BIOLOGY OF REPRODUCTION 59, 444–450 (1998)
Progesterone and Estrogen Regulation of Rat Decidual Cell Expression of
Proliferating Cell Nuclear Antigen1
Thomas F. Ogle,2 Philip George, and Donghai Dai
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912
ABSTRACT
stromal cell cycle of the DB during pregnancy and to examine hormone actions that initially favor proliferative processes in early pregnancy (Days 8–12) yet fail to promote
stromal cell proliferation after midpregnancy (Day 14 and
beyond). We used morphometric analysis and in situ expression of proliferating cell nuclear antigen (PCNA) to
evaluate changes in mesometrial stromal cell proliferation
during normal pregnancy. The effects of P4 and estradiol17b (E2) as well as of antiprogestin (RU-486) on stromal
cell proliferation were also examined.
PCNA, a 36-kDa protein, is an essential replication factor that binds DNA polymerase-d and D-cyclins to initiate
cell cycle progression [13–15]. PCNA is synthesized beginning in G1 and has a long half-life, accumulating in the
nucleus until mitosis [16]. Thus, PCNA nuclear immunoreactivity is a useful marker of cell proliferation and correlates with other parameters of proliferation [17, 18].
This study was an examination of the role of progesterone
(P4) and estradiol-17b (E2) as stromal cell mitogens in the decidua basalis (DB) of the rat during pregnancy. Pregnant rats
were ovariectomized (Ovx) on Days 8 and 12 of pregnancy,
treated with P4, E2, or both, and killed on Days 10 and 14, which
correspond to times of stromal cell proliferation and regression,
respectively. In some experiments, rats received pellets of the
anti-progestin RU-486 on Day 9 and were killed 6, 12, and 24
h later. The mitotic index (MI) and in situ image analysis of expression of proliferating cell nuclear antigen (PCNA) were used
to assess cell cycle progression. Highest expression of PCNA occurred on Days 8–12 of pregnancy, and MI was maximum; MI
became zero and PCNA expression decreased dramatically
thereafter (i.e., Days 14, 17, 21). Percentage of cells expressing
intense PCNA on Day 10 (40%) declined to 5% after Ovx and
Ovx 1 E2 (p , 0.05), whereas Ovx 1 P4 maintained PCNA. By
Day 14, only 1% of stromal cells expressed intense PCNA,
which was not significantly altered by Ovx, Ovx 1 E2, or Ovx
1 P4 but increased after Ovx 1 P4 and E2 (p , 0.05). By 6 h of
RU-486, MI declined 3-fold, and intense PCNA expression was
essentially lost. These changes preceded loss of histological integrity of the DB. Cells with undetectable PCNA steadily increased from 8% at 6 h to 28% by 24 h (p , 0.05). Thus RU486 appeared to block cell cycle progression and enhanced
PCNA turnover. P4 was essential for stromal cell proliferation
during early pregnancy (Days 8–10), but this action was lost by
Day 14.
MATERIALS AND METHODS
Animal Model
Adult female Holtzman rats weighing 225–250 g were
obtained from Harlan Sprague Dawley, Inc. (Indianapolis,
IN) and bred in the laboratory animal care facility of our
institution. All animal care and use were in accordance with
NIH Guidelines for the Care and Use of Laboratory Animals and a protocol approved by the Medical College of
Georgia Committee on Animal Use in Research and Education. Pregnancy (Day 1) was identified by presence of
vaginal sperm after overnight exposure to a fertile male.
Rats were killed at 0800–0900 h on Day 8 through Day 21
of pregnancy (term is on Day 22). Animals were also killed
at other times as indicated for specific experiments.
INTRODUCTION
Progesterone (P4)-induced differentiation of the endometrium is a prerequisite for implantation of a fertilized
ovum and the establishment of a successful pregnancy. In
the rat this involves a decidual reaction in which small fibroblast-like stromal cells undergo differentiation of function and acquire the decidualized phenotype [1–4]. Proliferation of endometrial stromal cells plays an essential part
in the decidual reaction and depends on the action of P4
[5, 6]. The mitogenic action of P4 in certain P4-responsive
tissues such as mammary gland and P4-dependent breast
cancer cells is cell cycle phase-specific through enhancement of G1 progression [7–9]. However, little is known
about the mitogenic action of P4 in mesometrial stromal
cells (i.e., decidua basalis, DB) during pregnancy. The role
of P4 in the progression of the stromal cell cycle appears
complex and is made more difficult to understand because
the DB regresses in midpregnancy in the absence of corresponding changes in progesterone receptor (PR) and PR
mRNA and in circulating levels of the hormone [10–12].
The aims of this study were to better understand the
Histology
The uterus was removed from the animal and transferred
to the surface of a glass plate held at 0–48C by an underlayer of ice and then trimmed of fat and mesometrium.
Razor blades were used to cut the uterus in cross section
at each implantation site (i.e., embryo and decidualized area
of uterus); these cross sections were fixed in neutral buffered 10% formaldehyde for 10–12 h and embedded in paraffin wax. Implantation sites from animals in advanced
stages of pregnancy (Days 12–21) were further dissected to
remove the amnion and embryo. All tissues were sectioned
in a midsagittal plane at 4 mm (routine histology) or 6 mm
(immunohistochemistry). Tissue sections stained with hematoxylin and eosin were used for morphometric studies.
Tissue from at least three animals from each stage of pregnancy or each treatment group was prepared by these methods and analyzed.
Accepted April 7, 1998.
Received March 4, 1998.
1
This work was supported by NIH Grant HD29843 (T.F.O.).
2
Correspondence: T.F. Ogle, 1120 15th Street, Medical College of
Georgia, Augusta, GA 30912–3000. FAX: (706) 721–7299;
e-mail: [email protected]
Mitotic Index
To determine the stromal cell mitotic index (MI), an ocular grid containing 400 squares (20 3 20) was randomly
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PCNA EXPRESSION IN DECIDUAL CELLS
moved about the tissue section at 31000 magnification, and
every stromal cell was counted. A minimum of 1000 cells
were counted in each section, and the percentage of cells
exhibiting mitotic figures was taken as the MI.
Immunohistochemistry
For immunolocalization of PCNA, paraffin-embedded
tissues were sectioned at 6 mm, mounted onto Probe-Plus
slides (Fisher Scientific, Pittsburgh, PA), and subjected to
microwave irradiation. This and subsequent procedures
were as previously described [12]. Briefly, the PCNA antibody (Novocastra Laboratories Ltd., New Castle upon
Tyne, UK) was diluted 1:300 and incubated with tissue sections for 24 h at room temperature in a humidified chamber.
The conjugated second-antibody and staining procedures
utilized Vectastain Elite ABC kits and diaminobenzidinenickel chloride as substrate for horseradish peroxidase
(Vector Laboratories, Burlingame, CA). Tissue sections
were not counterstained. Specificity of staining was
checked by incubating tissue sections with a similar concentration of preimmune mouse serum at the same concentration as antibody (1:300). Nonspecific staining was observed in some vascular spaces but never in nuclei.
In Situ Analysis of PCNA Expression
PCNA expression was determined by densitometric image analysis (Analytical Imaging Concepts, Irvine, CA) in
individual nuclei under 3400 magnification. To calibrate
and standardize the light intensity of the instrument prior
to each analytic session, a red glass filter was placed on the
stage of the microscope and the average gray value (AGV)
set to 139.5–140.5 transmittance units. The density of reaction product (0 AGV for no light transmittance and 255
AGV for complete transmittance) in each nucleus was measured for at least 1000 stromal cells per tissue section and
stored in a computer database. This procedure was repeated
until DB from 3–6 pregnant rats were analyzed for each
treatment group. AVG values ranged from about 5.0 to 210
in nuclei stained for PCNA. Nuclei of control sections incubated with preimmune serum instead of anti-PCNA had
AGV of about 131–210.
The density of reaction product was divided into four
AGV ranges: , 26 AGV, intense staining—nucleus uniformly black with no structures discernible; 26 to , 76
AGV, moderate staining—nuclear structures discernible,
e.g., nucleolus, nuclear membrane; 76 to , 131 AVG, weak
staining—light staining of nuclear matrix and nuclear membrane; and $ 131, undetectable staining—transmittance
equal to that of preimmune-treated tissue sections. The
number of stromal cells in each category for each DB was
summed, and those in similar treatment groups were averaged and values expressed as mean 6 SEM. Although all
staining categories were analyzed for changes with pregnancy and hormone treatments, only the percentage cells
expressing the intense level of PCNA (AVG , 26) and
those with undetectable PCNA (AVG $ 131) were significantly altered during pregnancy and hormone treatments.
Therefore, data from the two intermediate categories are
not presented.
E2 and P4 Treatment Regimens
Pregnant animals were ovariectomized (Ovx) on Day 8
or Day 12 of pregnancy and treated immediately with one
of the following hormonal regimens: 1) Ovx1Veh: this
TABLE 1. Mitotic index (MI, percentage stromal cells in mitosis) of decidual stromal cells during pregnancy.*
Day of pregnancy
8
10
12
14
17
21
MI
1.4 6 0.2a
2.0 6 0.4a
0.3 6 0.1
0
0
0
* Values are means 6 SEM, n 5 4–6.
a
Means differ from those for other stages of pregnancy (p , 0.05; statistical analysis by ANOVA followed by Student-Newman-Keuls multirange
test).
group received 0.2 ml corn oil (vehicle) daily, s.c.; 2)
Ovx1E: this group received 2.0 mg E2 dissolved in 0.2 ml
corn oil, s.c.; 3) Ovx1P: a 25-mg pellet of crystalline P4
was placed i.p. via the dorsal incision made for ovariectomy
to maintain pregnancy levels of serum P4 (a single injection
of P4 [0.5 mg, saline/20% ethanol, i.p.] was also given immediately after surgery to ensure continuous high levels of
P4 for the first several hours after ovariectomy; corn oil
vehicle was given daily s.c. [0.2 ml]). An Ovx1PE group
received both hormones as described above. Ovx1Veh and
Ovx1E groups killed on Day 10 were Ovx at 0800–0830
h on Day 9 (instead of on Day 8 as for the P4 and PE
treatment groups) because tissue harvest was very limited
due to extensive degeneration in absence of P4. Since vehicle and E2 treatments in Ovx animals have shown similar
effects on serum P4, P4 binding, and DB histology, we used
Ovx1E treatment groups as controls for administration of
P4 and PE [19–21]. We previously reported that the serum
levels of P4 produced by these regimens are consistent with
those in normal pregnancy; however, E2 induced serum levels about 3-fold higher than normally found on Days 10
and 14 [19, 20].
Antiprogestin (RU-486) Treatment
Pellets (25 mg) of crystalline RU-486 (Mifepristone,
11b-(4-dimethylaminophenyl)-17b-hydroxy-17a(prop-1ynyl)-estra-9,9-dien-3-one; kindly provided by Dr. R. Deraedt at Roussel-Uclaf, Romainville, France) were placed
i.p. in otherwise normal pregnant rats at 0830–0900 h on
Day 9. Rats also received 0.5 mg RU-486 in saline/40%
ethanol i.p. immediately after implantation of the pellet to
ensure rapid onset of RU-486 action. Animals were killed
at intervals of 3, 6, 12, and 24 h later. This compound also
has appreciable affinity for glucocorticoid receptors [22].
This regimen of RU-486 treatment does not alter serum P4
[19].
Expression of Data and Statistical Analysis
Values are reported as means 6 SEM, n 5 3–6. Statistical analysis was done by one-way ANOVA after testing
for homogeneity of variance. When ANOVA indicated significant treatment effects (F-ratio, p , 0.05), the StudentNewman-Keuls multirange test was employed to compare
individual treatment means.
RESULTS
Mitotic Index
Table 1 shows that the highest rate of stromal cell division occurred on Days 8 and 10 of pregnancy (1–2%)
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446
OGLE ET AL.
FIG. 1. Midsagittal sections of DB immunostained for PCNA. Representative sections of DB are shown from normal pregnancy, after ovariectomy and
hormone replacement, and after RU-486 treatment. Nuclear staining indicates immunoreactive PCNA. Tissues were not counterstained. A) Decidualized
stromal cells near the implantation site in DB on Day 10. The density of the immunoreaction obscures nuclear structures in these stromal cells, providing
good examples of the intense level of PCNA expression. B) Stromal cells in the antimesometrial decidua on Day 10. C) Uterine gland epithelial cells
from Day 9 DB are negative for PCNA. D) Day 10 stromal cells 24 h after Ovx1E. Note lack of normal histological organization and shrunken
appearance of nuclei. E) Day 10 stromal cells 48 h after Ovx1P. Photograph taken near outer edge of DB near myometrium where decidualization is
less advanced and stromal cells more fibroblast-like. Most nuclei exhibited intense PCNA staining. F) DB on Day 14 showing the border of the junctional
zone (J). Stromal cell nuclei show large variations in PCNA staining from undetectable to moderate staining, i.e., nuclear structures still visible.
Arrowhead: two nuclei of trophoblast giant cells. G) Day 14 stromal cells 48 h after Ovx1E. Notice loss of tissue organization and weak to undetectable
PCNA staining. H) Day 14 stromal cells 48 h after Ovx1P. I) Day 10 stromal cells 6 h after RU-486. Most nuclei exhibit weak or undetectable PCNA
staining. Note that the histological organization of the DB is similar to that of controls (A). J) Day 10 stromal cells 12 h after RU-486. PCNA staining
is similar to that in I but histological organization has been disrupted and resembles that in D and G in the absence of P4. K) DB control section
incubated with preimmune mouse serum instead of PCNA antibody and otherwise treated identically. Details of the hormone regimen and other
procedures are described in Materials and Methods. Photomicrographs originally 3250 (A–C, E, G–K ) or 3125 (D, F) (reproduced at 71%).
during invasion of trophoblastic cells. No stromal cells
were observed in mitosis after Day 12.
Stromal Cell PCNA Expression during Pregnancy
Between Days 8 and 10 of pregnancy, stromal cell nuclei
were densely stained throughout the DB. Decidualized stromal cells in the region of chorioallantoic invasion, as well
as peripheral fibroblasts localized near the myometrium, not
yet decidualized, expressed very high levels of PCNA. Antimesometrial decidual cells expressed similarly high levels
of PCNA whereas uterine glandular epithelial cells were
always negative (Fig. 1, A–C). On Day 12, decidualization
was complete and stromal cell PCNA expression varied
from high levels to undetectable (data not shown). The
heavily granulated natural killer cells found in DB and the
metrial gland expressed variable levels of PCNA; most had
low to undetectable levels, but a few found in the metrial
gland exhibited intense PCNA (data not shown). Most stromal cells of the metrial gland were strongly stained, but the
same variability existed as in DB. By Days 14 and 17, most
stromal cells expressed low levels of PCNA (Fig. 1F). On
the other hand, the trophoblast cells of the junctional zone
(J) and labyrinth zone expressed moderate to high levels of
PCNA; however, some populations of spongiotrophoblast
cells of the junctional zone were negative (Fig. 1F). By Day
21 (about 24 h prior to term) trophoblast cells of the junctional zone were strongly stained, but stromal cells of DB
exhibited moderate to weak staining (data not shown).
Figure 2 summarizes the changes in PCNA expression
during pregnancy. On Days 8, 10, and 12, 22–35% of stromal cells expressed intense levels of PCNA expression,
which were greater than at any other time during gestation
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PCNA EXPRESSION IN DECIDUAL CELLS
447
FIG. 2. Summary analysis of PCNA immunostaining in DB during pregnancy. Values are means 6 SEM, n 5 4–6. Intense PCNA and undetectable PCNA staining categories are shown. See Materials and Methods for
definitions and explanations. Statistical analysis by ANOVA followed by
Student-Newman-Keuls multirange test. aMeans differ from all others but
not each other, p , 0.01. bMean differs from that for Day 12, p , 0.05.
(p , 0.05). On the other hand, by Day 14 and thereafter
this proportion decreased to less than 8%, and the proportion of cells exhibiting undetectable levels of PCNA increased from less than 12% on Days 8–12 to 20–37% thereafter. There were no differences in the proportion of cells
expressing moderate or low levels of PCNA during gestation (data not shown).
Effects of P4 and E2 on PCNA Expression
We next examined the roles of E2 and P4 in regulation
of PCNA expression. The rationale for these experiments
was based on our previous findings that P4 was able to
maintain and up-regulate transcriptionally active PR (PR-A
and PR-B) and estrogen receptor (66 kDa) proteins on Day
10 but not on Day 14 [21]. Thus, we predicted that P4
would up-regulate PCNA expression on Day 10 but be less
able to do so on Day 14. This hypothesis was tested in
animals Ovx on Day 8 or 12 and given 48 h of hormone
replacement therapy before being killed on Days 10 or 14.
Figure 3A summarizes the results of the effects of hormone treatments on PCNA expression on Day 10. Treatment with E2 only (Fig. 1D) or corn oil alone (not shown)
resulted in an 88% decline in the percentage of stromal
cells expressing intense levels of PCNA compared to normal Day 10 controls (p , 0.05). The percentage cells with
undetectable PCNA increased after Ovx compared to that
in Day 10 controls (p , 0.05). PCNA expression was maintained at Day 10 control levels by P4 (Fig. 1E). Figure 3B
shows the influence of hormone administration after Ovx
on Day 12. In the Day 14 intact control group, only about
1% of stromal cells expressed intense levels of PCNA, and
this was not substantially altered by Ovx1Veh or estrogen
treatment (Fig. 1G); P4 increased the value to 8% and to
FIG. 3. Summary analysis of PCNA immunostaining in DB after Ovx.
Immediately after surgery, animals were given daily injections of 2 mg E2
in corn oil, s.c. (E) or were implanted with a 25-mg P4 pellet and given
0.5 mg P4 in saline, i.p. (P), or both (P1E). Values are means 6 SEM, n
5 3–4. Intense PCNA and undetectable PCNA staining categories are
shown. A) Pregnant rats were Ovx on Day 8 (Day 9 for the Ovx1E group).
All animals were killed on Day 10. Statistical analysis as in Figure 2.
a
Mean differs from all others, p , 0.05. bMean differs from that for
Ovx1E, p , 0.05. B) Pregnant rats were Ovx on Day 12. All animals
were killed on Day 14. aMean differs from those for control and Ovx1E
groups, p , 0.05. bMean differs from that for Ovx1PE group, p , 0.05.
The control groups in A and B represent untreated rats killed on Days 10
and 14, respectively. See Materials and Methods for detailed definitions
and explanations.
12% in the presence of E2 (PE, p , 0.05). The proportion
of cells expressing undetectable PCNA was greatest in the
Ovx1E treatment group (44%, p , 0.05) but was not influenced by the P4 treatments in comparison to Day 14
intact controls (Fig. 3B and compare Fig. 1, F–H). In normal pregnancy a 94% decline in the intense PCNA staining
cell population was noted at Day 14 as compared to Day
10 and was associated with a corresponding 76% increase
in the cell population exhibiting no detectable PCNA (compare Fig. 3, A and B; Fig. 1, A and F). This Day 14 pattern
could not be altered by P4 (Fig. 3B and Fig. 1H).
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OGLE ET AL.
Effects of Antiprogestin (RU-486) on MI and PCNA
Expression
The antiprogestin RU-486 was used to ascertain more
directly the role of P4 in maintaining PCNA expression on
Day 10. RU-486 was administered as described in Materials and Methods on Day 9 and rats were killed 3, 6, 12,
and 24 h later. Untreated rats at Days 9 and 10 of gestation
were needed as controls because extensive remodeling of
DB occurs during this period of active decidualization. Figure 4A shows that RU-486 caused more than a 2-fold decline in MI between 3 and 6 h that became essentially zero
by 12 h and 24 h (p , 0.01). Figure 4B summarizes the
effects of RU-486 on PCNA expression. Essentially no
stromal cells expressed intense PCNA by 6 h of RU-486
exposure, and the proportion of cells with undetectable
PCNA steadily increased from about 8% at 6 h to 28% by
24 h (p , 0.05). Thus, there was a rapid and drastic loss
of cell division and PCNA expression by RU-486; the 6-h
effects preceded loss of histological integrity of the DB
(Fig. 1, A, I, and J).
DISCUSSION
Many studies have investigated stromal cell proliferation
in deciduoma during the periimplantation period (reviewed
by Glasser and McCormack [5] and Clarke and Sutherland
[6] [23]), but there is a lack of information about stromal
cell proliferation of the mesometrium during DB growth
and regression in pregnancy. We used the MI and PCNA
expression in situ as measures of cell proliferation. In general, there was close agreement between the MI and the
proportion of cells expressing intense levels of PCNA.
These parameters showed that proliferation was extensive
on Days 8 and 10 and declined abruptly by Day 12, marking the end of stromal cell proliferation. Day 14 was characterized by a lack of stromal cell mitoses, a 95% decline
in the cell population that expressed intense levels of
PCNA, and a 5-fold increase in the cell population expressing no PCNA. That the period of active proliferation was
P4-dependent was shown in experiments using RU-486 administration and Ovx to effectively withdraw P4 action
from the DB.
RU-486 treatment to otherwise intact pregnant rats was
associated with a rapid decline in MI and in the population
of stromal cells expressing intense PCNA. The percentage
cells expressing no PCNA steadily increased throughout the
24-h treatment period to about 5-fold that of Day 10 controls. Additionally, only the administration of P4 to pregnant rats Ovx on Day 8 was able to maintain PCNA abundance equal to that in the intact control group, whereas E2
treatment by itself caused an 87% decline in the percentage
cells expressing intense PCNA. The combination treatment
(PE) was not different from P4 administered by itself. However, P4 was unable to counter the drastic decline in the
percentage cells expressing intense PCNA on Day 14.
Thus, P4 appears to be an essential mitogen for stromal
cell proliferation during early pregnancy (Days 8–10), but
this action is lost at midpregnancy (Day 14). The loss of
P4 sensitivity corresponds to the onset of internucleosomal
DNA fragmentation in DB [24]. Furthermore, we have previously noted that proteins promoting cell death also become dominant. For example, the Bax-Bcl2 ratio (cell death
and cell survival proteins, respectively [25]) increased 3fold by Days 14–17 (compared to Day 10) [26]. Taken
together these observations suggest the existence of a phys-
FIG. 4. Summary analysis of the effects of RU-486 on stromal cell proliferation. Values are means 6 SEM, n 5 3–6. Day 9 pregnant rats received a 25-mg RU-486 pellet, i.p. followed by 0.5 mg RU-486 in saline/
ethanol i.p. and were killed at various times thereafter. D9 are mean
values for untreated control animals at the time of RU-486 administration
(Day 9). D10 represents the untreated control group at the last time-point
of the experiment (Day 10). Statistical analysis as in Figure 2. A) MI calculated as the percentage cells exhibiting mitotic figures as described in
Materials and Methods. aMeans differ from those for 6, 12, and 24 h of
RU-486 treatment but not from each other, p , 0.01. B) Image analysis
of PCNA immunostaining in DB after RU-486 treatment. Intense and undetectable PCNA staining categories are shown. aMean differs from all
others, p , 0.05. bMean differs from all others, p , 0.05. cMean differs
from that for the 12-h group, p , 0.05.
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PCNA EXPRESSION IN DECIDUAL CELLS
iological mechanism that withdraws P4 action in midpregnancy and promotes stromal cell death.
The rapid and almost complete loss of mitotic cells and
PCNA expression within 6 h of exposure to RU-486 indicates that the antiprogestin not only blocked cell cycle progression but also increased the rate of PCNA turnover. The
nuclear protein normally has a long half-life of about 20 h
[18], extending well beyond the 6- and 12-h periods of our
observed effects. It is unlikely that the original population
of stromal cells died within this short span of time because
apoptotic (apoptotic bodies) or necrotic (inflammatory response) changes were absent in our histologic preparations.
Histological manifestations of RU-486-induced effects did
not become evident until after 12 h of exposure (Fig. 1, I
versus J). By 24 h the histological organization of the DB
and placenta was entirely lost, and individual stromal cells
resided in the uterine lumen (data not shown). However,
cell death signals markedly increased as indicated by a
markedly increased Bax-Bcl2 ratio within 6 h of RU-486
[26].
These changes are closely associated with marked declines in the concentration of nuclear P4-binding sites and
in the expression of specific PR isoforms after RU-486
treatment on Day 9, as well as after Ovx in the absence of
P4 [21, 26]. P4 administration to Ovx animals maintained
PR-A and PR-B protein expression on Day 10 (the most
biologically relevant isoforms [27–29]) but not on Day 14
[21]. Likewise, in this study P4 was able to maintain PCNA
expression on Day 10 but not on Day 14. It should be noted
that a PR-C isoform was abundantly expressed throughout
pregnancy and not affected by RU-486 or Ovx [21]. This
isoform has been shown to be biologically inactive by itself
in a transfection system, but formation of PR-C/A and PRC/B heterodimers exhibited unique activities [30]. Thus, we
propose that the loss of these isoforms or the changing ratios between PR-A and PR-B to PR-C may effectively withdraw stromal cell sensitivity to the mitogenic action of P4
at Day 14.
Although we have used PCNA expression as a marker
for proliferating cells and P4 action in DB, it should not be
inferred that P4 or RU-486 necessarily exerted its proliferative and antiproliferative effects, respectively, directly on
PCNA synthesis. P4 action is undoubtedly multifaceted, operating at many levels to regulate the cell cycle. For example, P4 promotes cell passage through G1 to S-phase by
stimulating D1 cyclin mRNA and cyclin D1-kinase activity
and suppressing p21 abundance, all of which are blocked
by RU-486 [8, 9]. PCNA is an essential component of Dcyclin/cyclin-dependent kinase complexes and may also be
up-regulated by P4 [15]. P4 also exerts more indirect actions
on proliferation by regulating growth factors, growth factor
receptors, and cell survival factors as part of the decidualization process [1, 3, 24, 26, 31]. Thus, P4 probably regulates the cell cycle in early pregnancy at multiple control
points to promote mitosis and cell survival. However, these
actions are clearly impaired in the second half of pregnancy
and may be related to differential expression of PR isoforms.
ACKNOWLEDGMENT
We gratefully acknowledge the expert technical assistance of Ms. Veronica McCloud.
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