doi:10.1093/humrep/dei075
Human Reproduction Vol.20, No.9 pp. 2509–2516, 2005
Advance Access publication May 19, 2005
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. E-mail: [email protected]
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
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, 1991; Abel and Hannigan, 1995; Livy et al., 2003;
Martı́nez-Frı́as et al., 2004). Many of the adverse effects of
ethanol persist after birth and extend into adolescence (Amini
et al., 1996). Jones and Smith first described the clinical
characters of FAS in 1973 (Jones and Smith, 1973), 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,
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,
a-fetoprotein, transferrin, ferritin, albumin, pre-albumin,
fibronectin and a1-antitrypsin (Jones and Jauniaux, 1995),
as well as various enzymes involved in digestion and energy
metabolism such as acid phosphatase, galactosidase, lactic
dehydrogenase, g-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
q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Y.Xu et al.
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.
Materials and methods
Experimental animals
Virgin female C57BL/6J mice were housed under controlled conditions of temperature (22 ^ 0.58C), 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) 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 (568C for 30 min)
and filter sterilized, and was supplemented with 100 U/ml penicillin
G and 100 mg/ml streptomycin. The embryos were incubated for
48 h at 37.5 ^ 0.58C 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 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
2510
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-mm 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 mm
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 (GibcoBRL; 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).
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.
Ethanol suppresses visceral yolk sac development
Table I. Primer sets used for RT–PCR
Gene
Primers (upper, sense; lower, antisense)
Product length (bp)
Reference
GAPDH
VEGF
Flk1
Flt1
Ang1
Tie1
Tie2
Ang2
50 -TGAAGGTCGGTGTGAACGGATTTGGC-30 50 -CATGTAGGCCATGAGGTCCACCAC-30
50 -GCCGTCCTGTGTGCCGCTGATG-30 50 -GCCCTCCGGACCCAAAGTGCTC-3
50 -AGAACACCAAAAGAGAGGAACG-30 50 -GCACACAGGCAGAAACCAGTAG-30
50 -TGTGGAGAAACTTGGTGACCT-30 50 -TGGAGAACAGCAGGACTCCTT-30
50 -GGCACGGAAGGCAAGCGCTG-30 50 -CAAGCATGGTGGCCGTGTGG-30
50 -GGGTGGTGCTGGCGCGCGGC-30 50 -CCAGAGGGGCAGACGCAGCC-30
50 -AAGACATACGTGAACACCACACT-30 50 -ACTCTAGAGTCAGAACACACTGCAGAT-30
50 -CGCATTCGCTGTATGATCAC-30 50 -GCACTTCCTGATGTGGAAAG-30
961
536
382
505
413
696
258
447
Chen et al. (2004)
Bi et al. (1999)
Fong et al. (1995)
Shalaby et al. (1995)
Bi et al. (1999)
Bi et al. (1999)
Fong et al. (1995)
Wang et al. (2002)
Table II. Effect of ethanol on the development of mouse visceral yolk sac
Ethanol
concentration (mg/ml)
Na
Yolk sac
diameter (mm)
Vitelline
vesselsb
Yolk sac
protein (mg)c
Yolk sac
DNA (mg)c
Control
1.0
2.0
4.0
20
20
20
20
5.64 ^ 0.19
4.91 ^ 0.24**
4.37 ^ 0.17**
4.22 ^ 0.20**
4.92 ^ 0.08
3.92 ^ 0.26*
2.92 ^ 0.42**
1.83 ^ 0.35**
141.26 ^ 4.71
129.11 ^ 2.38*
111.02 ^ 3.28**
101.02 ^ 6.54**
5.01 ^ 0.12
4.78 ^ 0.09*
4.25 ^ 0.24**
3.82 ^ 0.27**
Data are presented as means ^ SE.
a
The total numbers of embryos in each group whose yolk sac diameter and vitelline vessels development were evaluated.
b
The evaluation of vitelline vessels was according to the morphological scoring system of Van Macle-Fabry et al. (1990).
c
Ten embryos in each group were applied for protein and DNA measurements as mentioned in the Materials and methods.
*P , 0.05 versus control; **P , 0.01 versus control.
Results
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).
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, 1986; Qu
et al, 2000; Costa et al., 2002). Figure 2 shows some representative malformations found in the alcohol-exposed embryos.
Figure 1. Effect of ethanol on the development of mouse visceral
yolk sac in vitro (28£). (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.
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Y.Xu et al.
Table III. Effect of ethanol on the development of mouse embryos
Ethanol
concentration (mg/ml)
n
Crown–rump
length (mm)
Head length
(mm)
Somite
number
Total morphological
scorea
No. neural
tube and/or heart
abnormity/
No. total embryosb
Control
1.0
2.0
4.0
20
20
20
20
4.56 ^ 0.14
4.11 ^ 0.20*
3.42 ^ 0.15**
2.97 ^ 0.24**
2.18 ^ 0.11
2.02 ^ 0.13
1.73 ^ 0.13*
1.33 ^ 0.13**
30.50 ^ 0.38
28.42 ^ 0.31
27.58 ^ 0.47*
21.58 ^ 1.06**
58.75 ^ 1.32
56.28 ^ 1.95
42.75 ^ 2.76**
34.75 ^ 2.94**
0
0
4/20
17/20
Data are presented as means ^ SE.
a
Evaluation of the embryonic development was according to the morphological scoring system of Van Macle-Fabry et al. (1990) as mentioned in the Materials
and methods. The total morphological score here was the sum of each separately evaluated organ’s score.
b
The abnormal neural tube involves delayed neural fold fusion and neural fold malformations as mentioned in the results; the abnormal heart involves retarded
development of the three cardiac bulbs and inflated pericardial cavity.
*P , 0.05 versus control; **P , 0.01 versus control.
Figure 2. Ethanol induced abnormalities in the mouse embryos (42£ ). (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.
2512
Ethanol suppresses visceral yolk sac development
Figure 3. Effect of ethanol on the histological alterations of mouse visceral yolk sac (200 £ ). (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.
Figure 4. SEM of mouse visceral yolk sac endodermal cells (bar
represents 2 mm). (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.
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.
2513
Y.Xu et al.
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).
Discussion
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.
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, 1995; 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, 1990; Chan and Ng, 1995).
The mitochondria of endodermal cells in the control group
were normally shaped, the inner membrane ridges of which
were intact (Figure 5E).
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 concentrationrelated 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
2514
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
Ethanol suppresses visceral yolk sac development
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 17.5day 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 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, 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.
Acknowledgements
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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Submitted on December 20, 2004; resubmitted on April 6, 2005; accepted on
April 18, 2005