/. Embryol. exp. Morph. Vol. 27, 3, pp. 543-553, 1972
Printed in Great Britain
543
Effect of yolk-sac antibody on rat
embryos grown in culture
By D. A. T. NEW 1 AND R. L. BRENT 2
From the Physiological Laboratory, Cambridge
SUMMARY
Rat embryos, explanted with their embryonic membranes during the early stages of
organogenesis (H-l (H days gestation), were grown in culture in roller tubes.
Yolk-sac antibody (sheep anti rat yolk-sac gamma globulin), known to be teratogenic when
injected into pregnant rats, was added to the culture medium. At concentrations of 01 mg/ml
or more the antibody caused gross retardation of growth and differentiation.
Injection of antibody into the amniotic cavity so that it had direct contact with the embryo,
or between the amnion and yolk sac so that it was in contact with the mesodermal surface of
the yolk sac, had little or no effect on development of the embryo or its membranes.
These in vitro experiments indicate that yolk-sac antibody has an effect on development
independent of any immunological reaction of the mother, and the primary action is probably
on the visceral yolk-sac endoderm.
It has been shown that congenital malformations can be produced in rats
by injection into the pregnant female of rat kidney antiserum (Brent, Averich &
Drapiewski, 1961; Brent, 1966a, b) or heterologous placental antiserum (Brent,
1966tf, b, 1967). The teratogenic factor has been localized in the gammaglobulin fraction of the serum (Brent, 19666).
As with all teratogenic agents, the primary effect could be (1) directly on the
embryo, (2) on the maternal organism, or (3) on the extra-embryonic membrane
derivatives (chorio-allantoic placenta, yolk-sac placenta or amnion). In practice,
it has proved difficult to distinguish between these possibilities. Slotnick &
Brent (1966) and Brent (1969, 1971) showed by 125I and fluorescein labelling
that the teratogenic antibodies became localized in the Reichert membrane
and visceral yolk sac but not in the embryo. This suggested the yolk sac as
the primary site of antigenic action, a hypothesis that was strengthened when
it was found that rat yolk-sac antiserum was teratogenic (Brent, Johnson &
Jensen, 1971).
However, it still remains possible that the embryonic malformations are
caused by trace amounts of teratogenic antibody acting directly on the embryo,
or by immunological disease of the mother which secondarily affects the
1
Author's address: Physiological Laboratory, Cambridge CB2 3EG, U.K.
Author's address: Stein Research Center, Jefferson Medical College, Philadelphia,
Pennsylvania 19107, U.S.A.
2
544
D. A. T. NEW AND R. L. BRENT
embryo. These possibilities might be confirmed or eliminated by examining
the effects of the antibody on embryos grown in culture. Techniques have been
developed in recent years (New, 1971) for growing rat embryos, explanted with
their embryonic membranes, in culture for up to two days during the period
of organogenesis and have been used in at least one study of the effect of an
antiserum on embryonic development (Berry, 1971). The following report
describes the results of exposing explanted embryos and their membranes to
the yolk-sac antiserum. The results show a direct embryopathic effect of the
antiserum independent of any reaction of the mother. Furthermore, injection
of antibody into different parts of the explant has suggested the visceral yolk-sac
endoderm is the primary site of the antigenic action.
MATERIAL AND METHODS
Rat yolk-sac antibody was obtained from sheep, as described by Brent et al.
(1971). Whole yolk sacs from Wistar term foetuses were pooled, lyophilized,
ground into a fine powder and stored at - 20 °C. Sheep were immunized with
four weekly injections of the lyophilized yolk sac resuspended in 1 ml of
distilled water and 1 ml of Freund's incomplete adjuvant. After the fourth
injection serum was collected weekly from the immunized animals for several
months, pooled and lyophilized. Gamma globulin was prepared from the
immunized sheep and from non-immunized sheep by ammonium sulphate
precipitation and DEAE Sephadex chromatography.
The sheep anti rat yolk-sac gamma globulin used in this study has the
following embryopathic effects when injected into pregnant rats on the 9th day
of gestation. Fifty per cent of the embryos will not survive if the teratogenic
gamma globulin is administered at a dose of 25 mg/kg. Eighty-five per cent of
the surviving embryos are malformed. An estimate of the peak concentration
of this dose of gamma globulin in the maternal rat's extracellular compartment
is approximately 1 mg/ml. Thus the range of concentration of sheep anti
yolk-sac gamma globulin used in the in vitro experiments was based on the
estimated in vivo concentration of teratogenic gamma globulin when an LD50
dosage is administered.
Rat embryos, together with the embryonic membranes, were explanted at
head-fold ( 9 | days gestation) or early somite (10^ days) stages. The Reichert
membrane of each embryo was torn open before incubation but the visceral
yolk sac, amnion, and allantoic placenta were left intact. The culture medium
was homologous serum; this was continuously circulated within the culture
chamber but by a simpler method than those (New, 1967, 1971) described
previously. The culture chambers were small cylindrical glass specimen tubes,
2-5x5 cm, with silicone rubber stoppers. About half the available volume in
each tube was filled with serum (10 ml) and the other half with a gas mixture
of 5 % CO2 in air (Fig. 1). Three to five explants were placed in each tube. The
Effect of yolk-sac antibody on rat embryos
545
5% CO 2 in air
Embryo and
membranes
Serum
Fig. 1. Culture tube. The embryos float in serum, which halffillsthe tube. The space
above isfilledwith 5 % CO2 in air. The tube is rotated at 60 rev/min during incubation.
Fig. 2. Injection sites in explants at 9i and 10£ days gestation. A, Amniotic cavity.
B, Space between amnion and yolk sac (extra-embryonic coelom). Heavy stipple,
embryo. Medium stipple, allantois. Light stipple, ectoplacental cone.
tubes were laid horizontally on rollers and rotated at 60 rev/min during incubation. This promotes oxygenation of the serum by continuously exposing
a fresh layer to the gas phase; it also keeps the explants gently swirling about
in the serum, thereby assisting respiration and giving maximum exposure of
the explants to any antibody in the serum. In such cultures embryo, yolk sac
and amnion continue to develop but the allantoic placenta does not.
Three types of experiment were made.
35
E M B 27
546
D. A. T. NEW AND R. L. BRENT
Fig. 3. Embryo explanted at 10i-days gestation with glass
injection pipette inserted.
(1) Incubation of the embryos in rat serum to which anti yolk-sac gamma
globulin or control gamma globulin had been added in various concentrations.
(2) Incubation of the embryos after injection of anti yolk-sac gamma globulin
into the amniotic cavity or between the amnion and yolk sac (Figs. 2, 3).
Injections were made with a Beaudouin suction and force pump connected by
silicone tubing filled with mineral oil to a glass micro-pipette drawn out to
a tip diameter (external) of about 6 jam. No antibody was added to the culture
serum.
(3) A short period of exposure of the explanted embryos to serum containing
anti yolk-sac gamma globulin, followed by incubation in antibody-free serum.
In all the experiments each litter of embryos was distributed as evenly as
possible among the various treatments to minimize any differences between
litters.
Following incubation, the embryos were examined for size, malformation
and for the condition of the heart beat and blood circulation. The amount of
expansion of the yolk sac was noted. Some of the embryos were dissected free
of the embryonic membranes and the protein content of the embryo and of the
membranes was determined by the method of Lowry, Rosebrough, Farr &
Randall (1951).
Effect of yolk-sac antibody on rat embryos
547
Table 1. Development of embryos and membranes in control rat serum, in serum
with 1 mgjml sheep gamma globulin, and in serum with different concentrations
of yolk-sac antibody {sheep anti yolk-sac gamma globulin)
(Figures give mean diameters of yolk sacs in millimetres. Development of each embryo is
recorded in four grades, from + , indicating normal growth and differentiation, to
,
indicating no growth or differentiation.)
Yolk-sac antibody
Control
serum
Sheep
GG
(1 mg/ml)
9*
1-8+
1-6+
.
.
13-
1-2--
9*
21+
+
+
.
.
.
.
.
.
.
.
.
20—
-
1-6--
9*
1-9+
2-1+
2-2+
1-9-
1-5--
9*
•
1-8+
1-8+
1-8+
1-5-
10-
5
10*
.
3-8+
.
.
3-2-
2-7-
6
10*
40+
.
7
10*
31+
•
•
3-2+
2-7-
2-6--
10*
3-1+
.
.
3-2+
2-6-
2-6-
Exp.
no.
I
Age of
embryos
(days)
0001
mg/ml
.
001
mg/ml
.
01
mg/ml
.
10
mg/ml
- -
10
mg/ml
0-5x•
0-9-
1-6--
.
1-5
35-2
548
D. A. T. NEW AND R. L. BRENT
D
Fig. 4. Embryos explanted at 91 days gestation and grown for 24 h in (A) control
serum, (B) serum with 0-1 mg/ml yolk-sac antibody, (C) 1-0 mg/ml antibody, (D)
10 mg/ml antibody. Fixed and stained in boraxcarmine.
Fig. 5. Embryos explanted at 101 days gestation and grown in (left) serum with
1 mg/ml control sheep gamma globulin, (centre) 0-1 mg/ml yolk-sac antibody, (right)
1 -0 mg/ml antibody. The embryos have been dissected from their membranes after
culturing, then fixed and stained.
Effect of yolk-sac antibody on rat embryos
180
±12
549
Membranes
+ 11
160-
+ 11
140 -
+7
120-
100-
+ 10
+ 12
Embryo
120 100 80-
60-
+4
+3
0
0.01
0.1
1.0
Yolk sac antibody (mg/ml)
Fig. 6. Increase of protein of embryo (lower histogram) and of embryonic membranes
(upper histogram) in different concentrations of yolk-sac antibody. Each rectangle
gives the mean of 11 explants (Exps 7 and 8 in Table 1). Figures above each rectangle
are standard errors. The embryos were explanted at 101 days gestation and initial
protein content was about 40 ji<g in the embryo and 80 /*g in the membranes.
RESULTS
(1) Incubation in serum containing yolk-sac antibody
Table 1 summarizes the results of eight experiments with embryos cultured
in different concentrations of antibody. At explanation the 10^-day embryos
were at early somite stages, with a beating heart and yolk sac 2-2-5 mm diameter;
after 24 h in culture those in the control serum had mostly developed to typical
ll+-day stages with anterior limb-buds, 20-25 somites and yolk sac 3-4 mm
diameter. The 9+-day embryos were at head-fold stage in oval vesicles about
0-5x1-5 mm; after 24 h in the control serum most of them had developed to
typical 10^-day stages.
Growth and development were unaffected by the control gamma globulin
added to the culture serum at a concentration of 1 mg/ml, or by yolk-sac
antibody at concentrations of 0001 mg/ml or 0-01 mg/ml (Table 1). But in
550
D. A. T. NEW AND R. L. BRENT
10mg/ml antibody the explants were rapidly killed; there was no embryonic
development and by the end of the culture period the yolk sac was collapsed
and disintegrating. At the intermediate concentrations, 0-1 and l-0mg/ml, the
explants continued to develop but growth was retarded (Fig. 4). At the end
of the culture period the embryos were smaller than controls, with fewer somites
and retarded formation of neural folds and tube. Folding-off of the body wall
from the yolk sac, turning from the dorsally concave to ventrally concave
position, and extension of the allantois to the ectoplacental cone were all
delayed. Theie was frequently failure of heart beat and blood circulation. The
yolk sac was smaller. However, conspicuous malformations were rare, and
although the embryos were retarded they were mostly well formed (Fig. 5). The
gross visible effects of 1-0 mg/ml antibody were little, if any, more severe than
0-1 mg/ml.
Fig. 6 shows the increase of protein in the embryo, and in the embryonic
membranes, during the culture period. The rate of protein increase in the
membranes falls steadily with increasing concentrations of antibody in the
culture serum. This probably results mainly from an effect on the yolk sac
because the amnion contained relatively little material and the allantoic placenta
fails to differentiate in culture. Protein increase by the embryo rapidly approaches
zero when the antibody concentration is raised to 01—1-0 mg/ml, although
such embryos continue to differentiate, i.e. growth ceases while differentiation
is only retarded.
(2) Incubation after injection of yolk-sac antibody
(i) Five 9^-day explants were each injected between the amnion and the
yolk sac with 004-0-06/A saline containing 2-3fig anti yolk-sac gamma
globulin.
(ii) Eight 10^-day explants were injected in the amniotic cavity with 0-2 fi\
saline containing 10 fig anti yolk-sac gamma globulin.
(iii) Fifteen 10^-day explants were each injected between the amnion and
the yolk sac with 1-0 [A saline containing 50 fig anti yolk-sac gamma globulin.
Control embryos were injected with similar amounts of saline containing
50 figlfil control gamma globulin.
Particular care was taken to ensure that the injected material remained in
the explant and did not leak out through the injection hole. Initially the injected
fluid could be seen within the explant by its difference in refractive index. The
injection hole was kept as small as possible (< 10 fim diameter) and no injection
fluid was seen to escape when the micro-pipette was withdrawn. Closure and
healing of the hole was evidently very rapid because the amnion and yolk sac
remained 'blown up' and showed no sign of contraction either immediately
following the injection or during the subsequent incubation.
Growth and differentiation of the explants appeared to be little, if at all,
affected by the injected yolk-sac antibody. At the end of the culture period
Effect of yolk-sac antibody on rat embryos
551
Fig. 7. Embryos explanted at 10| days gestation and grown for 24 h in culture.
Left: control, not injected. Right: 10fig yolk-sac antibody injected into the
amniotic cavity at explantation.
size and stage of development of the embryo (Fig. 7), condition of the blood
circulation and diameter of the yolk sac were all the same as in controls injected
with control gamma globulin or grown in culture without injection. (In some
of the lO^-day explants injected with antibody between the yolk sac and
amnion the hinder part of the embryo failed to turn to the ventrally concave
position, but the numbers were too small to determine whether this was caused
by the antibody.)
It is remarkable that the injected antibody had so little effect in view of its
high concentration within the explant. The total volume of each 9^-day explant
was about 0-5 ji\ at the beginning of incubation and about 2 fi\ at the end. The
10^-day explants were about 7 [A at the beginning and 25 [A at the end. If the
injected antibody diffused through the whole volume, and remained unchanged
during the culture period, the concentration would decrease from about 5 to
1-2 mg/ml in the 9^-day explants; from 1-4 to 0-4 mg/ml in the 10^-day
explants, series (ii); and from 7 to 2 mg/ml in the 10^-day explants, series (iii).
As probably only a part of the whole volume is available for diffusion, because
it is divided by the amnion and occupied by the embryo, the actual concentrations
in the injected parts are likely to be two or three times higher. This suggests
concentrations in the range 10-200 times that (0-1 mg/ml) causing gross
retardation of development when present in the culture medium.
552
D. A. T. NEW AND R. L. BRENT
(3) Incubation after preliminary exposure to yolk-sac antibody
Five 9^-day explants and seven 10^-day explants were immersed for 1-2 h,
some at room temperature and some at 37 °C, in serum containing 1 mg/ml
anti yolk-sac gamma globulin. They were then transferred to serum without
antibody and incubated. All developed as well as controls grown in antibody-free
serum throughout.
DISCUSSION
One object of this study was to establish whether yolk-sac antibody has
a direct effect on the development of the embryo or embryonic membranes,
independent of any possible reactions of the mother. The use of explanted
embryos, growing in culture, has clearly demonstrated such a direct effect.
Anti yolk-sac gamma globulin added in concentrations of 0-1 mg/ml or more
to the nutrient medium caused gross retardation of growth and differentiation.
That the action was antigenic in nature was shown by the absence of any effect
when control gamma globulin was added to the medium. This direct action
on the conceptus is probably the main cause of the malformations produced
by the antibody in vivo (Brent et al. 1971), though the possibility cannot yet
be eliminated that there may be an additional effect from a maternal reaction.
The result of adding antibody to the culture medium was in striking contrast
to that of injecting it into the amniotic cavity or into the extra-embryonic
coelom. In the injected explants either the mesodermal (inner) layer of the
yolk sac, or the amnion, or the embryo itself was exposed to antibody at
over 10 times the concentration that retarded development when present in
the culture serum. But the injections had little or no effect. The tissue directly
in contact with the culture serum is the visceral endoderm (outer) layer of the yolk
sac and it seems probable that only this tissue is sensitive to the antibody. The
harmful effect on the embryo itself of antibody in the nutrient serum apparently
results from a primary action on the visceral endoderm. Several studies in
recent years (e.g. Padykula, Deren & Wilson, 1966; Beck, Lloyd & Griffiths,
1967) have emphasized the capacity of this layer of the yolk sac to absorb
macromolecules, particularly protein, and it is probable that after digestion
these are transported, together with other nutrient substances, to the embryo
by the yolk-sac blood circulation. The yolk sac would appear to be the main
nutritive organ of the embryo before the allantoic placenta becomes functional,
and the work of Payne & Deuchar (1972) has recently indicated that it also
has an important role in regulating the volume of extraembryonic fluids. It is
to be expected therefore that any interference with yolk-sac function at this
stage will have very harmful effects on development of the embryo.
Histological studies, by light and electron microscopy, and for fluorescent
localization of the antibody, are now being made and will be reported in a later
publication.
Effect of yolk-sac antibody on rat embryos
553
This work was done while R.L.B. was a Royal Society of Medicine Travelling Fellow
supported by Research Grant HD630 and Training Programme HD 370.
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(Manuscript received 28 January 1972)