The differential sensitivity of cultured chick mesodermal cells to actinomycin D.

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.

Successful culture of rat embryos on human serum: use in the detection of teratogens.

Growth of head-fold-stage rat embryos cultured with human serum for 48 hours was enhanced by supplementation with glucose. Embryo growth (protein and DNA contents) varied with the source of the serum. Serum from 16 of 19 untreated subjects produced normal embryos. Serum from five subjects undergoing cancer chemotherapy and six subjects receiving anticonvulsants was either lethal or teratogenic.

Polysome activity in relation to growth and protein starvation in brains and hearts of cultured early chick embryos.

In previous studies, brains but not hearts of intact early chick embryos were found to be sensitive to protein starvation. In this study, the in vitro protein synthetic activity of polysomes isolated from brains was found to be greater than those isolated from hearts. Starvation reduced the protein synthetic activity of polysomes in vitro but the extent of the reduction was approximately the same for both brains and hearts. A reduction in the amount of ribosomes as polysomes may have contributed to the lower synthetic activity of polysomes from tissues of starved embryos but not to the differences in synthetic activities between brains and hearts. In addition, neither the stability of isolated polysomes nor ribosome-associated ribonuclease activity appeared responsible for the differences observed in polysome synthetic activities. In direct relationship to the differential sensitivity of brains and hearts to starvation observed in the intact embryo, ribosomes isolated from brains of both growing and starved embryos were more readily degraded during in vitro incubation than those from hearts.

The use of whole rat embryo cultures to identify and characterize causes of reproductive failure.

The most important problem facing human teratology today is to identify the actual causes of this health problem. We have used cultures of whole rat embryos to address this problem using blood sera from individuals at risk as embryo culture media for this purpose. Through serum fractionations and nutrient supplementations to the serum we have studied drugs (dilantin, valproic acid), nutrient deficiencies (methionine) and an embryotoxic autoantibody to the protein laminin. In addition to identifying these factors it has been possible to address their mechanisms of action and to provide recommendations for treatment.

Autoantibodies to laminin and other basement membrane proteins in sera from monkeys with histories of reproductive failure identified by cultures of whole rat embryos.

Head-fold-stage rat embryos cultured on sera taken from monkeys with histories of reproductive failure had an abnormality frequency of 97% compared with only 7% on sera taken from monkeys with excellent reproductive histories. For a group of these poor reproducers, the toxicity of their sera was associated with the immunoglobulin G (IgG) fraction. These IgG fractions bound to Reichert's membrane and other basement membranes of the embryo. For one monkey, the IgG specifically reacted with a 41 kDa polypeptide of Reichert's membrane, while for two others binding was to laminin, type IV collagen, and several other minor polypeptides of Reichert's membrane. For serum from one monkey, the toxicity to cultured rat embryos was eliminated by absorption with laminin but not type IV collagen.

Role of laminin autoantibodies on the embryo toxicity of sera from mercuric chloride treated brown Norway rats.

In previous studies, antilaminin antibodies were found to be toxic to cultured rat embryos. In order to extend these studies, Brown Norway rats were treated with mercuric chloride, which led to the production of laminin autoantibodies. Sera samples from brown Norway rats treated with mercuric chloride were found to be teratogenic as well as lethal to cultured rat embryos. This embryotoxicity was not associated with sera mercury levels, but was related to the levels of antilaminin antibodies in sera. Affinity purified laminin antibodies from these mercuric chloride treated Brown Norway rats, when added to control sera, were found to be teratogenic but not lethal. These antibodies were found to bind to the laminin sequences IKVAV (A chain) and YIGSR (B1 chain), but not RGD (A chain) or YD (B1 chain). These observations suggested the possibility that an environmental pollutant such as mercury could cause the formation of embryotoxic autoantibodies that could persist in the body as embryotoxic factors for extended periods of time.

Serum protein synthesis in the early chick embryo.

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringer's solution. The presence of serum transferrin, two embryo-specific alpha-globulins, and a prealbumin were demonstrated by acrylamide gel analysis. Yolk-sacs from embryos explanted at 11-13 somites (40 hr preincubation) and cultured for 48 hr secreted in addition a protein with the mobility of serum albumin. Incubation of yolk-sacs in the presence of radioactive valine indicated that serum proteins were synthesized as early as the primitive streak stage. By incubating isolated yolk-sacs and embryos from 48-hr explants in the presence of radioactive valine, the synthesis of serum proteins was found to be restricted to the yolk-sac at this stage of development. Culturing explants on various nutrient proteins as well as protein starvation medium altered the relative synthesis of several serum proteins. We have proposed that morphological and biochemical changes in embryos resulting from altered nutrition may be mediated by the proteins of the serum.

Identification of a teratogenic drug-protein complex in sera of phenytoin-treated monkeys.

Whole rat embryos were cultured for 48 h on sera drawn from monkeys before and 10 h after phenytoin gavage (275 mg/kg body weight). Sera from treated monkeys caused exencephaly, anophthalmia, microcephaly, and incomplete ventral curvature when used as culture media, whereas sera drawn from the same monkeys before treatment supported normal embryonic development. To identify the cause of serum teratogenicity, isolated constituents of teratogenic sera were added to nonteratogenic sera for testing by embryo culture. Serum extracts containing free phenytoin and its free metabolites were not teratogenic. Teratogenicity was found associated with serum proteins. Using polyacrylamide gel electrophoresis (PAGE) and an antibody that recognized phenytoin and its metabolites, we were able to demonstrate that phenytoin was bound to a protein of 80,000 daltons. Addition of this same antibody to teratogenic sera from dosed monkeys improved the development of cultured embryos and provided additional support for this complex as the proximal teratogen. Use of the antibody to follow the uptake and distribution of phenytoin in cultured embryos suggested that only the phenytoin-protein complex (and not phenytoin itself) was able to pass through the yolk sac and reach the tissues of the embryo proper. These results suggested that a drug-protein complex may serve to transport drugs from their site of activation in the maternal liver to the developing embryo.

Methionine decreases the embryotoxicity of sodium valproate in the rat: in vivo and in vitro observations.

Methionine provided in the drinking water of pregnant rats injected with sodium valproate reduced the frequency of resorptions but did not improve embryo growth. Rats drinking methionine supplemented water had approximately twice the level of serum-free methionine and consumed only one-half the volume of water of controls. Using whole rat embryo cultures, the simultaneous addition of methionine and sodium valproate to the medium provided no protection from neural tube defects, nor did the addition of methionine to a medium of serum obtained from rats previously dosed with sodium valproate. However, protection from the teratogenic effects of sodium valproate was afforded by methionine when the culture medium was sera from rats consuming methionine and was particularly striking when embryos for culture were taken from pregnant rats that had been consuming methionine. These observations along with those of others indicated the importance of dietary and culture media methionine levels in evaluating experimental and regulatory teratology studies and suggested the possibility that methionine may play an important role in human teratology where multifactorial causes have been implicated in problems such as neural tube closure defects.

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells.

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.

Methionine and neural tube closure in cultured rat embryos: morphological and biochemical analyses.

When headfold-stage rat embryos were cultured on cow serum, their neural tubes failed to close unless the serum was supplemented with methionine. Methionine deficiency did not appear to affect the ability of the neural epithelium to fuse as a type of fusion was observed between anterior and posterior regions of the open neural tube in methionine-deficient embryos. Although methionine deficiency reduced the cell density and mitotic indices of cranial mesenchyme and neural epithelial cells, this did not appear to be a factor in failure of the neural tube to close. For example, embryos cultured on diluted cow serum also had fewer mesenchymal cells yet could complete neural tube closure if provided with methionine. Examination of the tips of the neural folds suggested that microfilament contraction could be involved; in the absence of methionine the neural folds failed to turn in. This possibility was supported by the reductions in neurite extension of isolated neural tubes cultured without methionine and by the reductions in microfilament associated methylated amino acids contained in embryo neural tube proteins.

In vitro and in vivo embryo toxicity of antilaminin antibodies in the rat.

Rats mated after laminin immunization had higher frequencies of resorptions (57%) than those immunized with bovine serum albumin (20%) and had sera that were toxic to cultured rat embryos. In addition, sera from rats immunized with laminin A chains but not B chains were toxic to cultured embryos. The toxicity of sera to embryos was related to the reactivity of sera to specific laminin fragments rather than to sera IgG levels against intact laminin. In addition, resorptions in pregnant rats immunized with laminin were not related to the sera antilaminin IgG levels. However, levels of uterine antilaminin IgA, the predominant uterine isotype, increased considerably during the first 3.5 days of pregnancy, while sera antilaminin IgG remained constant. (Am J Reprod Immunol.

Metabolic activation of cyclophosphamide by yolk sac endodermal cells of the early chick embryo.

Endodermal cells isolated from the yolk sacs of day 3 chick embryos were able to activate metabolically cyclophosphamide. This was demonstrated by the cytotoxicity of cyclophosphamide to the endodermal cells themselves as well as by the ability of endodermal tissue to mediate a cytotoxic response in coculture with KB cells, a human tumor cell line unable to activate cyclophosphamide. Yolk sac endodermal cells from day 3 embryos were sensitive to cyclophosphamide when the drug was added immediately after the start of culture, but not when the drug was added after 24 hr of culture. The ability to metabolize cyclophosphamide by the day 3 embryo appeared limited to the endodermal cells of the yolk sac as cells derived from neither the embryo proper nor yolk sac mesoderm-ectoderm tissue were positive in these tests. Using whole blastoderms, cyclophosphamide activation was detected as early as 12 hr of egg incubation.

Toxicity of sera from individuals with Chagas' disease to cultured rat embryos: role of antibodies to laminin.

In previous studies antilaminin antibodies in the sera of immunized monkeys and rats were found to be toxic to cultured rat embryos. In order to extend these studies to humans, head-fold stage rat embryos were cultured for 48 hours on ten different serum samples from individuals with Chagas' disease. All embryos (n = 20) cultured on these sera were found to be abnormal. Using ELISA, Western immunoblot, and indirect immunofluorescence it could be shown that antibodies in these sera reacted with laminin. That these antilaminin antibodies were, at least in part, responsible for the toxicity was indicated 1) by reduced cultured embryo toxicity for six of seven serum samples after pre-absorption with purified laminin, 2) by demonstrating a relationship between the amount of affinity-purified antilaminin IgG added to control serum for culture and the severity of embryo abnormalities seen at the end of culture, and 3) by the sera's failing to react with other basement membrane proteins, including type IV collagen, fibronectin, osteonectin, and heparan sulfate proteoglycan.

Serum protein depletion by cultured rat embryos (1).

Cultures of 10-day rat embryos depleted a protein band with a molecular weight of approximately 125,000 from the rat serum medium. Delayed centrifuged serum differed from immediately centrifuged serum by a reduction in the staining intensity of the 125,000 molecular weight protein band and by the absence of two high molecular weight (greater than 200,000) protein bands.

The use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide.

Head fold stage rat embryos were cultured for 48 hours in vitro on serum taken at various intervals from rats that had been injected ip with either cadmium or cyclophosphamide. Their response was compared to that of embryos cultured for the same period on control serum to which these substances were added directly. One and 4 hour sera from cadmium-injected rats (2.13 mgCd++/kg) were lethal. Eight hour serum allowed survival but embryos were exencephalic and contained reduced amounts of protein and DNA. The response to direct cadmium was characteristically different and was related to dosage and the extent to which zero-time embryos had progressed through the head fold stage. At 1.6 micro M, Cd++ susceptible embryos were hemorrhagic, though not exencephalic. One hour serum from rats given cyclophosphamide (180 mg/kg) was lethal. On 4 hour serum, embryos survived but were exencephalic and contained less protein and DNA than controls. Embryos were resistant to direct cyclophosphamide up to 800 micrograms per ml of medium. At this concentration, embryos appeared morphologically normal but contained reduced amounts of protein.