Marcela Jensen

Production of congenital malformations using tissue antisera.

Abstract Rabbit anti-rat Reicherts membrane (RM) serum, when injected into pregnant rats on the ninth day of gestation, produced embryolethality and stunting but a surprisingly low incidence of malformations. Immunofluorescent studies revealed that the maternally injected RM antiserum localizes in RM and in the basement membrane of some maternal kidney tubules but not in the maternal glomerulus or in the cytoplasm of the visceral yolk sac (VYS) cells as had teratogenic VYS and kidney antisera. When antiserum against rat RM was injected into mice, localization occurred in the glomerular basement membranes, thus confirming the findings of Pierce and co-workers. When teratogenic term VYS antiserum is adsorbed with RM, it still localizes in the VYS, does not localize in RM, and is still teratogenic. When this same antiserum is adsorbed with VYS, the antiserum is no longer teratogenic. It appears that the localization of antibodies in the VYS cells is the major contributor to the production of congenital malformations because localization of antisera in RM alone was associated with a very low incidence of teratogenesis. Since both structures participate in a multitude of biochemical processes, which include adsorption, transport, catabolism, and anabolism of nutrients, nonnutrients, and waste products, there are many plausible hypotheses to explain the nature of the parietal and visceral yolk sac dysfunction.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 1. Relative contributions of free amino acids and of proteins

Rat conceptuses on the 10th day of gestation were cultured for 27 h in whole rat serum. An addition of either [3H]leucine or [3H]leucine-labelled rat serum proteins was made once during the culture period, and the acid-soluble and acid-insoluble radioactivities of embryo and visceral yolk sac measured at harvesting. The extent of radiolabel incorporation into embryonic and yolk-sac proteins increased linearly with the duration of exposure of the conceptus to the radiolabelled leucine or radiolabelled serum proteins, indicating roughly constant rates of incorporation, per unit mass of tissue, throughout the culture period. The incorporation rates, expressed as clearances, were 0.73 and 0.78 microliter/mg tissue protein/h for embryo and yolk sac, respectively, when the source was [3H]leucine; and 1.8 and 1.3 microliters/mg tissue protein/h, for embryo and yolk sac, respectively, when the source was [3H]leucine-labelled serum proteins. It is estimated, from the known leucine and protein concentrations in serum, that protein contributed over 99 per cent of the leucine supplied to the conceptus for its protein synthesis. In parallel experiments, measurements were made on cultures conducted in the presence of an antiserum against rat visceral yolk sac (100 micrograms/ml). Antiserum profoundly inhibited incorporation of radioactivity into embryo and yolk-sac proteins, when the source was 3H-labelled protein, a result consistent with the known ability of the antiserum to inhibit pinocytosis in the yolk sac. Antiserum also decreased incorporation from [3H]leucine in the yolk sac, suggesting that a proportion of the free leucine entering the yolk sac does so by pinocytosis. The failure of antiserum to affect incorporation of [3H]leucine into the embryo probably indicates that leucine can enter the embryo without the mediation of yolk-sac pinocytosis. The primacy of protein, as a source of amino acids for the organogenesis-stage embryo, is consistent with the serious effects, in terms of embryonic death and malformation, that result from the interruption of amino acid supply when either pinocytosis or lysosomal proteolysis in the yolk sac is inhibited.

The Embryopathologic Effects of Teratogenic Yolk Sac Antiserum

In 1961, our laboratory reported that the injection of rabbit anti-rat kidney serum into pregnant rats resulted in a high incidence of congenital malformations in the offspring (Brent et al., 1961). Attempts to demonstrate an indirect mechanism of teratogenesis secondary to maternal immunologic or metabolic disease were not successful (Brent, 1964, 1966a, 1966b). It was demonstrated that the teratogenic factor was IgG and that the teratogenic process following exposure to kidney antibody was not dependent on complement fixation (Brent, 1966b; Bragonier et al., 1970). Furthermore, pretreatment with cortisone or antihistamines did not prevent the teratogenic effect of the kidney antiserum (Brent, 1966b). In 1966, Slotnick and Brent demonstrated that teratogenic antisera prepared against rat kidney or chorioplacenta localized in the developing yolk sac placentae of the rat. These results indicated that the mechanism of action of teratogenic antisera was not due to an indirect effect of induced maternal disease or a direct effect on the embryo, but possibly the induction of yolk sac dysfunction. In 1971, Brent and associates demonstrated that antiserum prepared against yolk was a more potent teratogen than was kidney antiserum. Yolk sac antiserum also localized in the visceral and parietal yolk sac (Brent et al., 1971). A direct primary effect was further supported by the work of New and Brent (1972). In their experiments, rat embryos were dissected from the embryonic sites on the 9th day of development and were cultured for 48 hours.

Immunological Aspects of Development

There are many areas of developmental biology in which the techniques and knowledge of immunology may be utilized to solve some of the unanswered questions. The areas where immunology and developmental biology interrelate include (1) the embryological development of immune mechanisms in normal embryos; (2) the role of macromolecular surface interactions (antigen-antibody-like reactions) in normal development; (3) the clinical and experimental significance of antibodies against gametocytes, gonads, or hormones; (4) the immunological aspects of fetal-maternal interaction in mammalian development; and (5) the effect of antibodies and autoimmune phenomena on the developing embryo. Only the last two categories will be dealt with in this chapter. The other areas have been reviewed by many authors (Auerbach, 1976; Billingham, 1964; Brambell, 1970; Brent, 1965, 1966c, 1971; Beer and Billingham, 1976; Haskova, 1961; Lanman et al., 1962, 1964; Lanman, 1965; Lanman and Herod, 1965; Schlesinger, 1962; Simmons, 1969). There are reports as early as 1918 that deal with the production of malformations using tissue antiserum. The reports of Guyer and Smith (1918, 1920) proved to be in error but were the stimulus for more recent investigations. They reported that heterologous lens antiserum produced eye malformations when injected into pregnant rabbits and that these eye malformations were hereditary.

THE EFFECTS OF TERATOGENIC ANTISERA ON PROTEIN SYNTHESIS AND DEGRADATION BY THE CULTURED RAT CONCEPTUS

Rat embryo culture was initiated on the 10th day of gestation to study embryonic site protein synthesis in normal and pathologic states. Three culture media were employedsrat serum, serum plus teratogenic sheep anti-rat visceral yolk sac (VYS) serum, or rat serum plus normal sheep serum. Two studies were performed: a) 3H-leucine was added to produce incubation periods of 20 min. to 24 hr.; and after an initial period of culture in the presence of 3H-leucine to obtain a constant precursor specific radioactivity, culture was continued in the absence of 3H-leucine for a total of 1-30 hr. Embryos, VYS and culture media were analysed for total radioactivity and 3H-leucine specific radioactivity in the protein-bound and free amino acid fractions. The results suggest that: a) anti-VYS serum inhibited protein synthesis by the embryo and VYS but had no detectable effect on protein degradation; b) anti-VYS serum increased intracellular labeled leucine concentration. The teratogenic mechanism of anti-VYS serum appears to involve both the suppression yolk sac amino acid nutritive support of the embryo and the inhibition of the utilization of amino acids by the embryo for protein synthesis. These techniques provide us with the ability to determine the amino acid requirements which will support normal organogenesis and to quantitate the amino acid deprivation which will result in embryopathology. (Supported by NIH)

CONGENTIAL MALFORMATIONS INDUCED MY MONOCLONAL ANTI-BODIES AGAINST RAT VISCERAL YOLK SAC

Polyclonal antisera against rat visceral yolk sac (VYS) produces severe congenital malformations when injected into pregnant rats on the 9th day of gestation. To define and characterize the teratogen-stimulating antigen(s) from the VYS, monoclonal antibodies (MCA) against rat VYS were prepared. Spleen cells from BALB/c mice, hyperimmunized with VYS antigens purified by IEF and gel filtration were fused with two myeloma cell lines SP2/0-Ag 14 and P3x63 Ag. 8.653. Hybrids specific for VYS by indirect immunofluorescence were selected and cloned by limiting dilution and further expanded as ascitic tumor in BALB/c mice. More than 25 immunofluorescent hybrids for VYS have been selected and their biological activity defined. The first 12 clones were not teratogenic but since last year (1986) ascitic fluid from B-3 clone (IgG 2b type) induced CNS malformations and the clone D-4 (IgG 2a type) induced growtn retardations when injected into pregnant rats on the 9th day of gestation. Each teratogenic MCA stained the apical portion of VYS endodermal cell, tubular kidney brush border and failed to react with kidney glomeruli, Reicherts membrane, long, lens capsule and liver tissue by immunofluorescence. Both teratogenic MCAs failed to recognize the VYS antigens by Western blots. Further studies will be directed toward using the teratogenic MACs to study the mechanism of teratogenesis and the quantitative aspects of embryonic nutrition during early organogenesis. (Supported by NIH)

THE EFFECT OF TERATOGENIC ANTISERA ON RAT FETAL DEVELOPMENMT WHEN ADMINISTERED LATE IN DEVELOPMENT

In 1961 we reported that heterologous antisera prepared in the rabbit against rat kidney proved to be teratogenic when injected into pregnant rats (Brent et al, Proc. Soc. Exp. Biol. Med.). The teratogenic antiserum localized in the yolk sac and yolk sac antisera proved to be a potent rat teratogen. Metabolic studies have indicated that the yolk sac dysfunction is due to the antiserums ability to interfere with the endocytosis of macromolecules, thus depleting the embryo and yolk sac of required quantities of amino acids. Two important questions were asked. At what stage of rat gestation does teratogenic antiserum no longer have an embryotoxic effect on the developing embryo or fetus arid does the teratogenic antiserum affect pinocytosis late in rat development. Pregnant rats were exposed to teratogenic antiserum and while the most dramatic effects were produced during early organogenesis, embryonic and fetal effects were observed during the early fetal period several days after trypan blue no longer affected the embryo. Mortality or growth retardation were not observed when pregnant rats were administered teratogenic antiserum the last 5 days of rat gestation, in spite of the fact that the antisera does depress pinocytosis in vitro in the yolk sac late in gestation. (Supported by NIH)

1279 SPECIFICITY OF RAT YOLK SAC ANTIBODIES IN PRODUCING CONGENITAL MALFORMATIONS, EMBRYONIC MALNUTRITION AND REDUCED PINOCYTOSIS

In a series of experiments first published in 1961, we reported that antibodies prepared against rat kidney were potent teratogenic agents when administered to pregnant rats (Brent 1961). It was determined that antisera which were teratogenic localized in the developing yolk sac (Slotnick and Brent, 1966). Antisera prepared against VYS proved to be the most potent teratogens, and these teratogenic antisera reduced the endocytic uptake of proteins by the VYS (Brent et al, 1972, Freedman et al, 1983). Utilizing isolectric focusing techniques, various proteins were isolated from the VYS and antisera were prepared against these individual proteins. A large number of antisera were not teratogenic and did not localize in the developing yolk sac. A smaller group of antisera were found to localize in the VYS These antisera were studied with several techniques: Determination of the endocytic index utilizing (14C) sucrose, in vivo yolk sac localization utilizing fluorescent labelled antibody and teratogenic potency. It was found that yolk sac localization of antibody specific for yolk sac antigens does not prove that the antibodies have teratogenic potency. On the other hand, if the antibodies also reduce the endocytic index, then the antibodies will invariably have teratogenic potency. Thus, the reduction of endocytosis is the first method of accurately predicting teratogenic potential without having to perform time-consuming in vivo bioassays.