Effect of ethanol on the development of visceral yolk sac

Yajun Xu, Rong Xiao and Yong Li1

Department of Nutrition & Food Hygiene, Laboratory of Molecular Toxicology & Developmental Molecular Biology, School of Public Health, Peking University, Beijing 100083, China

1 To whom correspondence should be addressed. Email: liyong{at}bjmu.edu.cn


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Prenatal ethanol exposure can cause development retardation and malformations in human offspring. Before the formation of chorioallantoic placenta, yolk sac plays an important role in transporting nutrients from the mother to the embryo. Functional suppression of yolk sac is found to be relevant to the malformations in mammalian embryos. METHODS: Female 8.5-day C57BL/6J mouse embryos were cultured in vitro and exposed to different doses of ethanol. The development of visceral yolk sac (VYS) was examined with light and electron microscopes. The expression profiles of some vasculogenesis-related genes were detected with reverse transcription–PCR. RESULTS: A dose-dependent toxicity to the VYS was found, including reduced diameter, decreased protein and DNA contents, and suppressed development of vitelline vessels. The hypogenesis of VYS agreed with the retarded development and/or malformations found in the embryos. Histological and functional alterations were found in the ethanol-exposed VYS endodermal cells. The expressions of vasculogenesis-related genes, fetal liver kinase 1 (Flk1) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2), were repressed by ethanol. CONCLUSIONS: Impaired structural and functional development of VYS may contribute to the teratogenic action of ethanol in mice, which may also provide a clue to the study of fetal alcohol syndrome in humans.

Key words: developmental toxicity/ethanol/mouse embryos/yolk sac


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human clinical studies and animal research programs have established that maternal ethanol consumption during pregnancy can produce developmental anomalies of the fetuses, which in human is known as fetal alcohol syndrome (FAS), or in less severe forms fetal alcohol effects (FAE) (Abel and Sokol, 1991Go; Abel and Hannigan, 1995Go; Livy et al., 2003Go; Martínez-Frías et al., 2004Go). Many of the adverse effects of ethanol persist after birth and extend into adolescence (Amini et al., 1996Go). Jones and Smith first described the clinical characters of FAS in 1973 (Jones and Smith, 1973Go), but the mechanism of FAS is still not entirely clear. In this research, we explored the effect of ethanol on the structure and function of mammalian yolk sac, hoping to provide a new clue to the mechanism of FAS in human.

The wall of the human yolk sac is formed by a mesothelial layer composed of flattened cells and vessels, and an endodermal layer made of columnar cells (Jauniaux and Moscoso, 1992Go; Enders and King, 1993Go). In the early period of human pregnancy, the yolk sac surrounds the developing embryo and acts as a metabolic active barrier between the mother and the embryo (Jauniaux and Moscoso, 1992Go; Jones and Jauniaux, 1995Go). Although the exact function of human yolk sac is not very clear, many relevant studies have agreed on its role in embryonic nutrition, biosynthesis and hematopoiesis (Gonzalez-Crussi and Roth, 1976Go; Moore, 1982Go; Jones and Jauniaux, 1995Go). Similar structure and functions have been found in other mammalian yolk sacs, such as those of rodents (reviewed by Jollie, 1990Go).

Before the formation of the chorioallantoic placenta, the yolk sac plays a role in the uptake and transport of nutrients from the mother to the developing embryo (Cross et al., 1994Go). Additionally, the endodermal layer synthesizes important proteins including apolipoproteins A1 and B, {alpha}-fetoprotein, transferrin, ferritin, albumin, pre-albumin, fibronectin and {alpha}1-antitrypsin (Jones and Jauniaux, 1995Go), as well as various enzymes involved in digestion and energy metabolism such as acid phosphatase, galactosidase, lactic dehydrogenase, {gamma}-glutamyl transferase and choline phosphotransferase (Buffe et al., 1993Go). The mesoderm layer of yolk sac is considered to be the first site of blood cells production during human and murine ontogenesis (Haar and Ackerman, 1971Go). Blood islands are first formed in the mesoderm layer, from which vitelline vessels develop under the co-regulation of some vasculogenesis-related factors, such as vascular endothelial growth factor (VEGF) and its receptors fetal liver kinase 1 (Flk1) and fms-like tyrosine kinase 1 (Flt1) (Fong et al., 1995Go; Shalaby et al., 1995Go; Carmeliet et al., 1996Go; Ferrara et al., 1996Go). Fully developed vitelline vessels can transport nutrients to the embryonic circulation and take away embryonic waste more efficiently (Jollie, 1990Go).

Nogales et al. (1993)Go noted an association between spontaneous abortion and a reduction in the size or complete absence of the yolk sac, suggesting a relation between yolk sac anomalies and embryonic development. Later researchers found that some agents which suppress yolk sac pinocytotic activity, such as Trypan Blue (Beck and Lloyd, 1966Go), yolk sac antibody (Brent et al., 1971Go) and excess glucose (Pinter et al., 1986Go), could induce development retardation and malformations of the rodent embryos. In this study, we have used a murine FAS model to explore the effect of ethanol on yolk sac development and the relevance to embryonic malformations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Experimental animals
Virgin female C57BL/6J mice were housed under controlled conditions of temperature (22±0.5°C), humidity (50±10%) and lighting (12/12 h light/dark cycle), and were provided with food and water ad libitum. The mice were mated overnight and pregnancies were confirmed the following morning by the presence of a vaginal ‘plug’, and this was considered as gestation day (GD) 0. The use of animals in this research was in accordance with the Guiding Principles in the Care and Use of Animals (DHEW publication, NIH, 80-23).

Whole-embryo culture
In-vitro post-implantation whole embryo culture was carried out according to the method developed by New (1978)Go and adapted by Van Macle-Fabry et al. (1990)Go. Briefly, on GD 8.5, the gravid uteri were removed from the dams and placed in sterile Hank's solution (pH 7.2). Maternal decidual tissue was removed, leaving the visceral yolk sac (VYS) intact. Embryos displaying three to five somite pairs were selected for culture. Culture medium was 100% male rat serum that was immediately centrifuged, heat-inactivated (56°C for 30 min) and filter sterilized, and was supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin. The embryos were incubated for 48 h at 37.5±0.5°C in sealed 50 ml glass bottles (three embryos/bottle, one embryo/ml culture medium), rotated at 40 rev/min. The culture medium was initially pre-gassed for 5 min with 5% O2:5% CO2:90% N2. Subsequent re-gassings occurred at 20 h (20% O2:5% CO2:75% N2) and 30 h (40% O2:5% CO2:55% N2). Ethanol (chromatography reagent; SABC Co.) was added at 1.0, 2.0 and 4.0 mg/ml, respectively. Equivalent sterile phosphate-buffered saline (PBS) was added to the culture medium of the control group.

Morphological evaluations of the VYS and embryos
At the end of the 48 h culture period, the embryos were removed from the culture medium into a plate with pre-warmed sterile Hank's solution (pH 7.2). Morphological evaluation was carried out under a Motic X40 (Germany) stereomicroscope, according to the morphologic scoring system of Van Macle-Fabry et al. (1990)Go. Briefly, scores of 0–6 were used to assess the development of each VYS and embryo. Higher scores represented better developmental status. A total morphological score was finally calculated for each embryo as a general indicator of the overall embryonic development. The VYS diameter, embryonic crown–rump length and head length (defined as the longest distance from the anterior part of forebrain to the dorsal part of midbrain) were also measured. Somite number of each embryo was recorded.

Histological examination
After morphological evaluation, 10 VYS randomly selected from each group were prepared for examinations by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Samples were removed from the same region (equatorial area) of each VYS and cut into three parts with a sterilized scalpel for each of the above three examinations. For light microscopy, the sampled tissue was fixed with fresh 4% paraformaldehyde in sterilized PBS for 24 h. Then, the fixed tissue was embedded in paraffin flatwise, and sectioned to produce 8-µm thick sections, which were mounted on slides, stained with haematoxylin and eosin (H&E), and examined under a Nikon E-400 (Japan) light microscope. For TEM and SEM examinations, the sampled tissue was fixed with 2.5% glutaraldehyde for 24 h, then washed with 0.1 mol/l cacodylate buffer (pH 7.2) and post-fixed with 1% osmium tetroxide in cacodylate buffer (pH 7.2). For TEM, the post-fixed tissue was then washed again with cacodylate buffer, dehydrated in ethanol solutions of serially increasing concentrations and embedded in Epon618. The embedded tissue was cut into sections ~0.5 µm thick with an ultramicrotome (LKB 2088/Uitrotome V; LKB, Japan), and stained with uranyl acetate and lead nitrate before examination with a JEM-100CXII (Japan) TEM. For SEM, the post-fixed tissue was critical-point dried, sputter-coated with gold, and examined with a Hitachi S-450 (Japan) SEM.

Measurements of protein and DNA contents
The remainng 10 VYS of each group were prepared for analysis of protein and DNA content. The amount of total protein per VYS was measured using the method of Bradford (1976)Go. DNA content per VYS was determined according to Lavarca and Paigen (1980)Go.

RNA preparation and semi-quantitative reverse transcription (RT)–PCR
Total RNA of each yolk sac was extracted using TRIzol (Gibco-BRL; Grandisland, NY, USA) according to the manufacturer's recommendations. RNA content was measured with a UV-photometer under 260 nm and normalized before reverse transcription. Two micrograms total RNA was reverse transcribed to first strand complementary DNA (cDNA) using oligodT (12–18) and M-MLV reverse transcriptase (Promega). The primers for PCR and the corresponding gene products are listed in Table I. The expression of the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH), was also assayed to semi-quantify the mRNA abundance in different cDNA samples. The densities of DNA bands were measured with Quantity One 4.4.1 software (Bio-Rad, USA).


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Table I. Primer sets used for RT–PCR

 
Statistical analysis
All analyses were performed using the Statistical Package for Social Sciences for Windows version 11.0 (SPSS Inc., Chicago, IL, USA). Parameters were calculated for each embryo and the data were presented as mean±SE. Between group differences were analysed with one-way analyses of variance. Significant data were further tested with the post-hoc analysis to evaluate the differences between data sets. P≤0.05 was taken as the level of significance for all analyses.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Morphological examination, DNA and protein contents
Ethanol exposure produced concentration-related effects on the growth and development of both the VYS and the embryos. VYS diameter and DNA and protein contents were all decreased by ethanol exposure (Table II), implying that the development of VYS was suppressed by ethanol. Under the stereomicroscope, the vitelline vessels of the control group were thick and fully extended (Figure 1A). Big and serpentine vessel branches were clearly apart from one another. Blood flow could be seen in the lumen. However, the vitelline vessels became thinner and less branched in the 1.0 mg/ml ethanol exposure group (Figure 1B), although blood flow could still be seen. Fewer shaped vitelline vessels were discriminated in the 2.0 mg/ml ethanol exposure group; scattered or circled blood islands were found instead (Figure 1C). The VYS of the 4.0 mg/ml ethanol exposure group appeared opaque under the stereomicroscope, with few or no blood islands (Figure 1D).


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Table II. Effect of ethanol on the development of mouse visceral yolk sac

 


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Figure 1. Effect of ethanol on the development of mouse visceral yolk sac in vitro (28x). (A) Control; (B) 1.0 mg/ml ethanol; (C) 2.0 mg/ml ethanol; (D) 4.0 mg/ml ethanol. VYS of control group showed well-developed blood vessels. The vessels in the 1.0 mg/ml ethanol exposure group became thin and less branched. Fewer shaped vessels were found in the 2.0 mg/ml group; blood islands took their place (indicated with arrows). The VYS exposed to 4.0 mg/ml ethanol appeared opaque and blood islands were seldom found.

 
The embryonic crown–rump length, head length and somite number were all decreased by ethanol exposure (Table III). The total morphologic score, which represented the general development of the embryonic major organs, was reduced in 2.0 and 4.0 mg/ml ethanol exposure groups (Table III). Unclosed neural tube and heart abnormity were the most frequent event found in the ethanol-exposed embryos. Uncompleted fusion of neural fold could be a sign of delayed growth of the embryos. However, there were cases of cephalic fold oedema or atrophy, or both oedema and atrophy existing in the same embryo, which could be malformations cause by ethanol. Delayed cardiac tube development mostly accompanied the neural tube malformations. In the control group, the three bulbs of atrium, ventricle and bulbus aortae could be clearly discriminated in each embryo. However, in the 2.0 and 4.0 mg/ml ethanol exposure groups, cases of undivided heart, which was represented as just one symmetric tube, were found. In addition, pericardial cavity inflation was detected. All these features were in accordance with the previously reported characteristics of FAS (Samson, 1986Go; Qu et al, 2000Go; Costa et al., 2002Go). Figure 2 shows some representative malformations found in the alcohol-exposed embryos.


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Table III. Effect of ethanol on the development of mouse embryos

 


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Figure 2. Ethanol induced abnormalities in the mouse embryos (42x). (A) Normal embryo with completed neural folds fusion and well developed primary heart. (B) Embryo with undivided cardiac tube and inflated pericardial cavity. (C) Embryo with undeveloped forebrain and disclosed midbrain. (D) Embryo with cephalic fold oedema on one side and atrophy on the other. FB=forebrain; MB=midbrain; HB=hindbrain; H=heart.

 
Histological examination of the VYS
H&E-stained tissue sections showed that VYS of the control group consisted of a layer of endodermal cells and a layer of mesodermal tissue. The mesodermal layer contained vitelline vessels (Figure 3A) (Jones and Jauniaux, 1995Go; Shimono and Behringer, 2003). Under SEM, numerous long microvilli with blunt tips were found extending from the apical surface of the endodermal cells (Figure 4A). Further examination of the endodermal cells with TEM showed that there were numerous pinocytotic invaginations of the plasma membrane at the base of the microvilli. A number of lysosomes of different size were near the pinocytotic invaginations and large storage vesicles were found in the cytoplasm (Figure 5A). These phenomena indicated that active pinocytosis was taking place (Jollie, 1990Go; Chan and Ng, 1995Go). The mitochondria of endodermal cells in the control group were normally shaped, the inner membrane ridges of which were intact (Figure 5E).



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Figure 3. Effect of ethanol on the histological alterations of mouse visceral yolk sac (200x). (A) Control: the visceral yolk sac consists of a layer of endodermal cells and a layer of mesodermal tissues containing vitelline vessels (indicated with arrow). Blood cells can be seen in the lumen. (B) In the 1.0 mg/ml ethanol group, the arrangement of endodermal cells was not as orderly as that of control. (C) In the 2.0 mg/ml ethanol group, the arrangement of endodermal cells was disorderly and large intracellular vacuoles (black arrow) were found in the subapical area. (D) In the 4.0 mg/ml ethanol group, intracellular vacuoles (black arrows) and nuclear pycnosis (white arrow) are found. EC=endodermal cells; MC=mesodermal cells.

 


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Figure 4. SEM of mouse visceral yolk sac endodermal cells (bar represents 2 µm). (A) Control; (B) 1.0 mg/ml ethanol; (C) 2.0 mg/ml ethanol; (D) 4.0 mg/ml ethanol. Numerous long microvilli with blunt tips extend from the apical surface of the endodermal cells of control group. However, a concentration-related reduction in the number and sharpening of microvilli on the endodermal cells was found in the ethanol-exposed groups.

 


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Figure 5. TEM of mouse visceral yolk sac endodermal cells. (A) Control group: numerous long microvilli extend from the apical surface of the endodermal cell; plenty of lysosomes are distributed in the top part of cytoplasm, indicating active pinocytosis; pinocytotic invaginations and large storage vesicles were seen beneath the cell surface. (B) Ethanol exposure group: microvilli, lysosome, pinocytotic invaginations and storage vesicles were all decreased in number. (C) Control group: normal nucleus of VYS endodermal cell. (D) In the 4.0 mg/ml ethanol exposure group: nuclear pycnosis. (E) Control group: normal mitochondria with intact inner membrane ridges. (F) In the 4.0 mg/ml ethanol exposure group: mitochondria swell, and the inner membrane ridges disappear. N=nucleus; S=storage vesicles; M=mitochondria; L=lysosome.

 
The VYS endodermal tissue of ethanol-exposed groups was characterized by cell rupture, combined with a collapse of the tissue structure and the appearance of intracellular vacuoles in the subapical area (Figure 3B–D). In the 4.0 mg/ml ethanol exposure group, nuclear pycnosis was found in some endodermal cells, paralleling the structural degeneration and vacuolation of the cells (Figure 3D). SEM examination of the endodermal cells showed concentration-related quantity reduction of microvilli on the apical surface (Figure 4B–D). The number of pinocytotic invaginations was also reduced in the 1.0 and 2.0 mg/ml ethanol exposure groups, and was seldom observed in the 4.0 mg/ml group. The numbers of lysosomes and large storage vesicles were also dramatically decreased (Figure 5B). Condensed chromatin beneath the nuclear envelope was observed in some of the endodermal cells (Figure 5D). Most of the mitochondria in these cells swelled, with the inner membrane ridges disappearing (Figure 5F). These above signs implied that these cells were undergoing apoptosis.

Semi-quantitative RT–PCR
The expressions of several genes required for normal vascular development were assayed by RT–PCR. RNA isolated from the entire VYS was used. The housekeeping gene GAPDH showed similar cDNA loading abundance in this experiment. Reduced expressions of Flk1 and Tie2 were detected. The expression of other genes was not significantly affected by ethanol (Figure 6).



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Figure 6. Semi-quantitative RT–PCR on vasculogenesis- and angiogenesis-related genes. Expression of several genes required for normal vascular development was assayed by RT–PCR. RNA from the entire visceral yolk sac was used. Reduced expression of Flk1 and Tie2 was detected.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this research, we used a murine FAS model to explore the effect of ethanol on the yolk sac development in the organogenesis period. VYS morphological and functional alterations were found in the ethanol exposure groups. In addition, the hypogenesis of VYS agreed with the growth retardation and organ malformations found in the embryos. VYS diameter and total protein and DNA contents were decreased by ethanol exposure. Reasons for this might be that ethanol suppressed VYS cell proliferation and growth, as well as induced excessive apoptosis, which was indicated by the H&E-stained tissue sections and TEM examination in this study.

Yolk sac endodermal cells uptake nutrients by pinocytosis, and transfer them in the form of storage vesicles. In this study, morphological changes were found in the endodermal cells after ethanol exposure. Compared with those in the control group, the endodermal cells in the ethanol exposure groups were arranged in a disorderly manner, combined with a collapse of the tissue structure and the appearance of intracellular vacuoles. Under normal physiological conditions, VYS endodermal cells have apical tight junctions, so as to keep regular arrangement and carry out efficient intercellular communications. The disarrangement and morphological alterations of endodermal cells found in the ethanol exposure group might be induced by the protein denaturation effect of ethanol, which caused collapse of the intercellular tight junction complexes and damage of the cell membrane. As a result, intercellular communications between endodermal cells would be disturbed, and some cellular activities might be lowered. With the aid of SEM and TEM, ultrastructural alterations of the endodermal cells were found in the ethanol exposure groups: microvilli were decreased in number and became sharper; the quantities of pinocytotic invaginations and storage vesicles were reduced; and signs of apoptosis such as nuclear pycnosis and mitochondria swelling appeared. Microvilli are the membrane extension of endodermal cells that enlarge the absorption area. Therefore, the decrease in number and morphological sharpness of microvilli will potentially reduce the total absorption area so as to decrease the efficiency of histiotrophic nutrition. Reduced pinocytotic invaginations and storage vesicles found with the TEM might be a sign of suppressed pinocytotic activity. Although histiotrophic nutrition is a dynamic process and the complete assessment of functional end points related to pinocytosis and vesicle trafficking still needs further experiments, our results at least implied that the initial and important step of VYS histiotrophic nutrition was affected by ethanol. Impaired function of histiotrophic nutrition will lead to retarded growth and malformations of the embryo, which has been reported by previous studies (Balkan et al., 1989Go; Hunter et al., 1991Go; Ambroso and Harris, 1993Go). We considered that the reasons for such alterations might be: (i) the free radicals produced by ethanol metabolism might cause lipid peroxidation of the cell membrane (Kotch et al., 1995Go; Chen and Sulik, 1996Go); (ii) ethanol might inhibit the activity of the ATPase located in the microvilli side cell membrane and mitochondrial membrane (Rodrigo, et al., 1998Go; Sepulveda and Mata, 2004Go), and so effect pinocytosis and storage vesicle transport; and (iii) ethanol might disturb the transmission of pinocytosis related biochemical signals.

Development of the vitelline circulation allows the embryo to shift from reliance on diffusion-dependent nutrient delivery to a more efficient system of vascular conduits (Jollie, 1990Go). In this study, the development of vitelline vessels was also effected by ethanol, which paralleled the delayed growth and malformations of the embryonic cardiovascular system, suggesting that ethanol might have some adverse effects on the vasculogenesis mechanism. The expression of a group of vasculogenesis- and angiogenesis-related genes in VYS were investigated in this research. We found that the expression of Flk1 and Tie2 were suppressed by ethanol, which might contribute to the hypogenesis of VYS blood vessels. It should be mentioned that Flk1 is also crucial in hematopoiesis (Shalaby et al., 1995Go). Whether the ethanol-induced down-regulation of Flk1 might have some effects on the hematopoiesis of the embryos is still to be investigated.

In-vitro ethanol exposure in the early organogenesis period caused morphological and functional alterations of mouse VYS in this study. Although there is a difference between human and mouse yolk sacs, and comprehensive study of human yolk sac is difficult to carry out for ethical reasons, various studies have reported structural and functional similarities between human and murine yolk sacs and confirmed the importance of yolk sac in early human embryo development (reviewed by Jones and Jauniaux, 1995Go). Steventon and Williams (1987)Go found that the pinocytic function of 17.5-day rat VYS could be inhibited by ethanol. Day 17.5 is a relatively late stage of gestation in rats. As ethanol's teratogenicity is widely known, few pregnant women drink alcohol after they know they are pregnant (except for alcoholics). However in some countries, the annual rate of FAS or FAE is still increasing (Eustace et al., 2003Go). One important reason is that far more women drink alcohol in their first trimester, especially the first 8 weeks, when they are not aware of their pregnancy. Therefore, to investigate the effect of ethanol on yolk sac development during the early organogenesis period makes some sense. The first 3–8 weeks of human pregnancy is the embryo organogenesis period. In this period, embryo growth and development take place in the absence of fully developed internal organs (Jones and Jauniaux, 1995Go), so histiotrophic nutrition would seem especially important at this stage. As showed by this study, ethanol could impair the early development and histiotrophic function of yolk sac in mice. We speculate that ethanol may also have adverse effect on human yolk sac development, which might be relevant to the teratogenic action of ethanol in human.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by a grant from the National Natural Sciences Foundations of the People's Republic of China (No. 30271364) and the Major Basic Research Development Program of People's Republic of China (2001CB510305).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 20, 2004; resubmitted on April 6, 2005; accepted on April 18, 2005.





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