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Smad 3 regulates proliferation of the mouse ovarian surface epithelium.

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Mouse House
THE ANATOMICAL RECORD PART A 273A:681– 686 (2003)
Smad 3 Regulates Proliferation of the
Mouse Ovarian Surface Epithelium
DANIEL SYMONDS,1 DRAGANA TOMIC,1 CHRISTINA BORGEEST,1
ELIZABETH MCGEE,2 AND JODI A. FLAWS1*
1
Department of Epidemiology and Preventive Medicine, Program in Toxicology,
University of Maryland School of Medicine, Baltimore, Maryland
2
Ob/Gyn and Reproductive Sciences, Magee Women’s Research Institute, University
of Pittsburgh, Pittsburgh, Pennsylvania
ABSTRACT
Smad 3 is a signaling intermediate for the transforming growth factor beta (TGF␤)
family; however, little is known about the role this protein plays in the regulation of the
ovarian surface epithelium (OSE). Using a transgenic mouse model, we found that in the
absence of Smad 3 there was a distinct morphological alteration of OSE cells. Wild-type (WT)
OSE was flat with thin cells, while Smad 3-deficient (Smad 3 –/–) OSE was thick with plump
cuboidal cells. WT OSE had less immunostaining for proliferating cell nuclear antigen
(PCNA) and estrogen receptor alpha (ER␣) than Smad 3 –/– OSE. However, there were no
differences in the number of apoptotic cells or Bax and Bcl-2 levels between WT and Smad 3
–/– OSE. Although WT mice had higher levels of serum estradiol than Smad 3 –/– mice, WT
and Smad 3 –/– mice had similar levels of progesterone. These data suggest that Smad 3
regulates OSE morphological appearance and proliferation in the absence of high serum
estradiol levels or alterations in progesterone levels. Anat Rec Part A 273A:681– 686, 2003.
©
2003 Wiley-Liss, Inc.
Key words: Smad 3; ovary; surface epithelium; mouse
Although ovarian epithelial-derived tumors are a major
cause of cancer mortality in women (Auersperg et al.,
2001), very little is known about the basic biology of the
ovarian surface epithelium (OSE). Insights into the native
control mechanisms of the OSE could be valuable in elucidating the origin and progression of ovarian epithelial
cancers. A complex autocrine system known as the transforming growth factor beta (TGF␤ ) signaling pathway has
emerged as a major cellular control mechanism of the OSE
(Havrilesky et al., 1995). The TGF␤ signaling pathway
has multiple complex intermediate components. One of
the most important of these components is a family of
tumor suppressors known as the Smads (Zimmerman and
Padgett, 2000). These proteins are divided into three disparate groups: the receptor-regulated Smads (Smads 1, 2,
3, 5, and 8), the co-partner Smad 4, and the anti-Smads
(Smads 6 and 7).
A powerful tool used in elucidating the role of Smad 3 is
the transgenic mouse model deficient in Smad 3 protein
(Weinstein et al., 2000). Investigations using this model
have revealed a role for Smad 3 in the development of
metastases (Zhu et al., 1998), wound healing (Ashcroft et
al., 1999), T-cell activation (Yang et al., 1999), and mucosal immunity (Yang et al., 1999). A role for Smad 3 in
ovarian physiology is suggested by the presence of high
levels of Smad proteins in the granulosa cells of the mouse
©
2003 WILEY-LISS, INC.
ovary (Kano et al., 1999). Further, a recent study showed
that oocytes in primordial and primary ovarian follicles,
and granulosa and theca cells in preantral and small
antral ovarian follicles contain high concentrations of
Smad 3 (Xu et al., 2002). Recently, work from our laboratory showed that Smad 3 plays an important role in the
regulation of ovarian follicle growth and fertility, in that
deletion of Smad 3 appears to slow the growth of primordial follicles to the antral stage and to reduce fertility in
mice (Tomic et al., 2002).
The effect Smad 3 has on the OSE is unknown. Since
Smad 3 is an important intermediary in the TGF␤-mediated process of epithelial growth inhibition, one goal of
Grant sponsor: NIH; Grant number: HD38955; Grant sponsor:
Lalor Foundation.
*Correspondence to: Jodi Flaws, Ph.D., Department of Epidemiology and Preventive Medicine, 660 W. Redwood St., Howard
Hall 133B, Baltimore, MD 21201. Fax: (410) 706-1503.
E-mail: jflaws@epi.umaryland.edu
Received 29 April 2003; Accepted 2 May 2003
DOI 10.1002/ar.a.10090
682
SYMONDS ET AL.
this study was to test the hypothesis that Smad 3 regulates proliferation and/or apoptosis of the OSE. In addition, since the Smad complex migrates to the nucleus,
where it can modulate expression of genes regulating cell
proliferation and apoptosis (Massague et al., 2000), we
evaluated a downstream marker for proliferation (known
as proliferating cell nuclear antigen (PCNA)), and downstream markers for apoptosis (known as Bax and Bcl-2).
We also measured serum levels of estradiol and progesterone to determine whether any potential differences in
the OSE between wild-type (WT) and Smad 3 –/– mice
were mediated by differences in the levels of these hormones, since these hormones are thought to regulate proliferation or apoptosis in the OSE (Rodriguez et al., 1998;
Bai et al., 2000). Lastly, we evaluated levels of estrogen
receptor alpha (ER␣) to determine whether differences in
the OSE between WT and Smad 3 –/– mice are attributed
to differences in receptor endowment, and because ER␣ is
the predominant ER subtype in the OSE (Pelletier et al.,
2000).
MATERIALS AND METHODS
Animals
The animals used in this study were generated as described previously (Yang et al., 1999), and were generously provided by Dr. Chuxia Deng (National Institute of
Diabetes and Digestive and Kidney Diseases, National
Institutes of Health). The University of Maryland School
of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia, and tissue collection.
Mice heterozygous for Smad 3 (Smad 3 ⫹/–) were
mated, and the pups were genotyped to determine
whether they were WT, Smad 3 ⫹/–, or Smad 3 –/– mice.
Briefly, DNA was extracted from 3-mm ear punches of
tissue. The tissues were then subjected to polymerase
chain reaction (PCR) using the following primers: 1) 5⬘CCACTTCATTGCCATATGCCCTG-3⬘, 2) 5⬘-CCCGAACAGTTGGATTCACACA-3⬘ , and 3) 5⬘-CCAGACTGCCTTGGGAAAAGC-3⬘. The conditions for PCR were as
follows: 94°C for 3 min, followed by 30 cycles of 94°C for 30
sec, 60°C for 30 sec, 72°C for 90 sec, and a final extension
at 72°C for 10 min. PCR products were subjected to agarose gel electrophoresis at 120 V for approximately 90
min, and visualized under UV light. The presence of a 400
base pair band indicated WT mice, while the presence of a
250 base pair band indicated Smad 3 –/– mice. The presence of both bands indicated Smad 3 ⫹/– mice. Only WT
and Smad 3 –/– mice were used in each experiment.
Histologic and Morphometric Evaluations of
the OSE
At postnatal days (PND) 18 and 90, the animals were
killed and their ovaries (including the bursa) were removed and fixed in Kahle’ s solution (4% formalin, 28%
ethanol, and 0.34 N glacial acetic acid). PND18 was selected because it is a time when mice are sexually immature and have not begun to experience estrous cycles.
PND90 was selected because it is a time when mice are
sexually mature, and before Smad 3 –/– mice die.
Tissues were dehydrated in gradient alcohols, cleared in
xylene, and embedded in paraffin. Sections were cut at 5
␮m and stained with Weigert’ s hematoxylin-picric acid
methyl blue. The cytologic features of the OSE, including
cellular polarity and stratification, were assessed for animals of unknown genotype using a 40⫻ objective. The
height of the OSE was measured with an occular micrometer and a 40⫻ objective. Measurements were made at
12, 3, 6, and 9 o’clock positions on serial sections. A total
of 60 consecutive observations were recorded for each
sample. The average was multiplied by a calibration factor
from a micrometer scale to arrive at the average height in
microns.
Demonstration of Smad 3 Protein in the OSE
Immunohistochemistry was performed on 5-␮m sections
from WT and Smad 3 –/– ovaries (PND18) using antiSmad 3 antibody (Zymed Laboratories, Inc., San Francisco, CA) as the primary antibody at 1:100 dilution, after
epitope retrieval with 0.01 M citric acid (pH 6.0) at 100°C
for 10 min and incubation for 60 min. The biotinylated
streptavidin Histomouse™-SP system (Zymed Laboratories, Inc. ) with background blocking was used with 3-amino-9-ethylcarbazole (AEC) chromogenic visualization and
Mayer’s hematoxylin counterstain. Negative controls (no
primary antibody) were run in parallel to assess background staining.
Measurement of Proliferation in the OSE
Ovaries were removed from WT and Smad 3 –/– mice
(PND18), fixed in Kahle’s solution, dehydrated in gradient
alcohols, cleared in xylene, and embedded in paraffin.
Sections were cut at 5 ␮m and subjected to staining for
PCNA using commercially available monoclonal antibody
(1:100 dilution; Oncogene Research Products, Boston, MA)
as described previously (Tu et al., 1993; Tomic et al.,
2002). Epitope retrieval was carried out with 0.01 M citric
acid (pH 6.0) at 100°C for 10 min. The biotinylated
streptavidin Histomouse™-SP system (Zymed Laboratories, Inc., San Francisco, CA) with background blocking
was used with AEC chromogenic visualization and Mayer’s hematoxylin counterstain. In all experiments, negative controls (no primary antibody) were run in parallel.
To obtain an estimate of the percentage of proliferating
cells, the number of nuclei positively staining for PCNA
per 100 cells (Tu et al., 1993) was counted in at least six
different sections per ovary using at least five different
ovaries (one ovary per animal) per genotype.
Measurement of Apoptosis in the OSE
In situ hybridization was carried out on 5-␮m histologic
sections using an ApopTag Peroxidase kit (Intergen Co.,
Purchase, NY) according to the manufacturer’s protocol.
The number of dark-staining apoptotic nuclei was counted
per 100 OSE cells in at least six different sections per
ovary, using at least five different ovaries (one ovary per
animal) to obtain the percentage of apoptotic cells. As a
positive control, sections containing apoptotic granulosa
cells were utilized during each assay to verify staining of
apoptotic cells. As a negative control, sections were incubated without primary antibody.
Measurement of Bax, Bcl-2, and ER␣ Levels
Immunohistochemistry was carried out on 5-␮m sections using commercially available monoclonal antibodies
for Bax, Bcl-2, and ER␣ (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) as the primary antibodies at 1:100 dilutions, following published methods with minor modifica-
SMAD 3 REGULATES PROLIFERATION OF THE OSE
683
All data were analyzed using SPSS statistical software
(SPSS, Inc., Chicago, IL). Means ⫾ S.E.M. for the height of
the OSE , percentage of PCNA labeled or apoptotic cells,
and the number of pixels from immunostaining were calculated using multiple sections from at least five ovaries,
all of which were collected from different animals.
Means ⫾ S.E.M. for hormone levels were calculated using
at least three mice per group. Differences in means between genotypes were evaluated using Student’s t-test,
with significance at P ⱕ 0.05.
14.1 ⫾ 3.1 ␮m for Smad 3 –/– OSE (n ⫽ 8, P ⱕ 0.003). The
morphological abnormalities in the OSE also persisted at
PND90. At that time, the WT OSE was still flat, with
horizontally oriented cells, and the Smad 3 –/– OSE still
contained plump, cuboidal cells. While no neoplastic
changes in the OSE were observed in Smad 3 –/– mice, it
should be noted that these mice usually develop immune
problems and die after 3 months (Yang et al., 1999). Thus,
their life span may be too short to allow for neoplastic
progression.
There was a low level of staining for apoptotic cells in
the OSE of both WT and Smad 3 –/– mice. In both cases,
⬍3% of cells were apoptotic and there were no demonstrable differences between WT and Smad 3 –/– ovaries. In
contrast, there was considerable staining for PCNA in the
OSE from both WT and Smad 3 –/– OSE (Fig. 1e and f).
This staining differed between WT and Smad 3 –/– ovaries: staining was present in 20.8% ⫾ 1.8% of OSE cells
from WT mice, and in 51.0% ⫾ 1.6% of OSE cells from
Smad 3 –/– mice (n ⫽ 5, P ⱕ 0.001).
Staining for ER␣ demonstrated strong, uniform nuclear
and cytoplasmic reaction products in OSE of WT mice, but
weaker staining in OSE of Smad 3 –/– mice, particularly
in the nuclei (Fig. 1g and h). There was no background
staining in the negative controls, and the immunostaining
was predominantly localized to OSE and the ovarian interstitial cells. Microscopic densitometry confirmed
weaker staining in the OSE of Smad 3 –/– mice compared
to that of WT mice. The average pixel intensity was 21.9 ⫾
1.38 in the OSE of WT mice, and 14.1 ⫾ 0.94 in the OSE
of Smad 3 – /– mice (n ⫽ 9, P ⱕ 0.03).
Immunostaining for Bcl-2 and Bax resulted in moderate
staining of the OSE (Fig. 2a– d). This staining was similar
in both WT and Smad 3 –/– ovaries, and there was no
staining in the negative controls. For Bcl-2 immunostaining, the average pixel intensity was 218.3 ⫾ 10.3 in the
OSE of WT mice and 200.7 ⫾ 11.9 in the OSE of Smad 3
–/– mice (n ⫽ 5, P ⱕ 0.33). For Bax immunostaining, the
average pixel intensity was 221.0 ⫾ 12.3 in the OSE of WT
mice and 230.4 ⫾ 2.5 in the OSE of Smad 3 –/– mice (n ⫽
5, P ⱕ 0.37).
WT mice had significantly higher levels of serum estradiol compared to Smad 3 –/– mice, but WT and Smad 3 –/–
mice had similar levels of serum progesterone on PND18.
Serum hormone analysis resulted in estradiol levels of
245 ⫾ 18 pg/ml in WT animals and 167 ⫾ 16 pg/ml in
Smad 3 –/– animals (n ⫽ 4 –7, P ⱕ 0.02). Progesterone
levels averaged 12.9 ⫾ 1.96 ng/ml in WT mice and 11.3 ⫾
3.0 ng/ml in Smad 3 –/– mice (n ⫽ 7, P ⱕ 0.5).
RESULTS
DISCUSSION
Our data indicate that WT OSE had strong immunohistochemical staining for Smad 3 protein (Fig. 1a), which
was absent in Smad 3 –/– OSE (Fig. 1b). Differences were
found in the histologic appearance of the OSE compared to
WT and Smad 3 –/– animals. The OSE of WT animals was
flat and thin, with horizontally oriented cells (Fig. 1c). In
contrast, the OSE of Smad 3 –/– animals was thick, with
plump, cuboidal cells that were often pseudostratified
(Fig. 1d). When the thickness of the OSE was measured in
WT and Smad 3 –/– mice on PND18, the surface epithelial
height in WT animals was only 6.6 ⫾ 0.5 ␮m, while in
Smad 3 –/– mice it was 10.9 ⫾ 1.4 ␮m (n ⫽ 8, P ⱕ 0.013).
This significant difference in OSE height persisted in
PND90 animals, with a height of 6.2 ⫾ 0.3 ␮m for WT and
The results of this study indicate that absence of the
Smad 3 gene induces morphologic changes in the surface
epithelium of the ovary that are indicative of proliferative
cellular events. Morphometric quantitation indicated that
the OSE in the Smad 3 –/– mouse is composed of increased
numbers of enlarged cells compared to the OSE of the WT
mouse. In addition, immunostaining with the cell cycle
marker, PCNA, indicated increased cell division in the
OSE of Smad 3 –/– mice compared to that of WT mice.
Moreover, we found the growth stimulatory effect of Smad
3 deficiency to be unaccompanied by evidence of enhanced
apoptosis. This is consistent with a previous report showing that TGF␤ does not induce apoptosis in a cell line of
normal ovarian epithelial cells, even though it does inhibit
tions (Pelletier et al., 2000; Tomic et al., 2002). All antibodies were confirmed to be specific by the manufacturer
as well as in preliminary studies using blocking peptides.
Epitope retrieval was carried out with 0.01 M citric acid
(pH 6.0) at 100°C for 10 min. The biotinylated streptavidin
Histomouse™ -SP system (Zymed Laboratories, Inc., San
Francisco, CA) with background blocking was used with
AEC chromogenic visualization and Mayer’ s hematoxylin
counterstain. Negative controls (no primary antibody)
were run in parallel to assess background staining. Bright
red cellular reaction products were qualitatively graded as
slight, moderate, or strong stain. To quantify differences
in red cellular reaction products, image analysis was conducted using software from AlphaImager Vr 5.5 (Alpha
Innotech Corp., San Leandro, CA). The appropriate histologic field under 40⫻ magnification with a blue filter was
captured in the spot-densitometry program with background correction. Aggregates of approximately 10 – 20
ovarian surface epithelial cells were manually outlined
and the field was exposed. The software program calculated area and pixel intensity, and corrected for background staining, giving a result of average pixels per unit
area for the OSE aggregates. The average number of pixels per unit was then compared in the OSE of WT and
Smad 3 –/– mice. This analysis was conducted using at
least six sections per ovary and at least five ovaries (one
ovary per animal) per genotype.
Hormonal Assays
17-␤-estradiol and progesterone assays were performed
using enzyme-linked immunoassay (ELISA) kits obtained
from Diagnostic Systems Laboratories, Inc. (Webster, TX).
The supplied protocol was followed without modifications
and all samples were run in duplicate in a single assay.
The average coefficient of variation was 4.2% for the 17␤–
estradiol assay and 4.3% for the progesterone assay.
Statistical Analysis
Fig. 1. The OSE in WT and Smad 3 –/– ovaries. a: Smad 3 staining
(red precipitate) in the OSE of a WT mouse. b: the absence of Smad 3
staining in the OSE of a Smad 3 –/– mouse. a and b: Area between the
black and white arrows represents the OSE. c: The morphological appearance of WT OSE. d: The morphological appearance of Smad 3 –/–
OSE. c and d: The area between the black and white arrows represents
the OSE. Ovary of a WT mouse (e) and (f) ovary of a Smad 3 –/– mouse
after staining for PCNA. e and f: White and black arrows indicate nuclei
in the OSE staining darkly for PCNA, and the area below the white
arrows indicates ovarian interstitial cells and granulosa cells staining for
PCNA. Ovary of a WT mouse (g) and (h) ovary of a Smad 3 –/– mouse
after staining for ER␣. G and H: White and black arrows indicate cells in
the OSE staining for ER␣. Original magnification ⫽ 560⫻. Bar (a) ⫽ 10
␮m.
SMAD 3 REGULATES PROLIFERATION OF THE OSE
685
Fig. 2. Bcl-2 and Bax staining in WT and Smad 3 –/– ovaries . Ovary of a WT mouse (a), and (b) ovary of a
Smad 3 –/– mouse after staining for Bcl-2. Ovary of a WT mouse (c) and (d) ovary of a Smad 3 –/– mouse after
staining for Bax. a and b: White and black arrows indicate cells in the OSE staining for Bcl-2. c and d: White and
black arrows indicate cells in the OSE staining for Bax. Original magnification ⫽ 560⫻. Bar (a) ⫽ 10 ␮m.
(3H) thymidine uptake in the cells (Havrilesky et al.,
1995). It is also consistent with the finding that TGF␤ by
itself has no effect on apoptosis in rat thecal interstitial
cells (Pehlivan et al., 2001). Further, it is consistent with
a recent report that Smad 3 is functional in some ovarian
cancers (Dunfield et al., 2002). The complexity of Smad 3
multistep activation may permit opportunities for Smad 3
dysfunction to play a role in the unrestricted cell growth of
neoplastic progression. Taken together, these data suggest that one major effect of Smad 3 activity on the OSE
may be the inhibition of cell proliferation. Inhibition of cell
proliferation is central to the TGF␤ response in epithelial
cells, and escape from this response is a hallmark of many
cancer cells. In addition, previous data suggest that at
least two classes of antiproliferative gene responses are
involved in TGF␤-mediated growth arrest: c-myc downregulation and cdk-inhibitory responses, including the induction of p15 and p21 and the downregulation of cdc25A
(Massague et al., 2000). Whether Smad 3 –/– mice could
develop ovarian tumors, and whether antiproliferative
gene responses are altered in the OSE of Smad 3 –/– mice
are unknown and should be examined in future studies.
Since Smad 3 –/– mice die relatively early during adulthood (Yang et al., 1999), it may be important to develop
mice with a conditional deletion of Smad 3 so that the mice
live long enough for investigators to determine whether
Smad 3 deficiency leads to ovarian tumors.
The absence of enhanced apoptosis in the OSE of Smad
3 –/– mice is of interest in view of our previous detection of
high levels of the pro-apoptotic protein Bax, and low levels
of the anti-apoptotic protein Bcl-2 in granulosa cells of
ovarian follicles from Smad 3 –/– mice (Tomic et al., 2002).
One explanation for this difference in the regulation of
pro-apoptotic and anti-apoptotic proteins may stem from
the fact that Smad 3 activity is tissue- or cell-type-dependent. For example, others have shown that Smad 3 mediates the growth inhibitory effects of TGF␤ in T-cells (Yang
et al., 1999), partially mediates growth in keratinocytes
(Ashcroft et al., 1999), and does not mediate growth in
B-cells and mammary epithelial cells (Yang et al., 2002).
Thus, we speculate that Smad 3 may regulate anti- and
pro-apoptotic protein levels in the granulosa cells of ovarian follicles, but not in the OSE. This potentially different
role of Smad 3 in the ovarian follicles and OSE may be due
to differential expression of downstream factors or receptors in the two tissues. An example of the phenotypic
differences in receptors between the OSE and granulosa
cells in the ovarian follicle is that each predominantly
expresses a different type of estrogen receptor: ER␣ is
highly expressed in the OSE, while ER␤ is highly expressed in granulosa cells (Pelletier et al., 2000).
The reasons for the increased proliferation in the OSE of
Smad 3 –/– mice compared to the OSE of WT mice are
unknown. We initially considered that the increased proliferation in Smad 3 –/– mice might be due to increases in the
levels of estradiol and/or progesterone in the Smad 3 –/–
mice compared to WT mice, because these hormones are
thought to regulate proliferation in a variety of other tissues
686
SYMONDS ET AL.
(Shi et al., 1994; Biegel et al., 1998). However, our data do
not support this hypothesis because we did not observe any
differences in progesterone levels in immature Smad 3 –/–
mice and WT mice. Furthermore, our data, along with previous findings (Yang et al., 2002), indicate that estradiol
levels are actually lower in Smad 3 –/– mice than in WT
mice. Indeed, we confirmed that the OSE is an estrogen end
organ by detecting the presence of estrogen receptors in this
tissue. Our findings are in contrast to other studies that
suggest that estradiol may increase the proliferation of the
OSE (Bai et al., 2000; Choi et al., 2001). For example, some
laboratory studies indicate that IOSE-29EC ovarian tumor
cell cultures exposed to estradiol have an increased uptake of
(3H) thymidine, a marker for proliferation (Choi et al., 2001),
and that cells from rabbit OSE cultured with estrogen undergo proliferation (Bai et al., 2000). Our findings are somewhat consistent with one study that showed that low concentrations of steroid hormones have no effect on
proliferation of the OSE in rhesus monkeys (Wright et al.,
2002). The discrepancies between these studies may be due
to differences in species, in vitro vs. in vivo study conditions,
or ages of the animals.
We also found that the OSE from Smad 3 –/– mice had
lower levels of ER␣ compared to the OSE from WT mice.
Our findings are consistent with recent studies indicating
that there is cross-talk between Smad 3 and hormone
receptor activity (Hayes et al., 2001; Matsuda et al., 2001).
Matsuda et al. (2001) reported that estrogen receptors
suppress Smad 3 activity in cell culture systems. Hayes et
al. (2001) found that Smad 3 is able to suppress transcriptional activation of the androgen receptor during TGF␤
signaling. We are uncertain why the OSE of Smad 3 –/–
mice contains lower levels of ER␣ compared to that of WT
mice. We initially expected that it would have higher
levels of ER␣ than the OSE of WT mice because this would
make the tissue more responsive to estrogen, a hormone
that is thought to increase cellular proliferation (Bai et al.,
2000). We now think that the OSE from Smad 3 –/– mice
may be less responsive to estrogen than that of WT mice
because it has lower levels of ER␣. Collectively, these data
suggest that there may be two pathways for stimulation of
the OSE: a fundamental nonhormonally-based pathway in
which Smad 3 participates, and an additional pathway in
which estrogen plays a significant role. Whether the two
pathways are related by modification of Smad 3 activity in
mature animals, and whether Smad 3 acts directly to
diminish ER␣ in the OSE should be examined in future
studies. Such studies may improve our understanding of
the native control mechanisms of the OSE, which could be
valuable in elucidating the origin and proliferation of
ovarian carcinomas.
ACKNOWLEDGMENTS
The authors thank Mrs. Janice Babus for her technical
help, Dr. Kimberly Miller for her constructive comments,
and Mrs. Lynn Lewis for assistance with the manuscript.
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