Allen S. Goldman

Malformations in Infants of Diabetic Mothers Occur Before the Seventh Gestational Week: Implications for Treatment

In the present study we used a developmental morphologic approach to fix the latest time in development at which the malformations commonly reported in infants of diabetic mothers could occur. Developmental morphologic dating shows that the significantly more common congenital malformations in infants of diabetic mothers occur before the seventh week of gestation. This suggests that any therapeutic intervention aimed at decreasing the incidence of congenital malformations must be instituted during the critical early period.

Myo-inositol and prostaglandins reverse the glucose inhibition of neural tube fusion in cultured mouse embryos

SummaryNeural tube defects in infants of diabetic mothers constitute an important and frequent cause of neonatal mortality/morbidity and long-term chronic handicaps. The mechanism by which normal neural tube fusion occurs is not known. The failure of rostral neural tube fusion seen in mouse embryos incubated in the presence of excess-D-glucose can be significantly prevented by the supplementation of myo-inositol to the culture medium. This protective effect of myo-inositol is reversed by indomethacin, an inhibitor of arachidonic acid metabolism leading to prostaglandin synthesis. Prostaglandin E2 added to the culture medium completely protects against the glucose-induced neural tube defect. These data suggest that the failure of neural tube fusion seen in diabetic embryopathy is mediated through a mechanism involving abnormalities in both the myo-inositol and arachidonic acid pathways, resulting in a functional deficiency of prostaglandins at a critical time of neural tube fusion.

Meticulous Control of Diabetes During Organogenesis Prevents Congenital Lumbosacral Defects in Rats

Lumbosacral defects occur in the offspring of the diabetic rat in a fashion analogous to those occurring in infants of diabetic human mothers. To test the hypothesis that control of diabetes during the critical period of organogenesis can affect the incidence of these defects, diabetes was produced by streptozotocin in pregnant rats on day 6 of gestation. One group of pregnant rats was treated with saline (group 2), while another (group 4) received insulin twice daily between days 6 and 13. The incidence of lumbosacral malformations in the fetuses was then compared between these groups, with a pregnant, nondiabetic control group (group 1), and with a group of rats in whom diabetes was produced by streptozotocin on day 12, after the critical period of fusion of the lumbosacral spine (group 3). Two types of lumbar and/or sacral malformations were noted: a fusion defect and an ossification defect. In the nondiabetic animals (group 1), only 1 out of 59 fetuses had an abnormality. Diabetes produced after the period of fusion of the neural tube in the lumbosacral area (group 3) had no effect on the incidence of lumbosacral malformations (0 out of 54). Diabetes treated with saline during the period of organogenesis (group 2) was associated with lumbosacral malformations in 25 of 146 fetuses, which was significantly increased (P < 0.005) when compared with the nondiabetic pregnant animals. Insulin treatment during the period of organogenesis (group 4) significantly reduced (P < 0.005) the number of lumbosacral defects (5 of 106) from that observed in the diabetic animals treated with saline. When the results of the insulintreated diabetic group were further analyzed, the incidence of malformations (5 of 58) in the animals that exhibited intermediate hyperglycemia during the period of organ differentiation [two or more serum glucose values greater than 200 mg/dl, (group 4A)] was not significantly different from that observed in the saline-treated diabetic group (P > 0.1). This occurred despite a mean glucose concentration during the period of insulin treatment that was significantly lower than the saline-treated diabetic controls (180 ± 16 mg/dl vs. 348 ± 16, P < 0.001). In contrast, the incidence of lumbosacral malformations (0 of 48) observed in the fetuses of those diabetic animals in whom insulin therapy produced normal serum glucose concentrations (122 ± 9 mg/dl) during the period of organogenesis (group 4B) was sharply reduced from that seen in the fetuses taken from the saline-treated diabetic mothers who had not received insulin (P < 0.001). These data are consistent with the thesis that (1) uncontrolled diabetes produces lumbosacral malformations only during the period of organ differentiation, (2) control of diabetes during the period of organ differentiation reduces the incidence of malformations, and (3) the “quality” of that control is quite important, because normal to near-normal serum glucose concentrations must be achieved in order to be effective.

PGE2 Prevents Anomalies Induced by Hyperglycemia or Diabetic Serum in Mouse Embryos

Both a high level of D-glucose in the medium and serum from a diabetic rat can induce neural-tube fusion defects and growth retardation in cultured mouse and rat embryos. To test our hypothesis that a deficiency of PGs may be involved in the mechanism of hyperglycemia- and diabetic serum-induced teratogenesis and growth retardation, we added PGE2 to the medium of a whole mouse embryo culture containing either normal rat serum and 52.7 mM D-glucose (hyperglycemic) or diabetic rat serum and 22.2 mM D-glucose (diabetic). After a 24-h culture, 94% of hyperglycemic embryos and 81% of diabetic embryos had neural-tube fusion defects; in addition, the number of somites, the morphological score, and the protein content of the embryos were significantly lower than those of controls. Supplementing the medium with PGE2 at concentrations of 0.028–28.4 nM (hyperglycemic) or 28.4 nM (diabetic) significantly reduced the incidence of neural-tube defects and increased the number of somites, the morphological score, and the protein content. These results strongly support the hypothesis that the teratogenicity of diabetic serum, as well as the teratogenic action of hyperglycemic culture, are mediated through a deficiency of PGs.

Inhibition of Programmed Cell Death in Mouse Embryonic Palate in Vitro by Cortisol and Phenytoin: Receptor Involvement and Requirement of Protein Synthesis

Abstract In an in vitro model Cortisol and phenytoin inhibit the precisely timed process of palatal development, the lysosomally mediated cell death of the medial edge palatal epithelium. This inhibition of programmed cell death of the palatal midline epithelium by each drug is virtually completely blocked by the antiglucocorticoid, cortexolone, whose blocking action results from competitive binding of the glucocorticoid receptor site. The inhibition produced by each of these drugs is prevented by the protein synthesis blocker, cycloheximide. Thus, blockade of programmed cell death by each of these drugs involves the glucocorticoid receptor site and requires protein synthesis.

Is feminine differentiation of the brain hormonally determined

The androgen insensitive, genetically male rat pseudohermaphrodite displays neither masculine or feminine sexual behavior when primed with the appropriate sex hormones. although in the absence of androgen imprinting the animal develops anatomically as female, our results suggest that feminine differentiation of the brain requires active imprinting by perinatal hormone(s), possibly adrenal progesterone.

Chapter 10. Biochemical Mechanism of Glucocorticoid-and Phenytoin-Induced Cleft Palate

The production of cleft palate by glucocorticoids and phenytoin is a complicated interference in a complex developmental program involving many genetic and biochemical processes. The H-2 histocompatibility region includes genes which affect (1) susceptibility to glucocorticoid- and phenytoin-induced cleft palate; (2) glucocorticoid receptor level in a variety of tissues including maternal and embryonic palates, adult thymuses, and lungs; and (3) the degree of inhibition of prostaglandin and thromboxane production by glucocorticoids and phenytoin in thymocytes. A gene linked to a minor histocompatibility locus (H-3) on the second chromosome also influences susceptibility to glucocorticoid- and phenytoin-induced cleft palate. Phenytoin is an alternate ligand for the glucocorticoid receptor affecting prostaglandin and/or thromboxane production. The capacity of glucocorticoids to induce cleft palate is correlated with their anti-inflammatory potency. At least some of the anti-inflammatory effects of glucocorticoids can be explained by the inhibition of prostaglandin and/or thromboxane release, which in turn could be caused by inhibition of arachidonic acid release from phospholipids. Similar mechanisms may be involved in cleft palate induction, as exogenous arachidonic acid injected into pregnant rats and mice at the same time as glucocorticoids reduces the teratogenic potency of the steroids, and indomethacin, an inhibitor of cyclooxygenase, blocks the corrective action of arachidonic acid. Glucocorticoids and phenytoin cause a delay in shelf elevation, and this delay is promoted by fetal membranes and the tongue. However, the cells of the medial edge epithelium are programmed to die whether contact is made with the apposing shelf or not. Glucocorticoids and phenytoin interfere with this programmed cell death, and this interference by both drugs seems to be glucocorticoid receptor mediated, to require protein synthesis, and to be related to arachidonic acid release.

Separation of multiple dihydrotestosterone receptors in rat ventral prostate by a novel micromethod of electro-focusing Blocking action of cyproterone acetate and uptake by nuclear chromatin

Abstract Six dihydrotestosterone-binding proteins in rat ventral prostate nuclei and cytosol have been separated and identified by a microelectrofocusing procedure based on their isoelectric points: pI 3.7, α; 4.6, β; 5.6, γ; 6.7, δ; 7.8, e and 8.5, ζ. Further resolution of these peaks accomplished by expansion of the pI range of electrofocusing separates one or more labelled proteins from these peaks: pI 3.6, α 1 ; 4.1, α 2 ; 4.7, β 1 ; 5.1, β 2 ; 5.4, γ 1 ; 5.8, γ 2 ; 6.2, δ 1 ; 6.5, δ 2 ; 6.8, δ 3 ; 7.1, e 1 ; 7.4, e 2 ; 7.8, e 3 ; 8.3, ζ 1 and 8.7, ζ 2 . Components at pI 3.6, α 1 ; 4.1, α 2 and 5.8, γ 2 are specifically bound by dihydrotestosterone but of these only γ 2 is taken up by nuclear chromatin and the binding of dihydrotestosterone to γ 2 is blocked by cyproterone acetate. Three other components, pI 6.8, δ 3 ; 7.8, e 3 ; and 8.3, ζ are also taken up by chromatin and blocked by the anti-androgen but also bind testosterone or progesterone ( δ 3 , e 3 and ζ 1 ) or estradiol-17β ( δ 3 ). These four components can be extracted from the nuclei ( δ 3 , e 3 and ζ 1 by 0.5 M KCL and γ 2 by 1 M KCL) and all these nuclear extracts except ζ 1 retain their capacity for absorption as dihydrotestosterone complexes by nuclear chromatin. Thus, there are four dihydrotestosterone-binding proteins in rat prostate cytosol which fulfill the criteria for receptor proteins, 5.8, γ 2 ; 6.8, δ 3 ; 7.8, e 3 and 8.3, ζ 1 , and it is possible that androgen action may have to be explained by multiple receptor proteins.

Evidence for a role of arachidonic acid in glucocorticoid-induced cleft palate in rats.

Abstract We have shown that arachidonic acid significantly reduces the production of cleft palate in rats by dexamethasone and that this corrective effect of arachidonic acid is blocked by indomethacin, an inhibitor of cyclooxygenase. Moreover, by using [3H]-arachidonic acid as a tracer we have shown that dexamethasone treatment depresses significantly the free [3H]arachidonic acid available to the microsomal cyclooxygenase in the fetal upper and lower jaws including the palate at the critical period of development. These observations suggest that glucocorticoids produce their palatal teratogenicity by limiting the release and consequently the availability of arachidonic acid at the critical period of development.

Influence of H-2-linked genes on glucocorticoid receptors in the fetal mouse palate

The binding of 3H-dexamethasone to cytosolic receptors in fetal jaws and in cytosols and nuclei of primary cell cultures of fetal palates was studied in various congenic strains of mice. The amount of specific binding was greater in palatal tissues from B10.A and BlO.A(2R) mice than in B10 or B10.A(5R) preparations. These differences were not observed in the liver. Since the strains with higher levels of glucocorticoid receptor are known to be more susceptible to cortisone-induced cleft palate than the strains with low receptor levels, it is suggested that quantitative variation in receptor levels may be involved in determining H-2-linked differences in cleft-palate susceptibility. Whether or not this is the case, it appears that an H-2-linked gene affects the quantity of a cytosolic glucocorticoid-binding protein which translocates to the nucleus.