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Sexually Dimorphic and Estrogen-Dependent Expression of Estrogen Receptor ß in the Ventromedial Hypothalamus during Rat Postnatal Development

Laboratory of Endocrinology (Y.I., S.H.), Graduate School of Integrated Science, Yokohama City University, Kanazawa-ku, Yokohama 236-0027, Japan; and Comprehensive Reproductive Medicine (A.N.) and Section of Molecular Embryology (A.N., M.-A.I.), Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8549, Japan

Address all correspondence and requests for reprints to: Yayoi Ikeda, Laboratory of Endocrinology, Graduate School of Integrated Science, Yokohama City University, 22–2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. E-mail: yayoi{at}yokohama-cu.ac.jp.

	Abstract

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The ventromedial hypothalamus (VMH) is a sexually dimorphic region of the brain related to female reproductive behavior. The effect of estrogen in the adult rat VMH is thought to be mediated predominantly via estrogen receptor (ER), because this receptor is expressed at considerably higher levels than ERß. The present study revealed, using in situ hybridization and immunohistochemistry, that both ERß mRNA and protein were expressed in the ventrolateral portion of the caudal VMH, at remarkably higher levels during early postnatal development than in adulthood. In addition, the expression was sexually dimorphic, with females having significantly more ERß-immunoreactive (-ir) cells than males, between postnatal d 5 (P5) and P14, although the sex difference was not significant by P21. Double-label immunofluorescence revealed that 66% of ERß-ir cells coexpressed ER in the caudal VMH of the P5 female rat. Furthermore, neonatal treatment with E2 benzoate down-regulated ERß mRNA in the female rat VMH at P5 and decreased VMH ERß-ir cells during the period between P5 and P14. In contrast to females, no differences in expression of ERß mRNA or protein were detected between control and E2 benzoate-treated males. These results suggest that estrogen is involved in regulating the sexually dimorphic expression of ERß in the VMH during early postnatal development of the rat.

	Introduction

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ESTROGEN IS THOUGHT to be critical for regulating sex differentiation of the rodent brain (reviewed in Refs.1, 2, 3). The action of estrogen is mediated via the major estrogen receptor (ER) subtypes ER and ERß (reviewed in Refs.4, 5). The DNA binding regions of both are highly homologous, but the N-terminal transactivation and C-terminal ligand binding domains differ, suggesting that the transcriptional activities of the two ERs differ, depending on the ligands and the promoters of target genes (4, 5, 6, 7). In vitro studies have found that ER-ß heterodimers can be formed (8, 9) and that ERß can modulate ER transcriptional activity as well as responses to ligands in cells coexpressing both ERs (10). The distribution of ERß and ER overlaps in several regions of the brain (11, 12, 13), supporting the notion that ER-ß heterodimers are present in vivo. Estrogen significantly decreases the number of cells coexpressing both ERs, compared with vehicle, in some regions of the brain of the ovariectomized rat (12). Thus, the three types of dimers, which are ERß- and ER-homodimers and ER-ß heterodimers, might play different roles in hormone-regulated brain functions, via a complex regulatory mechanism (14).

The sexually dimorphic development of specific brain regions is affected by the presence or absence of sex hormones during the critical developmental period (15, 16, 17). The ventromedial hypothalamus (VMH) is sexually dimorphic in structure and function and is an important site of estrogen action in the regulation of reproductive behaviors (18). The ER is expressed at high levels in the VMH in a sexually dimorphic manner, with females expressing more than males from early postnatal life until adulthood (19, 20, 21, 22). Moreover, ER is involved in regulating the estrogen-induced sexually dimorphic expression of progesterone receptor (PR), an estrogen-responsive gene that plays important roles in development and function in this region (23). These studies have established that ER plays important roles in the sexually dimorphic function and development of the VMH.

The distribution of ERß differs between males and females in several regions of the adult rat brain (24). Furthermore, ERß expression varies during physiological conditions such as estrus cycle, pregnancy, and lactation (25) and during aging (26). Because steroid hormone levels differ between the sexes and during those conditions, and because ERß expression in several brain regions is affected by sex steroids (12, 13), steroid hormones might regionally and temporally regulate ERß in the brain of adult rats. An early study using semiquantitative PCR found that ERß expression in the mouse hypothalamus/preoptic region is higher in male than in female rats during the period between E17 and P15, although the sex dimorphism in individual regions was unclear (27). Recently, Orikasa et al. (13) demonstrated that levels of ERß are higher in the anteroventral periventricular nucleus of the preoptic area of female than in male rats from P7 through adulthood and this difference can be reversed depending on steroid hormone levels. These studies suggest that ERß, as well as ER, is involved in regulating the sexual differentiation of the brain.

A similar sex difference in the ratio of ERß to ER has been identified in the sheep VMH, although the functional importance was unclear because of low expression (28). Early studies, using in situ hybridization and immunohistochemistry, found only very low levels of ERß mRNA or protein in the VMH of adult female rats (24, 29, 30), and sex differences were undetectable (24). Thus, ER was considered the predominant ER subtype in the VMH of the adult rats (31, 32, 33). However, Temple et al. (34) demonstrated that the sexually dimorphic responses to estrogen of ER and PR are reversed in several brain regions, including the VMH of adult ERß knockout mice, suggesting a role for ERß in the estrogen regulation of these two genes. Recent immunohistochemical studies have discovered that ERß is localized to the ventrolateral portion (vl) of the caudal part of the VMH of adult male (35) and female mice (36). Nomura et al. (35) have further demonstrated that the numbers of ERß immunoreactive (-ir) cells in the VMH do not differ between ER knockout and wild-type male mice, but the effects of estrogen on the ERß expression in this region differ between the two genotypes. These results suggest that estrogen regulates ERß expression in the VMH and that ER is involved in this regulation.

The spatial and temporal distribution, expression level, and sex difference of ERß in the developing VMH has remained obscure. We therefore analyzed the developmental expression profiles of ERß in the rat VMH from E19 to adulthood, using in situ hybridization and/or immunohistochemistry. Double-label immunofluorescence determined the cellular colocalization of ERß with ER in the developing VMH. Furthermore, to determine whether estrogen is involved in the regulation of ERß expression, we examined the effect of E2 benzoate (EB) on ERß expression in the VMH of neonatal rats.

	Materials and Methods

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Animal treatment and tissue preparation
Sprague Dawley rats, maintained under standard conditions, were mated; and the day when copulatory plugs were observed was considered embryonic d 0 (E0). Newborn rats received daily sc injections of EB (10 µg/0.02 ml olive oil; Sigma Chemical Co., St. Louis, MO) or an equal volume of vehicle, from the day of birth [postnatal d 0 (P0)] to P4. This dose of EB and the timing of administration reduce ER expression levels in the VMH of females (20). Fetuses (E19), EB- and vehicle-treated pups (P5, P7, P14, and P21), and adult female rats (3 months old) were deeply anesthetized and transcardially perfused with ice-cold 4% paraformaldehyde in 0.1 M PBS. The brains were removed, placed in fixative overnight at 4 C, and embedded in paraffin. Serial coronal sections (10-µm thickness), cut through the forebrain and mounted on silane-coated slides, were processed for in situ hybridization, immunohistochemistry, or double-label immunofluorescence as described below. All animals were handled in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

In situ hybridization
We generated ³⁵S-labeled sense and antisense riboprobes for ERß (37) and ER (38), using T7, T3, and SP6 RNA polymerases and a SureSite T7 RNA Probe Kit (Novagen Inc., Madison, WI). Serial VMH sections from P5 and adult female rats were analyzed by in situ hybridization using a SureSite Hybridization Reagent Kit (Novagen). Emulsion-coated slides were exposed for 1 or 2 wk at 4 C. In situ hybridization was performed at least twice per tissue sample with each probe. Control experiments used sense probe, and signals remained below background.

Immunohistochemistry
We performed immunohistochemistry on brain sections collected from male and female rats at E19, P5, P7, P14, and P21 and from adult females. Sections were dewaxed in xylene and rehydrated in graded ethanols, and antigen was retrieved by microwave heating in 10 mM citrate buffer (pH 6.0) at full power for 5 min. The sections were incubated in 0.1 M PBS containing 5% BSA (blocking solution) at room temperature for 30 min and subsequently incubated with primary antibody diluted with blocking solution overnight at 4 C. The primary antibodies were rabbit anti-ERß (Z8P; Zymed Laboratories, Inc., South San Francisco, CA; 1:2000 dilution) and mouse anti-ER (1D5; Dako Corp., Kyoto, Japan; 1:5000 dilution). Biotinylated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA) were detected using a Vectastain ABC Elite kit (Vector) and a VectaDAB substrate kit (Vector). Immunoreactivity for both ERß and ER was localized to the nucleus and was undetectable in the cytoplasm. Negative controls were prepared by substituting the primary antibody with normal rabbit or mouse IgG and by omitting the primary and/or the secondary antibody. Negative control staining remained at background levels. To minimize staining variations, sections from control and treated animals of the same age were mounted on the same slide.

Double-label immunofluorescence for ERß with ER
After dewaxing, antigen retrieval, and incubation in blocking solution as described above, sections were incubated overnight at 4 C with mouse anti-ER (1D5) and rabbit anti-ERß (Z8P) in blocking solution. Sections were then incubated in 3% H₂O₂ in 0.1 M PBS for 5 min. After three washes in PBS, the sections were incubated with Alexa Fluor 488 goat antimouse IgG (Molecular Probes, Inc., Eugene, OR) and Alexa Fluor 594 goat antirabbit IgG (Molecular Probes) secondary antibodies for 60 min at room temperature. The sections were rinsed three times for 5 min in PBS and coverslipped with PermaFluor Aqueous Mounting Medium (Shandon, PA). Digitized pictures of the same microscopic field were captured with two bandpass filters specific for the two Alexa Fluors, and cellular nuclei labeled with Alexa Fluors 488 and 594 appear as green and red, respectively. Cellular nuclei immunoreactive for both factors are labeled yellow in merged confocal images. We selected three sections of the VMH of each animal, and the numbers of cells expressing ER or ERß alone and those coexpressing both ERs in each animal were calculated. A simultaneous examination of negative controls (without primary antiserum) confirmed the absence of nonspecific immunofluorescent staining, cross-immunostaining, or fluorescence bleed-through.

Quantitative evaluation of ERß-ir cells in the VMH
ERß-ir cells, counted in every 10th serial section of an entire region of the VMH, are indicated as the sum. The operator who counted the cells was unaware of the group assignment. Sections from three to five animals for each treatment group were examined (see Fig. 7, in which columns represent the mean ± SEM per group). The tissue for each age was analyzed at different times. Because one-way ANOVA (P < 0.05) revealed that the average numbers of ERß-ir cells in left and right hemispheres did not statistically differ, we combined the values from both hemispheres. At each developmental stage, data regarding sex differences and the effects of EB were analyzed by a two-way ANOVA, with planned post hoc tests (Fisher’s protected least significant difference test and Scheffé’s multiple comparison test). A significant difference was established at P < 0.05.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Effect of estrogen on VMH ERß expression during postnatal development. Female and male rats, treated with either 10 µg EB or oil vehicle (control) from P0–P4, were killed on P5 (A), P7 (B), P14 (C), or P21 (D). Graph shows quantitative analysis of ERß-ir cells. Data are presented as means (bars) ± SEM (lines; n = 3–5/group). Bars with different letters are statistically different from each other (P < 0.05).

	Results

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The distribution of ERß mRNA in the VMH was analyzed at the rostral, middle, and caudal levels along the rostro-caudal axis, and a hybridization signal for ERß mRNA was detected in the vl, but not in the dorsomedial portion (dm), at all levels (Fig. 1, A–D). Hybridization signals were faint at the rostral and middle levels (Fig. 1, B and C) but intense at the caudal level (Fig. 1D). Cells expressing ERß mRNA were identified as clusters of silver grains at levels above background over single cells. Higher magnification views demonstrated that cells expressing ERß mRNA were scattered in sections of the rostral and middle levels (Fig. 1, B' and C') and were densely concentrated in the caudal section (Fig. 1D'). Consistent with a previous report (20), intense hybridization signals for ER mRNA were found in the vl at all VMH levels (Fig. 1, E–G), although the number of cells expressing ER mRNA was highest at the caudal level (Fig. 1G). The intensity of the hybridization signals for ER mRNA was higher than that for ERß mRNA at all VMH levels examined.

View larger version (134K): [in this window] [in a new window]   FIG. 1. ERß mRNA is expressed in VMH of neonatal rat brain. Serial coronal sections of VMH from a P5 female rat were analyzed by in situ hybridization with ERß and ER cRNA probes. A, Drawings show the dm and vl of rostral (left), middle (middle), and caudal (right) levels of the VMH. B–D, Detection of ERß mRNA in representative sections cut at rostral (B), middle (C), and caudal (D) levels of VMH. B'–D', Higher magnification views of areas within rectanglesin B–D, respectively; arrows, representative cells with intense hybridization signal. ERß mRNA is localized to vl and attenuated in dm of this region. Hybridization signals are most intense at caudal level (D) but are faint in rostral (B) and middle levels (C). E–G, Expression of ER mRNA in serial sections adjacent to those shown in B–D. H and I, Detection of ERß (H) and ER (I) mRNAs in the PVN. arc, Arcuate nucleus; 3v, third ventricle. Scale bars, 200 µm.

Immunohistochemistry revealed that ERß-ir cells were localized to the vl of the VMH (Fig. 2, A–C). ERß-ir cells were scattered in the rostral and middle VMH sections (Fig. 2, A and B) and concentrated in the caudal section (Fig. 2C). Thus, the distribution of ERß-ir cells closely paralleled that of cells containing ERß mRNA. We further compared expression levels between neonatal and adult rats. In situ hybridization did not identify any signals for ERß mRNA in the VMH of the adult female rat (data not shown). A few ERß-ir cells were detected at all levels of the adult VMH examined, with slightly more in the caudal VMH section (Fig. 2, D–F and D'–F'). These results demonstrated low levels of ERß expression in the adult rat VMH as reported (22, 24, 29, 30) and that neonatal rats expressed considerably more than adult rats.

View larger version (109K): [in this window] [in a new window]   FIG. 2. Comparative expression of ERß protein in VMH between neonatal and adult rats. Serial coronal sections of VMH from a P5 (A–C) and adult (D–F) female rats, analyzed by immunohistochemistry using ERß antibody. Expression of ERß protein in sections cut at rostral (left panels), middle (middle panels), and caudal (right panels) levels of VMH. A'–C' and D'–F', Higher magnification views of areas within rectanglesin A–C and D–F, respectively. Arrows in D' and E', Representative cells with intense immunoreactivity for ERß. Scale bars, 200 µm.

Colocalization of ERß and ER in the VMH of neonatal rats

View larger version (26K): [in this window] [in a new window]   FIG. 3. ERß is coexpressed with ER in some cells in neonatal rat brain VMH. Representative photomicrographs of double-label immunofluorescence for ERß and ER in vl of caudal VMH of a P5 female rat. ERß-ir nuclei (labeled with Alexa Fluor 488) and ER-ir nuclei (labeled with Alexa Fluor 594) are shown in red (left) and green (middle), respectively; and double-labeled cell nuclei are yellow in the merged confocal image (right). Scale bar, 200 µm.

The spatial distribution pattern of ERß-ir cells in the VMH did not differ, at all developmental stages examined or between sexes. At E19, intensely labeled cells were more abundant in females than males (Fig. 4A), but the total number of ERß-ir cells did not significantly differ between the sexes (P = 0.68). The number of ERß-ir cells in the VMH was noticeably larger in females than in males at P7 (Fig. 4B) and P14 (Fig. 4C), and quantitative analyses confirmed that the sex difference was significant between P5 and P14 (P < 0.05; see Fig. 7, A–C). At P21, the number of ERß-ir cells in the VMH tended to be greater in females than in males (Fig. 4D), but the sex difference was not statistically significant (P = 0.14; Fig. 7D).

View larger version (91K): [in this window] [in a new window]   FIG. 4. Developmental expression profile of ERß in VMH of male and female rats. Coronal sections, cut at caudal level of VMH from female (left) and male (right) rats at different developmental stages, analyzed by immunohistochemistry using ERß antibody. A, E19; B, P7; C, P14; D, P21. Scale bars, 100 µm.

The VMH of control females contained more ERß mRNA than did control males (Fig. 5, A and B), and neonatal estrogen exposure decreased ERß mRNA in this region of females to a level lower than that of control males at P5 (Fig. 5, B and C). The expression of ERß mRNA in the VMH of male rats was not changed by EB (data not shown). In agreement with a previous report (20), the number of ER-ir cells in the VMH of females was reduced by exposure to EB at P5 (Fig. 6, A and B). Similarly, the number of ERß-ir cells in the VMH of females was decreased by EB (Fig. 6, C and D). At P5, two-way ANOVA revealed a significant main effect of treatment [F(1, 5) = 203.17, P < 0.05] and a significant sex X treatment interaction [F(1, 5) = 237.08, P < 0.05]. Post hoc tests revealed that the ERß-ir cell number of EB-treated females was significantly lower, compared with control females (P < 0.05) and males (P < 0.05) (Fig. 7A). At P7, two-way ANOVA revealed a significant main effect of sex [F(1, 5) = 7.60, P < 0.05] and a significant main effect of treatment [F(1, 5) = 82.80, P < 0.05], as well as a significant sex X treatment interaction [F(1, 5) = 63.42, P < 0.05]. Post hoc tests revealed that the ERß-ir cell number of EB-treated females was significantly lower than that of control females (P < 0.05) but was not significantly different from that of control males (P > 0.05) (Fig. 7B). At P14, two-way ANOVA revealed a significant main effect of sex [F(1, 5) = 713.48, P < 0.05] and a significant main effect of treatment [F(1, 5) = 64.20, P < 0.05], as well as a significant sex X treatment interaction [F(1, 5) = 59.25, P < 0.05]. Post hoc analyses revealed that the ERß-ir cell number of EB-treated females was significantly lower than that of control females (P < 0.05) and was significantly higher than that of control males (P < 0.05) (Fig. 7C). At P21, no significant difference was observed among groups (P > 0.05) (Fig. 7D). In contrast to females, the number of ERß-ir cells of males did not differ significantly between control and EB-treated groups, at all developmental stages examined (P > 0.05) (Fig. 7, A–D).

View larger version (98K): [in this window] [in a new window]   FIG. 5. Effect of neonatal exposure to estrogen on ERß mRNA expression in VMH of P5 rats. Coronal sections, cut at caudal level of VMH from control female (A), control male (B), and EB-treated female (C) rats at P5, analyzed by in situ hybridization with the ERß probe. Scale bars, 200 µm.

View larger version (65K): [in this window] [in a new window]   FIG. 6. Effect of neonatal exposure to estrogen on ER and ERß protein expression in VMH of P5 rats. Coronal sections, cut at caudal level of VMH from control female (A and C) and EB-treated female (B and D) rats at P5, analyzed by immunohistochemistry using ER and ERß antibodies. Scale bars, 200 µm.

	Discussion

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The expression of ERß and ER differed in the developing VMH. The intensity of hybridization signal for ERß mRNA was weaker, compared with ER mRNA, in both neonatal and adult rats. This is consistent with the immunohistochemical results showing less ERß-ir than ER-ir cells. Thus, both the intensity of the in situ hybridization signals and the number of immunoreactive cells indicate that the expression levels of ERß mRNA and protein are lower than those of ER in the VMH. Although mRNA and protein for both ERß and ER were highest in the vl, some ER mRNA and protein were expressed in the medial and central portions, whereas ERß mRNA and protein were located essentially in the vl, with minimal expression in other portions. These results indicate that the distribution of both ERß mRNA and protein is confined to the ventrolateral and caudal VMH.

Double-label immunofluorescence revealed that the caudal VMH of neonatal rats contains cells expressing either ER or ERß, as well as cells expressing both ERs. This is in contrast to previous results using double-label in situ hybridization/immunohistochemistry, which detected only a few cells containing both ERß mRNA and ER protein in the adult rat VMH (11). Although the techniques used in the two studies differed, the number of cells coexpressing both ERs in the VMH seemed to be greater during the early postnatal period than in adulthood. In vitro studies have shown that ER-ß heterodimers can be formed in vitro and that the transcriptional activities of ER and ERß differ, depending on which dimer is formed (8, 9, 10). Our results suggest that the developing VMH is a brain region containing ER-ß heterodimers as well as ER- and ERß-homodimers.

The expression of both ERß mRNA and protein was sexually dimorphic at higher levels in females than in males in the developing VMH. The sex difference was evident between P5 and P14 but was not significant by P21. Zhang et al. (35) described the sexually different expression of ERß in several brain regions of adult rats, but the VMH was not among them. Because ERß is expressed at very low levels in this region of adult rats, the sex difference may be very small or insignificant.

A recent immunohistochemical study found that gonadectomy increases, and estrogen decreases, ERß expression in the VMH of adult male mice (35), indicating that ERß in the VMH of adult animals is responsive to sex steroids. The present study demonstrated that neonatal exposure to estrogen caused down-regulation of ERß in the developing VMH of female rats, suggesting that ERß in the neonatal rat VMH is also responsive to estrogen. The levels of ERß mRNA and protein in the EB-treated female VMH were below those in control females and similar to those of males between P5 and P14, suggesting that neonatal estrogen can alter the ERß expression profile in the VMH of female rats to that of males. In contrast to females, VMH ERß expression in males was not affected by EB. Because the gonads were intact, ERß-ir cells in the VMH of male rats might already have been reduced through metabolic estrogen derived from neural aromatization of testicular testosterone during the prenatal period, and this region would have been insensitive to estrogen by the time of EB action. Alternatively, ERß in the developing VMH of male rats might be regulated by testosterone but not by estrogen. Meanwhile, ERß in the female VMH during this period should be sensitive to estrogen, because the ovary does not produce estrogen around the time of birth. Thus, our results suggest that estrogen is involved in sexually dimorphic ERß expression in the VMH during early postnatal development of the rat.

In vitro studies have proposed that ERß can act as a dominant negative regulator of ER when the two ERs are coexpressed, and that the relative expression level of the two ERs is critical for cellular responses to ligands (10, 14). The effects of estrogen on the ratio of colocalization have been studied in several brain regions of ovariectomized, adult female rats. The results supported the notion that the actions of estrogen differ (depending on whether a cell expresses ER, ERß, or both) and that ERs differently function in mediating actions of estrogens (depending on which type of dimer is formed in each cell) (12). Several recent reports have suggested that the two receptors interact in the VMH. Temple et al. (34) have demonstrated that the sexually dimorphic responses to estrogen of ER and PR in the VMH are affected in adult ERß knockout mice, indicating that ERß is involved in the estrogen regulation of these two genes. Nomura et al. (35) have reported that the effects of estrogen on VMH ERß expression differ between ER knockout mice and their wild-type littermates, suggesting that ER is involved in ERß regulation by estrogen in this region. The present study found that ERß in the developing VMH is expressed in a sexually dimorphic manner, partially colocalized with ER, and is reduced by neonatal exposure to estrogen. Thus, ERß seems to function in regulating the estrogen-induced, sexually dimorphic development of the VMH, and the two ERs might interact during such regulation. To understand how the two ERs function in controlling cellular sensitivity to hormones in the developing VMH, studies should examine whether the ratio of coexpression differs according to sex and whether the ratio of coexpression for each sex varies after estrogen exposure.

	Acknowledgments

 
We thank Dr. J.-A. Gustafsson for providing rat ERß cDNA. We also thank H. Koibuchi for technical assistance.

	Footnotes

 
This work was supported in part by Grants-in-Aid for Scientific Research from Yokohama City University, Japan (to Y.I.).

Abbreviations: dm, Dorsomedial portion; E, embryonic day; EB, E2 benzoate; ER, estrogen receptor; -ir, immunoreactive; P, postnatal day; PR, progesterone receptor; PVN, paraventricular nucleus; vl, ventrolateral portion; VMH, ventromedial hypothalamus.

Received February 28, 2003.

Accepted for publication July 14, 2003.

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