81 results on '"T. W. Sadler"'
Search Results
2. Inhibitors of choline uptake and metabolism cause developmental abnormalities in neurulating mouse embryos
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Melanie C. Fisher, Steven H. Zeisel, Mei-Heng Mar, and T. W. Sadler
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Male ,Embryology ,medicine.medical_specialty ,Phosphodiesterase Inhibitors ,Health, Toxicology and Mutagenesis ,Biology ,Toxicology ,Choline ,Embryonic and Fetal Development ,Mice ,chemistry.chemical_compound ,Organ Culture Techniques ,Pregnancy ,Internal medicine ,medicine ,Animals ,Neural Tube Defects ,Anti-Dyskinesia Agents ,Embryogenesis ,Neural tube ,Abnormalities, Drug-Induced ,Phospholipid Ethers ,Deanol ,Embryo culture ,Embryo ,Embryo, Mammalian ,Teratology ,Endocrinology ,Neurulation ,medicine.anatomical_structure ,chemistry ,Female ,Acetylcholine ,Developmental Biology ,medicine.drug - Abstract
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.
- Published
- 2001
3. Susceptible periods during embryogenesis of the heart and endocrine glands
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T W Sadler
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medicine.medical_specialty ,Time Factors ,Health, Toxicology and Mutagenesis ,Cellular differentiation ,Morphogenesis ,Physiology ,Biology ,Embryonic and Fetal Development ,Pregnancy ,Endocrine Glands ,Internal medicine ,medicine ,Humans ,Endocrine system ,Heart development ,Embryogenesis ,Public Health, Environmental and Occupational Health ,Cell Differentiation ,Heart ,Teratology ,Pregnancy Trimester, First ,Teratogens ,Endocrinology ,Female ,Stem cell ,Research Article ,Endocrine gland - Abstract
One of the original principles of teratology states that, "Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence" [Wilson JG. Environment and Birth Defects. New York:Academic Press, 1973]. The time of greatest sensitivity encompasses the period of organ formation during weeks 3-8 following fertilization in human gestation. At this time, stem cell populations for each organ's morphogenesis are established and inductive events for the initiation of differentiation occur. Structural defects of the heart and endocrine system are no exception to this axiom and have their origins during this time frame. Although the function and maturation of these organs may be affected at later stages, structural defects and loss of cell types usually occur during these early phases of development. Thus, to determine critical windows for studying mechanisms of teratogenesis, it is essential to understand the developmental processes that establish these organs. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12
- Published
- 2000
4. Mechanisms of neural tube closure and defects
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T. W. Sadler
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Neural fold ,Cell division ,Neural tube ,Ectoderm ,Biology ,Neuroepithelial cell ,Neuropsychology and Physiological Psychology ,medicine.anatomical_structure ,Neurulation ,Pediatrics, Perinatology and Child Health ,Notochord ,medicine ,Neuroscience ,Neural plate ,Genetics (clinical) - Abstract
Neural tube closure (neurulation) is a complex process involving many cell phenomena. Both extrinsic and intrinsic forces are essential to elevate the neural plate into the neural folds and to bring the folds into apposition for closure. Extrinsic forces involve the underlying mesenchyme cells and extracellular matrix, nonneural ectoderm, the gut tube and notochord, and cell surface glycoproteins. Intrinsic forces involve cytoskeletal elements and microtubles, region-specific variations in cell cycle times, positioning of daughter cells during cell division, and rearrangement of neuroepithelial cells. Closure itself begins in the cervical region and proceeds in rostral and caudal directions. In the mouse, multiple additional closure sites occur in the cranial region, but in humans there appears to be only one additional site, at the rostralmost tip of the forebrain. Differences in closure exist in cranial vs. caudal regions, and these variations may play a role in the types of neural tube defects that occur. In this regard, virtually all of the cell phenomena involved in closure present targets for insults leading to abnormalities, although specific mechanisms responsible for neural tube defects have not been well-defined. It is also not clear what the role of folic acid is in normal neural tube closure, although the vitamin may be important for DNA synthesis and/or methylation of macromolecules, such as DNA and protein. Until more is learned about the regulation of neural tube closure at the genetic and cellular levels, understanding how defects occur and developing methods for their prevention will be limited. MRDD Research Reviews 1998;4:247–253. © 1998 Wiley-Liss, Inc.
- Published
- 1998
5. Growth and differentiation factors
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Patricia F. Maness, T. W. Sadler, Jean M. Lauder, Harold C. Slavkin, Phil Mirkes, Kathy Sulik, Elwood Linney, and Melissa B. Rogers
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Toxicology ,In Vitro Techniques ,Research areas ,Biology ,Neuroscience ,Embryonic stem cell ,Function (biology) ,Additional research - Abstract
The work group identified a number of research areas where they felt there were significant data gaps where additional research was critical to better understanding of the origins of birth defects and to developing ways for their prevention. These included: 1. 1. Studies designed to determine the role of growth and differentiation factors during pre- and postimplantation stages of development. These investigations could include descriptive studies involving the localization and timing of genes expressed and their products, but must also emphasize and include studies involving the function of these molecules and their interactions in normal and abnormal development. 2. 2. Studies designed to develop and utilize models for investigating normal and abnormal development. These approaches could include in vivo and in vitro techniques, such as creation of genetically defined systems and cell, organ, and whole embryo cultures. These technologies should emphasize ways to study the functions of growth and differentiation factors and the effects of environmental factors. 3. 3. Studies designed to identify environmental agents and their targets in embryonic, extraembryonic, and maternal tissues that may play a role in producing developmental abnormalities through perturbations of growth and differentiation factors. 4. 4. Studies designed to determine cellular and molecular mechanisms for protection and recovery from environmental insults.
- Published
- 1997
6. Mouse embryonic cardiac metabolism under euglycemic and hypoglycemic conditions
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T. W. Sadler and Jennifer H. Peet
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Embryology ,medicine.medical_specialty ,education.field_of_study ,Heart disease ,Heart malformation ,Health, Toxicology and Mutagenesis ,Population ,Oxidative phosphorylation ,Metabolism ,Biology ,Hypoglycemia ,Toxicology ,medicine.disease ,Endocrinology ,Lactic acidosis ,Internal medicine ,medicine ,Glycolysis ,education ,Developmental Biology - Abstract
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. © 1996 Wiley-Liss, Inc.
- Published
- 1996
7. Antisense inhibition ofEngrailed genes in mouse embryos reveals roles for these genes in craniofacial and neural tube development
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Edison T. Liu, Karen A. Augustine, and T. W. Sadler
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Embryology ,animal structures ,Health, Toxicology and Mutagenesis ,Molecular Sequence Data ,Nerve Tissue Proteins ,Biology ,Nervous System Malformations ,Toxicology ,Nervous System ,Facial Bones ,Embryonic and Fetal Development ,Mice ,Pregnancy ,Culture Techniques ,Notochord ,medicine ,Animals ,Homeodomain Proteins ,Genetics ,Mice, Inbred ICR ,Base Sequence ,Primitive streak ,Skull ,Genes, Homeobox ,Neural tube ,Neural crest ,Gastrula ,Oligonucleotides, Antisense ,Thionucleotides ,Embryo, Mammalian ,engrailed ,Cell biology ,Gastrulation ,Somite ,Phenotype ,medicine.anatomical_structure ,Neurulation ,embryonic structures ,Female ,Developmental Biology - Abstract
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.
- Published
- 1995
8. Effects of short-term exposure to ethanol on mouse embryos in vitro
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T. W. Sadler, J.A. Tugman, Kathleen K. Sulik, and E.S. Hunter
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Embryogenesis ,Neural tube ,Embryo ,Embryo culture ,General Medicine ,Biology ,Toxicology ,Teratology ,Andrology ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,In vivo ,Toxicity ,medicine ,Xenobiotic - Abstract
The adverse developmental effects of ethanol consumption have been documented in humans and in animal models. In animal models, the organ system affected by ethanol administration is dependent on the point in gestation at which the xenobiotic is administered. Previous studies have shown that an exposure of 24–48 hr beginning at the early somite stage in rodent conceptuses alters neural tube closure in vitro. However, the concentration and time dependency of this effect have not been fully defined. Whole embryo culture was therefore used to expose 3–6-somite mouse conceptuses (ICR strain) to ethanol at 300, 450, 600 and 800 mg/dl. The higher concentrations were selected to approximate the peak serum ethanol concentrations that have been shown to be teratogenic in vivo. A 24-hr exposure produced a concentration-dependent increase in neural tube defects (NTDs) and concomitant growth retardation. When shorter exposure periods were used (8, 10, 12 or 20 hr) the incidence of NTDs was dependent on the ethanol concentration and exposure period. At the 600 and 800 mg/dl concentrations an exposure of 8 hr or more produced NTDs, but shorter periods (4 and 6 hr) did not affect neural tube closure when evaluated at the end of a 24-hr culture period. At the 450 mg/dl concentration a 20-hr exposure induced NTDs, but a 12-hr exposure to this level did not. Exposure of conceptuses to ethanol for periods similar to their half-life in vivo did not induce NTDs and the highest concentration produced only a trend towards a reduction in protein content. When the incidence of NTDs was plotted against the area under the time and concentration curve (AUC) the correlation coefficient was 0.5779. An analysis of covariance indicated that the relationships between NTDs and AUC were similar at the 300 and 450 mg/dl concentrations and also at the 600 and 800 mg/dl concentrations. In contrast, the relationships between embryonic protein content and AUC did not differ at the 300, 450 and 600 mg/dl concentrations, but all differed from that at the 800 mg/dl level. These results indicate that ethanol-induced NTDs do not appear to be due solely to embryonic growth retardation. Additionally, ethanol-induced neural tube defects are a function of duration of exposure as well as of peak serum concentration.
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- 1994
9. Diabetic teratogenesis. In vitro evidence for a multifactorial etiology with little contribution from glucose per se
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T. A. Buchanan, K. M. Denno, G. F. Sipos, and T. W. Sadler
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Endocrinology, Diabetes and Metabolism ,Internal Medicine - Published
- 1994
10. Perturbations in choline metabolism cause neural tube defects in mouse embryosin vitro
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Steven H. Zeisel, Mei Heng Mar, T. W. Sadler, and Melanie C. Fisher
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Central Nervous System ,Phosphorylcholine ,Models, Neurological ,Biology ,Ceramides ,Biochemistry ,Choline ,Diglycerides ,Embryonic and Fetal Development ,Mice ,chemistry.chemical_compound ,Phosphatidylcholine ,Genetics ,medicine ,Animals ,Molecular Biology ,Cells, Cultured ,Phosphocholine ,Phosphatidylethanolamine ,Phospholipid Ethers ,Deanol ,Gastrula ,Embryo, Mammalian ,Choline acetyltransferase ,Acetylcholine ,Sphingomyelins ,Betaine ,chemistry ,Phosphatidylethanolamine N-methyltransferase ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin ,Biotechnology ,medicine.drug - Abstract
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.
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- 2002
11. Effects of Altered Maternal Metabolism during Gastrulation and Neurulation Stages of Embryogenesis
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K. M. Denno, E. S. Hunter, and T. W. Sadler
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Blood Glucose ,Central Nervous System ,Phenylketonurias ,Organogenesis ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Embryonic and Fetal Development ,History and Philosophy of Science ,Pregnancy ,medicine ,Animals ,Humans ,Amino Acids ,Yolk sac ,General Neuroscience ,Embryogenesis ,Embryo ,Gastrula ,Cell biology ,Pregnancy Complications ,Gastrulation ,Chorioallantoic membrane ,Diabetes Mellitus, Type 1 ,Neurulation ,medicine.anatomical_structure ,embryonic structures ,Female - Abstract
In summary, many congenital malformations are produced during gastrulation and neurulation stages of embryogenesis at a time when no definitive chorioallantoic placenta has been established. In rodents, altered maternal metabolism may have a direct impact on the embryo or an indirect impact via disruption of the nutritive function of the visceral yolk sac. If similar mechanisms operate in human embryos, these factors probably alter functions of the trophoblastic shell. In any case, it is crucial to remember that the metabolic status of the embryo is rapidly changing and during early stages of organogenesis may respond to alterations in nutrients quite differently during the first four weeks of gestation than at later stages of organogenesis and the fetal period.
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- 1993
12. The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro
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T. W. Sadler and E. S. Hunter
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Male ,Embryology ,medicine.medical_specialty ,Ratón ,Health, Toxicology and Mutagenesis ,Biology ,Sulfur Radioisotopes ,Toxicology ,Congenital Abnormalities ,Hemoglobins ,Mice ,Pregnancy ,Culture Techniques ,Internal medicine ,medicine ,Animals ,Humans ,Yolk sac ,Yolk Sac ,Mice, Inbred ICR ,Glucose tolerance test ,medicine.diagnostic_test ,Embryogenesis ,Neural tube ,Embryo culture ,Embryo ,Fetal Diseases ,Somite ,Glucose ,medicine.anatomical_structure ,Endocrinology ,Hyperglycemia ,Pinocytosis ,Female ,Developmental Biology - Abstract
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.
- Published
- 1992
13. Hypothermia: Teratogenic and protective effects on the development of mouse embryos in vitro
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T. W. Sadler and Ida W. Smoak
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Embryology ,medicine.medical_specialty ,Health, Toxicology and Mutagenesis ,Hypothermia ,In Vitro Techniques ,Hypoglycemia ,Biology ,Toxicology ,Congenital Abnormalities ,Mice ,Limb bud ,Pregnancy ,Internal medicine ,medicine ,Animals ,Mice, Inbred ICR ,Embryogenesis ,Proteins ,Embryo culture ,Embryo ,medicine.disease ,Teratology ,Glucose ,Neurulation ,Endocrinology ,Lactates ,Female ,medicine.symptom ,Developmental Biology - Abstract
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.
- Published
- 1991
14. Recovery by mouse embryos following teratogenic exposure to ketosis
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T. W. Sadler and L. Shum
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medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Hydroxybutyrates ,Biology ,Mice ,Organ Culture Techniques ,Pregnancy ,Internal medicine ,Ectoderm ,Mitotic Index ,Internal Medicine ,medicine ,Animals ,Mice, Inbred ICR ,3-Hydroxybutyric Acid ,Embryogenesis ,Neural tube ,Brain ,Embryo ,Ketosis ,Embryo, Mammalian ,Teratology ,Neuroepithelial cell ,Teratogens ,medicine.anatomical_structure ,Endocrinology ,Neurulation ,Forebrain ,Microscopy, Electron, Scanning ,Ketone bodies ,Female - Abstract
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.
- Published
- 1991
15. Altered visceral yolk sac function produced by a low-molecular-weight somatomedin inhibitor
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Lawrence S. Phillips, T. W. Sadler, E. S. Hunter, and Steven Goldstein
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Embryology ,medicine.medical_specialty ,Health, Toxicology and Mutagenesis ,Pregnancy in Diabetics ,Protein degradation ,Biology ,Toxicology ,Congenital Abnormalities ,Mice ,Pregnancy ,Somatomedins ,Internal medicine ,medicine ,Animals ,Humans ,Yolk sac ,Yolk Sac ,chemistry.chemical_classification ,Mice, Inbred ICR ,Pinocytosis ,Embryogenesis ,Proteins ,Trophoblast ,Embryo culture ,Embryo ,Amino acid ,Cell biology ,Molecular Weight ,Diabetes Mellitus, Type 1 ,Teratogens ,medicine.anatomical_structure ,Endocrinology ,chemistry ,embryonic structures ,Female ,Developmental Biology - Abstract
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.
- Published
- 1991
16. Activity profiles of developmental toxicity: Design considerations and pilot implementation
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John M. Rogers, Richard E. Morrissey, Elizabeth T. Owens, H. Frank Stack, Michael D. Waters, Gary L. Kimmel, Frank Welsch, T. W. Sadler, Jacqueline A. Greene, and Robert J. Kavlock
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Embryology ,Databases, Factual ,Health, Toxicology and Mutagenesis ,Concordance ,Developmental toxicity ,Ethylenethiourea ,Computational biology ,Biology ,Toxicology ,Embryonic and Fetal Development ,In vivo ,Caffeine ,Animals ,Humans ,Hydroxyurea ,Cyclophosphamide ,Pilot implementation ,Mutagenicity Tests ,Data Collection ,Effective dose (pharmacology) ,Methotrexate ,Investigation methods ,Toxicity ,Biological Assay ,Developmental Biology - Abstract
The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100–500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment II and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positive/negative) and quantitative (e.g.), IC50, highest ineffective dose (HID), lowest effective dose (LED)) results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships.
- Published
- 1991
17. Biochemical basis for D,L,-beta-hydroxybutyrate-induced teratogenesis
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T. W. Sadler and L. Shum
- Subjects
Embryology ,Health, Toxicology and Mutagenesis ,Hydroxybutyrates ,Mice, Inbred Strains ,Biology ,Toxicology ,Pentose Phosphate Pathway ,Andrology ,Mice ,Culture Techniques ,medicine ,Animals ,Neural Tube Defects ,Yolk sac ,3-Hydroxybutyric Acid ,Muscles ,Embryogenesis ,Embryo ,Embryo culture ,Teratology ,Somite ,Pyrimidines ,Teratogens ,Neurulation ,medicine.anatomical_structure ,Biochemistry ,embryonic structures ,Pyrimidine metabolism ,Developmental Biology - Abstract
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.
- Published
- 1990
18. Prevention of fumonisin B1-induced neural tube defects by folic acid
- Author
-
Ping Wang, Victoria L. Stevens, M. Cameron Sullards, Alfred H. Merrill, Elaine Wang, and T. W. Sadler
- Subjects
Embryology ,Time Factors ,Health, Toxicology and Mutagenesis ,Leucovorin ,Biology ,Pharmacology ,Toxicology ,Fumonisins ,chemistry.chemical_compound ,Folinic acid ,Mice ,Folic Acid ,Organ Culture Techniques ,In vivo ,Fumonisin ,medicine ,Animals ,Neural Tube Defects ,Fumonisin B1 ,Dose-Response Relationship, Drug ,Neural tube ,food and beverages ,Teratology ,medicine.anatomical_structure ,Teratogens ,chemistry ,Biochemistry ,Folate receptor ,Neural Crest ,Toxicity ,Microscopy, Electron, Scanning ,Developmental Biology ,medicine.drug - Abstract
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.
- Published
- 2002
19. Embryology of the sternum
- Author
-
T W, Sadler
- Subjects
Mesoderm ,Sternum ,Osteogenesis ,Funnel Chest ,Humans - Abstract
Development of the sternum during the growth of the embryo is described. Anterior body wall defects in the thoracic region may be severe, leading to ectopia cordis, or mild, as in skin-covered sternal clefts. The embryologic basis for other sternal abnormalities, such as pectus excavatum and pectus carniatum, is not clear; however, abnormalities of rib morphogenesis and growth are the most likely causes.
- Published
- 2000
20. Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development
- Author
-
L, Foerst-Potts and T W, Sadler
- Subjects
Homeodomain Proteins ,MSX1 Transcription Factor ,Mice, Inbred ICR ,Skull ,Gene Expression Regulation, Developmental ,Oligonucleotides, Antisense ,Eye ,DNA-Binding Proteins ,Embryonic and Fetal Development ,Mice ,Pregnancy ,Animals ,Female ,Body Patterning ,Transcription Factors - Abstract
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.
- Published
- 1997
21. Mouse embryos in culture: models for understanding diabetes-induced embryopathies and gene function
- Author
-
T W, Sadler
- Subjects
Disease Models, Animal ,Embryonic and Fetal Development ,Fetal Diseases ,Mice ,3-Hydroxybutyric Acid ,Pregnancy ,Culture Techniques ,Gene Targeting ,Pregnancy in Diabetics ,Animals ,Hydroxybutyrates ,Female ,Oligonucleotides, Antisense - Abstract
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.
- Published
- 1997
22. Targeted Gene Disruptions as Models of Abnormal Development
- Author
-
T. W. Sadler, E. T. Liu, and K. A. Augustine
- Subjects
Genetics ,Mutation ,Mutant ,medicine ,Congenital malformations ,Embryo ,Biology ,medicine.disease_cause ,Phenotype ,Gene ,Organ system ,Teratology - Abstract
For years teratologists have had access to mice with spontaneously occurring mutations resulting in unique phenotypes. Animals with defects in many organ systems are available and provide valuable information about the origin of specific congenital malformations. However, most of these mutations involve recessive genes, and the genes themselves are unknown (Kalter 1980). Consequently, there are no markers available to identify mutant embryos before they overtly exhibit an altered phenotype. Thus it is difficult to investigate the earliest cellular events and mechanisms responsible for abnormal phenotypes, since only 25% of all embryos in a litter are affected and there is no way to identify which embryos they are before they exhibit grossly abnormal morphology. As a result, the amount of information provided by these models has been limited, and interpretations of the cellular mechanisms of teratogenesis somewhat speculative.
- Published
- 1997
23. Mouse embryonic cardiac metabolism under euglycemic and hypoglycemic conditions
- Author
-
J H, Peet and T W, Sadler
- Subjects
Blood Glucose ,Mice, Inbred ICR ,Myocardium ,Heart ,Carbon Dioxide ,Hypoglycemia ,Oxidative Phosphorylation ,Mice ,Adenosine Triphosphate ,Organ Culture Techniques ,Pregnancy ,Animals ,Female ,Lactic Acid ,Glycolysis - Abstract
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.
- Published
- 1996
24. Effects of cocaine and cocaine metabolites on mouse developmentin vitro
- Author
-
E.S. Hunter and T. W. Sadler
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Stereochemistry ,Alkaloid ,Metabolite ,Developmental toxicity ,General Medicine ,Biology ,Toxicology ,Teratology ,chemistry.chemical_compound ,Endocrinology ,chemistry ,In vivo ,Internal medicine ,Toxicity ,medicine ,Benzoylecgonine ,Ecgonine - Abstract
The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 μ m or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 μ m . EME also induced defects at concentrations of 400 μ m or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 μ m or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 μ m , exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDs in vitro is higher than the teratogenic concentration in vivo . Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reported in vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.
- Published
- 1996
25. Effects of cocaine administration during early organogenesis on prenatal development and postnatal growth in mice
- Author
-
E. Sidney Hunter, L. E. Kotch, Robert C. Cefalo, and T. W. Sadler
- Subjects
Male ,Narcotics ,medicine.medical_specialty ,Litter Size ,Toxicology ,Kidney ,Kidney morphogenesis ,Route of administration ,Embryonic and Fetal Development ,Mice ,Fetus ,Cocaine ,Pregnancy ,Internal medicine ,Medicine ,Animals ,Fetal Viability ,Fetal Death ,Mice, Inbred ICR ,business.industry ,Body Weight ,Embryo, Mammalian ,Prenatal development ,Teratology ,Endocrinology ,medicine.anatomical_structure ,Animals, Newborn ,Maternal Exposure ,Prenatal Exposure Delayed Effects ,Toxicity ,Gestation ,Female ,business ,Injections, Intraperitoneal - Abstract
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.
- Published
- 1995
26. A teratologist to extinction?
- Author
-
T W, Sadler
- Subjects
Teratology ,Research Design ,Research ,Infant, Newborn ,Abnormalities, Drug-Induced ,Animals ,Humans ,Infant ,Female ,Forecasting - Published
- 1995
27. Interactions of Wnt-1 and Wnt-3a are essential for neural tube patterning
- Author
-
Karen A. Augustine, Edison T. Liu, and T. W. Sadler
- Subjects
Embryology ,Neural tube patterning ,Health, Toxicology and Mutagenesis ,Molecular Sequence Data ,Down-Regulation ,Wnt1 Protein ,Biology ,Toxicology ,Nervous System ,Wnt3 Protein ,Mice ,Proto-Oncogene Proteins ,Wnt3A Protein ,medicine ,Animals ,Drosophila Proteins ,Genetics ,Homeodomain Proteins ,Mice, Inbred ICR ,Base Sequence ,S100 Proteins ,Wnt signaling pathway ,Neural tube ,Genes, Homeobox ,Gene Expression Regulation, Developmental ,Proteins ,DNA ,Oligonucleotides, Antisense ,Zebrafish Proteins ,engrailed ,Cell biology ,Neuroepithelial cell ,Wnt Proteins ,Somite ,medicine.anatomical_structure ,Neurulation ,Insect Hormones ,Developmental Biology ,Transcription Factors - Abstract
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.
- Published
- 1995
28. Effects of the biguanide class of oral hypoglycemic agents on mouse embryogenesis
- Author
-
Kelly M. Denno and T. W. Sadler
- Subjects
Embryology ,medicine.medical_specialty ,medicine.drug_class ,Health, Toxicology and Mutagenesis ,Phenformin ,Toxicology ,chemistry.chemical_compound ,Embryonic and Fetal Development ,Mice ,Oral administration ,Pregnancy ,Internal medicine ,Diabetes mellitus ,Culture Techniques ,medicine ,Animals ,Hypoglycemic Agents ,Mice, Inbred ICR ,Biguanide ,business.industry ,Embryo culture ,medicine.disease ,Teratology ,Metformin ,Endocrinology ,chemistry ,Toxicity ,Female ,business ,Developmental Biology ,medicine.drug - Abstract
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.
- Published
- 1994
29. Serotonin and cardiac morphogenesis in the mouse embryo
- Author
-
T. W. Sadler, Jean M. Lauder, H. Tamir, Dana L. Shuey, and Mark Yavarone
- Subjects
Nialamide ,Embryology ,medicine.medical_specialty ,Serotonin ,Monoamine oxidase ,Health, Toxicology and Mutagenesis ,Immunocytochemistry ,Morphogenesis ,Biology ,In Vitro Techniques ,Toxicology ,Mice ,Endocardial cushion formation ,Fetal Heart ,Cell Movement ,Pregnancy ,Internal medicine ,Fluoxetine ,Sertraline ,medicine ,Animals ,Mice, Inbred ICR ,Embryogenesis ,Embryo culture ,Immunohistochemistry ,Cell biology ,Endocrinology ,1-Naphthylamine ,embryonic structures ,cardiovascular system ,Female ,Carrier Proteins ,Cell Division ,Selective Serotonin Reuptake Inhibitors ,Developmental Biology ,medicine.drug ,Signal Transduction - Abstract
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.
- Published
- 1993
30. Postulated mechanisms underlying the development of neural tube defects. Insights from in vitro and in vivo studies
- Author
-
Kathleen K. Sulik and T. W. Sadler
- Subjects
Central Nervous System ,medicine.medical_specialty ,General Neuroscience ,Mutant ,Neural tube ,Embryo ,Biology ,General Biochemistry, Genetics and Molecular Biology ,In vitro ,Cell biology ,Disease Models, Animal ,medicine.anatomical_structure ,History and Philosophy of Science ,In vivo ,Molecular genetics ,medicine ,Microscopy, Electron, Scanning ,Animals ,Humans ,Nutritional Physiological Phenomena ,Neural Tube Defects ,Gene ,Function (biology) - Abstract
In recent years, use of animal models has resulted in acquisition of a significant amount of new information regarding normal and abnormal neural tube development. Studies of mutant and of teratogen-exposed mice are complementary, with each providing insights that promise to advance our understanding of the other. Analysis of teratogen-exposed embryos is best suited for identifying susceptible developmental stages and vulnerable populations. Advances in molecular genetics, with the ability to identify gene products, their cell/tissue location, and, potentially, to understand their function, will make naturally occurring as well as man-made mutants invaluable for understanding the heterogeneous mechanisms that underly NTDs.
- Published
- 1993
31. Serotonin uptake in the ectoplacental cone and placenta of the mouse
- Author
-
Jean M. Lauder, T. W. Sadler, Mark Yavarone, and Dana L. Shuey
- Subjects
medicine.medical_specialty ,Serotonin ,Serotonin uptake ,Placenta ,Immunocytochemistry ,Biology ,Immunoenzyme Techniques ,Mice ,Fetal membrane ,Pregnancy ,Internal medicine ,Culture Techniques ,medicine ,Animals ,reproductive and urinary physiology ,Mice, Inbred ICR ,Embryogenesis ,Obstetrics and Gynecology ,Embryo ,Biological Transport ,Staining ,Cell biology ,Culture Media ,Trophoblasts ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,Giant cell ,embryonic structures ,Female ,Selective Serotonin Reuptake Inhibitors ,Developmental Biology - Abstract
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.
- Published
- 1993
32. Antisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis
- Author
-
Edison T. Liu, T. W. Sadler, and Karen A. Augustine
- Subjects
Heart Defects, Congenital ,Central nervous system ,Molecular Sequence Data ,Gene Expression ,Hindbrain ,Biology ,Facial Bones ,Embryonic and Fetal Development ,Mice ,Culture Techniques ,Proto-Oncogenes ,Genetics ,medicine ,Animals ,RNA, Messenger ,Gene knockout ,Neural fold ,Mice, Inbred ICR ,Base Sequence ,Skull ,Neural crest ,Brain ,Heart ,Cell Biology ,DNA ,Oligonucleotides, Antisense ,Spinal cord ,Cell biology ,medicine.anatomical_structure ,Phenotype ,Spinal Cord ,GDF7 ,Forebrain ,Developmental Biology - Abstract
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.
- Published
- 1993
33. Serotonin and morphogenesis
- Author
-
H. Tamir, T. W. Sadler, Dana L. Shuey, and Jean M. Lauder
- Subjects
Central Nervous System ,Serotonin ,Embryology ,Serotonin uptake ,Rhombomere ,Hindbrain ,Biology ,Serotonergic ,Sensitivity and Specificity ,Epithelium ,Mice ,Culture Techniques ,Morphogenesis ,medicine ,Animals ,Yolk sac ,Craniofacial ,Rhombic lip ,Mice, Inbred ICR ,Myocardium ,Skull ,Cell Biology ,Embryo, Mammalian ,Immunohistochemistry ,Cell biology ,Nasal Mucosa ,medicine.anatomical_structure ,Face ,embryonic structures ,Nasal placode ,Anatomy ,Carrier Proteins ,Developmental Biology - Abstract
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.
- Published
- 1993
34. Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture
- Author
-
Kelly M. Denno and T. W. Sadler
- Subjects
Embryology ,medicine.medical_specialty ,Health, Toxicology and Mutagenesis ,Metabolite ,Phenylalanine ,Mice, Inbred Strains ,Biology ,Phenylacetic acid ,Toxicology ,chemistry.chemical_compound ,Mice ,Pregnancy ,Internal medicine ,Culture Techniques ,Phenylketonurias ,medicine ,Animals ,Neural Tube Defects ,Yolk sac ,Embryogenesis ,Embryo culture ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,Teratology ,Endocrinology ,medicine.anatomical_structure ,Teratogens ,chemistry ,embryonic structures ,Toxicity ,Female ,Developmental Biology - Abstract
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.
- Published
- 1990
35. Embryopathic effects of short-term exposure to hypoglycemia in mouse embryos in vitro
- Author
-
T. W. Sadler and Ida W. Smoak
- Subjects
medicine.medical_specialty ,Time Factors ,Ratón ,Pregnancy in Diabetics ,Hypoglycemia ,Two stages ,Congenital Abnormalities ,Embryonic and Fetal Development ,Mice ,Pregnancy ,Internal medicine ,medicine ,Animals ,Humans ,business.industry ,Obstetrics and Gynecology ,Embryo ,Gastrula ,medicine.disease ,Embryo, Mammalian ,In vitro ,Culture Media ,Endocrinology ,Neurulation ,Diabetes Mellitus, Type 1 ,Female ,business - Abstract
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.
- Published
- 1990
36. Serotonin and morphogenesis in the cultured mouse embryo
- Author
-
D L, Shuey, M, Yavarone, T W, Sadler, and J M, Lauder
- Subjects
Mice ,Serotonin ,Neural Crest ,Culture Techniques ,Skull ,Morphogenesis ,Animals ,Heart ,Facial Bones - Published
- 1990
37. Letter
- Author
-
Harold Kalter and T. W. Sadler
- Subjects
Embryology ,medicine.medical_specialty ,Pregnancy ,business.industry ,Health, Toxicology and Mutagenesis ,Embryo ,Hypoglycemia ,Toxicology ,medicine.disease ,In vitro ,Endocrinology ,Internal medicine ,Medicine ,business ,Developmental Biology - Published
- 1996
38. Letter from the editor
- Author
-
Gary C. Schoenwolf, J. W. Hanson, Mont R. Juchau, Kathleen K. Sulik, Philip E. Mirkes, Craig Harris, Thomas B. Knudsen, Lewis B. Holmes, Gary L. Kimmel, and T. W. Sadler
- Subjects
Embryology ,Actuarial science ,Extinction ,Geography ,Health, Toxicology and Mutagenesis ,Toxicology ,Developmental Biology ,Grant funding - Published
- 1996
39. Editor's letter: A teratologist to extinction?
- Author
-
T. W. Sadler
- Subjects
Embryology ,Extinction ,Health, Toxicology and Mutagenesis ,Astrophysics ,Biology ,Toxicology ,Infant newborn ,Developmental Biology - Published
- 1995
40. Ultrastructural alterations in neuroepithelial cells of mouse embryos exposed to cytotoxic doses of hydroxyurea
- Author
-
Robert R. Cardell and T. W. Sadler
- Subjects
Central Nervous System ,Nucleolus ,Abnormalities, Drug-Induced ,Embryo ,Biology ,Agricultural and Biological Sciences (miscellaneous) ,Neuroepithelial cell ,Andrology ,Mice ,Fetus ,Cytoplasm ,Immunology ,Ultrastructure ,Animals ,Hydroxyurea ,Cytotoxic T cell ,Anatomy ,Fragmentation (cell biology) ,Pyknosis - Abstract
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.
- Published
- 1977
41. Effects of hyperketonemia on mouse embryonic and fetal glucose metabolism in vitro
- Author
-
W. E. Horton, T. W. Sadler, and E. S. Hunter
- Subjects
Embryology ,medicine.medical_specialty ,Health, Toxicology and Mutagenesis ,Citric Acid Cycle ,Hydroxybutyrates ,Gestational Age ,Ketone Bodies ,Biology ,Carbohydrate metabolism ,Toxicology ,Andrology ,Mice ,Fetus ,Fetal Stage ,Pregnancy ,Internal medicine ,medicine ,Animals ,Glycolysis ,Lactic Acid ,3-Hydroxybutyric Acid ,Embryo ,Embryo, Mammalian ,Embryonic stem cell ,Glucose ,Endocrinology ,embryonic structures ,Lactates ,Ketone bodies ,Gestation ,Female ,Energy Metabolism ,Developmental Biology - Abstract
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.
- Published
- 1985
42. Role of the mouse visceral yolk sac in nutrition: Inhibition by a somatomedin inhibitor
- Author
-
Lawrence S. Phillips, T. W. Sadler, Wayne Balkan, and Steven Goldstein
- Subjects
medicine.medical_specialty ,Biology ,Mice ,Organ Culture Techniques ,Somatomedins ,Internal medicine ,medicine ,Protein biosynthesis ,Animals ,Conceptus ,Amino Acids ,Yolk sac ,Yolk Sac ,chemistry.chemical_classification ,Mice, Inbred ICR ,Embryogenesis ,Embryo ,Embryo culture ,General Medicine ,Somatomedin ,Amino acid ,Endocrinology ,medicine.anatomical_structure ,chemistry ,embryonic structures ,Animal Science and Zoology ,Chromatography, Thin Layer - Abstract
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.
- Published
- 1989
43. Culture of early somite mouse embryos during organogenesis
- Author
-
T. W. Sadler
- Subjects
medicine.medical_specialty ,Fetus ,Period (gene) ,Morphogenesis ,Embryo ,Organogenesis ,Biology ,Somite ,Endocrinology ,medicine.anatomical_structure ,In vivo ,Internal medicine ,embryonic structures ,medicine ,Ectogenesis ,Molecular Biology ,Developmental Biology - Abstract
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 + 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.
- Published
- 1979
44. A potential role for spectrin during neurulation
- Author
-
Keith Burridge, T. W. Sadler, and J. Yonker
- Subjects
Neuroepithelial cell ,Neural fold ,Neurulation ,Biochemistry ,Embryogenesis ,Spectrin ,Biology ,Cytoskeleton ,Microfilament ,Molecular Biology ,Actin ,Developmental Biology ,Cell biology - Abstract
An actin-myosin complex located in apical regions of the neurectoderm has been postulated to play a role in neurulation. Numerous studies have documented the presence of microfilaments in this area and confirmed their composition as actin. By necessity, if such a contractile system is to exert a force, these filaments must be anchored in some way to the cell membrane. In this study, the presence of the actin-binding protein, spectrin (fodrin), is demonstrated in the neurectoderm of neurulating mouse embryos using antispectrin antibodies and indirect immunofluorescent techniques. The patterns of spectrin localization correlate with the previously reported regions of increased numbers of microfilaments and also with the morphology of the neural folds. Thus, during the initial stages of cranial fold elevation, a process reportedly dependent on increased glycosaminoglycan synthesis, little spectrin is present in the neuroepithelial cells. Later as the folds begin to converge toward the midline, deposition of the protein, as demonstrated by the intensity of fluorescence, is increased in the apices of these cells, and is most prominent in regions of greatest bending in the neural folds. Caudal neural fold regions show a similar pattern of staining. Thus, the hypothesis that a cytoskeletal system assists in neurulation is supported by these results, which for the first time demonstrate the presence of a putative actin-membrane attachment protein in a morphogenetically active system.
- Published
- 1986
45. Biosynthesis of DNA, RNA and proteins by mouse embryos cultured in the presence of a teratogenic dose of chlorambucil
- Author
-
D. M. Kochhar and T. W. Sadler
- Subjects
animal structures ,Amnion ,DNA synthesis ,RNA ,Embryo culture ,Embryo ,Biology ,Molecular biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,Biosynthesis ,chemistry ,embryonic structures ,Protein biosynthesis ,medicine ,Molecular Biology ,DNA ,Developmental Biology - Abstract
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 chlora-bucil (15 μg/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.
- Published
- 1976
46. Distribution of surface coat material on fusing neural folds of mouse embryos during neurulation
- Author
-
T. W. Sadler
- Subjects
Central Nervous System ,Male ,Ruthenium red ,Macromolecular Substances ,Surface Properties ,Hindbrain ,Ectoderm ,Biology ,Midbrain ,Mice ,chemistry.chemical_compound ,Pregnancy ,medicine ,Animals ,Neural fold ,Epithelial Cells ,Anatomy ,Spinal cord ,Agricultural and Biological Sciences (miscellaneous) ,Cell biology ,Neuroepithelial cell ,Neurulation ,medicine.anatomical_structure ,chemistry ,Female ,Extracellular Space - Abstract
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.
- Published
- 1978
47. Culture of mouse embryos during neurulation
- Author
-
T. W. Sadler and D. A. T. New
- Subjects
Morphogenesis ,Neural tube ,Embryo ,Anatomy ,Biology ,Andrology ,Protein content ,Somite ,medicine.anatomical_structure ,Neurulation ,In vivo ,medicine ,Ectogenesis ,Molecular Biology ,Developmental Biology - Abstract
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.
- Published
- 1981
48. Effects of maternal diabetes on early embryogenesis: II. Hyperglycemia-induced exencephaly
- Author
-
T. W. Sadler
- Subjects
Embryology ,medicine.medical_specialty ,animal structures ,Health, Toxicology and Mutagenesis ,Pregnancy in Diabetics ,Exencephaly ,Biology ,Toxicology ,Mice ,Pregnancy ,Culture Techniques ,Internal medicine ,medicine ,Animals ,Mice, Inbred ICR ,Embryogenesis ,Neural tube ,Brain ,Embryo ,medicine.disease ,Neuroepithelial cell ,Somite ,medicine.anatomical_structure ,Endocrinology ,Hyperglycemia ,embryonic structures ,Female ,Pyknosis ,Developmental Biology - Abstract
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.
- Published
- 1980
49. Embryonic catch-up growth after exposure to the ketone body D,L,-beta-hydroxybutyrate in vitro
- Author
-
L. Shum and T. W. Sadler
- Subjects
Embryology ,Ratón ,Health, Toxicology and Mutagenesis ,Hydroxybutyrates ,Organogenesis ,Biology ,Toxicology ,Andrology ,Embryonic and Fetal Development ,Mice ,Reference Values ,medicine ,Animals ,Growth rate ,Mice, Inbred ICR ,3-Hydroxybutyric Acid ,Embryogenesis ,Neural tube ,Embryo ,Embryo culture ,Anatomy ,Embryo, Mammalian ,Teratogens ,medicine.anatomical_structure ,Microscopy, Electron, Scanning ,Ketone bodies ,Developmental Biology - Abstract
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.
- Published
- 1988
50. The role of pharmacokinetics in determining the response of rodent embryos to teratogens in whole-embryo culture
- Author
-
M. Kate Smith, C.W. Warner, L. Shum, and T. W. Sadler
- Subjects
animal structures ,Embryo ,Embryo culture ,General Medicine ,Pharmacology ,Biology ,Toxicology ,Embryonic stem cell ,In vitro ,Teratology ,Neurulation ,Pharmacokinetics ,In vivo ,embryonic structures - Abstract
The rodent embryo's response to teratogenic insult in whole-embryo culture during the period of neurulation was characterized by determining the role of pharmacokinetics and embryonic recovery in producing abnormal growth and development. Five known teratogens, hydroxyurea, cyclophosphamide, cadmium, diphenylhydantoin, and the ketone body β-hydroxybutyrate were employed. The dose and exposure times in vitro were designed to reproduce the peak serum concentrations and half-life of the compounds present following the administration of a teratogenic or maximum maternally tolerated dose of the agents in vivo . The results showed: first, that both the serum concentration and duration of exposure to an agent play a role in determining the embryonic response in vitro ; secondly, that compounds that do not produce effects during the period of neurulation or that require maternal metabolic activation are not teratogenic in culture; and thirdly, that embryos have the capacity to recover from certain teratogenic insults in vitro . Thus, both pharmacokinetics and the potential for embryonic recovery should be considered when applying the whole-embryo culture technique to studies in teratology and toxicology.
- Published
- 1988
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