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Both the metabolic studies on diabetes and the genetic studies using antisense oligodeoxynucleotides clearly demonstrate the importance and usefulness of rodent whole embryo culture. Without this technique, these studies would be impossible and, consequently, our knowledge of both normal and abnormal development would not be as advanced as it is today. The culture system fills a unique niche in studies in the fields of developmental biology and teratology and these sciences would have been less well served without Dr. New's contribution.

Mouse conceptuses at the 18-21-somite stage were grown for 2-24 h in vitro in the presence of a serum fraction (Mr = 800-1,080 daltons) possessing somatomedin-inhibitory activity (SI) isolated from diabetic rats. Following an 8-h exposure to the SI, DNA and incorporation of 3H-thymidine were reduced in the embryos while 12 h was required to observe a reduction in total protein and RNA. At the 24-h time point, the neurectoderm was thinner than in controls, and autoradiograms of this region showed a substantial decrease in grain density with 3H-thymidine, but not 3H-leucine or -uridine. Effects of the visceral yolk sac (VYS) preceded those on the embryo. The cytoplasm of the VYS endoderm cells from conceptuses exposed to the SI contained many vacuoles by 4 h, which were larger by 24 h. Total protein was greater than in controls from 4 h onward, although 3H-leucine incorporation, which had increased after 2 h of SI exposure, returned to control levels by 8 h. As seen by SDS-polyacrylamide gel electrophoresis, VYSs from conceptuses exposed to the SI for 4 or 24 h were enriched (compared to control VYSs) in four protein bands also present in the culture medium (primarily rat serum), suggesting that protein degradation and/or transfer of amino acids and peptides to the embryo was inhibited in these VYSs. Such a conclusion was supported by a quantitative decrease in proteins and amino acids in the exocoelomic fluid of conceptuses exposed to the SI for 24 h. The altered processing of proteins may therefore represent a primary cause of the SI-induced embryonic abnormalities.

The effects of hypoglycemia on mammalian embryos undergoing neurulation (third to fourth week of human development) were investigated. Mouse embryos were maintained for 28 hours in whole embryo culture in serum collected from rats that had received 50 units of 100 United States Pharmacopeia insulin units per milliliter. Glucose concentrations used were 40, 60, 80, and 147 mg/dl (normal blood glucose in the pregnant mouse is 125 mg/dl). After the culture period embryos were evaluated for malformations and growth and compared with those maintained under euglycemic conditions. The results demonstrate that glucose concentrations approximately 50% of normal maternal levels were teratogenic but not growth inhibitory, whereas concentrations in the range of 30% to 40% of maternal levels were lethal to the embryo. Furthermore, a 14-hour exposure to reduced blood sugar in either the first or second half of the culture period produced malformations.

Serum factors characteristically altered in the diabetic state, e.g., glucose, ketone bodies (beta-hydroxybutyrate), and somatomedin inhibitors, induce dysmorphogenesis with or without growth retardation in rodent embryos in whole-embryo culture. Furthermore, serum from diabetic animals, which contains combinations of altered factors, is teratogenic, thereby implying that the diabetic embryopathy is of a multifactorial origin. However, a detailed investigation with various combinations of factors at known concentrations to test this hypothesis has not been conducted. Therefore, we employed combinations of hyperglycemia (300 and 600 mg/dl; 16.6 and 33.3 mM), hyperketonemia (8 and 16 mM D-beta-hydroxybutyrate), and an 800- to 1000-Mr somatomedin-inhibitor serum fraction (at an amount equal to that found in 0.05 and 0.1 ml of serum from a diabetic rat per deciliter culture medium), which represented doses with low teratogenic potential, to determine if interactions of the factors could occur that would increase the risk of malformations in mouse embryos exposed in whole-embryo culture during gastrulation and neurulation. The results demonstrate that glucose and D-beta-hydroxybutyrate can act synergistically to produce growth retardation and additively to induce malformations. The addition of the somatomedin inhibitor exacerbates the induction of malformations produced by the ketone body and glucose. Thus, the origin of the diabetic embryopathy may be multifactorial, involving several altered maternal factors.

Through the use of the in vitro technique of mammalian whole embryo culture, the factors and mechanisms responsible for the increased incidence of congenital malformations among infants of diabetic mothers have been investigated. During early stages of embryogenesis, serum from streptozotocin-induced diabetic rats, hyperglycemia, hypoglycemia, hyperketonemia (beta-hydroxybutyrate), and low molecular weight somatomedin inhibitors are teratogenic or growth inhibitory, or both. Furthermore, combinations of these factors interact to increase the risk of a malformation occurring. In contrast, other factors, such as hyperinsulinemia and excess leucine, palmitic acid, and acetoacetate, have little or no teratogenic potential. Mechanisms of action of the factors are varied and may include alterations of arachidonic acid metabolism (hyperglycemia), glycolysis (hypoglycemia), DNA synthesis (beta-hydroxybutyrate), and embryonic nutrition (somatomedin inhibitors). The results demonstrate that the origin of the diabetic embryopathy is multifactorial, that many of the congenital defects are induced early in gestation before the diabetic woman may realize she is pregnant, and that insulin therapy reduces the risk.

The present study used the whole embryo culture technique to determine the effects of the ketone body, B-hydroxybutyrate (B-OHB), on organogenesis in mouse embryos. Embryos of two stages (3-4 and 5-6 somites) were exposed for 24 h to a racemic mixture of D-L B-OHB at levels of 8, 16, or 32 mM/L. An abnormal pattern of development resulted, consisting of growth reduction and inhibition or delay of neural tube closure in the cranial and/or caudal regions of the embryo. These effects were dose- and age-dependent, such that younger embryos were more frequently affected than older ones, and increasing doses produced a higher rate of malformation. Growth reduction as evidenced by embryonic protein showed similar relationships. Histologic analysis of embryos exposed to 32 mM/L B-OHB for 24 h revealed numerous cytoplasmic "vacuoles" widespread throughout the neuroepithelium, mesenchyme, and ectoderm. Ultrastructurally, these "vacuoles" proved to be mitochondria that had undergone high-amplitude swelling with a loss of matrix density and few identifiable cristae. No other consistent ultrastructural changes were noted, and the mitochondria of control tissues displayed a typical orthodox configuration. While this study does not conclusively limit the effects to the D-form of B-OHB, possible relationships exist between the ultrastructural alterations, ketone body metabolism, and abnormal morphogenesis.

Using the system of whole embryo culture the effects of insulin on early mouse embryogenesis were investigated. Three experimental approaches were employed in which (1) serum from diabetic rats receiving insulin therapy was used as culture medium; (2) insulin was added directly to serum collected from diabetic rats for use as culture medium; (3) insulin was added directly to medium (serum) collected from control rats. Mouse embryos were cultured in each of these media for 24 h and then evaluated for growth and malformations. The results demonstrated that insulin therapy was effective in reducing the incidence of malformations and growth retardation produced by serum from diabetic rats receiving no hormone supplement. However, addition of insulin directly to serum collected from diabetic rats was not effective in reducing the rate of abnormalities produced by this medium. Finally, addition of insulin at extremely high concentrations (10,000 muU/ml) produced no abnormal embryos. Thus, it appears that insulin therapy is successful in reducing malformation rates and that the hormone itself is not teratogenic in this system.