/. Embryo/, exp. Morph. Vol. 25, 2, pp. 203-212, 1971
203
Printed in Great Britain
The effects of an
antiserum to the contractile proteins of the
heart on the developing rat embryo
By COLIN L. BERRY 1
From the Institute of Child Health, Guilford Street, London
SUMMARY
An antiserum with specific reactivity against the contractile proteins of the rat heart has
been raised in the rabbit. Fractions of the serum have been shown to enter the isolated rat
embryo by electron-microscopic studies with ferritin, where they bind to the myocardium,
producing degenerative changes. Both IgG and IgM fractions are toxic, producing foetal
death in a large proportion of explanted embryos.
INTRODUCTION
The effects of anti-tissue antisera of varying specificity on developing animals
have been studied for many years (see Berry, 1970a, for bibliography). Most
experimental systems have shown toxic and growth inhibitory effects with little
evidence of specific target-organ damage. A number of difficulties attend such
experiments, particularly in the field of teratogenesis. The work of Guyer &
Smith (1918, 1920) and its refutation by Huxley & Carr-Saunders (1924) has
emphasized the importance of an adequate knowledge of defects normally
occurring in the animals under test, and Langman (1963), discussing the evaluation of previous work in this field, pointed out that precise information on three
points was essential; the presence and localization of the antigenic component
in the embryo during antibody exposure, the organ and species specificity of the
antibody, and the site and effects of the antigen-antibody interaction.
Studies by Brent and his co-workers have shown that antisera with basement
membrane reactivity are capable of inducing growth defects and malformation
in developing rat embryos if given at particular stages in development, and that
such defects are probably induced by the effect of the antiserum on the yolk sac
in which it localizes (Brent, Averich& Drapiewski, 1961; Brent 1964a, b, 1966;
Slotnick & Brent, 1966; Brent & Jensh, 1967). This work has been extended by
Barrow (1968).
Antisera of high specificity may alter the relative proportions of immunoglobulin allotypes present in the serum of mice (Dray, 1962; Lieberman & Dray,
1
Author's address: Department of Pathology, Guy's Hospital Medical School, St Thomas
Street, London, S.E.I.
14
EMB 25
204
C. L. BERRY
1964). Specific effects of this kind may therefore be obtained using antisera, and
this report concerns a study using an antiserum with specific affinity for the
contractile proteins of the heart, without basement membrane reactivity. The
effects of this antiserum on rat embryos have been investigated. Lanman &
Herod (1965) have shown that antibody may be transmitted to the early embryo
by 'diffusion', and Brambell and his co-workers have emphasized the role of the
yolk sac in antibody transmission in the rodent foetus (Brambell & Halliday,
1956). Therefore it seemed probable that a specific antibody could be transported
into the embryos and foetus via the yolk sac, and could be shown to bind to its
reactive site. Pathological changes might then be demonstrated, providing the
information considered necessary by Langman in the evaluation of the effects of
antisera.
The experiment was undertaken in the explanted rat embryo, since this eliminated maternal reaction to the antiserum. The biological effectiveness of the
antiserum was assessed by its effect on isolated rat heart tubes.
A
.
r
MATERIALS AND METHODS
Animals
(a) The rats used in all experiments were 'Wistar' strain, maintained as a
closed colony in a constant-temperature environment, on ' Oxoid' breeding diet
and water. No female was used for breeding until in excess of 14 weeks of age.
The morning on which vaginal plugs were found after mating was considered to
be 'day 0' of gestation.
(b) Rabbits were a local strain of Californians, hand-reared as part of another
experiment. They were used for the production of antisera.
Preparation of antigen
Actin was isolated by the methods of Straub (1943) and Carsten & Mommaerts
(1963). Since actin was not found to be antigenic in our hands (see below),
actin-myosin complexes were prepared by the method of Knieriem, Kao &
Wissler (1967). Immunization with both antigens was carried out by an injection
of 5 mg of protein, injected intravenously and intramuscularly with Freund's
complete adjuvant daily for 3 days. This pattern of injection was repeated at
10-day intervals for four courses.
The rabbits used were exsanguinated 5 days after the completion of the last
course of injections, the serum separated and subsequently fractionated on
Sephadex G200. IgG and IgM fractions were collected and freeze-dried after
dialysis to remove sodium azide. Before use, IgG was reconstituted in normal
saline and IgM in 6 M saline with subsequent dilution before use in the cultures.
This procedure was undertaken in order that an identical preparation could be
used in all experiments. Normal rabbit serum was similarly treated to obtain
' control' IgM and IgG.
Antiserum to heart contractile proteins
205
Evaluation of antiserum
Specificity. Fluorescence, with specific blocking, was sought using sections of
foetal myocardium after processing by the cold paraffin technique described by
Saint-Marie (1962). Conjugation of the serum with fluorescein isothiocyanate
was performed by the technique of Marshall, Eveland & Smith (1958).
In order that the biological effects of the antiserum might be assessed directly,
heart tubes were explanted from rat foetuses obtained on the eleventh day of
gestation, on to lens tissue in Eagle's medium with 10 % foetal calf serum added.
The cultures were incubated at 37 °C in 5 % carbon dioxide, 45 % nitrogen and
50 % oxygen. The heart rudiments were examined at intervals to see if they
continued to beat. To evaluate the effect of antiserum on myocardium contractility, 0-2 ml of reconstituted anti-globulin was added drop wise to each
culture of ten heart tubes. Cessation of contraction was taken as indicating a
toxic effect of the antiserum.
The passage of antibody through the yolk sac and its transport to the embryonic heart were determined by the use of ferritin-labelled immunoglobulin
and electron microscopy. Immunoglobulin was labelled by the technique of Sri
Ram, Tawde, Pierce & Midgley (1963) and electron microscopy performed
after fixation in 10% buffered formalin, followed by Araldite embedding and
sectioning with an L.K.B. ultramicrotome. Unstained sections were examined
with a Philips EM 200 microscope.
Embryo cultures
Explanted rat embryos were cultured using the technique of New (1966);
at the completion of the experiment they were stripped of all membranes and
examined by dissecting microscopy. A somite count was performed. Following
this, each embryo was stored at — 20 °C until protein estimation was carried
out by the technique of Lowry, Rosebrough, Farr & Randall (1951). In all
instances the viable embryos were regarded as those with a vigorous yolk-sac
circulation at the completion of the experimental period. Antisera were used
in these cultures in such a way that the antiserum added amounted to one-tenth
of the total volume of the fluid in which the rat embryo was suspended.
RESULTS
The separation pattern obtained with serum from rabbits immunized with
actin is shown in Fig. 1. In Fig. 2 the 'immune' type serum obtained with actinmyosin complexes as antigen is clearly seen. Normal rabbit serum exhibits a
pattern closely resembling that seen in Fig. 1.
Pooled IgG and IgM fractions demonstrated specific fluorescence with foetal
rat heart (Fig. 3). Blocking with unconjugated antiserum was effective. The
effects of antiserum on heart tubes may be seen in Table 1. After an initial fall
14-2
206
C. L. BERRY
_
Actin
Fig. 1. Pattern of optical density tracing of successive fractions, after separation
of rabbit serum from actin-injected animals, on Sephadex G 200.
1-0 i - Complex
0-8 -
10
0-8
06
05
04
0-3
0-2
0-1
Fig. 2. Pattern of optical density tracing of successive fractions, after separation
of serum from actin-myosin-complex injected rabbits, on Sephadex G200.
Fig. 3. Foetal rat myocardium. Section stained with anti-rabbit globulin conjugate, x 280.
Antiserum to heart contractile proteins
B
Fig. 4(A) Electron photomicrograph of unstained section. Considerable mitochondrial damage and ferritin labelling are seen, x 14000. (B) Higher magnification of ferritin-labelled myocardium, x 25000.
207
208
C. L. BERRY
in the number of beating tubes in control explants during a 24 h period it can
be seen that a relatively stable state is reached at 48 h, and for this reason
antiserum was added at this stage. The toxicity of the two components of
the serum is evident, IgM, in the presence of complement, proving the more
damaging fraction.
Figs. 4 A and B are electron photomicrographs of the heart of a 10-day-old
embryo exposed to labelled IgG for 8 h before fixation. Ferritin labelling of the
myocardium and considerable cellular damage are seen.
Survival rates of animals cultured after explanting on days 10 and 11 are seen
in Table 2, based on the extension of a previously reported series (Berry, 1968).
Comparison with Table 3 shows that the addition of non-immune rabbit serum
to the cultures did not significantly affect survival rates.
Table 1. Explanted heart tubes: M-day embryos
No.
explanted
Survivors
-*
48 h
,
24 h
Additive
s
72 h
Controls
101
—
76
67
65
—
28
28
8
0
Test
35
37
IgC
IgM + C
Table 2. Rat embryo, control series: in vitro survival
Survivors
Time in culture
No. explanted
(h)
6
12
24
Total
10-day explants
94
116
386
596
i
No.
%
79
106
256
441
84
91
66
76
5
92
3
.11 -day explants
6
24
Total
82
168
250
81
Table 3. Rat embryo, survival of 10-dfay explants in normal rabbit IgG
Survivors
Time in culture
(h)
No. of
explants
No.
0/
6
24
114
212
79
135
69
64
,o
Antiserum to heart contractile proteins
209
The effects of adding antiglobulin to the culture medium are seen in Tables 4
and 5 for 10- and 11-day explants. It can be seen, by comparison with Table 2,
that both immunoglobulin fractions are toxic. IgM with added complement is
more toxic to explants of either day. Immunoglobulin fractions were used neat
in 11-day explants since these animals were larger and had a more rapid and
vigorous circulation when examined at explantation.
The effects of antiserum on the form of killed embryos were stereotyped;
they became opaque, with disruption of the tail bud, ballooning of the cerebral
vesicles, and showed a tendency to fragmentation when manipulated. Gross
distension of the pericardial sac was often seen.
Table 4. Rat embryo, survival of 10-day explants in antiglobulin
Time in
Survivors
Additive
(h)
Explants
No.
%
IgG, .1/10 dilution
6
24
24
130
128
114
110
53
23
84
51
20
IgM, 1/10 dilution
+ complement
Table 5. Rat embryo, survival of 1 l-day explants in antiglobulin
Time
Additive
IgG neat
IgM neat
+ complement
IgG, 1/10 dilution
IgM, 1/10 dilution
+ complement
Survivors
(h)
Explants
No.
0/
6
24
6
24
6
24
6
41
16
54
10
26
126
47
12
0
3
0
6
1
0
34
/o
5
26
1
DISCUSSION
Growth in the explanted rat foetus is presumably limited by the efficiency of
yolk-sac metabolism. The sac forms a sphere, the surface area of which varies
as the square of the linear dimension, whilst its volume varies as the cube. In
this instance the embryo increases in volume more rapidly than the surface of
the yolk sac increases in area; hence the metabolic demands of the growing
animal may exceed the absorptive capacity of the yolk-sac epithelium. This
kind of inhibition of growth may be demonstrated in vivo using teratogens that
interfere with yolk-sac functions. The effectiveness of allantoic development in
removing this limiting factor has been demonstrated; embryos exposed to trypan
210
C. L. BERRY
blue show marked growth inhibition until somite numbers approach the midthirties, when 'catch-up' growth commences: although an allantoic circulation
exists from around twenty somites in the rat, it is probable that a functionally
effective placenta develops somewhat later (Berry, 19706).
Embryos grown in vitro gain protein, although the gain is less marked than
in vivo growth (Berry, 1970a). In the periods of culture studied, with the
exception of 24 h cultures of 11-day explants, it seems unlikely that the eventual
limitation of growth implicit in the system is a significant cause of death. It is
considered that damage to developing myocardium has reduced yolk-sac
perfusion and interfered with embryonic nutrition in this way.
The survival rate of untreated 10-day explants is high: about 90 % up to 12 h
and 66% at 24 h. For 11-day explants the very poor survival at 24 h (3%)
contrasts markedly with the good figures at 6 h (92 %). This is presumably
related to the rapid growth of the embryo that normally occurs at this stage, and
the necessity later that day for alternative sources of nutrition to those available
in our system.
Antisera to actin-myosin complexes were readily produced but tended to
disappear rapidly from the serum of immunized animals. Actin was not antigenic in our hands; its uniformity of structure in widely differing species makes
it a poor antigen (see Hatano & Oosawa, 1966 a, b).
The immunoglobulin fractions used reacted specifically with myocardium and
were effective in killing isolated heart tubes. It is perhaps surprising that IgG
is lethal in this system, although it has been demonstrated that this type of antibody may damage the cells in the absence of complement (Green, Barrow &
Goldberg, 1959; Jakobsson & Wahren, 1965). The antibody has been shown to
enter the embryo in a biologically active form, localizing in the myocardium and
producing cellular injury—demonstrated by fluorescence microscopy, with
successful 'blocking' tests, and immuno-electron microscopy. The extensive
cellular damage seen on electron microscopy is a morphological indicator of
poor myocardial function and inadequate yolk-sac perfusion.
Non-immune rabbit globulin had little effect on 10-day explants. There was
no significant difference in survival at 6 or 24 h when animals cultured in the
presence of rabbit globulin were compared with controls. The protein content
of animals treated in this way did not differ significantly from normal (Student's
t test) and somite numbers were within the expected range.
Immunoglobulin fractions had little effect on short-term cultures of 10-day
explants; 6 h survival is not affected by the addition of a 1:10 dilution of antiglobulin to the culture (this may be related to the necessity for a latent period
in which muscle damage occurs). Twenty-four hour cultures show a significantly
increased mortality if IgG is added to the culture. Relatively few animals
survived explanting into medium containing IgM and complement. It was
anticipated that this combination would be toxic because of the rapidity with
which cell lysis may occur in this system.
Antiserum to heart contractile proteins
211
In 11-day explants both dilutions of antiserum used were extremely toxic,
with 6 h survival of 34 % and 26 % compared with normal survival of 92 % of
embryos explanted.
The mode of entry of antibody into the foetus via the yolk sac in a biologically
active form necessitates a method of transport of the kind discussed by Brambell, Hemmings & Oakley (1959) and Brambell (1966). Brambell has suggested
that specific receptor sites in pinocytotic vesicles protect antibody from enzymic
degradation, and that these vesicles may transport the immunoglobulin across
the cell. Brambell and co-workers have studied both rat (1956) and rabbit (1959),
but this experiment represents a demonstration of biologically effective antibody
obtaining access to the rat embryo at an earlier stage than previously examined.
I wish to thank Mrs H. Davidson for technical assistance. This work is supported by
grants from the Central Research Fund of the University of London, The Royal Society,
and Action for the Crippled Child. C. L. Berry is the Gillson Scholar of the Worshipful
Society of Apothecaries of London.
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