Molecular Human Reproduction, Vol. 10, No. 1, pp. 7-14, 2004
© European Society of Human Reproduction and Embryology 2004
Differences in connective tissue gene expression between normally functioning, polycystic and post-menopausal ovaries
1Departments of Medical Biochemistry and Molecular Biology and 2Obstetrics and Gynaecology, Turku University Central Hospital, University of Turku, FIN-20520 Turku, and 3The Family Federation of Finland, Maariankatu 3 A, FIN-20100 Turku, Finland
4 To whom correspondence should be addressed at: The Family Federation of Finland, Maariankatu 3 A, FIN-20100 Turku, Finland. e-mail: marja-leena.anttila{at}vaestoliitto.fi
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ABSTRACT |
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Arrested follicular maturation is a characteristic feature of polycystic ovary syndrome (PCOS). Follicles mature in ovarian stroma composed of extracellular matrix (ECM). However, little is known of the expression of ECM genes in polycystic ovaries. The present study compares the expression levels of genes coding for collagens, matrix metalloproteinases (MMP), their inhibitors (TIMP) and cathepsins in polycystic ovaries using fertile and post-menopausal ovaries as controls. In northern analyses, the gene expression profiles of type I and III collagen of PCOS samples resembled those observed in normal follicular phase ovaries, while mRNA levels of pro
1(IV) collagen and TIMP-3 mRNA were significantly lower in polycystic than control ovaries. During the normal menstrual cycle, an increase was observed in MMP-9 gene expression during the luteal phase. In post-menopausal ovaries, mRNA levels for type I, III and IV collagens and osteonectin were reduced, while the MMP, TIMP (excluding TIMP-3) and cathepsins did not reflect this metabolic down-regulation. Immunohistochemical staining for MMP-9 and TIMP-4 suggested differences between polycystic and normally functioning ovaries. These data demonstrate that normal ovarian functions are associated with changes in production and degradation of ECM. The alterations observed in the production and/or distribution of type IV collagen, TIMP-3 and TIMP-4 suggest involvement of basement membranes in the pathogenesis of PCOS. Key words: Key words: extracellular matrix/human ovary/mRNA/polycystic ovary syndrome/post-menopause
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Introduction |
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of women of reproductive age, affecting 5–10% of women in this age group (Knochenhauer et al., 1998). It is characterized by menstrual irregularity due to anovulation and by evidence of hyper andogenism, at clinical and/or biochemical levels. Characteristic features of a polycystic ovary (PCO) are an arrested follicular maturation resulting in formation of multiple small ovarian cysts throughout the cortical surface of the ovary, increased volume and density of ovarian stroma, and thickening of the tunica albuginea (Clement, 2002). Despite intense research, the local mechanisms underlying the dysfunction of follicular development in PCOS remain obscure.
Collagens I, III and IV are the three main collagen types found in ovarian extracellular matrix (ECM) both in stroma and in tunica albuginea (Woessner, 1982; Auersperg et al., 1994; Kruk et al., 1994). They provide the structural strength to the ovaries and support the matrix where follicular maturation, ovulation, formation of corpus luteum and its regression take place (Woessner, 1982). Our previous studies on non-hormone-primed mouse ovaries have demonstrated cyclic variation in the mRNA levels for several connective tissue components, collagenolytic enzymes and their inhibitors during the natural estrous cycle (Oksjoki et al., 1999, 2001). Similar cyclic changes have been observed in the expression of ECM–associated genes in ovaries of other experimental animals (reviewed in Hulboy et al., 1997; McIntush and Smith, 1998; Curry and Osteen, 2001; Ny et al., 2002). This fluctuation suggests that in addition to providing structural strength to the ovaries, collagens and other ECM components may also have other roles connected with ovarian function and dysfunction.
The present study is based on a hypothesis that aberrations in connective tissue metabolism and alterations in gene expression patterns for ECM components, proteolytic enzymes and enzyme inhibitors could at least partly explain the follicular arrest in PCOS, and the subsequent formation of the ovarian phenotype in PCOS. Our hypothesis is supported by recent findings of Shalev et al. (2001), who observed a shift in the MMP/TIMP balance towards MMP-2 and -9 in luteinized granulosa cells from women with PCOS. Decreased expression of TIMP-1 in follicular fluid of patients with PCOS has been suggested to be part of the compensatory process to overcome the physical properties of the thick ovarian capsule (Lahav-Baratz et al., 2003).
The present study was performed on human ovarian samples collected by surgical removal of small specimens of ovarian tissue prior to electrocauterization. For controls, similar specimens were collected at hysterectomy or laparoscopic operations from fertile and post-menopausal controls. Although the samples did not contain the leading follicle or corpus luteum, consistent differences were detected in mRNA levels for ECM components between polycystic ovaries (PCO) and fertile and post-menopausal controls.
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Materials and methods |
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Subjects
The voluntary study groups consisted of 15 apparently healthy and regularly menstruating women aged 32–52 years (mean ± SD: 45.2 ± 5.5), 11 post-menopausal women aged 48–75 years (57.8 ± 9.1) and 13 women with PCOS aged 26–37 years (30.1 ± 3.3). None of the women had diagnosis of a connective tissue-associated disease or was under long-term anti-inflammatory medication. The diagnosis of PCOS was based on ultrasonographically assessed ovarian morphology, menstrual disturbances (oligo/amenorrhoea) and elevated androgen levels. All women with PCOS exhibited elevated serum LH levels, suffered from anovulatory infertility, and were resistant to clomiphene citrate. Ovarian samples from the first two groups were collected during hysterectomy or laparoscopic operations. Polycystic ovary samples were taken from patients treated with electrocauterization by Karl Stortz endoscope ovarian biopsy forceps. Typically, several small antral follicles were seen in the PCO samples. Most of the samples (10 fertile controls, eight post-menopausal and six PCOS) were used for gene expression analyses; however, when sufficient material was available, half of the sample was used for histology. An additional 15 samples were used for immunohistochemistry, western analysis and/or zymography. The samples analysed did not contain the leading follicle or corpus luteum, but represented an ‘averaged’ sample of the ovary containing surface epithelium, antral and preantral follicles as well as stroma. In the fertile controls, the day of the menstrual cycle at the time of the sample collection was noted and confirmed by LH, FSH, estradiol and progesterone measurements. This information was used to divide the samples into follicular and luteal phase groups. Post-menopausal ovaries were used as samples where the follicular pool was exhausted, although occasionally some primordial follicles and regressing corpora albicans were seen. The study design was approved by the Ethical Committee of Turku University and Turku University Central Hospital, and an informed consent was signed by all participants.
Extraction of total RNA and northern hybridization
Samples were frozen in liquid nitrogen and stored at –70°C. Total RNA was isolated by the guanidinium isothiocyanate method as described previously (Chirgwin et al., 1979). Aliquots (10 µg) of total RNA were denatured with glyoxal and dimethylsulphoxide, electrophoresed on two (nearly) identical 0.75% agarose gels, transferred by blotting onto Pall Biodyne membranes and hybridized with 32P-labelled cDNA inserts at 42°C for 20 h. The hybridizations and washes were performed as suggested by the supplier (Pall BioSupport Division, USA). After high stringency washes, the bound probes were detected and quantified on a Molecular Imager Phosphoimager (Bio-Rad, USA) and the signals corrected for variations in the 28S rRNA levels determined by hybridization. The cDNA clones used as probes are listed in Table I.
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Immunohistochemical studies
For histological analyses, human ovarian samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and stained with haematoxylin and eosin or processed for immunohistochemistry. For the latter, formalin-fixed, paraffin-embedded histological sections of human ovaries were deparaffinized and re-hydrated. For TIMP-3 and -4 immunostainings, tissue sections were pretreated by boiling them in 10 mmol/l citrate buffer (pH 6.0) for 10 min as recommended by the supplier. For immunostaining of MMP-9, no pretreatment was used. For immunohistochemical detection of TIMP-3 and TIMP-4, rabbit polyclonal antibodies (NeoMarkers, USA) in diluted 1:400 and 1:2000, and for MMP-9 detection, a rabbit polyclonal antibody (Chemicon International, USA) in diluted 1:2000 were used respectively. The secondary antibodies were visualized using the avidin–biotin method as recommended by the supplier (Histostain-Plus Kit; Zymed, USA). The brown colour was developed with diaminobenzinine (DAB-Plus Kit; Zymed), and sections were counterstained with haematoxylin and mounted. The specificity of the immunoreactions was controlled by omitting the primary antibody, by using non-immune sera, or (for TIMP-3) by incubating the antibody overnight with an excess of blocking peptide (NeoMarkers) at 4°C.
Gelatinase zymography
Ovarian samples representing three follicular phase ovaries, three post-menopausal ovaries and three PCO were collected into liquid nitrogen, homogenized and mixed with ice-cold extraction buffer containing cacodylic acid (10 mmol/l), NaCl (0.15 mol/l), ZnCl (20 mmol/l), NaN3 (1.5 mmol/l), and 0.01% Triton X-100 (pH 5.0). The homogenates were centrifuged, and the final protein concentration of the extracts determined by a standardized colorimetric assay (Bio-Rad Protein Assay). The pH of each sample was then raised to 7.6 using Tris buffer. Equal amounts of protein were mixed with a sample buffer [10% sodium dodecyl sulphate (SDS), 4% sucrose, 0.25 mol/l Tris–HCl, and 0.1% Bromophenol Blue, pH 6.8] in proportion 3:1. Samples were fractionated on 10% SDS–polyacrylamide gel electrophoresis containing 1 mg/ml gelatin (G-9382; Sigma, USA) and 0.5 mg/ml 2-methoxy-2,4-diphenyl-3(2H)-furanone at 4°C. The gels were then washed, activated for 24 h at 37°C, fixed in 50% methanol/7% acetic acid and stained with 0.2% Coomassie Blue as described previously (Ala-Aho et al., 2000). The proteolytic regions in each lane were quantified using a digital camera and image analysis.
Western blot analysis
Aliquots of 20–50 µg of protein extracts (see above) were run on 10% SDS–polyacrylamide gels and subsequently electroblotted onto Hybond ECL nitrocellulose membranes in a semi-dry electroblotter (Bio-Rad). The membranes were blocked overnight in Superblock Blocking Buffer (Pierce Biotechnology, USA) at +4°C and incubated with polyclonal antibodies for TIMP-4 (dilution 1:4000) and MMP-9 (dilution 1:15 000) for 1 h. After washing six times with T-TBS buffer (0.1% Tween, 0.5% Superblock in TBS buffer), horseradish peroxidase-conjugated anti-rabbit secondary antibody (Bio-Rad) (dilution 1:30 000 for TIMP-3 and 1:45 000 for MMP-9) was applied for 1 h, followed by six washes with T-TBS buffer and one wash with TBS buffer. The bound antibodies were detected by an ECL kit as recommended by the supplier (Pierce Biotechnology) and autoradiography.
Statistical analyses
One-way analysis of variance (ANOVA) for repeated measures and the unpaired Student’s t-test were used to analyse differences in mRNA levels between cycle phases. When ANOVA disclosed significant differences, t-tests were used to determine which means were different. P < 0.05 was considered statistically significant.
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Results |
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Northern analyses
ECM components
Although considerable individual variation was observed in the mRNA levels of type I and III collagens and osteonectin (SPARC), the averaged gene expression profiles in polycystic ovaries most closely resembled those seen in normal ovaries during the follicular phase (Figure 1a, b and d). In post-menopausal ovaries, the corresponding mRNA levels were significantly lower than in the follicular phase samples (P < 0.05, Figure 1a, b and d). These changes were in contrast to the mRNA levels for pro
1(IV) collagen, where polycystic ovaries most closely resembled post-menopausal ovaries (Figure 1c). In both polycystic and post-menopausal ovaries, pro1(IV) collagen expression levels were significantly lower than in normal ovaries during the follicular and luteal phases of the menstrual cycle (P < 0.05, Figure 1c). No statistically significant differences were observed in any collagen type or osteonectin mRNA levels in normal ovaries during the menstrual cycle, although there was a tendency for decreased levels in the luteal phase compared with the follicular phase.
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MMP and TIMP
In comparisons of mRNA levels for MMP and TIMP, PCO predominantly exhibited profiles consistent with those seen in normal ovaries in the luteal phase and post-menopausal samples, except for mRNA for MMP-2, TIMP-1 and -2, where great individual variation in the mRNA levels was seen in all study groups. In respect of MMP-9 mRNA levels, the samples from normal follicular phase ovaries exhibited lower values than post-menopausal (P < 0.01) or polycystic ovaries (Figure 2c). However, the difference between PCOS and control values did not reach statistical significance (P = 0.071). TIMP-3 mRNA levels were lower in post-menopausal (P < 0.01) and polycystic ovaries (P < 0.05) than in normal follicular phase samples (Figure 2f).
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During the normal menstrual cycle, the highest mRNA levels for MMP-1, MMP-2 and MMP-9 were found in the luteal phase; this difference was only statistically significant for MMP-9 (P < 0.01, Figure 2c). Transcripts for MMP-13 could not be detected in the ovary, and the levels of TIMP-4 mRNA were too low for reliable quantification.
Cathepsins
No statistically significant changes were observed in the mRNA levels for cathepsins B, H, K or L between the groups (Figure 2g–k). The expression profiles in PCOS samples resembled those in post-menopausal and normal luteal phase ovaries more closely than in normal early follicular phase ovaries. Cathepsin S levels were too low for reliable quantification.
Immunohistochemistry
Immunohistochemical localization was performed for MMP-9 and TIMP-3, which exhibited statistically significant differences in northern analyses between fertile and PCOS/post-menopausal groups, using ovarian samples from women with PCOS (n = 6), normal follicular phase ovaries (n = 4), normal luteal phase ovaries (n = 3) and post-menopausal ovaries (n = 2). Furthermore, TIMP-4, the most recently discovered MMP inhibitor, was studied similarly. The results are summarized in Table II and Figure 3.
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MMP-9 immunostaining was mainly localized in stromal cells, particularly beneath the surface epithelium in fertile ovarian samples (Figure 3B1; Table II). Faint staining was seen in individual luteal cells (Figure 3C1). No staining was observed in the developing follicles (Figure 3A1). In the PCO, immunostaining was similarly localized in stromal cells, but faint staining could also be seen in the non-luteinized granulosa cells (Figure 3D1). In post-menopausal ovaries, weak staining was seen in fibroblasts within some corpora albicans (not shown). In all samples, most vessel walls exhibited intense staining, but no staining was observed in the surface epithelium in any of the samples.
In all ovaries, the strongest staining for TIMP-3 was seen in individual granulosa cells; the proportion of positive cells being greater in antral than primordial follicles (Figure 3A2 and B2; Table II). In antral follicles, however, the basal layer of granulosa cells was negative (Figure 3A2). Intense staining was observed in thecal and luteal cells as well as in oocytes (Figure 3A2, B2 and C2). Staining for TIMP-3 was also seen in the walls of small blood vessels, in surface epithelium and beneath the epithelial cells. As in normal follicles, TIMP-3 staining was also present in the non-luteinized granulosa cells of follicules in PCO ovaries (Figure 3D2). Additionally, TIMP-3 staining was seen in the thecal layer consisting of luteinized cells surrounding PCO follicles (Figure 3D2). In post-menopausal ovaries, intense staining was seen in individual cells located in peripheral areas of regressing corpus albicans (not shown).
Immunostaining for TIMP-4 was most obvious in oocytes (Figure 3B3; Table II). Furthermore, the protein was localized in individual granulosa cells (Figure 3A3), luteal cells (Figure 3C3) and in the non-luteinized granulosa and thecal cells of the PCOS sample (Figure 3D3). Stromal cells (Figure 3B3), surface epithelium and blood vessels were negative for TIMP-4 immunostaining. No staining was observed in the post-menopausal ovary (not shown).
Antibody characterization
In western blotting, single bands of the expected molecular sizes (24 kDa for TIMP-4, and 88 kDa for the active form of MMP-9) were detected (Figure 4D and E). To demonstrate the specificity of TIMP-3 antibodies, a peptide blocking experiment was performed. No TIMP-3 immunostaining was observed after incubating the antibody with excess amount of the corresponding blocking peptide (Figure 4A–C).
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Gelatinase zymography
The activities of MMP-2 and MMP-9 in normal fertile, polycystic and post-menopausal ovaries were also studied by gelatinase zymography (Figure 5). The predominant gelatinase activity was observed at 72 kDa molecular weight, which corresponds to the latent form of MMP-2. Activities were also detected at 66 and 92 kDa, corresponding to active MMP-2 and latent MMP-9 respectively. As shown in Figure 5, the highest MMP-9 activities were seen in post-menopausal ovaries, but statistical significance was not reached, possibly due to the small number of samples available.
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Discussion |
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No commonly accepted explanation is available for the arrest in follicular maturation that results in accumulation of persistent mid-sized antral follicles in PCOS. The present experimental set-up was primarily designed to test whether connective tissue could participate in this phenomenon. Several novel observations were made, which include differences in the expression levels of pro
1(IV) collagen and TIMP-3, as well as changes in cellular localization of MMP-9 and in staining for TIMP-4 between polycystic and normally functioning ovaries. Apart from these differences, the gene expression profiles of structural ECM components of PCOS samples resembled those observed in normal follicular phase ovaries, which suggests otherwise normal collagen production in PCO. This is in agreement with earlier biochemical analyses on normal and PCOS ovaries (Mori et al., 1984).
In the present study, PCOS samples exhibited somewhat lower transcript levels for pro1(IV) collagen, an important basement membrane component, than normally functioning ovaries, but similar to post-menopausal ovaries. Type IV collagen is produced by cultured ovarian epithelial cells (Kruk et al., 1994) and by LH/hCG-stimulated granulosa cells (Yamada et al., 1999). Granulosa cell-derived type IV collagen has been proposed to be involved in the inhibition of their premature luteinization (Yamada et al., 1999). As premature luteinization is characteristic for granulosa cells obtained from small antral follicles of anovulatory women with PCOS (Willis et al., 1998), decreased type IV collagen production in PCOS observed in the present study could explain the premature response to LH in granulosa cells from women with PCOS. As the structure of the follicular basement membrane is known to change along follicular maturation and atresia, it probably participates in the regulation of survival, proliferation and differentiation of granulosa cells (Rodgers and Irving-Rodgers, 2002).
Recently, Shalev et al. (2001) reported higher MMP-2 and -9 levels in follicular fluid of PCO than in normal ovaries after gonadotrophin stimulation for IVF. They observed increased production of MMP-9 by cultured granulosa cells from PCO (Shalev et al., 2001). In our northern analyses, no statistically significant changes could be observed between polycystic and normal ovaries. By immunohistochemistry, MMP-9 was mainly localized to stromal cells. Only in PCO did some granulosa cells exhibit faint staining for MMP-9. During the normal menstrual cycle, an increase in the expression of MMP-9 was observed in luteal phase samples. Through its ability to remodel basement membranes, MMP-9 could participate in the initial recruitment of follicles during the luteal phase. Stromal localization of MMP-9 supports this view. By analogy, impaired breeding efficiency has been observed in mice lacking MMP-9 (Dubois et al., 2000).
In the present study, lower TIMP-3 mRNA levels were observed in PCO than in normal follicular phase ovaries. TIMP-3 binds firmly to the ECM, where it can regulate molecular and cellular movement through the basement membranes and stroma (Woessner, 2001). As TIMP-3 mainly inhibits MMP-9, decreased TIMP-3 could augment the basement membrane degrading effect of MMP-9 in PCOS and thereby participate in the development of the polycystic ovarian phenotype. In addition to regulating MMP, TIMP also function as growth factors, modulate angiogenesis and regulate steroidogenensis (Gomez et al., 1997). Altered reproductive cyclicity, but normal reproductivity, has been reported in TIMP-1 knockout mice (Nothnick, 2000). Although TIMP-3 has been shown to induce apoptosis in human tumour cells (Ahonen et al., 1998), TIMP-3 null mice exhibit markedly reduced lifespan (Leco et al., 2001), but apparently normal breeding capacity (Fata et al., 2001). In the present study, TIMP-3 immunostaining was observed in all cellular compartments with the strongest staining in granulosa cells of antral follicles. Similar findings have been reported in the human fetal ovary (Robinson et al., 2001).
TIMP-4, the newest member of TIMP family, effectively inhibits the activity of MMP with preference for MMP-2 and -9. In human fetal ovary, TIMP-4 has been localized by immunostaining in oocyte cytoplasm and vascular endothelium (Robinson et al., 2001). In the present study, intense TIMP-4 immunostaining was seen in PCO follicles, particularly in luteinized theca cells, while in normal ovaries, TIMP-4 immunostaining of granulosa cells increased upon follicular maturation. These distribution patterns are largely in agreement with our earlier work on TIMP-4 mRNA in the mouse ovary (Rahkonen et al., 2002).
In an attempt to understand the relationship between folliculogenesis and connective tissue metabolism, we also compared the transcript levels of ECM components between post-menopausal ovaries and normally functioning ovaries in the follicular and luteal phases of the menstrual cycle. Our observations of reduced mRNA levels for type I, III and IV collagens and osteonectin are novel and support the general belief that the overall metabolic activity of the post-menopausal ovary is reduced. A reduction in collagenase activity has been reported in the ageing ovary (Postawski et al., 1999), whereas no information exits about age-dependent changes in the rate of collagen production. Most earlier studies focus on ovarian tumours (e.g. Zhu et al., 1993) or function of ovarian surface epithelial cells (Auersperg et al., 1994; Kruk et al., 1994), but the results have not been correlated to the age of the cases. In the present study, the mRNA levels for the MMP, TIMP (excluding TIMP-3) and cathepsins studied did not reflect similar menopausal status-dependent down-regulation as collagens. Actually, the transcripts for MMP-9 were increased in the post-menopausal ovaries when compared with follicular phase samples. These observations are consistent with the suggested roles of MMP-9 and TIMP-3 in induction of follicular atresia (Huet et al., 1998; Khandoker et al., 2001) and apoptosis (Ahonen et al., 1998) respectively. We suggest that high MMP-9 and reduced TIMP-3 levels in peri- and post-menopausal ovaries contribute to the accelerated follicular loss, leading gradually to total depletion of the resting follicle pool (Faddy, 2000).
The alterations observed in the gene expression profiles in human PCO suggest involvement of basement membranes in the pathogenesis of the disease. At the present time, we do not know whether these changes are primary or secondary. As several other questions about the role of basement membranes during normal folliculogenesis await answers, further conclusions regarding these observations must be based on additional analyses, such as gene expression profiling using cDNA microarrays.
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Acknowledgements |
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This study was financially supported by the Academy of Finland (project no. 52940) and the Turku University Central Hospital (project no. 13449). The assistance of Dr Sofia Sallinen in the collection of control ovarian samples is gratefully acknowledged. The authors are grateful to the expert technical help of Ms Merja Lakkisto and Ms Tuula Oivanen, and the assistance of the personnel at the Ob/Gyn Department of the Turku University Central Hospital. Sanna Oksjoki and Otto Rahkonen have been recipients of a training grant from Turku Graduate School of Biomedical Sciences.
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Submitted on July 8, 2003; resubmitted on August 15, 2003; accepted on August 26, 2003.
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