademan, H. B. Kal, and D. G. de Rooij Involvement of the D-Type Cyclins in Germ Cell Proliferation and Differentiation in the Mouse Biol Reprod, December 1, 2000; 63(6): 1893 - 1898. [Abstract] [Full Text]
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N. A. Grieshaber, S. Boitano, I. Ji, J. P. Mather, and T. H. Ji Differentiation of Granulosa Cell Line: Follicle-Stimulating Hormone Induces Formation of Lamellipodia and Filopodia via the Adenylyl Cyclase/Cyclic Adenosine Monophosphate Signal Endocrinology, September 1, 2000; 141(9): 3461 - 3470. [Abstract] [Full Text] [PDF]
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I. J. Gonzalez-Robayna, A. E. Falender, S. Ochsner, G. L. Firestone, and J. S. Richards Follicle-Stimulating Hormone (FSH) Stimulates Phosphorylation and Activation of Protein Kinase B (PKB/Akt) and Serum and Glucocorticoid-Induced Kinase (Sgk): Evidence for A Kinase-Independent Signaling by FSH in Granulosa Cells Mol. Endocrinol., August 1, 2000; 14(8): 1283 - 1300. [Abstract] [Full Text]
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L. L. Espey, T. Ujioka, D. L. Russell, M. Skelsey, B. Vladu, R. L. Robker, H. Okamura, and J. S. Richards Induction of Early Growth Response Protein-1 Gene Expression in the Rat Ovary in Response to an Ovulatory Dose of Human Chorionic Gonadotropin Endocrinology, July 1, 2000; 141(7): 2385 - 2391. [Abstract] [Full Text] [PDF]
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K. L. Britt, A. E. Drummond, V. A. Cox, M. Dyson, N. G. Wreford, M. E. E. Jones, E. R. Simpson, and J. K. Findlay An Age-Related Ovarian Phenotype in Mice with Targeted Disruption of the Cyp 19 (Aromatase) Gene Endocrinology, July 1, 2000; 141(7): 2614 - 2623. [Abstract] [Full Text] [PDF]
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B. D. Murphy Models of Luteinization Biol Reprod, July 1, 2000; 63(1): 2 - 11. [Abstract] [Full Text]
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A. Hampl, J. Pacherník, and P. Dvorák Levels and Interactions of p27, Cyclin D3, and CDK4 During the Formation and Maintenance of the Corpus Luteum in Mice Biol Reprod, May 1, 2000; 62(5): 1393 - 1401. [Abstract] [Full Text]
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S Dupont, A Krust, A Gansmuller, A Dierich, P Chambon, and M Mark Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes Development, January 10, 2000; 127(19): 4277 - 4291. [Abstract] [PDF]
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T. N. Alliston, I. J. Gonzalez-Robayna, P. Buse, G. L. Firestone, and J. S. Richards Expression and Localization of Serum/Glucocorticoid-Induced Kinase in the Rat Ovary: Relation to Follicular Growth and Differentiation Endocrinology, January 1, 2000; 141(1): 385 - 395. [Abstract] [Full Text] [PDF]
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