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.

The effects of anti-tissue antisera of varying specificity on developing animals have been studied for many years (see Berry, 1970 a, 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 immuno-globulin allotypes present in the serum of mice (Dray, 1962; Lieberman & Dray, 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.

Animals

  • 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.

  • 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 G 200. 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.

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.

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 actin-myosin complexes as antigen is clearly seen. Normal rabbit serum exhibits a pattern closely resembling that seen in Fig. 1.

Fig. 1.

Pattern of optical density tracing of successive fractions, after separation of rabbit serum from actin-injected animals, on Sephadex G 200.

Fig. 1.

Pattern of optical density tracing of successive fractions, after separation of rabbit serum from actin-injected animals, on Sephadex G 200.

Fig. 2.

Pattern of optical density tracing of successive fractions, after separation of serum from actin-myosin-complex injected rabbits, on Sephadex G 200.

Fig. 2.

Pattern of optical density tracing of successive fractions, after separation of serum from actin-myosin-complex injected rabbits, on Sephadex G 200.

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 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.

Table 1.

Explanted heart tubes: 11-day embryos

Explanted heart tubes: 11-day embryos
Explanted heart tubes: 11-day embryos
Fig. 3.

Foetal rat myocardium. Section stained with anti-rabbit globulin conjugate. × 280.

Fig. 3.

Foetal rat myocardium. Section stained with anti-rabbit globulin conjugate. × 280.

Figs. 4A 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.

Fig. 4

(A) Electron photomicrograph of unstained section. Considerable mitochondrial damage and ferritin labelling are seen. × 14000. (B) Higher magnification of ferritin-labelled myocardium. × 25000.

Fig. 4

(A) Electron photomicrograph of unstained section. Considerable mitochondrial damage and ferritin labelling are seen. × 14000. (B) Higher magnification of ferritin-labelled myocardium. × 25000.

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 2.

Rat embryo, control series: in vitro survival

Rat embryo, control series: in vitro survival
Rat embryo, control series: in vitro survival
Table 3.

Rat embryo, survival of 10-day explants in normal rabbit IgG

Rat embryo, survival of 10-day explants in normal rabbit IgG
Rat embryo, survival of 10-day explants in normal rabbit IgG

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.

Table 4.

Rat embryo, survival of 10-day explants in antiglobulin

Rat embryo, survival of 10-day explants in antiglobulin
Rat embryo, survival of 10-day explants in antiglobulin
Table 5.

Rat embryo, survival of 11-day explants in antiglobulin

Rat embryo, survival of 11-day explants in antiglobulin
Rat embryo, survival of 11-day explants in antiglobulin

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.

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 blue show marked growth inhibition until somite numbers approach the mid-thirties, 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, 1970b).

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, 1966a, 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.

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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