Role of nitric oxide in chick embryonic organogenesis and dysmorphogenesis

BACKGROUND: Nitric oxide (NO), produced by the nitric oxide synthase family of enzymes, mediates multiple signaling functions, and when unchecked, NO causes pathological damage. Exposure of embryos to a variety of teratogens, including carbon monoxide (CO), has been shown to increase reactive intermediates, such as NO, and recent work showed that either the excess or absence of NO caused morphological defects. While endogenous NO is known to regulate many adult tissues, its role during embryonic organogenesis and/or in mediating responses to teratogen exposure has not been explored. METHODS: We have examined here the presence of NO during normal chick embryonic organogenesis, and investigated the teratogenicity of NO through the application of sodium nitroprusside (SNP), which mimics NO overproduction, and NG-monomethyl-L-arginine (L-NMMA), which inhibits endogenous NOS activity. RESULTS: Topical treatment with SNP or L-NMMA for 18 h resulted in morphological defects, specifically in the neural tube and somites, which corresponded to sites of altered apoptosis. The location of NO was histochemically correlated with the observed morphological defects. Coadministration of SNP or L-NMMA with CO showed functional coregulation and interaction between NO and CO in chick embryonic development. CONCLUSIONS: Our results showed that regulation of NO is essential for normal axial development, that sites of altered NO expression correlate to those of altered apoptosis and dysmorphogenesis, and that CO coadministration resulted in a rectification of normal NO expression. Collectively, these results suggest that alteration in endogenous NO/CO signaling is responsible, at least in part, for the observed NO-induced teratogenesis.     

Antioxidative treatment of pregnant diabetic rats diminishes embryonic dysmorphogenesis

BACKGROUND: Diabetic pregnancy is still associated with an increased rate of congenital malformations despite extensive clinical efforts to normalize the risk for the offspring. The etiology of diabetic embryopathy is not clear; however, experimental studies have suggested a role for oxidative stress in the teratogenicity of diabetic pregnancy. The antioxidants alpha-tocopherol and ascorbate have improved fetal outcome in diabetic rodent pregnancy when supplemented in moderate to high doses. In the present work we investigated if extremely high doses of either alpha-tocopherol or ascorbate might further improve fetal outcome in offspring of diabetic rats and, in addition, if such treatment may exert any adverse effects of fetal development. METHODS: Nondiabetic and streptozotocin diabetic female rats were fed 2, 5, 10, or 15% alpha-tocopherol or 4, 10, or 15% ascorbate in their diet. RESULTS: Both alpha-tocopherol and ascorbate treatment improved fetal morphology in offspring of diabetic rats. There was a dose-dependent improvement for the alpha-tocopherol supplementation, in which the higher doses diminished fetal dysmorphogenesis more than the 2% diet. The ascorbate supplementation was less dose-dependent; however, the higher doses tended to improve fetal outcome more than the lower doses. No adverse effects of the antioxidants were noted in the offspring with the exception of 1 case of agnathia in a fetus of a nondiabetic rat supplemented with 15% alpha-tocopherol. CONCLUSIONS: These results indicate that very high doses of dietary antioxidants may be needed to normalize the development of the offspring in experimental diabetic pregnancy, but that treatment with such high doses may also have adverse effects in nondiabetic pregnancy.     

Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.     

Inhibitory effects of tryptamine on tolbutamide-induced hypoglycemia in mice: Mediation by 5-HT receptors

Effects of tryptamine on tolbutamide-induced hypoglycemia were investigated in mice. Tryptamine significantly inhibited hypoglycemia elicited by tolbutamide. The inhibitory effects of tryptamine were strongly blocked by the 5-HT₁ and 5-HT₂ receptor antagonist methysergide and the 5-HT₂ receptor antagonist ketanserin, while the 5-HT₃ receptor antagonist ICS 205–930 was without effect. Tryptamine induced hyperglucagonemia in tolbutamide-treated mice, and this effect elicited by tryptamine was strongly inhibited by the 5-HT₂ receptor antagonist ketanserin. These results suggest that the inhibitory effects of tryptamine on tolbutamide-induced hypoglycemia are mediated by 5-HT₂ receptors and that tryptamine is involved in glucagon release.     

Teratogenic effects of bis-diamine on early embryonic rat heart: an in vitro study

BACKGROUND: Bis-diamine induces cardiac defects, including conotruncal anomalies in rat embryos when the agent is administered to the mother. To evaluate the teratogenic effects and mechanism of bis-diamine, we performed morphological and immunohistochemical analyses of early rat embryos cultured in medium containing bis-diamine. METHODS: The embryos were removed from mother rats on gestational day 10.5 and cultured in medium containing 1 mg of bis-diamine for 6 hr. The embryos were then cultured in medium only for another 6, 12, 18, and 42 hr, corresponding to embryonic day (ED) 11.0, 11.25, 11.5, and 12.5, respectively. Some embryos from the same mothers were used as controls and were cultured in medium only for the corresponding periods to the embryos exposed to bis-diamine. Some mother rats were given a single oral dose of 200 mg of bis-diamine on gestational day 10.5. Embryos from these pregnant rats were removed 6 hr after the oral administration of bis-diamine, and were also cultured in medium only for 6, 12, 18, and 42 hr. RESULTS: No cardiac abnormalities were detected in the controls at any stage of development. Thirty-three of 51 (65%) embryos exposed to bis-diamine and 15 of 20 (75%) embryos removed from bis-diamine-administered mothers showed abnormal cardiac development, including dilated ventricle, elongation of outflow tract, and pericardial defect on ED 11.5. Four of six (67%) embryos exposed to bis-diamine, and five of seven (71%) removed from bis-diamine-administered mothers also presented almost the same cardiac abnormalities on ED 12.5. No cardiac abnormalities were detected in bis-diamine-treated embryos before ED 11.5. In addition, the expression of neural cell adhesion molecule (N-CAM) was examined using immunohistochemical methods. Fewer N-CAM immunoreactive cells were detected in the third and fourth aortic arches in the bis-diamine-treated embryos than in controls on ED 11.5. However, more N-CAM immunoreactive cells were detected in the bis-diamine-treated embryos than in controls on ED 12.5. CONCLUSIONS: These results suggest that bis-diamine induces cardiac anomalies by delaying the migration of neural crest cells into the heart and by disturbing the proliferation of pericardial precursor during early cardiac development.     

Elevations in the endogenous levels of the putative morphogen retinoic acid in embryonic mouse limb-buds associated with limb dysmorphogenesis

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.     

Polyamines protect rat embryo in vitro from high glucose‐induced developmental delay and dysmorphogenesis

BACKGROUND : Pregnancy in mammals with diabetes mellitus results in low birth weight, malformations, and intrauterine death. Parenteral application of natural polyamines or their precursor, L ‐arginine, to diabetic pregnant rats partially prevents the alterations of development caused by diabetes mellitus. This experiment has been designed to understand if this preventive action also occurs in rat whole embryo in culture. MATERIALS AND METHODS : Rat embryos of gestational day 10 were cultured for 24 h in normal medium, high glucose medium, or high glucose medium supplemented with polyamines or L ‐arginine, and furthermore embryo growth and development were evaluated. RESULTS : L ‐arginine and putrescine partially prevents the dysmorphogenic effects of high glucose, whereas spermidine and spermine prevent these effects almost completely. CONCLUSIONS : Polyamines directly protect the embryo from the toxic effect of high glucose concentration on growth and development, although the mechanism remains to be elucidated. Birth Defects Res (Part B)86:58‐64, 2009 ©2009 Wiley‐Liss, Inc.     

Successful pig embryonic development in vitro outside a CO2 gas-regulated incubator: effects of pH and osmolality

We investigated the effects of HEPES in the medium (to maintain pH) and paraffin oil covering the medium (to maintain osmolality) on the developmental ability of porcine embryos produced in vitro using tightly closed glass tubes in the absence of a CO2 gas-regulated incubator. Putative porcine zygotes obtained by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes (day of IVF=Day 0) were cultured in 5% CO2 gas-equilibrated NCSU-37 media containing pyruvate and lactate during Days 0-2, and glucose during Days 2-6, in open glass tubes in a CO2 incubator or tightly closed glass tubes without a CO2 incubator at 38.5 degrees C. The following four media were used: (1) medium covered with paraffin oil and supplemented with HEPES; (2) medium covered with paraffin oil but with no HEPES supplementation; (3) medium not covered with paraffin oil but supplemented with HEPES; (4) medium not covered with paraffin oil and with no HEPES supplementation. As a control group, zygotes were cultured in medium with neither paraffin oil coverage nor HEPES supplementation using a four-well dish in a CO2 gas-regulated incubator. After culture, the osmolality in each of the four closed conditions was maintained at approximately 285-286 mOsm, lower (P<0.05) than that in the control (291 mOsm). In the two HEPES-supplemented media groups in the closed-tube system, the pH was maintained at 7.5-7.7, and the blastocyst development rates (15.5% in non-oil covered and 18.5% in oil covered group) did not differ significantly from that of the control (20.2%), although the mean cell numbers in the blastocysts in the two closed-tube condition groups (28.2 and 33.0) were lower (P<0.05) than in the control (43.5). In contrast, the pH was higher in the two groups without HEPES supplementation (approximately 8.0) than the control (7.4; P<0.05), and the blastocyst development rates (10.9% in non-oil covered and 7.5% in oil covered group) or total cell numbers in the blastocyst (24.8 and 28.7) in the two non-HEPES groups were drastically decreased (P<0.05) compared to those in the control (20.2% and 43.5). These results suggested that maintenance of pH is important for successful in vitro porcine embryo culture under closed-air conditions, whereas the range of osmolality that suits embryo development is not limited to a small range. Furthermore, blastocyst production was possible in a glass tube without a CO2 incubator, although blastocyst quality was lower compared to those produced in an incubator.     

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK‐3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β‐catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells‐c1 (NFATc1), both key regulators of valve development. Li‐exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK‐3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β‐catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up‐regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK‐3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Fertilization and embryonic development in vitro in cattle. Morphological and cytogenetic observations.

The presented ichonography provides a documentation of the changes occurring during maturation, fertilization of oocytes and embryonic development in vitro; it points out the correlations between morphological features and the stage of nuclear chromatin and gives the possibility to observe some abnormalities that frequently occur during these processes.     

Differential dysmorphogenesis induced by microinjection of an alkylating agent into rat conceptuses cultured in vitro

A technique of microinjection of small quantities of teratogens into extraembryonic compartments or specific organ primordium of rat conceptuses of pregnancy day 11 is described. Conceptuses microinjected with 50 nl tissue culture medium developed normally for 44-45 hr when cultured in homologous rat serum, indicating that the microinjection procedure itself did not produce any deleterious effects on growth and differentiation of embryos. Microinjection of an alkylating agent, phosphoramide mustard dissolved in tissue culture medium, into the exocoelom produced anomalous embryogenesis, consisting of retarded embryonic growth, anomalies of the neural tube, and general necrosis of various organ primordia. In contrast, the embryonic development remained relatively unaffected by microinjection of identical amounts of this alkylating agent into the amniotic cavity. However, neural-tube differentiation was markedly affected when phosphoramide mustard was injected into anterior neural-tube fluid, producing anencephalic or microcephalic embryos without significant effect on postcephalic organ differentiation. The morphogenesis of the anterior limb was unaffected by local injection of the agent into somitic tissues adjacent to the presumptive limb-bud region. Therefore, it appears that differential dysmorphogenesis could be induced by microinjection of an alkylating agent into different conceptus compartments. These results indicate that even during early embryogenesis various cell types are not equally susceptible to a given teratogen, and that the differential cytotoxicity of the teratogen toward specific embryonic or extraembryonic cells and tissues may account for embryonic anomalies characteristically produced by that agent.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

Induction of embryonic dysmorphogenesis by high glucose concentration, disturbed inositol metabolism, and inhibited protein kinase C activity

BACKGROUND: Exposure to a diabetic environment causes excess reactive oxygen species (ROS), decreased prostaglandin E(2) (PGE(2)) concentration, and increased embryonic maldevelopment. The aim of the present work was to study whether embryonic dysmorphogenesis is also dependent on alterations of inositol and associated intracellular metabolites. METHODS: Day 9 rat embryos were cultured for 24 or 48 hr and evaluated for gene expression. Day 10 and day 11 embryos from normal and diabetic rats were also examined. RT-PCR was used to study embryonic gene expression of protein kinase C (PKC) and cytosolic phospholipase A(2) (cPLA(2)). RESULTS: Embryos exposed to 30 mmol/L glucose (30G), 500 or 750 micromol/L of scyllo-inositol (500SI or 750SI) had higher malformation score than control embryos cultured in 10 mmol/L glucose (10G). Adding 1.6 mmol/L inositol to the 30G or 750SI culture medium partly corrected these embryos, and completely normalized 500SI embryonic development. Adding 0.5 mmol/L N-acetylcysteine (NAC) or 280 nmol/L PGE(2) protected, and failed to protect, the SI-exposed embryos, respectively. 10G embryos exposed to the PKC inhibitor GF-109203X displayed dose-dependent dysmorphogenesis. Addition of 1.6 mmol/L inositol or 0.5 mmol/L NAC to the PKC-inhibitor-exposed 10G embryos largely normalized the outcome, whereas PGE(2) again failed to protect embryonic development. 30G culture tended to decrease the expression of cPLA(2) after 24 hr in vitro. We also found decreased mRNA levels of cPLA(2) in offspring of diabetic rats on gestational day 10 and of PKC on day 11, as compared with normal offspring. CONCLUSIONS: High glucose concentration causes dysmorphogenesis in embryos by an interaction of oxidative stress and inositol depletion.     

Drosophila embryonic 'fibroblasts': Extending mutant analysis in vitro

The in vivo analysis of Drosophila using genetics, with almost a hundred year history, has produced an immense body of knowledge about biology. In vitro analysis, while arguably the poor cousin to its in vivo relative, has a utility-in biochemical analyses and in cell-based screening, for example, with RNAi. A major block to the development of in vitro analysis has been the lack of an efficient genetic method to derive cell lines from mutant Drosophila strains. We recently discovered that expression of activated Ras (Ras^V12) provides cells in vitro with both a survival and a proliferative advantage and hence promotes the generation of cell lines.¹ In this addendum, we provide new data describing the genesis of seven cell lines corresponding to a rumi mutant, which demonstrate that the method can be used to derive lines and study genetic mutants in vitro.     

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro

Summary Different organotypical culture methods are used to test the direct effects of serotonin and ketanserin, a S₂, α₁, and H₁ receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [³H]thymidine show that ketanserin, within a dose range from 0.05 to 5 μg/ml, stimulates proliferation. Serotonin at a concentration of 10 μg/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 μg/ml result in tritium activities that do not differ from control cultures. This investigation was financially supported by the National Fund of Scientific Research, Belgium, 3.0022.87.     

Disorders of post-squalene cholesterol biosynthesis leading to human dysmorphogenesis.

Insights in molecular developmental biology in animals and humans are facilitating the understanding of pathophysiologic mechanisms in dysmorphogenesis or abnormalities in normal embryologic structural development. A milestone was recognition of the role of shh in morphogenesis of craniofacial structures, especially the development of holoprosencephaly. The dependence of hedgehog morphogens on cholesterol modification for normal hedgehog signaling function has particular relevance to disorders of cholesterol synthesis which manifest dysmorphogenesis. Four human disorders of morphogenesis (Smith-Lemli-Opitz syndrome, desmosterolosis, X-linked chondrodysplasia punctata, CHILD syndrome) have recently been shown to be caused by sterol abnormalities resulting from cholesterol biosynthesis enzyme deficiencies. This review summarizes the clinical, biochemical and molecular data in these disorders with an emphasis on understanding the pathophysiology of dysmorphogenesis.