Norman W. Klein

Human serum teratogenicity studied by rat embryo culture: epilepsy, anticonvulsant drugs, and nutrition.

Summary: Epileptic women have a greater risk for spontaneous abortions and children with birth defects than do nonepileptics. In a unique approach to identifying causes of these problems, we have cultured whole rat embryos for 48 h on blood sera from epileptics. In the first part of the study, three embryos were cultured on each serum sample from 128 different epileptics being treated with either single anticonvulsants or no drug to compare the teratogenicity of these drugs. Sera from subjects receiving either phenobarbital or no drug had comparable frequencies of cultured embryo abnormalities, which were lower than those from subjects taking phenytoin, valproic acid, or carbamazepine. In the second phase of the study, attempts to identify causes for serum teratogenicity led to the finding that the abnormalities and reduced embryo growth produced by many serum samples could be completely overcome by adding vitamins and/or amino acids to the serum. Of 53 samples tested, 32 (60%) were corrected by supplementation (17 of 23 phenytoin, seven of nine phenobarbital, six of 12 carbamazepine, none of six valproic acid, and two of three no drug). Although the results of this study provided a general assessment of drug teratogenicity that agreed with other studies and emphasized the role of nutrition in fetal defects, the importance of individual differences in causes of teratogenicity was also noted.

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 Reicherts membrane and other basement membranes of the embryo. For one monkey, the IgG specifically reacted with a 41 kDa polypeptide of Reicherts membrane, while for two others binding was to laminin, type IV collagen, and several other minor polypeptides of Reicherts membrane. For serum from one monkey, the toxicity to cultured rat embryos was eliminated by absorption with laminin but not type IV collagen.

Growth of explanted chick embryos on a chemically defined medium and effects of specific amino acid deficiencies

Abstract Chick embryos of 11–13 somites were cultivated in vitro for either 24- or 48-hour periods. A net accumulation of both protein nitrogen and DNA was observed in embryos cultured on a chemically defined medium. Data are presented which indicate that the chick embryo requires both leucine and lysine, but not aspartic acid or proline. Protein nitrogen and DNA content of embryos cultured on a medium containing egg nutrients were higher than the levels observed for embryos after cultivation on a chemically defined medium.

ENHANCED GROWTH AND SURVIVAL OF EXPLANTED CHICK EMBRYOS CULTURED UNDER HIGH LEVELS OF OXYGEN.

Abstract Chick embryos of 11–13 somites were cultured on a whole egg homogenate medium for various periods under different combinations of oxygen, carbon dioxide, and air. A mixture of 75% air + 25% O 2 for 0–24 hours of in vitro cultivation followed by either 95% O 2 + 5% CO 2 or 100% O 2 resulted in embryo PN and DNA levels which were considerably greater than the levels previously obtained with air and the other gas mixtures investigated. All embryos provided with these high levels of oxygen survived 48 hours of cultivation, and 86% of the embryos provided with 95% O 2 + 5% CO 2 survived for 72 hours. Comparison of growth parameters and the presence of blood pigment for embryos explanted with somite numbers between 4 and 20 paris suggested that embryos at the 11–13 somite stage or less were particularly sensitive to high levels of oxygen. Growth of embryos provided with a chemically defined medium was not enhanced over that obtained with 95% air + 5% CO 2 when higher levels of oxygen were provided. Supplementing the chemically defined medium with horse serum and embryo extract or transferring embryos to the defined medium after an initial period of cultivation on the whole egg medium did not appreciably improve the response of these embryos to increased amounts of oxygen. Exposure of embryos inclubated in ovo to amounts of oxygen found to be most favorable for the explanted embryos did not result in greater growth than that obtained when air was provided.

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 Eagles 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.

ACTINOMYCIN D: SPECIFIC INHIBITORY EFFECTS ON THE EXPLANTED CHICK EMBRYO.

Chick embryos containing 11 to 13 somites were cultured for 48 hours on media containing various amounts of the antibiotic actinomycin D. Morphological observations as well as quantitative determinations of protein nitrogen, DNA, and RNA indicate that the antibiotic specifically inhibits the growth and development of the embryonic axis posterior to the 12th somite.

Yolk sac endoderm: exclusive site of serum protein synthesis in the early chick embryo

Abstract In order to determine which cell type or types synthesized serum proteins, yolk sacs from 4-day chick embryos were manually separated into ectoderm, mesoderm, and endoderm and incubated in the presence of radioactive valine. Analysis of the incubation media by polyacrylamide gel electrophoresis as well as by immunoprecipitation showed that all serum proteins were synthesized exclusively by the cells of the endoderm. These included transferrin and three embryo-specific serum proteins: α-globulin a, α-globulin b, and prealbumin.

Serum protein synthesis in the early chick embryo.

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringers 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.

The Direct Embryotoxicity of Immunoglobulin G Fractions From Patients With Systemic Lupus Erythematosus

PROBLEM: To determine if IgG fractions from sera of individuals with systemic lupus erthymatosus (SLE) were toxic to cultures of whole rat embryos.

A quantitative analysis of ovalbumin utilization by the cultured chick embryo and its relationship to growth regulation during development

Chick embryo explants of 11–13 somites were cultured on defined medium containing a 9:1 mixture of ovalbumin- 14C and conalbumin- 12C for periods up to 48 hr. After 24 hr of culture on labeled medium some explants were transferred to unlabeled medium for the remaining 24 hr of culture. The amounts of intact ovalbumin- 14C (antibody precipitable), ovalbumin- 14C as breakdown products (trichloroacetic acid soluble) and ovalbumin- 14C converted to embryonic proteins were measured in the various regions of the explants. The relatively greater quantities of intact ovalbumin in the area opaca region of the explant during the 48-hr culture period suggested that this region was the site of intact protein uptake. During the first 6-hr of culture on labeled medium, the rate of uptake of intact ovalbumin by the area opaca exceeded the rate of accumulation of ovalbumin breakdown products, and, after transfer to unlabeled medium, the rate of decrease in intact ovalbumin exceeded the rate of decrease of ovalbumin breakdown products. These results indicated that the ovalbumin was not transported intact to the embryo, but rather was degraded by the area opaca. The larger pool of ovalbumin breakdown products in the area opaca compared to that in the embryo suggested that these pools were not in direct equilibrium. Despite the different pool sizes, both the area opaca and the embryo accumulated embryonic protein derived from ovalbumin at similar rates. A model is proposed describing the flow of nitrogenous nutrients from the medium to the embryo proper.