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Background Choline is an essential nutrient in methylation, acetylcholine and phospholipid biosynthesis, and in cell signaling. The demand by an embryo or fetus for choline may place a pregnant woman and, subsequently, the developing conceptus at risk for choline deficiency. Methods To determine whether a disruption in choline uptake and metabolism results in developmental abnormalities, early somite staged mouse embryos were exposed in vitro to either an inhibitor of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), or an inhibitor of phosphatidylcholine synthesis, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Cell death following inhibitor exposure was investigated with LysoTracker Red and histology. Results Embryos exposed to 250–750 μM DMAE for 26 hr developed craniofacial hypoplasia and open neural tube defects in the forebrain, midbrain, and hindbrain regions. Embryos exposed to 125–275 μM ET-18-OCH3 exhibited similar defects or expansion of the brain vesicles. ET-18-OCH3-affected embryos also had a distended neural tube at the posterior neuropore. Embryonic growth was reduced in embryos treated with either DMAE (375, 500, and 750 μM) or ET-18-OCH3 (200 and 275 μM). Whole mount staining with LysoTracker Red and histological sections showed increased areas of cell death in embryos treated with 275 μM ET-18-OCH3 for 6 hr, but there was no evidence of cell death in DMAE-exposed embryos. Conclusions Inhibition of choline uptake and metabolism during neurulation results in growth retardation and developmental defects that affect the neural tube and face. Teratology 64:114–122, 2001. © 2001 Wiley-Liss, Inc.

The roles ofengrailed-1 (En-1) andengrailed-2 (En-2) have been investigated during gastrulation and neurulation in mouse embryos. Using antisense oligonucleotides and murine whole embryo culture, early somite embryos were injected with antisense phosphorothioated oligonucleotides directed againstEn-1 ofEn-2 transcripts and then grown in vitro for 48 hr. Inhibition ofEn-1 by antisense targeting during this period resulted in embryos with craniofacial abnormalities, specifically loss of mid-and hindbrain tissue and hypoplasia in associated neural crest derived areas such as the face and first and second pharyngeal arches. In addition, En-1 appeared to be essential in early patterning of the neural tube. Embryos removed from culture as 8 hr following injection exhibited undulations in the presumptive spinal cord. Histological analysis of the affected neural tubes at 48 hr showed disrupted cytoarchitecture and in some cases, apparent dorsal-ventral duplication of the neural tube and underlying notochord. Using S100 beta as a notochord marker, embryos removed from culture at 8 or 12 hr following injection exhibited loss of S100 beta expression in the notochord region subjacent to affected neural tube segments, suggesting that S100 beta, or other notochordal genes, may be downstream components of the En-1 regulatory cascade. Furthermore, antisense inhibition ofEn-1 induced caudal dysgenesis, suggesting disruption in primitive streak function. Antisense targeting of En-2 expression at early somite stages produced few alterations in development, although approximately one third of the embryos exhibited a series of lateral folds in the spinal cord at the level of the forelimb-bud. Combined injections withEn-1 andEn-2 antisense resulted in no novel phenotypes, suggesting that the genes do not possess functional redundancy. © 1995 Wiley-Liss, Inc.

A role for choline during early stages of mammalian embryogenesis has not been established, although recent studies show that inhibitors of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), produce neural tube defects in mouse embryos grown in vitro. To determine potential mechanisms responsible for these abnormalities, choline metabolism in the presence or absence of these inhibitors was evaluated in cultured, neurulating mouse embryos by using chromatographic techniques. Results showed that 90%-95% of 14C-choline was incorporated into phosphocholine and phosphatidylcholine (PtdCho), which was metabolized to sphingomyelin. Choline was oxidized to betaine, and betaine homocysteine methyltransferase was expressed. Acetylcholine was synthesized in yolk sacs, but 70 kDa choline acetyltransferase was undetectable by immunoblot. DMAE reduced embryonic choline uptake and inhibited phosphocholine, PtdCho, phosphatidylethanolamine (PtdEtn), and sphingomyelin synthesis. ET-18-OCH3 also inhibited PtdCho synthesis. In embryos and yolk sacs incubated with 3H-ethanolamine, 95% of recovered label was PtdEtn, but PtdEtn was not converted to PtdCho, which suggested that phosphatidylethanolamine methyltransferase (PeMT) activity was absent. In ET-18-OCH3 treated yolk sacs, PtdEtn was increased, but PtdCho was still not generated through PeMT. Results suggest that endogenous PtdCho synthesis is important during neurulation and that perturbed choline metabolism contributes to neural tube defects produced by DMAE and ET-18-OCH3.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h). In early somite stage embryos hyperglycemia (⩾ 33 mM) reduced both VYS uptake and embryonic accumulation of 35S-Hb during the last 12 h of a 48 h exposure period. However, there was no concentration dependent effect on the embryonic accumulation of 35S at the early somite stage. The time course for hyperglycemia-induced effects on morphology and VYS histiotrophic function are different in the early head fold versus early somite staged embryos. Thus, at the early head fold stage malformations were induced only after a 6-12 h exposure, while the histiotrophic function was reduced only during the 32-44 h of exposure. Therefore, our study indicates that hyperglycemia can alter the histiotrophic function of the VYS following long exposure periods. The time course for alterations in VYS histiotrophic function is not correlated with that for induction of malformations but may be related to the growth reduction produced by long-term exposure to hyperglycemia in vitro.

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.

Previous studies have shown that the ketone body D,L,-beta-hydroxybutyrate was teratogenic to mouse embryos exposed in culture during the period of neurulation. Inhibition of closure of the cranial and caudal neuropores was the most frequently occurring defect and these abnormalities were thought to be the forerunner of anencephaly and spina bifida, respectively. However, additional studies demonstrated that embryos could recover morphologically from these effects if the ketone body was removed from the culture medium and if the recovery period was of sufficient duration. In an attempt to define further the phenomenon responsible for this recovery and to determine the extent of the recovery process, the present study examining the cross-sectional area, cell number, and mitotic index of cranial neuroepithelial cells was conducted in mouse embryos cultured from the early somite stage under one of the following conditions: 1) control medium for 60 h; 2) medium containing 32 mmol/l D,L,-beta-hydroxybutyrate for 24 h followed by culture in control medium for an additional 36 h (recovery group); 3) medium containing 32 mmol/l D,L,-beta-hydroxybutyrate for 60 h (continuously exposed group). The results indicate that although neural tube closure occurred in the recovery group, complete recovery was limited to the ventral regions of the forebrain and that the remainder of the prosencephalon as well as the rhombencephalon failed to undergo complete catch-up growth. Thus, cell numbers in these areas were approximately 70% of control values. Therefore, while the gross anatomical disturbances produced by the ketone body may be compensated for, histological alterations in the affected tissues remain. Ultimately, these data suggest that neurological deficits may be an outcome of ketone body exposure during the early stages of embryogenesis.

A fraction from diabetic rat serum containing a low-molecular-weight (800-1000) somatomedin inhibitor (SI) alters growth and development in both neurulation and early limb bud staged mouse embryos in vitro. Previous studies suggested that an accumulation of serum proteins and morphological changes of the visceral yolk sac (VYS) were produced following exposure to the SI in early limb bud staged conceptuses. The morphological changes, characterized by the presence of large endosomes in the endodermal cells, suggested that the SI altered histiotrophic nutrition, whereby proteins are pinocytosed by the endodermal VYS cells and degraded to constituent amino acids. Therefore, the effects of the SI on pinocytosis and protein degradation by the VYS were evaluated using the whole embryo culture system. Results showed that the SI reduced fluid phase pinocytosis as determined by the uptake of [U-14C]sucrose, but that accumulation of [3H]leucine-labeled hemoglobin ([3H]Hb) by the VYS was greater following exposure to the SI than in controls. In contrast, the accumulation of 3H-labeled amino acids in the embryo (produced from the degradation of [3H]Hb by the VYS) was reduced by the SI. The extent of amino acid reduction in embryonic accumulation is dependent upon the concentration of SI in the culture medium and correlates with the incidence of malformations produced by the SI, i.e., high rates of malformations occur with large reductions in embryonic 3H-labeled amino acid accumulation. The apparent paradox of high [3H]Hb accumulation in the presence of decreased pinocytosis appears to be the result of altered processing of the [3H]Hb in the endodermal cells. The altered processing decreases the "elimination" of the proteins from the VYS and results in the decrease in 3H-labeled amino acid present in the embryo proper. Therefore, the SI appears to alter two processes of VYS histiotrophic function. (1) decreased pinocytosis and (2) altered protein processing, ultimately resulting in a decreased availability of substrates for the embryo. During the early stages of embryogenesis in the human, the trophoblast cells of the placenta are responsible for the transport of nutrients from the maternal to embryonic systems. Since these cells show high phagocytic and pinocytotic activities, the SI may also disrupt these processes in the chorioallantoic placenta and contribute to diabetes-induced embryopathies.

Previous investigations have demonstrated that a potential mechanism for D,L,-beta-hydroxybutyrate (BOHB)-induced teratogenesis in neurulating mouse embryos (5–6 somite stage) after 24 hours of exposure in vitro is mediated by an inhibition of the pentose phosphate pathway (PPP) (Hunter, et al. '87). Employing conceptuses of an earlier stage (2–3 somite stage), the biochemistry of BOHB-induced abnormalities was examined further by exposing embryos to 32 mM BOHB for 24 hour and comparing results with controls with respect to the rate of metabolism via the PPP, de novo pyrimidine biosynthesis (PB), and BOHB utilization. Moreover, the capability of these BOHB-exposed embryos to recover from such an insult was also assessed by transferring them to fresh control medium and allowing them to grow for an additional 36 hours. Both controls and BOHB-exposed embryos showed a progressive increase in rate of BOHB utilization between days 9 and 11.5 of gestation in vitro. Exposure to the ketone body produced a 100% rate of neural tube defects and a 25.2% decrease in total embryonic protein content. In contrast to results obtained at the 5–6 somite stage, no inhibition of the PPP in whole conceptuses, embryos, or visceral yolk sacs was observed in the group exposed to BOHB at the 2–3 somite stage. Furthermore, a 7.5 mM D-ribose supplement, an intermediate in the PPP, was unable to rescue the younger embryos from BOHB-induced abnormalities and growth retardation. On the other hand, BOHB produced a 34.3% decrease in pyrimidine biosynthesis in the 2–3 somite embryos, but not in the visceral yolk sac. In addition, embryos recovered biochemically after being transferred to control medium, demonstrating a 25.5% overshoot in pyrimidine biosynthesis. Therefore, the mechanism of BOHB-induced teratogenesis appears to differ depending on the stage of embryonic development at the time of initial exposure.

Background The mycotoxin fumonisin B1 (FB1) inhibits sphingolipid synthesis, blocks folate transport, and has been associated with increased incidences of cancer and neural tube defects. Results from reproductive studies in animal models in vivo and in vitro have demonstrated toxicity in some cases, but no specific terata after fumonisin exposure. No information is available about folic acid's potential to protect against this toxicity. Methods Neurulating mouse embryos were exposed to fumonisin or folinic acid in whole embryo culture and assessed for effects on growth and development. Results Fumonisin exposure inhibited sphingolipid synthesis, reduced growth, and caused cranial neural tube defects in a dose dependent manner. Supplemental folinic acid ameliorated the effects on growth and development, but not inhibition of sphingolipid synthesis. Conclusion Fumonisin has the potential to inhibit embryonic sphingolipid synthesis and to produce embryotoxicity and neural tube defects. Folic acid can reverse some of these effects, supporting results showing that fumonisin disrupts folate receptor function. Teratology 66:169–176, 2002. © 2002 Wiley-Liss, Inc.

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

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.

Children of mothers with insulin-dependent diabetic mothers (IDDM) have a 2-4 times higher incidence of congenital birth defects as compared to the general population, including cardiac abnormalities, of unknown etiology. Using rodent embryos to explore potential teratogenic factors of the altered IDDM metabolism, it has been shown that exposure to hypoglycemia in vitro results in a variety of defects, including cardiac malformations. Since pregnant diabetics experience frequent episodes of low blood glucose, it was hypothesized that hypoglycemia may play a role in the generation of heart abnormalities seen in children born to IDDM mothers. Several studies have indicated that during embryogenesis the heart is dependent on glucose for energy production such that under hypoglycemic conditions, insufficient amounts of ATP may be produced resulting in abnormalities. To test this hypothesis, cardiac ATP content was monitored in D10-D12 (plug day = D1) hearts. In addition, the contribution of glycolysis and the Krebs cycle to ATP production was monitored. D10 hearts exposed to euglycemic control conditions were found to be primarily dependent on glycolysis for ATP production from glucose before switching to the Krebs cycle and oxidative phosphorylation for energy production from this substrate on D11. Exposure to hypoglycemia did not alter the timing of this maturation process or deplete cardiac ATP content. However, cardiac lactate levels increased approximately twofold in the presence of hypoglycemia on d10. Since increased concentrations of lactate are harmful to many tissues and have been shown to be detrimental to the adult rat heart, lactic acidosis may explain the origin of cardiac defects produced by hypoglycemia, and not a deficiency of ATP.

Effects of Cocaine Administration during Early Organogenesis on Prenatal Development and Postnatal Growth in Mice. Hunter, E. S., III, Kotch, L. E., Cefalo, R. C., and Sadler, T. W. (1995). Fundam. Appl. Toxicol. 28, 177-186. Cocaine use has been associated with adverse developmental effects in humans. However, clinical reports both confirm and deny an association between cocaine use and malformations. Similarly, differences in species and strain, as well as route and timing of cocaine administration, have added to the difficulties in determining the teratogenicity of cocaine in animal models. This study was undertaken to compare the effects of dose, route, and timing of cocaine administration in ICR mice during early organogenesis. A single intraperitoneal (ip) administration of cocaine (⩾60 mg/kg) on Day 9 of gestation (plug day = 1) produced maternal lethality. The predominant developmental effect of cocaine administration was an increase in the percentage of litters exhibiting an enlarged renal pelvis. Despite a high incidence of affected pups at these doses, the enlargement was not severe. These results, in agreement with previous reports, provide further evidence that the developing urogenital system is sensitive to cocaine administration. When cocaine was administered using a subcutaneous route, pup weights were greater and the incidence of enlarged renal pelvis was lower than when an ip route was used. To better mimic human binge cocaine abuse, the toxicity of a "split dose" was determined. A 60 mg/kg dose was administered using one administration of 60 mg/kg, two treatments of 30 mg/kg, or three administrations of 20 mg/kg with 1 hr separating the treatments. The incidence of enlarged renal pelvis was similar when cocaine was administered as one or two but was decreased when cocaine was administered as three treatments. Both the route and split-dose studies suggest that high-peak serum concentrations are required to perturb development. There were no differences in the incidence or severity of enlarged renal pelvis when cocaine was administered on Day 8, 9, or 10 or on all 3 days of gestation. This suggested that the increase in enlarged renal pelvis may not be a specific teratogenic effect of cocaine administration but may be a delay of normal development induced by cocaine exposure during this early period of organogenesis. To address this hypothesis, cocaine was administered on Day 9 using an ip route and the pups were allowed to be naturally born. In pups whose mothers received cocaine there was an increase in postnatal deaths and a trend toward a reduction in pup body weight/litter at Postnatal Day 21. However, when renal morphology was assessed on Postnatal Day 21 no abnormal kidneys were seen. This supports the hypothesis that enlarged renal pelvis produced by cocaine administration during early organogenesis represents a developmental delay and not a persistent teratogenic defect. These studies suggest that high peak cocaine concentrations are required to delay normal kidney morphogenesis in mice.

Wnt-1 and Wnt-3a have been postulated to share functional redundancy in spinal cord morphogenesis due to their homologies in protein structure and overlapping expression patterns. In this study, antisense oligonucleotides and a murine whole embryo culture system were used to examine functional interactions of Wnt-1 and Wnt-3a in late gastrulation and neurulation. Early somite mouse embryos were injected with combinations of Wnt-1 and Wnt-3a antisense oligonucleotides and then grown in vitro for up to 48 hr. Simultaneous inhibition of Wnt-1 and Wnt-3a expression resulted in pattern loss in the presumptive spinal cord, which was apparent within 4 hr following antisense treatment. The neural tube was wavy, there was a reduction in the number of nuclear layers in the walls of the neural tube, and evidence of decreased cell adhesion between neuroepithelial cells by 12 hr postinjection. In addition, notochord and primitive streak abnormalities accompanied neural tube abnormalities. The existence of regulatory interactions between Wnt-1, Wnt-3a, and engrailed genes was also examined in this study. Antisense inhibition of Wnt-1 or Wnt-3a expression resulted in reduction of engrailed protein levels in the brain, somites, and spinal cord. However, simultaneous inhibition of both Wnt genes resulted in more complete loss of engrailed protein in these regions. Herein, we present data suggesting functional redundancy of Wnt-1 and Wnt-3a in neural tube patterning and in regulation of engrailed expression.

The incidence of birth defects among offspring of mothers with non–insulin dependent diabetes mellitus (NIDDM) is 2–3–fold higher than among infants of non diabetics. Since mothers with NIDDM are frequently given oral hypoglycemic agents, including sulphonylureas and biguanides, to control their disease and since these agents have been associated with the occurrence of congenital malformations in humans and animals, the embryotoxic effects of the most commonly employed biguanides, metformin and phenformin, were evaluated in whole embryo culture. Neurulating mouse embryos were exposed to therapeutic concentrations (metformin 500–2,550 mg per day; phenformin 50–400 mg per day, respectively) of the compounds for 24–48 h. Concentrations of metformin in culture ranged from 0.15 to 1.8 mg/ml and phenformin from 2.5 × 10−5 to 0.4 mg/ml. Cultures were terminated and scored for gross morphological alterations and total protein content. Metformin produced no alterations in embryonic growth and no major malformations. Approximately 10% of all embryos exposed to metformin regardless of dose, exhibited open cranial neuropores after 24 h of culture. However, this anomaly appeared to represent a delay in closure as opposed to an overt defect, since no embryos exposed to the highest concentration of the drug and cultured for 48 h showed open neural tubes. In contrast, phenformin produced dose dependent changes in incidence of malformations, protein content, and embryolethality. Malformations included neural tube closure defects, craniofacial hypoplasia, and reduction in size of the first and second visceral arches. Doses above 0.1 mg/ml produced embryolethality and all embryos were killed at the 0.4 mg/ml concentration. Thus, of the two biguanides phenformin has a greater toxicity in whole embryo culture, suggesting that metformin may be safer for use during pregnancy. © 1994 Wiley-Liss, Inc.

The possible involvement of the neurotransmitter serotonin (5-HT) and its binding protein (SBP) in cardiac morphogenesis was studied using mouse whole embryo culture (together with immunocytochemistry or 3H-thymidine autoradiography) and a cell migration assay. Embryos were cultured before and during the period of endocardial cushion formation, embryonic (E) days 9-12, in the presence of 5-HT, the monoamine oxidase (MAO) inhibitor nialamide, or an uptake inhibitor (fluoxetine or sertraline). For the migration assay, cells from the outflow tracts of E12 embryos were dissociated and placed in a chemotaxis chamber together with different concentrations of 5-HT. E9 embryos cultured in the presence of 10 microM 5-HT and nialamide exhibited intense 5-HT immunoreactivity (5-HT IR) throughout the myocardium. This staining was greatly diminished by fluoxetine, sertraline, or the absence of nialamide. As morphogenesis proceeded, myocardial staining in embryos exposed to 5-HT became restricted to developing endocardial cushion forming regions and was more completely blocked by uptake inhibitors. No evidence for 5-HT synthesis by myocardium was found at any age studied using the precursor L-tryptophan. SBP was present in endocardial cushions in cultured and uncultured embryos. 3H-thymidine autoradiography demonstrated that both fluoxetine and sertraline inhibited proliferation of cardiac mesenchyme, endocardium, and myocardium. These effects were most pronounced when exposure began at E9 (prior to cushion formation). Dose-dependent effects of 5-HT on migration of outflow tract cells were also observed. Taken together, these results suggest that 5-HT may play a role in cardiac morphogenesis during endocardial cushion formation.

Summary The neurotransmitter serotonin (5-HT) was localized in the ectoplacental cone (EPC) and placenta of the day 9–12 (E9–12) mouse embryo in vivo and in whole embryo cultures, using immunocytochemistry with a specific 5-HT antiserum. In uncultured conceptuses, 5-HT immunoreactivity (5-HT IR) was most intense in the EPC at E9 (2–7 somites), particularly in giant cells around the periphery. Nuclear staining was observed in lightly staining giant cells and in small cells in the core of the cone. By E10 (18–24 somites) 5-HT IR in the placenta was less intense and almost exclusively limited to giant cells, where it was localized to chromatin-like material in nuclei. The same pattern and level of 5-HT IR persisted through E12. In the placenta, 5-HT IR appeared to be most intense in giant cells located near aggregations of platelets in decidual blood vessels. 5-HT IR was enhanced in cultured conceptuses, and further increased when exogenous 5-HT was added to the culture medium. Immunoreactivity was greatly reduced by adding the 5-HT uptake inhibitor fluoxetine to the culture medium, or culturing conceptuses in medium containing 5-HT depleted rat serum. Thus, 5-HT was apparently taken up from the culture medium. In conceptuses exposed to exogenous 5-HT, immunoreactivity in the placenta appeared as a gradient from the giant cells to the inner layers, suggesting that these cells may transport 5-HT toward the embryo. No evidence of 5-HT synthesis by the EPC/placenta was found. These results suggest that 5-HT present in the EPC/placenta is due to uptake, not synthesis. Possible sources and functions of 5-HT in the developing placenta are discussed.

Wnt-1 and Wnt-3a proto-oncogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attenuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurally related to Wnt-1, targeted the forebrain and midbrain region, which were hypoplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nucleotides) make this antisense oligonucleotide/whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development.

This study describes the timecourse of expression of low-affinity serotonin uptake sites in the developing craniofacial region of the mouse embryo. Whole mouse embryos were incubated in the presence of various serotonergic compounds followed by immunocyto-chemical localization of serotonin (5-HT) and its binding protein. In the gestational day 9 embryo (3–5 somites), 5-HT uptake was observed in the myocardium of the heart, the visceral yolk sac and foregut. A specific and transient pattern of 5-HT uptake was observed in the hindbrain neuroepithelium from day 9.5–11, where it was localized in rhombomeres 2–5 in the day 9.5 embryo. By day 10, when rhombomeres were no longer evident, uptake was present in the dorso-lateral neuroepithelium surrounding the fourth ventricle (rhombic lip; cerebellar anlage). Uptake of 5-HT was initially observed in the surface epithelium of the craniofacial region at day 10 (20–25 somites) and was greatly increased at day 11. The invaginating lens, nasal placode epithelium and otocyst also took up 5-HT at day 11. During these stages a 45 kD serotonin-binding protein (SBP) was expressed in craniofacial mesenchyme, and became progressively restricted to regions subjacent to epithelial uptake sites. These staining patterns were shown to be specific for 5-HT and SBP by their absence in embryos stained using preabsorbed antisera. The timecourse of these patterns are correlated with critical events in craniofacial morphogenesis including (1) onset of inductive epithelial-mesenchymal interactions, (2) invagination and fusion of placodal structures, (3) presence of rhombomeres, and (4) regions of low proliferative activity.

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phe-nylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyllactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla. In summary, the results showed that the relative teratogenicity of Phe and its metabolites differs and suggested that a metabolite of Phe and/or a combination of Phe and one or more of its metabolites play a role in the phenylketonuric embryopathy.

The effect of short-term hypoglycemia was studied at two stages of development in postimplantation mouse embryos in vitro. Day 8 (gastrulating) mouse embryos were placed in hypoglycemic medium (60, 80, 100, or 110 mg/dl glucose) for 4 hours in which normoglycemia (120 to 150 mg/dl glucose) was restored for the remaining 44 hours of culture. Day 9 (neurulating) mouse embryos were exposed to hypoglycemia (20, 40, 60, or 80 mg/dl glucose) for 2, 4, 6, or 24 hours followed by normoglycemia for the remainder of 24 hours. At the end of culture embryos were evaluated for growth and malformations and compared with controls grown in normoglycemic medium. The results show that a 50% reduction in glucose for as little as 2 hours causes dysmorphogenesis in neurulating mouse embryos, whereas longer exposure times, more severe levels of hypoglycemia, or both are required to affect growth. Furthermore, gastrulating embryos are more sensitive to short periods of hypoglycemia than those undergoing neurulation.

Developing neuroepithelial cells from the cranial region of neural folds were examined at various intervals from 15 minutes to four hours after administration of teratogenic (250 mg/kg) or embryolethal (500 mg/kg) doses of HU to ICR/DUB mice on ninth day of gestation. Thirty percent of surviving fetuses showed extencephaly after the teratogenic dose. The initial ultrastructural change in some neuroepithelial cells occurred within 15 minutes to one hour after treatment with the teratogenic dose and involved a breakdown of polyribosomes into monosomes which were dispersed evenly in the cytosome. At one to two hours, condensation of the cytoplasm and chromatin was observed along with distorted nuclear shape. At two to four hours, cells exhibited extremely condensed cytoplasm and fragmentation of the cells occurred. The affected cells showed pyknotic nuclei and segregation of fibrillar and granular components of the nucleolus. In some nuclei, particularly after treatment with the higher dose of HU, a condensation of the nucleolus was observed. At this stage images were obtained which indicated that neighboring cells phagocytosed the cellular fragments derived from the HU affected cells.

The ketone body beta-hydroxybutyrate (B-OHB) has been shown to be teratogenic to early-somite mouse embryos, although the mechanism responsible for these defects has not been determined. In an attempt to define this mechanism, the present study investigated the normal pattern of both glucose and B-OHB utilization in the developing embryo and fetus. Furthermore, the metabolic interaction of these two substrates, i.e., the potential for B-OHB to inhibit glycolysis, was studied. All studies compared early and late embryonic periods of development as well as fetal stages. The results show that the early embryo relies almost exclusively on glycolysis for energy metabolism and suggests that there is an increasing importance of the Krebs cycle with increasing gestational age. Similarly, the early embryo has a low capacity to metabolize B-OHB, whereas later gestational stages display a greater rate of utilization. Finally, there appears to be no inhibition of glycolysis by B-OHB (via so-called "substrate interactions") during early embryonic stages. However, the compound significantly inhibits glycolysis during later embryonic and fetal stages. These studies suggest that the teratogenicity of B-OHB in the early embryo is not due to its effects on modulating glycolysis, although this mechanism may be operating at later periods of gestation.

A low molecular weight somatomedin inhibitory serum fraction (SI), obtained from streptozotocin-induced diabetic rats, causes morphological abnormalities and growth reduction in mouse embryos grown in whole embryo culture (WEC). These abnormalities are thought to be caused, at least in part, by a failure of the visceral yolk sac (VYS) to properly degrade proteins, a process that normally provides the conceptus with amino acids and peptides for de novo protein synthesis (histiotrophic nutrition). To test this hypothesis, embryos exposed to the SI were provided with a mixture of ten essential amino acids (supplemented group) in an attempt to circumvent SI-induced VYS dysfunction. Results showed that 82.4% (14/17) of embryos in the amino acid-supplemented group exhibited improved growth and development compared to those embryos exposed to medium containing the SI alone (unsupplemented group). Supplemented embryos showed greater expansion of the brain regions, improved visceral arch development, and increased protein content compared to nonsupplemented SI-treated embryos. However, these parameters were still reduced compared to controls. VYSs from both the unsupplemented and amino acid-supplemented groups were identical with respect to alterations in morphology and increased protein content compared to VYSs from conceptuses cultured in control medium (with or without amino acid supplementation). The improvement in embryonic growth and development due to amino acid supplementation in spite of VYS abnormalities supports the hypothesis that nutritional deprivation is one aspect of SI-induced teratogenesis.

Fusing and non-fusing regions of neural folds from mouse embryos were examined during neurulation for the distribution of extracellular macromolecules (surface coats) prior to and at the time of closure. Ruthenium red staining of 10th day ICR/DUB mouse embryos was used to detect the distribution of surface coat material. Light microscopic examination of fusing and non-fusing regions in the midbrain, hindbrain, and spinal cord showed a consistent increase in ruthenium red positive material immediately prior to closure. Heavy deposits of positive staining material were present along apical neural fold borders and overlying ectoderm cells. This staining pattern was consistent in the three regions examined, but the pattern of initial contact between opposing neural folds differed. In mid- and hindbrain areas contact was initiated by overlying ectoderm, whereas in spinal cord regions contact was first established by neuroepithelial cells. Once contact between opposing neural folds was initiated a decrease in stainable material was observed.

Teratogenic effects of hyperglycemia on early stages of embryogenesis were investigated using the whole embryo culture technique. Young (0–1 and 2–3 somites) and old (4–6 somites) mouse embryos were exposed for 24 h to diabetic levels of excess glucose equal to 5× and 8× the normal blood glucose levels. A high frequency of neural tube defects (exencephaly) was observed in embryos maintained at either concentration, and this effect was dose- and age-dependent. Thus, younger embryos (0–3 somites) were more frequently affected than older ones, and more embryos were affected at the higher dose than the lower one. Although malformations were observed, amounts of total protein were similar between control and treated cultures except for the youngest (0–1 somite) embryos, which had significantly lower amounts of protein than controls. Histological analysis of embryos maintained in 8× glucose for 4, 8, and 24 h after initiation of culture showed only a slight amount of pyknotic debris in the neuroepithelium and revealed no other causes for the abnormality. Inhibition of neural tube closure observed in this study is similar to that occasionally described in infants of diabetic mothers, and suggests that hyperglycemia may play a role in production of the malformation.

The capability of rodent embryos to recover in growth and development subsequent to exposure to an insult was investigated employing whole embryo culture. Early somite stage mouse embryos were exposed to 32 mM D,L,-beta-hydroxybutyrate (D,L,-beta OHB) for 24 hr (Period I), and were then transferred and maintained in control medium for an additional 36 hr maximum (Period II). Growth of this recovery group (Group B) was monitored at various time points of Period II and the results were compared with an unexposed control group (Group A) and another continuously-exposed reference group (Group C). At the end of Period I, 100% of D,L,-beta OHB-exposed embryos exhibited neural tube closure defects and were growth retarded. At 36 hr of Period II, cranial and caudal neural tube defects of Group B embryos were reduced to 3-7% and 0%, respectively. These embryos also demonstrated an excess in growth velocity during recovery so that at the end of Period II, total protein content was comparable to control values. In contrast, Group C embryos remained growth retarded and showed a significant increase in cranial and caudal neural tube defects (20 and 75%, respectively). Thus, neurulating mouse embryos were capable of catch-up growth following an embryotoxic exposure to D,L,-beta OHB. The progression of development of total protein values and morphological features such as elimination of neural tube defects was intimately related to the amount of time allowed for recovery. Moreover, an elevation of growth rate over normality, which is characteristic of catch-up growth, was observed.

Mouse embryos were exposed to cadmium (5.35 micrograms/ml) for either 0.5h or 1h and then transferred to fresh serum for the duration of the experiment. Embryos exposed for 0.5h developed numerous malformations that were primarily localized to the craniofacial region. Embryos exposed for 1h to the same level of cadmium all failed to thrive. In a separate series of experiments zinc (10 micrograms/ml) was added 15 minutes prior to the addition of cadmium. Embryos treated with cadmium for 30 min. after the addition of zinc, resulted in 15 of 16 embryos developing normally. The second group of embryos treated for 1h after preincubation with zinc resulted in 93% of the embryos surviving after 48h, albeit malformed. These results suggest that: 1) Exposure time to a teratogen is a critical parameter to consider when designing in vitro experiments 2) zinc is effective in protecting embryos against cadmium induced teratogenesis and 3) the whole embryo culture system is effective in demonstrating biological interactions between agents.

Serum factors characteristically altered in the diabetic state, e.g., glucose, ketone bodies (β-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-β-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-β-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.

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3–4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.

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 μ/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.

Offspring of diabetic humans and laboratory animals have been shown to have a higher incidence of congenital malformations with attendant growth retardation. These defects have been attributed to alterations in the intrauterine environment and specifically to changes in maternal serum factors, e.g., glucose and ketone bodies. Our investigation examines the potential teratogenicity of a low-molecular-weight (940) serum fraction with demonstrated somatomedin inhibitory activity isolated by column chromatography from streptozocin-induced diabetic rats. Mouse embryos were exposed to control or the inhibitor fraction at concentrations of 0.25–0.6% vol/vol (0.9–3.0 μg protein/ml culture medium) in whole embryo culture and evaluated for the presence of malformations and growth retardation. Embryos exposed to inhibitor during the period of neurulation (3–5 somites) exhibited neural tube and craniofacial defects, whereas those exposed during early limb bud stages (18–19 somites) exhibited abnormalities of the forebrain and face. In addition, both stages were growth retarded. Control fractions produced no abnormalities. These results demonstrate a potential role for somatomedin inhibitors in diabetic embryopathy and suggest that factors other than hyperglycemie and hyperketonemia may contribute to the higher incidence of malformations among infants of diabetic mothers.

DL-beta-Hydroxybutyrate (DL-BOHB) is teratogenic to rodent embryos in culture, but the biochemical mechanism(s) responsible for the induction of malformations has not been elucidated. Thus, to delineate a potential mechanism, the interaction of the ketone body with embryonic glucose metabolism was investigated. Mouse embryos were exposed in whole embryo culture to DL-BOHB or each isomer separately during the period of neurulation. The results demonstrate that DL-BOHB inhibits glucose oxidation by the pentose phosphate pathway (PPP) with no concomitant effect on Krebs cycle or glycolysis. Furthermore, decreased rates of embryonic uridine monophosphate and DNA synthesis were produced by DL-BOHB, whereas orotate synthesis was unaffected, thereby suggesting that the availability of ribose moieties synthesized by the PPP was compromised. This hypothesis was supported by the observation that addition of ribose to DL-BOHB-containing medium reduced the incidence of ketone body-induced neural tube defects and maintained the rates of DNA synthesis at control levels. Furthermore, these biochemical alterations are due to the synergistic effects of the D and L isomers and may be related to changes in redox states.

The ketone body B-hydroxybutyrate (B-OHB) produces malformations and ultrastructural alterations in mitochondria of mouse embryos exposed for 24 hours to the compound in whole embryo culture. The present study was conducted to establish the time-course of the mitochondrial changes to determine whether the changes are reversible, and to relate these changes to the malformations produced by the compound. Since mitochondria also play a key role in the metabolism of ketone bodies, the capacity of the early somite embryo to metabolize B-OHB was investigated in an effort to link the morphological alterations in the mitochondria to a biochemical process. Early somite embryos were cultured 4, 8, or 24 hours in the presence of 32 mM DL-B-OHB and then cultured for an additional 24 hours in control serum. Finally, embryonic tissue during the teratogenic period was assessed for its capability to oxidize B-OHB using D-(3-14C)-B-OHB. The treated embryos showed progressive alterations in the mitochondria, beginning at 4 hours with a loss of matrix density and culminating at 24 hours with high-amplitude swelling, complete loss of matrix density, and disappearance of cristae. These alterations were reversible following removal of the embryos after 24 hours of exposure to B-OHB and culturing for an additional 24 hours in control serum. Metabolism studies demonstrated that the early somite embryo possesses a limited capacity to oxidatively metabolize B-OHB. The biochemical implications of these findings are discussed with respect to the possible role of ketone bodies in the mechanism of diabetes-induced congenital malformations.

The morphogenesis of median facial clefts induced in mice by treatment with diazo-oxo-norleucine (DON) was studied. Following maternal treatment with 0.4 mg/kg DON on the 11th gestational day, 23.1% of fetuses recovered at the 18th day exhibited median facial clefts, which were characterized by a separation in the midline of normal midfacial components, i.e., the premaxilla, nasal bones, and nasal capsule. Malformed embryos could first be identified grossly 24 hr after DON administration by the presence of an abnormally wide separation between the two narrowed medial nasal processes. Evidence of cellular degeneration was observed in the mesenchyme of the nasal processes of DON-treated embryos 8 and 12 hr after treatment, but little or no pyknotic debris remained at 24 hr post-injection. Loss of cells due to cell death was reflected in decreased cell density observed in all areas of facial mesenchyme examined 24 hr after DON administration. It is suggested that DON-induced median facial clefts may be caused by a reduction in tissue volume, particularly in the midline, or by an interference with normal facial growth resulting in increased facial width and consequent failure of merging of the medial nasal processes.

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.

A comparison between static versus rotator culture systems and a variety of media (rat serum, new born calf serum, DMEM and Waymouth's) was made in an attempt to promote in vitro growth of mouse embryos from the beginning of neurulation (headfold stage) to the closure of the neural tube and formation of the limb buds (48 h). The results demonstrate that good development can be achieved for 48 h using a rotator system and that 80% of embryos cultured on rotators show growth and differentiation similar to that obtained for the same time period in vivo. Static cultures are less successful and embryos grown in this system show lower protein content and somite numbers than those maintained on rotators. Undiluted rat serum is superior to all other media tested and supports better growth and development as monitored by total protein and developmental abnormalities.

Hypoglycemia has been reported to induce congenital malformations and growth retardation in rodent embryos during the period of neural tube closure in vitro. However, the biochemical alterations responsible for the production of the dysmorphogenic effects have not been evaluated. Therefore, the rates of glucose metabolism by glycolysis, citric acid cycle, oxidative pentose phosphate pathway (PPP), and anabolic utilization were evaluated in mouse embryos and extraembryonic membranes using the whole embryo culture technique. Altered glucose metabolism by glycolysis and oxidative PPP, as well as altered anabolic synthesis, were produced by exposure to hypoglycemia. In embryos exposed to mild hypoglycemia (80 mg/dl) altered metabolism by the PPP and an associated effect on nucleic acid synthesis were in part responsible for the dysmorphogenic effects of this treatment. In contrast, severe hypoglycemia (40 mg/dl) appeared to have an immediate effect on glycolytic metabolism in addition to effects on the PPP and nucleic acid synthesis. Therefore, a multifactorial biochemical mechanism contributes to the induction of malformations by severe hypoglycemia in mouse embryos in vitro. Furthermore, the differential effects of moderate vs. severe hypoglycemia on glycolytic metabolism, and possibly energy production, may account for the differences in the severity of these treatments on embryonic growth and the incidence of malformations.

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3–4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30–45×103MT. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.

The effect of chlorambucil on the rates of DNA, RNA, and protein synthesis in mouse embryos was investigated using a system of whole embryo culture. Embryos were isolated on the 11th day of gestation (33 +/- 3 somites) and grown in culture media for periods of 4-8 h. Reichert's membrane and most of the placental tissue was removed leaving only the amnion and visceral yolk-sac surrounding the embryo. In the presence of teratogenic doses of chlorabucil (15 mug/ml) the rate of DNA synthesis was significantly decreased at 4 and 8 h. RNA and protein synthesis were not inhibited at either of these times. A trend toward decreasing rates of protein synthesis at some time beyond 8 h was noted, but not tested.

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.

Head-fold and early somite stages of mouse and rat embryos maintained in whole-embryo culture throughout much of the period of organogenesis demonstrate normal growth and morphogenesis. Embryos directly exposed to teratogens in the culture system and embryos cultured in serum from adult animals treated with toxic compounds develop congenital abnormalities that resemble malformations induced in vivo by these same agents. Furthermore, the defects are dose- and stage-dependent, such that higher doses produce a greater percentage of malformed embryos, and younger embryos are more susceptible than older ones. These results--together with the observations that 1) data are rapidly produced, 2) quantifiable endpoints can be measured in mammalian systems at costs considerably below those inherent in in vivo analyses, and 3) the potential exists for monitoring serum toxicity in humans and primates--suggest that the whole-embryo culture system may be useful as a screening technique for potentially teratogenic substances.

3H-concanavalin A and the concanavalin A-horseradish peroxidase staining technique were used to study the distribution of surface coat material on the epithelium of the nasal folds and nasal groove of mouse embryos. In stages shortly before and during epithelial fusion concanavalan A stained or labeled material was present at apical surfaces of epithelial cells of the nasal groove and nasal folds. Silver grains, representing bound 3H-concanavalin A, were counted in defined areas of the nasal groove and presumptive fusion area in both anterior and posterior regions of the nasal folds. For both stages examined there was a significant increase in the amount of 3H-concanavalin A bound by presumptive fusion areas in posterior regions of the nasal folds as compared with anterior regions; i.e., the amount of surface coat was greater in the region just prior to the point of contact between the nasal folds. This finding is consistent with results from investigations of palatal shelf and neural fold fusion which suggest that increased synthesis of surface coat material is associated with adhesion and fusion of epithelial folds and shelves.

Early somite (2-4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum and Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.

Two human serum fractions, one from normal individuals (Mr 1,150-1,310 daltons) and the other (Mr 800-1,100 daltons) from patients suffering with uremia (renal failure, azotemia), were added to the medium used to grow embryos in whole-embryo culture (WEC) beginning at the 3-5 (day 9) or 18-21 (day 10) somite stage. Both of these fractions possessed somatomedin (insulin-like growth factor) inhibitory activity. Day 9 embryos exposed to either of the serum fractions for 24 hr exhibited incomplete rotation and neural tube closure defects and were smaller than control embryos (decreased total protein content). Developmental abnormalities induced in day 10 embryos following 24 hr in culture included a marked decrease in expansion of the brain regions, hypoplasia of the first two branchial arches, and decreased amounts of total protein compared to controls. The visceral yolk sacs (VYSs) of somatomedin inhibitor (SI)-exposed conceptuses were opaque, and those from day 10 conceptuses contained significantly more protein than controls. Morphologically, the VYS endoderm cells from SI-exposed embryos contained a much higher density of "vacuoles" than controls. These results mimic those produced by exposure of conceptuses to an SI of Mr800-1,100 obtained from the serum of diabetic rats and suggest that similar substances and mechanisms are involved.

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.

A rotator whole embryo culture system was used to assess the growth and development of late-primitive-streak-stage (Theiler stage 9-10) mouse embryos to the limb-bud stage of organogenesis in a variety of media containing combinations of mouse serum (MS), rat serum (RS), and Tyrode's buffer (TB). The results demonstrate that embryonic growth and morphogenesis to the early limb-bud stage (20 somite pairs; 48-h total culture period) mimicked that in vivo when embryos were grown for 24 h in combinations of MS:RS:TB 1:2:1 or 2:1:1 (v/v/v) and then were transferred to fresh medium containing RS:TB 3:1 at the early somite stage. When the culture period was extended for an additional 24 h (total 72-h culture period) embryonic growth retardation was observed. Regardless of the medium employed, superior growth was observed in embryos transferred at the early somite stage when compared to embryos cultured continuously in the same medium for the entire 48- or 72-h culture period.