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, 1966a, b, 1967). The teratogenic factor has been localized in the gammaglobulin fraction of the serum (Brent, 1966b).

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 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.

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 ( days gestation) or early somite ( 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, 2·5×5 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 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.

Fig. 1.

Culture tube. The embryos float in serum, which half fills the tube. The space above is filled with 5 % CO2 in air. The tube is rotated at 60 rev/min during incubation.

Fig. 1.

Culture tube. The embryos float in serum, which half fills the tube. The space above is filled with 5 % CO2 in air. The tube is rotated at 60 rev/min during incubation.

Three types of experiment were made.

  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 μm. No antibody was added to the culture serum.
    Fig. 2.

    Injection sites in explants at 912 and 1012 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.

    Fig. 2.

    Injection sites in explants at 912 and 1012 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.

    Fig. 3.

    Embryo explanted at 1012-days gestation with glass injection pipette inserted.

    Fig. 3.

    Embryo explanted at 1012-days gestation with glass injection pipette inserted.

  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).

(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 explantation the -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 -day stages with anterior limb-buds, 20–25 somites and yolk sac 3–4 mm diameter. The -day embryos were at head-fold stage in oval vesicles about 0·5 × 1·5 mm; after 24 h in the control serum most of them had developed to typical 10½-day stages.

Table 1.

Development of embryos and membranes in control rat serum, in serum with 1 mglml sheep gamma globulin, and in serum with different concentrations of yolk-sac antibody (sheep anti yolk-sac gamma globulin)

Development of embryos and membranes in control rat serum, in serum with 1 mglml sheep gamma globulin, and in serum with different concentrations of yolk-sac antibody (sheep anti yolk-sac gamma globulin)
Development of embryos and membranes in control rat serum, in serum with 1 mglml sheep gamma globulin, and in serum with different concentrations of yolk-sac antibody (sheep anti yolk-sac gamma globulin)

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 0.001 mg/ml or 0.01 mg/ml (Table 1). But in 10 mg/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 1.0 mg/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. There 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 TO mg/ml antibody were little, if any, more severe than 0.1 mg/ml.

Fig. 4.

Embryos explanted at 912 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) 1 · 0 mg/ml antibody. Fixed and stained in boraxcarmine.

Fig. 4.

Embryos explanted at 912 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) 1 · 0 mg/ml antibody. Fixed and stained in boraxcarmine.

Fig. 5.

Embryos explanted at 1012 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.

Fig. 5.

Embryos explanted at 1012 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.

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 0·1–1·0 mg/ml, although such embryos continue to differentiate, i.e. growth ceases while differentiation is only retarded.

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 1012 days gestation and initial protein content was about 40 µg in the embryo and 80 µg in the membranes.

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 1012 days gestation and initial protein content was about 40 µg in the embryo and 80 µg in the membranes.

(2) Incubation after injection of yolk-sac antibody

  • Five -day explants were each injected between the amnion and the yolk sac with 0·04–0·06 µl saline containing 2–3µg anti yolk-sac gamma globulin.

  • Eight -day explants were injected in the amniotic cavity with 0·2 µlsaline containing 10 µg anti yolk-sac gamma globulin.

  • Fifteen -day explants were each injected between the amnion and the yolk sac with 1.0 µl. saline containing 50 µg anti yolk-sac gamma globulin.

Control embryos were injected with similar amounts of saline containing 50µg/µl 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 µm 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 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 -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.)

Fig. 7.

Embryos explanted at 1012 days gestation and grown for 24 h in culture. Left: control, not injected. Right: 10µg yolk-sac antibody injected into the amniotic cavity at explantation.

Fig. 7.

Embryos explanted at 1012 days gestation and grown for 24 h in culture. Left: control, not injected. Right: 10µg yolk-sac antibody injected into the amniotic cavity at explantation.

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 -day explant was about 0·5 µl at the beginning of incubation and about 2 µl at the end. The -day explants were about 7 µl at the beginning and 25 µl 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 -day explants; from 1·4 to 0·4 mg/ml in the -day explants, series (ii); and from 7 to 2 mg/ml in the -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.

(3) Incubation after preliminary exposure to yolk-sac antibody

Five -day explants and seven -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.

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.

Rat embryos, explanted with their embryonic membranes during the early stages of organogenesis ( 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 0·1 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.

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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