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
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Abstract |
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Key words: developmental toxicity/ethanol/mouse embryos/yolk sac
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Introduction |
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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, 1992; Enders and King, 1993
). 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, 1992
; Jones and Jauniaux, 1995
). 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, 1976
; Moore, 1982
; Jones and Jauniaux, 1995
). Similar structure and functions have been found in other mammalian yolk sacs, such as those of rodents (reviewed by Jollie, 1990
).
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., 1994). Additionally, the endodermal layer synthesizes important proteins including apolipoproteins A1 and B,
-fetoprotein, transferrin, ferritin, albumin, pre-albumin, fibronectin and
1-antitrypsin (Jones and Jauniaux, 1995
), as well as various enzymes involved in digestion and energy metabolism such as acid phosphatase, galactosidase, lactic dehydrogenase,
-glutamyl transferase and choline phosphotransferase (Buffe et al., 1993
). 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, 1971
). 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., 1995
; Shalaby et al., 1995
; Carmeliet et al., 1996
; Ferrara et al., 1996
). Fully developed vitelline vessels can transport nutrients to the embryonic circulation and take away embryonic waste more efficiently (Jollie, 1990
).
Nogales et al. (1993) 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, 1966
), yolk sac antibody (Brent et al., 1971
) and excess glucose (Pinter et al., 1986
), 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.
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Materials and methods |
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Whole-embryo culture
In-vitro post-implantation whole embryo culture was carried out according to the method developed by New (1978) and adapted by Van Macle-Fabry et al. (1990)
. 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). Briefly, scores of 06 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 crownrump 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). DNA content per VYS was determined according to Lavarca and Paigen (1980)
.
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 (1218) 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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Results |
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Semi-quantitative RTPCR
The expressions of several genes required for normal vascular development were assayed by RTPCR. 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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Discussion |
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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., 1989; Hunter et al., 1991
; Ambroso and Harris, 1993
). 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., 1995
; Chen and Sulik, 1996
); (ii) ethanol might inhibit the activity of the ATPase located in the microvilli side cell membrane and mitochondrial membrane (Rodrigo, et al., 1998
; Sepulveda and Mata, 2004
), 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, 1990). 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., 1995
). 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, 1995). Steventon and Williams (1987)
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., 2003
). 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 38 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, 1995
), 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.
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Acknowledgements |
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References |
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Submitted on December 20, 2004; resubmitted on April 6, 2005; accepted on April 18, 2005.
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