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. 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