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T W Sadler

Publications and source records attributed to T W Sadler.

At least 55 records · Page 3Linked to original sources
PubMed1989

Fuel-mediated teratogenesis: biochemical effects of hypoglycemia during neurulation in mouse embryos in vitro.

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

Animals↗
PubMed1989

Evidence for multifactorial origin of diabetes-induced embryopathies.

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

3-Hydroxybutyric Acid↗
PubMed1988

Improved growth and development of presomite mouse embryos in whole embryo culture.

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

Animals↗
PubMed1988

Potential role of somatomedin inhibitors in the production of diabetic embryopathies.

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

Abnormalities, Drug-Induced↗
PubMed1988

Somatomedin inhibitors from human serum produce abnormalities in mouse embryos in culture.

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

Animals↗
PubMed1988

Embryonic catch-up growth after exposure to the ketone body D,L,-beta-hydroxybutyrate in vitro.

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.

3-Hydroxybutyric Acid↗
PubMed1988

Effects of maternal diabetes on embryogenesis.

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

Animals↗
PubMed1988

Serotonin and morphogenesis. I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo.

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

5-Hydroxytryptophan↗
PubMed1987

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro.

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

3-Hydroxybutyric Acid↗
PubMed1987

Metabolism of D- and DL-beta-hydroxybutyrate by mouse embryos in vitro.

Rates of 14CO2 production by mouse conceptuses in vitro from D- and DL-[3-14C]-beta-hydroxybutyrate (beta OHB) were determined during the period of organogenesis (days 9 through 12 of gestation) in the presence of 4 to 32 mmol/L DL-beta OHB. During this time period the rates 14CO2 production from D-beta OHB metabolism are concentration-dependent, increase on each day of gestation, and the site of metabolism appears to shift from the visceral yolk sac placenta to the embryo proper. In contrast to fetal and neonatal tissues, the rates of 14CO2 production from DL-beta OHB oxidation is significantly greater than from D-beta OHB suggesting that the utilization of the L-isomer may be equal to or greater than that of the D-form.

Animals↗
PubMed1987

Hypoglycemia: how little is too much for the embryo?

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

Animals↗
PubMed1987

A potential mechanism of DL-beta-hydroxybutyrate-induced malformations in mouse embryos.

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

3-Hydroxybutyric Acid↗
PubMed1987

Somatomedins/insulin-like growth factors, their receptors and binding proteins are present during mouse embryogenesis.

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3-4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30-45 X 10(3) Mr. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.

Affinity Labels↗
PubMed1986

Somatomedin inhibitors from diabetic rat serum alter growth and development of mouse embryos in culture.

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

Animals↗
PubMed1986

A potential role for spectrin during neurulation.

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.

Animals↗
PubMed1985

Mitochondrial alterations in embryos exposed to B-hydroxybutyrate in whole embryo culture.

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

3-Hydroxybutyric Acid↗
PubMed1985

Effects of hyperketonemia on mouse embryonic and fetal glucose metabolism in vitro.

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.

3-Hydroxybutyric Acid↗