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Regular Article |
a Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201
b National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
c The Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201
d Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, follicle, follicular development, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, the Smad proteins, which were discovered through genetic studies in Drosophila [4] and Caenorhabditis elegans [5], have been shown to mediate the TGFß signaling pathway. The Smads are a family of nine related proteins that can be divided into three different groups: the receptor-regulated Smads (Smad 1, 2, 3, 5, 8, and 9), the common-partner Smads (Smad 4), and the inhibitory Smads (Smads 6 and 7) [6–8]. These proteins share two highly conserved regions known as MH-1 (located in the N-terminus region, which is important for DNA binding activity of the Smads) and MH-2 (located in the C-terminus region, which mediates receptor-Smad and Smad-DNA interaction) [8]. Smad 2 and 3 are structurally similar and mediate TGFß and activin signals [8, 9]. Smads 1, 5, and 9 mediate bone morphogenetic signals [10, 11], whereas Smad 4 forms heteromeric complexes with Smad 2 and 3 after TGFß or activin stimulation, or with Smad 1 and Smad 5 after bone morphogenetic stimulation [6].
Previous studies have shown that the Smad proteins mediate TGFß signaling through a cascade of ligand-induced phosphorylation. Briefly, TGFß proteins are thought to bind to type 2 receptors on the cell surface, causing the phosphorylation of type 1 receptors, and activation of type 1 receptor-kinases [7, 12]. Type 1 receptor kinases then may phosphorylate and activate receptor-regulated Smads [13, 14]. The receptor-regulated Smads then form heteromeric complexes in the cytoplasm with Smad 4 and translocate to the nucleus [15]. Once the Smads translocate to the nucleus, they may recruit transcription factors and stimulate expression of genes that regulate cell proliferation and apoptosis [3, 16–18].
Targeted disruption of the Smad family is beginning to provide insights into the role of Smad proteins in development and diseases. Mice with homozygous disruptions of Smad 2 and Smad 4 die during embryonic life, indicating an important role for the genes of Smad 2 and Smad 4 in early embryonic development [19–22]. In contrast to Smad 2 and Smad 4, the biological function of Smad 3 is unclear. Mice that harbor a deletion of the Smad 3 gene are viable and survive to adulthood [23–25]. Previous studies using Smad 3-deficient mice indicate that Smad 3 may play an important role in the development of metastatic colorectal cancer [23], wound healing [24], and in TGFß-mediated regulation of T cell activation and mucosal immunity [25]. Recent studies also suggest that Smad 3 may be an essential factor in the TGFß signaling pathway in ovarian cells, because high levels of Smad 3 protein are present in the granulosa cells of the rodent ovary [26] and in oocytes of the human ovary [27].
Despite this previous research, the role of Smad 3 in the ovary is still an enigma. Thus, the purpose of this study was to investigate the effect of Smad 3 on female fertility, the development of the ovary, and the growth of ovarian follicles. Specifically, the goals of these studies were to 1) test whether deletion of Smad 3 affects female fertility; 2) test whether deletion of Smad 3 affects the size of the primordial follicle pool at birth; and 3) determine whether deletion of Smad 3 affects the numbers of primordial, primary, and preantral/antral follicles in postnatal life. In addition, the goal of this study was to investigate whether Smad 3 affects levels of members of the B-cell lymphoma/leukemia-2 family of protooncogenes (Bcl-2, Bcl-x, and Bax), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk-2).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Smad 3-deficient mice (Smad 3-/-) were generated by disrupting exon 8 of the Smad 3 gene [25]. Smad 3-/-, Smad 3+/-, and wild-type (control) animals were housed in the University of Maryland School of Medicine Central Animal Facility and provided food and water ad libitum. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, killing, and tissue collection.
Screening and Genotyping Mice
Individually housed heterozygote females (Smad 3+/-) were mated with Smad 3+/- males. Offspring were genotyped to determine whether they were Smad 3-/-, Smad 3+/-, or wild-type, as previously described with minor modifications [25]. Briefly, ear punch tissue from pups was lysed in proteinase K buffer (50 mM Tris-HCl, 20 mM NaCl, 1 mM EDTA, and 1% SDS pH 8.0) containing 1 µl of 20 mg/ml proteinase K (Qiagen Inc., Valencia, CA). Digestion was carried out at 100°C for 3 min. The lysate then was subjected to the 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 94°C for 3 min of initial denaturation followed by 30 cycles at 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 then were subjected to agarose gel electrophoresis. The presence of a 400-base pair (bp) band indicated that the mice were wild type. The presence of a 250-bp fragment indicated that the mice were homozygous for the Smad 3 deletion, whereas the presence of 250- and 400-bp bands indicated that the mice were heterozygous for the Smad 3 deletion.
Histological Evaluation
At selected time points (Postnatal Days 2, 7, 18, and 90), Smad 3-/- and wild-type animals were killed by halothane inhalation, and their ovaries were collected for histological evaluation and immunohistochemical analysis as described below. Postnatal Day 2 was selected because it is right after birth and before the time when the primordial follicle pool is completely formed in the mouse. Postnatal Day 7 was selected because it is when the primordial follicle pool is fully formed in the mouse. Day 18 was selected because that is when follicular growth is in full process, and Day 90 was selected because that is when mice should be cycling adults [28].
Ovaries were fixed in Kahle solution (4% formalin, 28% ethanol, and 0.34 N glacial acetic acid), serially sectioned (8 µm), mounted on glass slides, and stained with Weigert hematoxylin-picric acid methyl blue. A stratified sample consisting of every 10th section was used to estimate the total number of primordial, primary, preantral, and antral follicles per ovary. The selected sections were randomized and the number of primordial, primary, preantral, and antral follicles was counted in each of the entire sections. Only follicles with a visible nucleolus in the oocyte were counted to avoid counting follicles twice. The number of follicles in the marked sections then was multiplied by 10 (because every 10th section was used) and subsequently by 8 (accounting for section thickness) to obtain an estimate of the total number of follicles in each ovary.
Ovarian follicles were categorized as described by Flaws et al. [29]. Follicles were counted as primordial if they contained an intact oocyte with a visible nucleolus surrounded by a one-layer ring of fusiform granulosa cells. Follicles were counted as primary if they consisted of an oocyte with a visible nucleolus and a single layer of cuboidal granulosa cells. Follicles were counted as preantral if they contained an oocyte with a visible nucleolus and more than one layer of granulosa cells. Finally, follicles were counted as antral if they contained an oocyte with a visible nucleolus, more than one layer of granulosa cells, and an antral space. All sections were evaluated without knowledge of the genotype and age of the animals.
Immunohistochemistry for Bax, Bcl-2, Bcl-x, PCNA, and Cdk-2 Protein
Immunohistochemistry was performed as previously described by Flaws et al. [29]. Briefly, ovaries were fixed in Kahle or Bouin solution for at least 24 h. Following fixation, the tissue was dehydrated, embedded in paraplast (VWR Scientific, Baltimore, MD), serially sectioned (5 µm), and mounted on glass slides. Commercially available monoclonal Bax antibody (1:100 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Bcl-2 antibody (1:100 dilution, Santa Cruz Biotechnology), Bcl-x antibody (1:100 dilution, Santa Cruz Biotechnology), PCNA (1:100 dilution; Oncogene Research Products, Boston, MA) and Cdk-2 (1:50 dilution, Santa Cruz Biotechnology) were used. The secondary antibody and visualization reagents came from the HistoMouse-SP kit (Zymed Laboratories, San Francisco, CA). As a control for background staining, some tissues were incubated with secondary antibody without primary antibody. Each stained section was counterstained with Mayer hematoxylin (Zymed Laboratories) for 30 sec to 1 min to help visualize ovarian tissue. As a result, the ovarian tissues were stained light blue, and the proteins of interest were stained red. All stained sections were visualized without knowledge of genotype, and each section was scored as having a light stain, a medium stain, or a heavy red stain. The relative intensity of staining then was compared in Smad 3-/- and wild-type ovaries.
Animal Fertility
Several breeding schemes were conducted to evaluate fertility: 1) wild-type females with wild-type males (n = 5 mating pairs), 2) Smad 3+/- females with Smad 3+/- males (n = 10 mating pairs), 3) Smad-/- females with Smad-/- males (n = 3 mating pairs), 4) Smad-/- females with Smad+/- males (n = 3 mating pairs), and 5) Smad-/- females with wild-type males (n = 3 mating pairs). In each scenario, a single female mouse (at least 5 wk old) was placed in a cage with a male mouse (at least 5 wk old) in the late afternoon. These mating pairs were housed together overnight. Every morning vaginal canals were gently checked for copulatory plugs. If a plug was found, the male was removed from the cage and the female was considered to be pregnant. These females were monitored every day for increases in weight, the presence of protruding bellies, birth, and numbers of dead and live pups. If a plug was not found, the male was kept in the cage and the female was checked every morning for the presence of a vaginal plug.
Statistical Analysis
All data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL). The mean number of primordial, primary, preantral, and antral follicles per ovary was calculated using ovaries from at least three different animals. The mean number of pups per litter was calculated from at least three different mating pairs per genotype, and mean body weights were calculated from at least five different pups per genotype. Differences between the means were evaluated by one-way ANOVA, with statistical significance assigned at P 
0.05. When a significant P value was obtained with ANOVA, the Scheffé test was used in the post hoc analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first noticeable phenotype in the Smad 3-/- animals was a significant decrease in their size and weight compared with wild-type and heterozygous littermates (Table 1). On Postnatal Day 2, control mice weighed 1.75 ± 0.09 g, whereas Smad 3-/- mice weighed only 1.36 ± 0.11 g (n = 9 for controls, n = 5 for Smad 3-/-, P 0.03). Although the weight of both Smad 3-/- and wild-type mice increased over time, the weight of Smad 3-/- mice remained significantly lower than wild-type mice at all time points (P 0.03).
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Effect of Smad 3 Deletion on Fertility
When wild-type females were mated with wild-type males (WT:WT), the mating produced 6.8 ± 1.08 pups per litter (n = 5). All the pups were viable and lived until they were killed for histological or immunohistochemical analyses. When Smad 3+/- females were mated with Smad 3+/- males (Smad 3+/-:Smad 3+/-), the mating produced a similar number of pups per litter as the WT:WT mating (Smad 3+/-:Smad 3+/- = 7.6 ± 0.8 pups per litter, n = 10). These matings followed normal inheritance patterns because they resulted in approximately 25% wild-type pups, 25% Smad 3-/- pups, and 50% Smad 3+/- pups per litter. All wild-type and Smad 3+/- mice were viable and lived until they were killed for histological or immunohistochemical analyses. In contrast, 7%–10% of Smad 3-/- pups died from intestinal abscesses by 3 mo of age. When Smad 3-/- females were mated with either wild-type, Smad 3+/-, or Smad 3-/- males (n = 3), no pups were produced, even though Smad 3-/- females were housed with Smad 3-/- males for 4–6 mo.
Effect of Smad 3 Deletion on Ovarian Morphology and the Size of the Primordial, Primary, and Preantral/Antral Follicular Pool
On Postnatal Day 2, ovaries from Smad 3-/- and wild-type animals were similar in appearance (Fig. 1, A and B). These ovaries consisted primarily of primordial follicles and some primary follicles, each of which contained an intact oocyte surrounded by a single layer of granulosa cells. At this time, there were no significant differences in the number of primordial follicles between Smad 3-/- and wild-type mice (Fig. 2A, P 0.701). The Smad 3-/- ovaries, however, contained significantly fewer primary follicles than did wild-type ovaries (Fig. 2B, P 0.016).
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By Postnatal Day 7, ovaries from Smad 3-/- mice began to differ morphologically from those of wild-type animals (Fig. 1, C and D). Although both Smad 3-/- and wild-type ovaries consisted of similar numbers of healthy primary follicles (Fig. 2B, P 0.550), there were more primordial follicles and fewer preantral follicles in Smad 3-/- ovaries than in those of wild-type mice (Fig. 2A, P 0.006 and Fig. 2C, P 0.005). At this time point, for example, wild-type ovaries contained 1640 ± 216 preantral and antral follicles, whereas Smad 3-/- mice contained only 40 ± 24 preantral and antral follicles per ovary (Fig. 2C, P 0.005).
By Postnatal Day 18, Smad 3-/- and wild-type ovaries were even more distinguishable from each other (Fig. 1, E and F). The ovaries isolated from Smad 3-/- mice contained approximately 61% more primordial follicles (Fig. 2A, P 0.002) and 40% fewer preantral and antral follicles (Fig. 2C, P 0.138) than ovaries isolated from wild-type animals.
By Postnatal Day 90, wild-type ovaries consisted mostly of large preantral and antral follicles, which contained an intact oocyte surrounded by numerous layers of morphologically healthy granulosa cells (Fig. 1G). Smad 3-/- ovaries contained mostly primordial and primary follicles and a few preantral and antral follicles (Fig. 1H). The ovaries isolated from Smad 3-/- mice contained 2.7-fold more primordial follicles (Fig. 2A, P 0.001) and 3-fold fewer preantral and antral follicles (Fig. 2C, P 0.009) than wild-type animals.
Immunohistochemical Analysis for Bax, Bcl-2, Bcl-x, Cdk-2, and PCNA in Ovarian Tissue
As shown in Figure 3, Bax staining was observed in the granulosa cells and oocytes of Smad 3-/- and wild-type ovaries. On Postnatal Day 7, Smad 3-/- and wild-type ovaries appeared to have similar levels of Bax (Fig. 3, A and B). By Postnatal Day 18, however, Smad 3-/- ovaries appeared to have higher levels of Bax in both granulosa cells and oocytes than did wild-type ovaries (Fig. 3, C and D).
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Bcl-2 positive staining was observed in the granulosa cells and oocytes of both Smad 3-/- and wild-type animals, but Smad 3-/- ovaries appeared to have less staining for Bcl-2 protein compared with wild-type ovaries (Fig. 3, E and F). In addition, similar levels of Bcl-x (Fig. 3, G and H), Cdk-2 (Fig. 4, A and B), and PCNA (Fig. 4, C and D) proteins were observed in granulosa cells of the ovaries isolated from both Smad 3-/- and wild-type animals. These proteins appeared to be present in primordial, primary, preantral, and antral follicles in the ovary.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data differ from the data obtained from mice with a targeted disruption of Smad 3 in exon 2 [23]. According to a report by Zhu et al. [23], adult homozygous mutant mice were fertile and produced homozygous litters. The reason that Smad 3 mutant mice harboring a targeted disruption in exon 2 have a distinct phenotype from those harboring a disruption in exon 8 is unknown. It is possible that in mice, a differential activation of downstream targets exists with a disruption in exon 2 and exon 8. This hypothesis is in agreement with previous in vitro studies, which indicate that different domains of the Smad 3 protein may be involved in activation of diverse downstream pathways [30, 31]. Future experiments, however, are required to fully understand why varying the site of the null mutation (exon 2 vs. exon 8) differentially affects female fertility.
The depletion of the preantral and antral follicular pool in Smad 3-/- mice during postnatal life may occur by two different mechanisms. First, the Smad 3 deletion may result in a slower growth of primordial ovarian follicles to the antral stage (i.e., more follicles remain in the immature primordial stage instead of growing to the more mature antral stage). In an alternative mechanism, the Smad 3 deletion may stimulate atresia of preantral and antral follicles in the ovary during postnatal life. Our data support the first hypothesis because we observed a higher number of primordial follicles in the Smad 3-/- animals compared with wild-type mice, suggesting that the primordial follicles remain in the primordial pool instead of growing to the antral stage. Our data also support the second hypothesis because on Postnatal Day 18, in comparison with ovaries of wild-type mice, those of Smad 3-/- mice had higher levels of Bax, a known proapoptotic protein [32, 33], and lower levels of Bcl-2, a known antiapoptotic protein [32, 33].
The mechanism by which Smad 3 regulates growth or atresia in ovarian follicles is unknown. Previous studies indicate that Smad 3 may mediate signaling of TGFß and activins in ovarian cells [8, 26, 27]. Previous work also suggests that activin and TGFß proteins play an autocrine/paracrine role in controlling the early growth of ovarian follicles [34–38]. It is believed that activin controls the induction of FSH receptors in ovarian follicular cells and that this in turn controls the FSH-induced growth of ovarian follicles [34, 35]. Thus, it is possible that a Smad 3 deletion results in slower follicular growth, increased atresia, or both, because it interferes with the ability of ovarian cells to respond to activin and thus to increase the number of FSH receptors.
In addition, it is possible that Smad 3 deletion results in slower follicular growth or increased atresia because it interferes with the ability of ovarian cells to respond to two members of the TGFß family known as growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15). Both GDF-9 and BMP-15 have been shown to regulate early follicular growth in the mouse ovary [39–42]. Recent evidence suggests that BMP-15 and GDF-9 may interact with each other (i.e., they form heterodimers) [43]. Thus, it is possible that deletion of Smad 3 interferes with the ability of GDF9, BMP-15, or both to regulate early follicular growth in the mouse ovary.
In conclusion, we have investigated the in vivo function of Smad 3 in the ovary using a Smad 3-deficient mouse model. This study is the first to show that 1) Smad 3-/- mice with a disruption in exon 8 have reduced fertility, 2) Smad 3 does not affect the size of the primordial follicle pool at birth but it may be important for the normal growth of the primordial follicles to the antral stage during postnatal life, and 3) Smad 3 may regulate the expression of the B-cell lymphoma/leukemia-2 family of protooncogenes in the mouse ovary.
Future studies of the Smad 3 signaling pathway in the ovary may give fresh insights into the biology of ovarian tissue and lead to a better understanding of the mechanisms that underlie normal follicular development. Furthermore, future studies using Smad 3-deficient mice may provide a useful model system for identifying molecular alterations that may lead to female infertility.
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FOOTNOTES |
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1 Supported by National Institutes of Health grants RO1 HD38955 to J.A.F., 5R01 CA83199-02 to L.H.M., and RO1 CA74904 to R.J.H. 
2 Correspondence: Jodi A. Flaws, University of Maryland School of Medicine, Department of Epidemiology and Preventive Medicine, 660 W. Redwood Street, Baltimore, MD 21201. FAX: 410 706 1503; jflaws{at}epi.umaryland.edu
Accepted: November 5, 2001.
Received: August 17, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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