J. Embryol. exp. Morph. Vol. 71, pp. 63-74,1982
Printed in Great Britain © Company of Biologists Limited 1982
63
The effect of teratogenic
antiserum on yolk-sac function in rat embryos
cultured in vitro
By STUART J. FREEMAN1, ROBERT L. BRENT2
AND JOHN B.LLOYD3
From the University of Keele and the
Thomas Jefferson University
SUMMARY
The teratogenicity of rabbit anti-rat visceral yolk-sac antiserum, injected into pregnant rats at
either 8-5 or 9-5 days of gestation, has been confirmed. Normal rabbit serum was found not to
be teratogenic.
When conceptuses from 9-5-day pregnant rats were cultured for 48 h in heat-denatured
homologous serum, to which antiserum was added for the final (or the penultimate) 6 h of
culture, embryonic development was normal. The protein contents of embryos and yolk sacs
(at harvesting) were however decreased.
When antiserum was present in cultures for the final 6 h, pinocytosis by the yolk sac, as
measured by the uptake of 125l-labelled polyvinylpyrrolidone (PVP), was decreased to an
extent related to the concentration of antiserum in the culture medium and to a minimum
level of about 40%. The presence of antiserum in cultures for the penultimate 6 h only, with
125
I-labelled PVP present for the final 6 h only, produced an identical result. No uptake of
radioactivity into the embryo was observed, in either the absence or presence of antiserum.
When conceptuses were cultured for the final 6 h in vitamin- and glucose-supplemented
dialysed homologous serum whose proteins were [3H]leucine-labelled, the presence of antiserum for either the final or penultimate 6 h again resulted in a decrease in the uptake of
radioactivity by conceptuses. Uptake of radioactivity into yolk sac and embryo was decreased by the same amount, indicating that proteolysis in yolk-sac lysosomes was not inhibited.
In parallel control experiments in which normal rabbit serum replaced rabbit anti-rat
visceral yolk-sac antiserum, no effects on embryonic development, on protein contents of yolk
sacs and embryos at harvesting, or on the uptake of radioactivity by conceptuses were
observed.
These results are interpreted as providing evidence that teratogenic antibodies decrease
pinocytosis of protein by visceral yolk sac at the early organogenesis stage and consequentially
decrease the availability of amino acids and thus protein synthesis in both yolk sac and
embryo. It is proposed that this effect constitutes the mechanism of action of teratogenic
antisera.
1
Author's address: Research Institute, The Hospital for Sick Children, 555 University
Avenue, Toronto, Ontario M5G 1X8, Canada.
2
Author's address: Stein Research Center, Jefferson Medical College, Philadelphia 19107,
U.S.A.
3
Author's address: Department of Biological Sciences, University of Keele, Keele,
Staffordshire ST5 5BG, U.K.
3-2
64
S. J. FREEMAN, R. L. BRENT AND J. B. LLOYD
INTRODUCTION
In a series of papers Brent and colleagues have reported the teratogenic effects
in rat of certain maternally administered antisera. The finding (Slotnick & Brent,
1966) that sheep anti-rat kidney antiserum, when administered to pregnant rats
at 8 days of gestation, localized strongly in the visceral yolk sac, together with
evidence that the yolk sac plays a nutritional role during early organogenesis
(Beck, Lloyd & Griffiths, 1967 a) led to a suggested mechanism of action. It was
proposed (Brent, Johnson & Jensen, 1971; Lloyd, 1970) that the antisera might
cause developmental anomaly by interfering with the uptake or digestion of
macromolecular nutrients by the epithelial cells of the visceral yolk sac. The
same authors drew attention to the parallels with teratogenesis by trypan blue,
which had been attributed (Beck, Lloyd & Griffiths, 1961 b) to the same proposed
mechanism. Further evidence that the visceral yolk sac was the primary target in
antiserum-induced teratogenesis came from experiments by New and Brent
(1972) using rat embryos cultured in vitro and by Jensen, Koszalka and Brent
(1975), who showed that adsorption by visceral yolk sac, but not by Reichert's
membrane, destroys the teratogenic potency of antisera. Kobrin & Brent (1973)
briefly reported that teratogenic antisera alter the distribution of radioactivity in
rat conceptuses following an injection of free and bound [14C]leucine; they
interpret their data as indicating that the antiserum interferes with intracellular
digestion of protein and/or the trans-yolk-sac passage of small molecules or
macromolecules.
Freeman, Beck & Lloyd (1981) have recently described experiments that
provide direct evidence that the 9-5- to 11-5-day rat embryo obtains amino acids
from proteins that are pinocytosed by the yolk sac and digested within that
tissue's lysosomes. The present paper describes the effects of a teratogenic
antiserum on this nutritional route in the early rat embryo.
MATERIALS AND METHODS
Rabbit anti-rat visceral sac antiserum
A New Zealand albino rabbit received four weekly injections each of 5-6 mg
of lyophilized 18-day rat visceral yolk sac, as described by Brent et al. (1971).
y-Globulin comprised 30 % of the final antiserum preparation.
Teratogenesis experiments
Pregnant Wistar rats received a single intraperitoneal injection of anti-rat
visceral yolk-sac antiserum or normal rabbit serum. Each injection contained
100 mg serum protein per kg body weight. At 20-5 days the uterus was removed,
and the numbers of resorption sites and live foetuses recorded. Each foetus was
weighed and examined for evidence of external malformation. Two out of
three foetuses were then fixed in Bouin's fluid, and sectioned freehand with a
Antiserum,
8-5 days
Normal
rabbit
serum,
8-5 days
Antiserum,
9-5 days
Normal
rabbit
serum,
9-5 days
timing
of
injections
Nature
87
39
84
49
4
8
4
No. of
implantations
8
No of
pregnant
rats
treated
6
0
3-82
0
48
3-65
3-73*
2-18*
No.
0
27-3
0
87-3
%of
surviving
foetuses
0
7-1
0
55-2
% of total
implantations
Foetuses
with external abnormalities
32
16
26
37
No. of
foetuses
sectioned
* Significantly different (P < 0-001) as judged by Student's /-test.
1(2%)
62(74%)
1(3%)
32(37%)
Average
weight
nf
Ui
surviving
foetuses
Resorptions (g)
1(3%)
14(88%)
1 (4%)
37(100%)
Foetuses
sectioned
showing
internal/
external
abnormalities
16
6
12
18
1(6%)
4(67%)
3(25%)
i-
•5
g
Si
Ci'
1
17(94%) %
with
stained
alizarin showing
defects
red S
ctai-npH
No. of
foetuses
Table 1. Teratogenic effects of intraperitoneal injection of rabbit anti-rat yolk <mtiserum or normal rabbit serum
Tera\
66
S. J. FREEMAN, R. L. BRENT AND J. B. LLOYD
Table 2. Abnormalities detected after rabbit anti-rat yolk-sac
antiserum or normal rabbit serum treatment
Antiserum
Day of injection
Normal rabbit serum
8-5
9-5
8-5
9-5
53 (55)
31 (48)
13(48)
15(48)
1(55)
1(55)
2(55)
1(55)
10 (22)
9(16)
1(16)
3(16)
—
—
—
—
—
—
—
1(6)
4(6)
1(6)
1 (38)
—
—
—
—
—
—
—
—
—
—
3(12)
—
—
1 (32)
—
—
—
—
—
Abnormalities
Anophthalmia
Hydrocephalus
Kidneys (absent or malformed)
Gonads (absent or malformed)
Exencephaly
Limb defects
Spina bifida
Ventral hernia
Facial defects
Tail defects
Jaw defects
Spine
Sternum (totally or partly absent)
Ribs
2(55)
2(55)
2(18)
8(18)
17(18)
8(18)
—
—
—
1(16)
—
Figures in brackets indicate the size of the population examined for the relevant malformation.
razor blade to detect soft tissue malformations, each third foetus being cleared
with KOH and stained with alizarin red-S to reveal skeletal anomalies, following
the procedure of Wilson (1965).
Cultures of 9-5-day rat egg cylinders in the presence of radiolabelled macromolecules
The techniques were as described by Freeman et al. (1981). The total culture
period in all cases was 48 h, with antiserum or normal rabbit serum present
either for the final 6 h or for the penultimate 6 h (i.e. from 36 to 42 h from the
start of the culture).
(/) 125I-LabeIled polyvinylpyrrolidone (PVP) as substrate. The radiolabelled
substrate (2-3 /*g/ml) was present only for the final 6 h of culture. Where
antiserum or normal rabbit serum was present for the final 6 h, serum and
125
I-labelled PVP were simply added together at 42 h from the start of the culture.
Where antiserum was present for the penultimate 6 h, it was added at 36 h from
the start of the culture, and at 42 h the conceptuses were transferred to fresh rat
serum (without antiserum) containing 125I-labelled PVP.
(ii) [3H]Leucine-labelled serum proteins as substrate. When antiserum or
normal rabbit serum were present for the final 6 h of culture, conceptuses were
cultured for 42 h in normal rat serum and then transferred to vitamin and
glucose-supplemented dialysed [3H]leucine-labelled rat serum (Freeman et al.
Teratogenic antiserum-effect on rat embryo cultures
67
Table 3. Protein contents (mean ± S.D.) of yolk sacs and embryos after incubation of 9-5-day rat conceptuses in vitro for 48 h, with added rabbit serum present
for the final 6 h
(Values are from at least six determinations.)
Concentrations of
Owl
Protein content (/tg) of tissue
lilll
added
Normal
rabbit
serum
(jig protein/ml)
Embryo
Yolk sac
0
83
167
333
182-3 ±18-4
179-1 ±22-3
165-8 ±27-3
189-7 ±15-4
176-2 ±12-8
177-2±17-1
188-0±21-0
199-2 ±16-6
189-3±21-3
185-8 ±24-6
160-7 ±14-9
156-2 ± 8-6
129-3 ±13-2
132-4 ±16-7
108-5 ±22-6
120-8+17-8
121-0± 7-6
120-1 ± 9-2
138-1 ±10-2
119-8± 8-6
109-5 ± 8-3
1000± 5-9
94-6 ± 9-4
84-5 ± 6-6
1667
3333
Rabbit
anti-rat
visceral
yolk-sac
antiserum
0
83
167
333
1667
3333
Table 4. Radioactivity associated with yolk sacs and embryos after incubation of
9-5-day rat conceptuses in vitro for 48 h with 125I-labelled PVP present for the
final or the penultimate 6 h
(Values are expressed as clearances and are mean ±S.D. for five determinations.)
Nature of
and timing of
exposure to
rabbit serum
Normal serum
(final 6 h)
Concentration of
added serum
(/tg protein/ml)
Radioactivity
in yolk sac
(^wl/mg protein)
Radioactivity
in embryo
(/tl/mg protein)
0
83
167
333
6-5 ±1-57
7-9 ±0-77
7-511-25
7-9 ±0-86
7-1 ±0-79
60 ±101
8-2 ±0-69
5-9 ±0-54
5-0 ±0-77
4-7 ±0-46
3-9 + 0-50
3-5 ±0-58
7-6 ±0-52
5-8 ±0-90
4-4 ±0-50
0-5 ±0-38
1-1 ±0-39
0-4 ±0-38
0-5 ±0-35
0-5 ±0-46
0-4 ±0-37
0-7 ±0-23
0-3±0-15
0-2±0-19
0-5 ±0-27
0-4 ±0-20
0-2 ±0-31
0-4±0-16
0-4 ±0-23
0-5 ±0-27
1667
3333
Anti-rat
visceral yolksac antiserum
(final 6 h)
0
83
167
333
1667
3333
Anti-rat
visceral yolksac antiserum*
(penultimate 6 h)
0
83
333
* Values are derived from ten determinations.
68
S. J. FREEMAN, R. L. BRENT AND J. B. LLOYD
A
m
f 9
t>
8
I
?
•o
6
s
5
4
i
0
83
333
Antiserum concentration (/ig protein/ml)
15
1
S
14
13
^
12
2P 11
I 10
9
%
I 8
o
0
7
1 6
I
^
^
I
5
1 4
0
83
333
Normal rabbit serum concentration
(/jg protein/ml)
Fig. 1. Radioactivity associated with yolk sac (hatched) and embryo (plain) after incubation for the final 6 h of culture in vitamin- and glucose-supplemented dialysed
serum whose proteins were [3H]leucine-labelled, containing either anti-rat visceral
yolk-sac anti-serum (1 A) or normal rabbit serum (IB). Each left-hand column expresses uptake as a function of yolk-sac protein content, and each right-hand column
expresses uptake as a function of embryo protein content. Values are means (with S.D.)
of at least six determinations.
Teratogenic antiserum-effect on rat embryo cultures
I
13
|
10
1
3
H
2
69
1 12
i
fJ
1
0
0
83
333
Antiserum concentration (jug protein/ml/
Fig. 2. Radioactivity associated with yolk sac (hatched) and embryo (plain) after
incubation for the penultimate 6 h of culture in the presence of anti-rat yolk-sac antiserum, and for the final 6 h of culture in vitamin- and glucose-supplemented serum
containing [3H]leucine-labelled proteins. Each left-hand column expresses uptake as
a function of yolk-sac protein content and each right-hand column expresses uptake
as a function of embryo protein content. Values are means (with S.D.) of at least eight
determinations.
1981) containing antiserum or normal rabbit serum. Where antiserum was
present for the penultimate 6 h, it was added at 36 h from the start of a culture
in normal rat serum, and at 42 h the conceptuses were transferred to vitaminand glucose-supplemented [3H]leucine-labelled rat serum (without antiserum).
RESULTS
Teratogenicity of anti-rat visceral yolk-sac antiserum
Table 1 is a summary of the results obtained and Table 2 an analysis of the
types of malformation detected.
Treatment with normal rabbit serum at either 8-5 or 9-5 days of gestation
resulted in a very low incidence of foetal resorption and malformation. In
contrast, anti-rat visceral yolk-sac antiserum caused high rates of foetal resorption and abnormality. Following injection at 8-5 days, anti-rat visceral yolk-sac
antiserum caused malformations in all foetuses surviving to 20-5 days. The
average weight of these foetuses was significantly lower than the average weight
of foetuses surviving maternal injection of normal rabbit serum at 8-5 days.
Treatment at 9-5 days with anti-rat yolk-sac antiserum was lethal to more than
70
S. J. FREEMAN, R. L. BRENT AND J. B. LLOYD
Table 5. Comparison of the effects of various concentrations of
anti-rat visceral yolk-sac antisera
Concentration
of antiserum in
culture media or
rat extracellular
fluid
33 /Mg/ml
83 /*g/ml
125/tg/ml
250 /Ag/ml
312/*g/ml
Teratogenic effects
, Incorporation of
protein-derived
[3H]leucine
fNew & Brent, 1972) (Present experiment)
A
In vitro
In vivo
(Brent et al. 1971;
Jensen et al. 1975)
—
—
No effect
Foetal LD50
Growth retardation
Malformation
—
No effect
—
—
—
—
—
Foetal LD 70
Growth retardation
Malformation
333/^g/ml
3*3 mg/ml
No effect
Growth retardation
Growth retardation
and death
Decreased by
approx. 40 %
70% of foetuses. Of the surviving foetuses, nearly all were malformed. Table 2
shows that the relative frequency of the different malformations produced by
antiserum was similar whether treatment was at 8-5 or 9-5 days of gestation.
Abnormalities of the eyes, the cerebral ventricular system, the urogenital system
and the axial skeleton were particularly common.
Effects of teratogenic antiserum on uptake ofradiolabelledmacromolecules in vitro
The inclusion of antiserum or normal rabbit serum in the culture medium for
either the final or penultimate 6 h of a 48 h culture did not decrease the yield of
viable conceptuses or cause developmental deviations. However, the protein
contents at harvesting of yolk sacs and embryos of conceptuses incubated for the
final 6 h of culture in a medium containing antiserum were decreased (Table 3).
Higher concentrations of antiserum were needed to effect a significant decrease
in embryonic than in yolk-sac protein. The presence of normal rabbit serum had
no effect on protein content at harvesting (Table 3).
(i) Uptake of125I-labelledPVP. Table 4 shows the effect of various concentrations of antiserum or normal rabbit serum on the uptake of 125I-labelled PVP
by conceptuses. As previously found (Freeman et al. 1981), little or no radioactivity was detectable in the embryo. With antiserum present in the culture
medium for the final 6 h of a 48 h culture, radioactivity in the yolk sac was 61 %
of the control value, at an antiserum concentration of 167/tg/ml. At higher
concentrations of antiserum, uptake was decreased further but less sharply,
Teratogenic antiserum-effect on rat embryo cultures
71
tending to a minimum value of about 40 % of the control. No effect was seen
with normal rabbit serum.
For all subsequent experiments two concentrations only of antisera (83 and
333 /*g/ml) were used, those inducing intermediate and near-maximal inhibition
of uptake of 125I-labelled PVP. When conceptuses were exposed to these antiserum concentrations for the penultimate 6 h period, the extent to which the
uptake of radioactivity into the visceral yolk sac was inhibited (Table 4) was
virtually identical to that seen when antiserum and radiolabelled substrate were
present in the culture medium at the same time.
(//) Uptake of [3H]leucine-labelled serum proteins. Freeman et al. (1981)
showed that, if conceptuses are cultured in vitamin- and glucose-supplemented
dialysed [3H]leucine-labelled rat serum, uptake by the visceral yolk sac is
followed by intracellular digestion of labelled protein and the accumulation in
both visceral yolk sac and embryo of proteins that have incorporated [3H]leucine.
Figure 1 shows that antiserum (83 fig serum protein/ml) present in the culture
medium for the final 6 h of a 48 h culture period did not affect either the total
uptake of radioactivity or the distribution of radiolabel between the embryo and
the visceral yolk sac. The percentage of tissue radioactivity that was soluble
in 6-7% (w/v) trichloroacetic acid (TCA) was also unchanged (results not
shown). However, at a concentration of 333 /*g/ml, antiserum, but not normal
rabbit serum, decreased the total amount of radioactivity taken up by conceptuses to a level of 62 % of the control value. The distribution in the conceptus
of radiolabel, and the proportion that was TCA-soluble, was not altered.
When conceptuses were exposed to antiserum for 6 h before being transferred to
serum containing [3H]leucine-labelled serum proteins and cultured for a further
6 h, essentially identical data were obtained (Figure 2). The lower concentration
(83 /Ag/ml) of antiserum had no effect on the incorporation of radiolabel into
the tissues of conceptuses, whereas the higher concentration (333 /*g/ml) decreased the total uptake of radioactivity by conceptuses to 55 % of the control
value. As before, the distribution of radiolabel and the proportion that was
TCA-soluble were not altered.
DISCUSSION
Our first concern was to demonstrate the teratogenic potency of anti-rat
visceral yolk-sac antiserum in the strain of Wistar rat to be used for the subsequent experiments on rat embryos cultured in vitro. The observed incidence
of foetal resorptions and malformations and the nature of the malformations
induced are entirely consistent with earlier reports (Brent et al. 1971; Jensen
et al. 1975).
When Cockroft (1979) showed that 9-5-day rat embryos could grow and
develop in vitro in a medium comprising dialysed rat serum to which only
glucose and certain vitamins had been added, it was apparent that the amino
72
S. J. FREEMAN, R. L. BRENT AND J. B. LLOYD
acids needed for synthesis of embryonic proteins must be derived from protein
and not from free amino acids. Our recent experiments (Freeman et al. 1981)
identify the source and supply route: exogenous serum proteins are captured by
pinocytosis into the visceral yolk-sac epithelial cells, whereupon they are digested
within the lysosomes to amino acids, which are used for protein synthesis in both
the visceral yolk sac and the embryo.
The present experiments have shown that a teratogenic antiserum to visceral
yolk sac decreases the pinocytic uptake of macromolecules by the visceral yolk
sac of early-organogenesis-stage rat embryos cultured in vitro. When [3H]leucinelabelled protein was the macromolecule used, antiserum (at the higher concentration) decreased the extent of incorporation of [3H]leucine into both
embryo and visceral yolk sac. These effects were not seen in conceptuses exposed
to normal rabbit serum. The data afford convincing evidence in support of the
hypothesis that teratogenic antibodies perturb normal embryogenesis by
inhibiting uptake of macromolecular nutrients by the visceral yolk sac and in
consequence decreasing the supply of nutrients to the embryo at a critical stage
of its development. The precise target in the yolk sac appears to be the process
of pinocytosis: the data indicate that the intralysosomal digestion of ingested
macromolecules and the incorporation of digestion products into the embryo
proceeds normally, although at a decreased rate owing to the inhibition of
pinocytosis. The proposed mechanism of action precisely parallels that advanced
to explain the teratogenic action of trypan blue (Williams et al. 1976).
It is of interest to compare the effective concentrations of antiserum in the
present experiments with those reported to exert teratogenic effects in vivo
(Brent et al. 1971; Jensen et al. 1975), and in vitro (New & Brent, 1972). In vivo,
no malformations, embryonic death or growth retardation were observed in
pregnant rats given less than 50 mg/kg of teratogenic yolk-sac antiserum. The
concentration of teratogenic antiserum in the extracellular fluid when administered at 50 mg/kg (foetal LD0), 100 mg/kg (LD50) and 125 mg/kg (LD70) are
125/*g/ml, 250/tg/ml and 312/tg/ml. In vitro, the no effect concentration
was 33/ig/ml and the first concentration that produced embryonic growth
retardation was 333 /^g/ml of culture media. Table 5 summarizes these data and
shows that there is good correspondence between concentrations of antiserum
that are teratogenic and those that markedly decrease the incorporation of
protein-derived [3H]leucine into embryonic protein in vitro.
There is an apparent inconsistency in the data from experiments using the
lower concentration (83 /ig protein/ml) of teratogenic antiserum. Although
there was no effect on uptake of 3H-labelled serum proteins by the yolk sac or on
the incorporation of radiolabel into the embryo, this concentration of antiserum
inhibits pinocytosis of 125I-labelled PVP into the visceral yolk sac. At present we
have no explanation for this anomaly.
It is interesting that the effectiveness of teratogenic antibody in inhibiting
pinocytosis by the visceral yolk sac is as great if the antibody is present in the
Teratogenic antiserum-effect on rat embryo cultures
73
penultimate 6 h of culture as if it is present, together with the radiolabelled
macromolecule, for the final 6 h. Antibody is ingested by the visceral yolk-sac
cells, presumably by pinocytosis into the lysosomes, where it remains undigested
for several days (Brent et al. 1971). The demonstration (Schneider, Tulkens, De
Duve & Trouet, 1979) that membrane recycling between lysosomes and plasma
membrane can carry with it anti-plasma membrane antibody probably explains
how an intralysosomal material can continue to affect an event at the plasma
membrane. It could also explain how antibody injected into rats at 2-5 or 4-5
days of gestation induces malformations whose nature indicates an effect on
embryonic development several days later (Brent, 1966). The mechanism by
which antibody inhibits pinocytosis remains to be resolved.
The antibody used in our experiments was prepared using whole rat visceral
yolk sac as antigen. Experiments with antibody prepared against various subcellular fractions of the visceral yolk sac (M. Jensen, T. R. Koszalka & R. L.
Brent, unpublished data) indicate a wide distribution of the antigen or antigens.
We thank the British Medical Research Council and the United States National Institutes
of Health for grants in support of this work. We also thank Dr T. R. Koszalka and Mrs
M. Jensen for their collaborative efforts on this project.
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{Received 14 December 1981, revised 3 May 1982)