From the Departments of Molecular Genetics and
§ Physiology and Biophysics and ¶ Research Resources
Center, University of Illinois College of Medicine,
Chicago, Illinois 60607
Received for publication, February 4, 2003, and in revised form, February 24, 2003
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ABSTRACT |
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The timing of cellular exit from the
cell cycle during differentiation is specific for each cell type or
lineage. Granulosa cells in the ovary establish quiescence within
several hours after the ovulation-inducing luteinizing hormone surge,
whereas they undergo differentiation into corpora lutea. The expression
of Cdk inhibitors p21Cip1/Waf1 and
p27Kip1 is up-regulated during this process, suggesting
that these cell cycle inhibitors are involved in restricting
proliferative capacity of differentiating granulosa cells. Here we
demonstrate that the lack of p27Kip1 and
p21Cip1 synergistically renders granulosa cells extended an
proliferative life span. Immunohistochemical analyses demonstrated that
corpora lutea of p27Kip1, p21Cip1
double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27Kip1
single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21Cip1-deficient mice
ceased proliferation similarly to those in wild-type mice.
Interestingly, granulosa cells isolated from p27Kip1,
p21Cip1 double-null mice exhibited markedly prolonged
proliferative life span in culture, unlike cells with other genotypes.
Cultured p27Kip1, p21Cip1 double-null granulosa
cells maintained expression of steroidogenic enzymes and gonadotropin
receptors through 8-10 passages and could undergo further
differentiation in responses to cAMP accumulation. Thus, the
cooperation of p27Kip1 and p21Cip1 is critical
for withdrawal of granulosa cells from the cell cycle, in
concert with luteal differentiation and possibly culture-induced senescence.
For tissue homeostasis, differentiation is usually coordinated
with exit from the cell cycle. Proliferation stimulatory and inhibitory
signals regulate the G1 phase of the cell cycle, governing the transition between proliferation and quiescence (1).
Cyclin-dependent kinases
(Cdks)1 form the central
machinery of cell cycle progression (2-4). In G1
regulation, cyclin D- and cyclin E-dependent
kinases play interacting roles. D-type cyclins, D1, D2 and
D3, activate Cdk4 or Cdk6, whereas cyclin E activates Cdk2. Cyclin
D-Cdk phosphorylates some sites of retinoblastoma protein followed by
cyclin E-Cdk2-mediated phosphorylation of other specific sites (5). The
sequential retinoblastoma protein phosphorylation converts the E2F
transcription factor from a repressor to activator form, leading to
transactivation of a number of S-phase specific genes (6, 7). For
withdrawal from the cell cycle, proper inactivation of the
G1 Cdks is required, which largely depends on physical
association with the Cdk inhibitor proteins. The Cdk inhibitor family
consists of the Ink4-type Cdk4/Cdk6 inhibitors such as
p16Ink4a, p15Ink4b, p18Ink4c, and
p19Ink4d and the Kip/Cip-type Cdk2 inhibitors such as
p21Cip1/Waf1/Sdi1/Cap20, p27Kip1, and
p57Kip2 (8, 9).
Granulosa cells in the adult ovary undergo dynamic regulation of cell
cycle progression during folliculogenesis, ovulation, and luteinization
(10-12). The importance of the cell cycle control for reproductive
function is recapitulated by the observations that deficiency for
cyclin D2, Cdk4, or p27Kip1 renders female mice infertile.
Cyclin D2(Ccnd2)-null ovaries exhibit a block of folliculogenesis at
the secondary follicle stage because of impaired proliferation of
granulosa cells (13, 14). In contrast, ovarian function of Cdk4-null
mice is intact, but maintenance of corpora lutea is perturbed by
insufficient prolactin secretion from the pituitary (15, 16).
Intriguingly, p27Kip1(Cdkn1b)-null ovaries show
hyperproliferation of granulosa cells during luteinization, which is at
least partly responsible for the sterility (17, 18). Thus, the
G1-Cdk regulatory system plays critical roles in the
homeostasis of granulosa/luteal cells. During luteal differentiation,
the expression of both p21Cip1 and p27Kip1 is
up-regulated (14, 18). Unlike p27Kip1-null mice,
p21Cip1(Cdkn1a)-null mice show normal fertility (19, 20).
To further investigate the significance of G1-Cdk
down-regulation during luteal differentiation, we generated
p21Cip1, p27Kip1 double-null mice by
cross-breeding. Here we demonstrate that differentiating granulosa
cells in p21Cip1, p27Kip1 double-null mice
undergo more prolonged proliferation relative to those in
p27Kip1-single null mice. Moreover, granulosa cells
isolated from p21Cip1, p27Kip1 double-null mice
are capable of dividing through a number of passages in culture,
exhibiting markedly prolonged proliferative life spans. These data
suggest that p21Cip1 and p27Kip1
synergistically cooperate for the exit of differentiating granulosa cells from the cell cycle.
Animals--
p27Kip1-null mice,
Cdkn1btm1Kiyo, and p21Cip1-null mice,
Cdkn1atm1Tyj, were created as described previously (18,
19). p21Cip1 Isolation and Culture of Granulosa Cells--
Mice at 25 days of
age were injected intraperitoneally with 5 international units of PMSG
at noon, and granulosa cells were collected by punctuation 40 h
later. The ovaries were then trimmed of the oviduct and fat and
incubated at 37 °C for 15 min with 6 mM EDTA in
DMEM/F-12 medium (Cambrex, Walkersville, MD). Ovaries were then
incubated with 0.5 M sucrose in DMEM/F-12 at 37 °C for 5 min. Granulosa cells were released by punctuation using a 25-gauge needle. Cells were collected and washed twice in DMEM/F-12 without serum. Cells were counted, plated in serum-coated culture dishes, and
maintained in DMEM/F-12 supplemented with 2% fetal bovine serum,
antibiotics, and antimycotics at 37 °C, 5% CO2. To
determine the growth curve of granulosa cells, 200,000 freshly isolated granulosa cells were plated in a 12-well plate (3.8 cm2/well). The numbers of cells were counted every day for
10 days. To determine the cumulative growth curves, 100,000 granulosa
cells were plated in a 35-mm dish (8.2 cm2), and passage
was done every 5-6 days. For the immunofluorescent staining of
P450scc, 40,000 granulosa cells were plated into a chamber of 4 chamber
slides and incubated at 37 °C, 5% CO2 until reaching
80% confluency before the start of cAMP treatment procedure. Cells
were further incubated in the medium without serum for 24 h and
then treated with 8-bromo-cAMP at the final concentration of 0.5 mM for 48 h.
RT-PCR--
Total RNA from 8-bromo-cAMP (0.5 mM)
treated and untreated granulosa cells was extracted using TRIzol
Reagent (Invitrogen). Contaminated DNA was eliminated by incubation
total RNA with RQ DNase (Promega, Madison, WI). Reverse transcriptions
were done using Thermoscript RT-PCR system (Invitrogen). PCR reactions
were performed with 0.75 units of platinum Taq polymerase
(Invitrogen), 20 pmol of each primer, 0.25 mM dNTPs, 1.5 mM MgCl2 in a 12.5-µl reaction at 94 °C
for 1 min, 62 °C for 30 s, 72 °C for 45 s. To determine
linear ranges of amplification of target cDNA, various amounts of
total RNA (0, 125, 250, 500, 1000 ng) were reverse-transcribed and
amplified by PCR in a series of cycle numbers. The optimized cycle
numbers using 250 ng of total RNA were 23 cycles for 3 Immunohistochemistry and Double-immunofluorescent
Staining--
Tissue samples were fixed at 4 °C overnight in 10%
buffered-formalin (Sigma) Fixed samples were then dehydrated,
paraffin-embedded, and sectioned in 5-µm thickness by microtome using
standard procedures. Immunohistochemistry for BrdUrd was previously
described (18). Double immunofluorescent staining of PCNA and P450scc
was done using anti-PCNA (PC10) mouse antibody (Neomarkers, Fremont,
CA) and a polyclonal rabbit antiserum to rat P450 side-chain cleavage cytochrome (P450scc) (21), respectively. The signals were visualized by
fluorescein-conjugated anti-mouse IgG for PCNA and Texas red-conjugated anti-rabbit IgG for P450scc (Vector Laboratories, Inc., Burlingame, CA). Double immunofluorescent staining of BrdUrd and P450scc was performed using anti-BrdUrd mouse antibody (Roche Molecular
Biochemicals) and the polyclonal rabbit antiserum to rat P450scc. The
stained slides were examined under a fluorescent microscope (Zeiss
microimaging, Thornwood, NY). The number of PCNA- and BrdUrd-positive
luteal cells was determined by counting cells that were stained
positive for both P450scc + PCNA and P450scc + BrdUrd, respectively,
from 40× merge pictures. Three counting fields within corpora lutea were chosen randomly from one ovary, and three individual mice were
examined. To examine the expression of P450scc and the morphology of
granulosa cells in culture, cells inoculated in chamber slides were
washed by phosphate-buffered saline and fixed in 10% buffered-formalin (Sigma) for 20 min at room temperature. The slides were then washed for
5 min, 3 times in phosphate-buffered saline and permeabilized with 1%
bovine serum albumin (Sigma) + 0.2% Triton X-100 (Fisher) in
phosphate-buffered saline for 5 min on ice. They were then washed again
with phosphate-buffered saline plus 1% bovine serum albumin 3 times, 5 min each at room temperature. Cells were stained using the
polyclonal anti-P450scc and Texas Red-conjugated anti-rabbit IgG.
Analysis of Apoptosis--
Apoptotic cells were detected in
paraffin-embedded tissue sections by the TdT-mediated dUTP nick-end
labeling assay (TUNEL assay) using Apoptosis Detection System,
Fluorescein (Promega), according to the manufacturer's protocol.
Statistics--
Data are expressed as means ± S.E. The
differences between groups were evaluated with Student's t
test and analysis of variance, with a significance level of
p < 0.05.
Hyperplastic Ovaries of p21Cip1, p27Kip1
Double-null Mice--
To determine whether p21Cip1 and
p27Kip1 cooperate for the cell cycle control in granulosa
cells, we generated p21Cip1, p27Kip1
double-null (p21Cip1 Hyperproliferation of Luteinizing Granulosa Cells in
p21Cip1, p27Kip1 Double-null
Mice--
Ovulation and fertilization occur normally in
p27Kip1 single-null females. However, implantation is
perturbed with sub-optimal steroid regulation (17). Examinations of
oviducts after superovulation demonstrated that ovulation occurred in
the p21Cip1
Within 4-6 h post-LH surge, luteinizing granulosa cells in wild-type
mice ceased DNA replication, whereas p21Cip1 expression was
up-regulated, and cyclin D2 expression was suppressed (14). Ovulation
occurs normally around 12 h post-LH, and the expression of
p27Kip1 accumulates between 12 and 24 h. We previously
reported that luteinizing granulosa cells in ovaries of
p27Kip1-null mice remain proliferative even at 48 h
after hCG treatment (17), suggesting p27Kip1 plays a
rate-limiting role in cessation of proliferation. To further
investigate the possible cooperation of p21Cip1 and
p27Kip1, we examined proliferation and differentiation of
granulosa cells in p21Cip1, p27Kip1 double-null
mice in comparison with those in p21Cip1 or
p27Kip1 single-null mice. Mice at 25 days of age were
superovulated by PMSG and hCG. To visualize cells undergoing DNA
replication, mice were injected with BrdUrd 2 h before sampling
ovaries. Proliferation and differentiation of ovarian follicles were
analyzed by immunohistochemistry for incorporated BrdUrd, PCNA, and
P450scc (Fig. 2). At 48 h post-hCG, many cells in corpora lutea of p27Kip1 single-null mice
showed BrdUrd incorporation (Fig. 2C), as demonstrated previously (17). A majority of p27Kip1-null luteal cells
expressed high levels of PCNA expression (Fig. 2B), which is
consistent with the proliferative capacity of the cells. Ovaries of
p21Cip1 Increased Apoptosis in Corpora Lutea of p21Cip1,
p27Kip1 Double-null Mice--
Continued proliferation of
p21Cip1, p27Kip1 double-null luteal cells
beyond 96 h post-hCG suggested that the deficiency of the two Cdk
inhibitors might immortalize or transform granulosa/luteal cells.
However, we observed no spontaneous ovarian tumors in
p21Cip1 Prolonged Proliferative Life Span of Cultured p21Cip1,
p27Kip1 Double-null Granulosa Cells--
The continuous
proliferation of luteal cells in superovulated p21Cip1 Differentiation of Cultured p21Cip1,
p27Kip1 Double-null Granulosa Cells--
To confirm that
proliferating cells in p21Cip1
To determine the differentiation status of proliferating
p21Cip1 Cessation of proliferation is normally coordinated with
progression of differentiation essentially in all cell types. Multiple Cdk inhibitors are involved in the down-regulation of Cdk activities during differentiation (8, 27). Here we show that p27Kip1
and p21Cip1 play cooperative roles in quiescence of
differentiating granulosa/luteal cells. p27Kip1 single
deficiency prolongs proliferation during luteal differentiation, and
p27Kip1, p21Cip1 double deficiency further
enhances the proliferative capacity. In contrast, loss of
p21Cip1 alone shows little effects on proliferation of
luteinizing cells. These observations suggest that p27Kip1
is a major rate-limiting factor for the exit of differentiating granulosa cells from the cell cycle. In contrast, the cooperative role
for p21Cip1 in the timely establishment of quiescence is
dispensable by itself. In normal rodent ovaries, an increase in
p21Cip1 expression is observed within 6 h after
luteinization-inducing LH surge, preceding a marked increase in
p27Kip1 expression after 12 h (14). The up-regulation
in P450scc expression, an indicator of differentiation, occurs within
4-6 h after the LH receptor signal, regardless of the status of the
p27Kip1 and p21Cip1 genes (data not shown).
Although previous studies suggest a reciprocal correlation between
differentiation and proliferation in various cell types, including
granulosa cells (24), our studies indicate that luteal differentiation
and cell cycle exit are regulated by distinct mechanisms and can be
uncoupled. As another example of such uncoupling between cell cycle
progression and differentiation, cyclin D2-null mice exhibit defective
proliferation with intact luteinization (13, 14).
Both p27Kip1 and p21Cip1 bind to cyclin D-Cdk4
(or Cdk6) and cyclin E (or A)-Cdk2 complexes (9). In
proliferating cells, the association of the Kip/Cip proteins with
cyclin D-Cdk is not inhibitory to the kinase activity, whereas their
association with cyclin E (or A)-Cdk2 always inhibits the catalytic
subunit. Indeed, p27Kip1 and p21Cip1 promote
the assembly of D-type cyclins and Cdk4 (28). Consistently, MEFs lacking p27Kip1 and p21Cip1 exhibit
markedly decreased levels of cyclin D-Cdk complexes and aberrantly high
activity of cyclin E-Cdk2 (29). In MEFs lacking Cdk4, the ternary
p27Kip1-cyclin E-Cdk2 complexes are increased, leading to a
higher inhibitory threshold to the Cdk2 activation and delayed
G0-S transition (30). Thus, cyclin D-Cdk complexes titrate
p27Kip1 and p21Cip1, regulating the
availability of the inhibitors to inactivate Cdk2. We observed that the
ovaries of superovulated p27Kip1, p21Cip1
double-null mice have aberrantly high Cdk2
activity,2 suggesting that
continuous proliferation of luteinized granulosa cells depends on
elevated Cdk2 activity. In addition, cyclin D2-dependent kinase, which is required for proliferation of undifferentiated granulosa cells (13, 14), could also play a role in the prolonged proliferative life span. The large dose of 8-bromo-cAMP, which promotes
further differentiation of cultured p27Kip1,
p21Cip1 double-null granulosa cells, also inhibits cell
cycle progression (data not shown), with concomitant suppression of
cyclin D2 expression (Fig. 6B). Although the assembly of
cyclin D and Cdk4 is diminished in MEFs lacking p27Kip1 and
p21Cip1, the activities of cyclin
D-dependent kinases are detectable, and the
cells are sensitive to p16INK4a-induced G1
arrest (31, 32). These observations imply that in the absence of
p27Kip1 and p21Cip1, cyclin D-Cdk complexes are
still required for proliferation.
Most Cdk inhibitors are ubiquitously expressed, and cooperation between
multiple Cdk inhibitors seems to be critical for development and
function of various tissues. We have recently demonstrated that
increased p21Cip1 expression plays a role in maintaining
quiescence of hepatocytes in adult p27Kip1-deficient mice
(33). Cooperation between p27Kip1 and p57Kip2
in development of the lens and placenta has been demonstrated in mice
deficient for these two Cdk inhibitors (34). p21Cip1 and
p57Kip2 cooperate in differentiation of skeletal muscle and
alveoli in the lung (35). The two Ink4-type inhibitors,
p18Ink4c and p19Ink4d, cooperate for
spermatogenesis (36). Thus, the negative regulation of cell cycle
progression, particularly during development, could depend on various
combinations of Cdk inhibitors in cell type-specific manners.
The present study is unique in demonstrating that the absence of
multiple Cdk inhibitors prolongs the proliferative life span of primary
rodent cells ex vivo. p27Kip1,
p21Cip1 double-null granulosa cells could continue
proliferation up to 10 passages, whereas wild-type and
p21Cip1 or p27Kip1 single-null granulosa cells
cease proliferation almost immediately when placed in culture.
Consistently, a previous study showed that primary rat granulosa cells
can divide no more than once in culture (24). It has been thought that
the culture environment rapidly leads primary granulosa cells to
spontaneous differentiation. Deficiency for p27Kip1 and
p21Cip1 overcomes the cell cycle inhibitory effect of
culture, but these cells are neither immortal nor transformed.
p27Kip1, p21Cip1 double-null granulosa cells
continue to express granulosa cell-specific genes such as P450scc,
3 Continuous passages of primary fibroblasts or epithelial cells in
culture induce replicative senescence with the characteristic flat
enlarged morphology (37). Senescence involves the
Arf-p53-p21Cip1 and p16Ink4-retinoblastoma
protein tumor-suppressive pathways (26, 38, 39). MEFs lacking p53 or
Arf display an immortal phenotype ex vivo devoid of
senescence (26, 40). In contrast, MEFs deficient for
p16Ink4a or p21Cip1 show normal senescence in
culture (41-43). MEFs lacking p27Kip1 also exhibit a
normal senescence response.3
Wild-type granulosa cells develop a senescence phenotype within several
days in culture, sometimes displaying a bi-nucleated morphology similar
to senescent fibroblasts (Fig. 5C) (24). The markedly prolonged proliferative life span of p27Kip1,
p21Cip1 double-null granulosa cells implies that these two
inhibitors may cooperate not only in differentiation-associated
withdrawal from the cell cycle but also in inducing senescence to
maintain irreversible quiescence. However, it should be noted that
p27Kip1, p21Cip1-null granulosa cells are not
completely devoid of senescence. Continuous culture beyond
passage 10 results in dominance of the senescent morphology, and most
cells undergo apoptosis thereafter. This crisis-like cell death may be
associated with enhanced apoptosis of corpora lutea in
p27Kip1, p21Cip1 double-null mice (Fig. 4),
although the mechanism of apoptosis is unclear.
In summary, the absence of the two Cdk inhibitors p27Kip1
and p21Cip1 creates a unique cellular condition that
extends the proliferative life span of granulosa cells without losing
the potential of differentiation. This experimental model provides a
useful tool for the study of cell cycle control during differentiation
and senescence.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
p27Kip1
/
mice were
produced by crossing p21Cip1
/
, p27Kip1+/
males and females. p21Cip1
/
, p27Kip1
/
and p21Cip1
/
p27Kip1
/
female mice and
wild-type female littermates were genotyped by PCR using tail DNA. Mice
were kept at 25 ° C with a 13-h light, 11-h dark cycle; they were
fed a pelleted diet ad libitum. To stimulate follicle growth
and ovulation, 25-day-old females were treated with the standard
superovulation scheme, injected with 5 international units of pregnant
mare serum gonadotropin (PMSG) (Calbiochem) at noon and 46 h later
with 5 international units of human CG (hCG) (Calbiochem). To evaluate
ovulation and mating, injected mice were then moved to a cage housing a
wild-type male. Vaginal plugs were detected in the morning of the next
day indicating mating. Oviducts were then dissected to examine ova
under a dissection microscope. To label the cells in S phase, mice were
injected by intraperitoneally with 50 µg of bromodeoxyuridine
(BrdUrd)/gram of body weight at 2 h before sacrificing. Animals
were used in compliance with the National Institutes of
Health/Assessment and Accreditation of Laboratory Animal Care
guidelines and with the approval of the animal care committee of the
University of Illinois.
-HSD, 25 cycles for P450scc and L19, 28 cycles for luteinizing hormone receptor
and prolactin receptor (long form), 38 cycles for cyclin D2, 40 cycles
for aromatase, and 42 cycles for prolactin receptor short form. The
primers used are as follows: 3
-HSD, 5'-ACT GGC AAA TTC TCC ATA GCC-3
and 5'-TTC CTC CCA GCT GAC AAG TGG-3', product size 330 bp; P450scc,
5'-CAA CCT TTC CTG AGC CCT ACG-3' and 5'-TCT TGG TTT AGG ACG ATT CGG
TC-3', product size 415 bp; L19, 5'-CTG AAG GTC AAA GGG AAT GTG-3' and
5'-GGA CAG AGT CTT GAT GAT CTC-3', product size 195 bp; cyclin D2,
5'-GGA AGC ACT CCC CGA CTC TC-3' and 5'-TCC TTC TGC ACG CAC TTG AA-3',
product size 248 bp; luteinizing hormone receptor, 5'-CGC TTT CCA AGG
GAT GAA TA-3' and 5'-CTG GAG GGC AGA GTT TTC AG-3', product size 285 bp; prolactin receptor long form, 5'-ATA CTG GAG TAG ATG GAG CCA GGA
GAG TTC-3' and 5'-CTT CCG TGA CCA GAG TCA CTA CTG TCG GGA TCT-3',
product size 422 bp; PRLP short form, 5'-ATA CTG GAG TAG ATG GAG CCA
GGA GAG TTC-3' and 5'-CTA TTT GAG TCT GCA GCT TCA GTA GTC A-3', product
size 332 bp; P450arom, 5'-TGC ACA GGC TCG AGT ACT TTC-3' and 5'-ATT TCC
CAC AGG TGC CTG TCC-3', product size 268 bp. Product were then run on a
2% agarose gel and analyzed by Gel Doc image analysis system
(Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
p27Kip1
/
) mice.
Mating of double-null females with wild-type males indicated that
p21Cip1
/
p27Kip1
/
females are sterile
with no exception, although mating behaviors and vaginal plugs were
observed. This phenotype is analogous to that of p27Kip1
single-null female mice (17, 18). p21Cip1 single-null mice
exhibit normal fertility (19, 20), but their ovarian function has not
been characterized previously. Examinations of prepubertal mice at 25 days of age showed that p27Kip1 single-null mice displayed
gigantism (Fig. 1A), as
described previously (18, 22, 23). Their body weights were 44% higher than those of wild-type mice. No further increase in body weights was
observed p21Cip1
/
p27Kip1
/
mice,
suggesting no cooperation of p21Cip1 and
p27Kip1 in body growth. Ovaries of p27Kip1
single-null mice were enlarged, weighing 2.5-fold more than wild-type ovaries (Fig. 1, B and C). Ovaries of
p21Cip1
/
p27Kip1
/
mice were even
heavier, showing a 3.3-fold increase over wild-type weights. In
contrast, p21Cip1 single-null mice did not show significant
enlargement of bodies or ovaries. These observations indicate that
double deficiency for the two Cdk inhibitors results in more remarkable
effects on ovary tissue growth than p27Kip1-single
deficiency.
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Fig. 1.
Deficiency for p21Cip1 and
p27Kip1 in mice synergistically enlarges the ovary.
A, the body weights of 25-day-old wild-type (WT),
p21Cip1 /
, p27Kip1
/
, and
p21Cip1
/
p27Kip1
/
(double-null) mice.
B, the wet weights of ovaries. C, the ovary
weights normalized as ratios to the body weights. D, ovaries
from 25-day-old WT and p21Cip1
/
p27Kip1
/
mice at 18 h after superovulation (see
"Experimental Procedures"). The quantified data are expressed as
the mean ± S.E. (n = 3).
/
p27Kip1
/
mice with
slightly reduced numbers of ova compared with those in wild-type mice
(30.0 ± 6.0 versus 38.8 ± 1.3, mean ± S.E., n = 4). After mating with normal fertile males,
embryos in oviducts appeared intact with proper fertilization.
/
p27Kip1
/
mice displayed even
higher numbers of BrdUrd+ or PCNA+ cells in
corpora lutea compared with p27Kip1
/
ovaries. The
proliferative characteristics were observed in p21Cip1
/
p27Kip1
/
ovaries beyond 96 h post-hCG. It should
be noted that replicating DNA was pulse-labeled with BrdUrd for 2 h, so only a fraction of proliferating cells was detected by BrdUrd
immunoreactivity. Double immunofluorescent staining showed that high
levels of PCNA expression and BrdUrd incorporation were observed in
p21Cip1, p27Kip1-null luteinized cells that
express abundant P450scc (Fig. 3), confirming that the proliferating cells detected here were luteal cells. In contrast, p21Cip1-null ovaries showed minimum
proliferation of luteal cells at 48 h post-hCG (Fig. 2,
B and C). These data suggest that
p27Kip1 plays a limiting role in terminal divisions of
luteinizing granulosa cells. p21Cip1 cooperates in this
regulation, which becomes obvious especially in the absence of
p27Kip1.
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Fig. 2.
Hyperproliferation in corpora lutea of mice
deficient for p21Cip1 and p27Kip1.
A, immunohistochemistry for cholesterol side chain
cleavage (P450scc), PCNA, and BrdUrd (BrdU). Mice at
25 days of age were superovulated, and ovaries were harvested 96 h
post-hCG administration. BrdUrd was injected 2 h before sampling.
B, quantification of PCNA+ cells. C,
quantification of BrdUrd+ cells in wild-type
(WT, open columns), p21Cip1 /
(dotted columns), p27Kip1
/
(hatched
columns), and p21Cip1
/
p27Kip1
/
(closed columns) mice. Ovaries were sampled at the indicated
times after hCG administration. Data from three independent animals are
expressed as mean ± S.E. *, p < 0.05.
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Fig. 3.
Cells proliferating in corpora lutea of
p21Cip1 /
p27Kip1
/
mice are luteinizing. Mice at 25 days of age were superovulated,
and ovaries were harvested 96 h post-hCG administration. BrdUrd
was injected 2 h before sampling. BrdUrd (BrdU) or PCNA
(green) and P450scc (red) were visualized by
double immunofluorescent staining.
/
p27Kip1
/
mice during
15-month-long observations. To investigate the fate of proliferating
luteal cells, we examined apoptosis in ovaries after superovulation
using the TUNEL assay (Fig. 4). Apoptosis was readily detectable in corpora lutea of p21Cip1
/
p27Kip1
/
ovaries at 48 h post-hCG and appeared
more remarkable at 96 h. In contrast, wild-type ovaries did not
display appreciable apoptosis in corpora lutea until 72 h. At
96 h, apoptosis was detected in wild-type corpora lutea,
presumably because of the lack of luteotropins without mating.
Therefore, highly proliferative luteinized granulosa cells in
p21Cip1
/
p27Kip1
/
mice undergo robust
apoptosis, possibly as a secondary response to the perturbed cell cycle
control.
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Fig. 4.
Enhanced apoptosis in ovaries lacking
p21Cip1 and p27Kip1. A,
Wild-type (WT) and p21Cip1 /
p27Kip1
/
mice at 25 days of age were superovulated, and
ovaries were harvested at the indicated times after hCG administration.
Cells undergoing apoptosis were visualized by the fluorescein-based
TUNEL assay. B, quantification of TUNEL-positive cells in
corpora lutea at 48 and 96 h after hCG administration from
wild-type (open columns) and p21Cip1
/
p27Kip1
/
(closed columns) mice. Numbers of
TUNEL-positive cells per field (40× magnification) were counted.
Fields were randomly chosen within each corpus luteum, and three
corpora lutea per mouse were analyzed. Data are expressed as mean ± S.E. from three individual mice. *, p < 0.05.
/
p27Kip1
/
ovaries prompted us to examine whether this
phenotype is because of a change intrinsic to the granulosa/luteal cell
or a change in the extracellular environment. We isolated granulosa
cells from ovarian follicles of p21Cip1,
p27Kip1-null mice at 40 h after PMSG administration.
Isolated cells were cultured under standard conditions, and
proliferation was quantified by cell counts. Wild-type granulosa cells
ceased proliferation after one passage (Fig.
5, A and B) and
exhibited a flat enlarged phenotype after the first passage (Fig.
5C). The volume of each postmitotic cell was gradually
increased through 8-10 days of continuous culture, and some cells
became bi-nucleated, suggesting that these cells undergo senescence, as
described previously (24). This is consistent the view that primary
mouse cells are quite sensitive to culture stress-induced senescence
(25, 26). p21Cip1 single-null granulosa cells displayed a
proliferation kinetics similar to wild-type cells (Fig. 5, A
and B) and underwent senescence (not shown).
p27Kip1 single-null cells showed slow
proliferation up to the second passage and then stopped dividing (Fig.
5B). In contrast, p21Cip1
/
p27Kip1
/
cells continued to proliferate beyond passage
7. However, p21Cip1
/
p27Kip1
/
granulosa
cells were not completely immortal in vitro, as they slowed
down and underwent senescence and/or cell death around passage 10 (data
not shown).
View larger version (38K):
[in a new window]
Fig. 5.
Markedly prolonged proliferative life span of
p21Cip1 /
p27Kip1
/
granulosa cells in culture. A, growth curves of
granulosa cells at the first passage. Cells were isolated from
preovulatory follicles in PMSG-treated mice with the indicated
genotypes. Culture was initiated at the density of 2 × 105 cells/2.2-cm well (3.8 cm2) as described
under "Experimental Procedures." B, cells were passed
every 5 days, and cumulative cell numbers are shown. Data are shown as
the averages of three independent preparations. C, the
morphology of cultured granulosa cells after one passage. The
asterisks indicate a senescence-like morphology, and the
arrows show luteinized cells. WT, wild
type.
/
p27Kip1
/
cultures are indeed granulosa/luteal cells,
the expression of P450scc was examined by immunostaining (Fig.
6A). Essentially all cells in p21Cip1
/
p27Kip1
/
cultures examined at passage 5 expressed
readily detectable levels of P450scc, indicating their steroidogenic
activity. In contrast, human osteosarcoma U2OS cells, examined as a
control, exhibited no significant staining for P450scc. Moreover,
normal mouse embryonic fibroblasts (MEFs) displayed neither P450scc
immunoreactivity (data not shown) nor P450scc mRNA in RT-PCR assays
(Fig. 6B). These data indicate
that p21Cip1
/
p27Kip1
/
cells
continuously proliferating in culture are steroidogenic granulosa/luteal cells.
View larger version (27K):
[in a new window]
Fig. 6.
Granulosa cells lacking p21Cip1
and p27Kip1 retain differentiation responses to cAMP after
prolonged culture. A, P450scc immunostaining and
4,6-diamidino-2-phenylindole (DAPI) nuclear staining with
cultured p21Cip1 /
p27Kip1
/
granulosa
cells (GC) at passage 5 and human osteocarcinoma U2OS cells
(U2OS). B, the expression of granulosa/luteal cell-specific
genes in p21Cip1
/
p27Kip1
/
cells at
passage 4 was examined by RT-PCR after 48 h of incubation with 0.5 mM 8-bromo-cAMP. Data from two different cell preparations
are shown as duplicates. MEFs and whole ovaries from 6-week-old
wild-type mice were used as negative and positive controls.
LHR, luteinizing hormone receptor; PRLR,
prolactin receptor; P450arom, aromatase; L19,
ribosomal RNA L19. C, the morphology and the expression of
P450scc in p21Cip1
/
p27Kip1
/
cells at
passage 4 treated with or without 0.5 mM 8-bromo-cAMP for
48 h.
/
p27Kip1
/
granulosa cells, we
examined the expression of genes characteristic to the granulosa/luteal
cell type by semi-quantitative RT-PCR (Fig. 6B). The
expression of cyclin D2 detected in cells at passage 4 was 40% reduced
by treatment of cells with 0.5 mM 8-bromo-cAMP. The
expression of LH receptor, which was relatively low in untreated cells,
was up-regulated by 8-bromo-cAMP. Low levels of follicle-stimulating hormone receptor were also detected, which did not significantly respond to cAMP (data not shown). Prolactin receptor expression was
undetectable in untreated proliferating cells, but both long and short
forms of the transcripts were induced by 8-bromo-cAMP. P450scc
expression was detectable in untreated cells and, remarkably, up-regulated by cAMP. In contrast, 3
-HSD expression was not
significantly affected by 8-bromo-cAMP. Immunocytochemistry for P450scc
confirmed that treatment with 8-bromo-cAMP increased expression of
P450scc (Fig 6C). Cells became more round in morphology upon
the cAMP treatment, which is similar to the differentiation response in rat primary granulosa cells (24). Taken together, relatively high
levels of P450scc and 3
-HSD suggest that p21Cip1,
p27Kip1 double-null granulosa cells, which exhibit markedly
prolonged proliferative life span in vitro, may be partially
differentiated. The cells are still capable of undergoing further
differentiation in response to cAMP accumulation, showing induction of
prolactin receptor, accumulation of P450scc and P450arom, and
down-regulation of cyclin D2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD, follicle-stimulating hormone receptor, and LH receptor,
through 8-10 passages. Relatively high expression of P450scc and low
expression of follicle-stimulating hormone receptor suggest that these
proliferating cells are differentiated (luteinized) to some extent,
presumably in response to the culture environment. However, treatment
with 8-bromo-cAMP induces prolactin receptors and P450arom and further
increases P450scc and LH receptors. Progesterone secretion into the
culture medium is also increased by cAMP treatment (data not shown).
These data indicate that p27Kip1,
p21Cip1-deficient granulosa cells are capable of further
differentiation even after several passages in culture. The large
dose of 8-bromo-cAMP also inhibits proliferation of
p27Kip1, p21Cip1-deficient granulosa cells with
a reduction in cyclin D2 expression. Thus, the absence of the two Cdk
inhibitors does not create complete resistance to
differentiation-associated cell cycle arrest, despite its
proliferation-enhancing effect.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Tyler Jacks for providing p21Cip1-null mice, Asgi Fazleabas, Joseph Orly, JoAnne Richards, and members of the Kiyokawa laboratory and the Reproductive Endocrinology Research Group at the University of Illinois for helpful discussions and suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the National Institute of Health Grant HD38085 (to H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Molecular Genetics, University of Illinois College of Medicine, 900 S. Ashland Ave., M/C 669, Chicago, IL 60607-7170. Tel.: 312-355-1601; Fax:
312-413-2028 or 413-0353; E-mail: kiyokawa@uic.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M301206200
2 D. Moons and H. Kiyokawa, unpublished observations.
3 H. Kiyokawa, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Cdk, cyclin-dependent kinase;
PMSG, pregnant mare serum
gonadotropin;
hCG, human chorionic gonadotropin;
LH, luteinizing
hormone;
P450scc, cholesterol side chain cleavage cytochrome P450;
P450arom, aromatase cytochrome P450;
BrdUrd, bromodeoxyuridine;
PCNA, proliferating cell nuclear antigen;
MEF, mouse embryonic fibroblast;
3-HSD, 3
hydroxysteroid dehydrogenase;
DMEM, Dulbecco's modified
Eagle's medium;
RT, reverse transcription;
TUNEL, TdT-mediated dUTP
nick-end labeling.
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