The effects of high-sucrose diets and of maternal diabetes on the ultrastructure of the visceral yolk sac endoderm in rat embryos developing in vivo and in vitro

The ultrastructure of the visceral yolk sac endoderm of in vivo developing 9- to 13-day-old embryos from 2 diabetic rat models (streptozotocin diabetes and Cohen--genetically determined--diabetes) and from nondiabetic rats fed high sucrose diets have been studied. This was compared to yolk sacs from 9.5-day-old embryos cultured for 48 h in sera from diabetic and nondiabetic rats fed a high-sucrose diet. Light-microscopic, TEM and SEM studies showed that the pathological cellular changes in the visceral yolk sac endoderm from diabetic rats were first observed on day 9 and were most severe among 11-day-old embryos. In vitro culture of control rat embryos in serum from experimental animals induced a reduction in the number of microvilli, of vacuolar intracellular inclusions and an increase in the number of degenerated endodermal cells. SEM studies showed that in addition to disappearance of microvilli, the majority of cells were collapsed and had degenerated cell membranes. Culture of embryos from diabetic animals in control serum only slightly reversed the pathological changes in the visceral yolk sac endoderm. A good correlation exists between the rate of embryonic malformations in diabetic rats and an index of endodermal-cell damage in the visceral yolk sac.     

Nitroxide radicals protect cultured rat embryos and yolk sacs from diabetic-induced damage

BACKGROUND: Diabetic teratogenicity relates, partly, to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We investigated the effects of superoxide dismutase-mimics nitroxides, 2,2,6,6-tetramethyl piperidine-N-oxyl (TPL) as an effective antioxidant, on diabetes-induced embryopathy. METHODS: Embryos (10.5 day old) and their yolk sacs from Sabra female rats were cultured for 28 h in the absence or in the presence of nitroxides at 0.05-0.4 mM in control, diabetic subteratogenic, or diabetic teratogenic media, and monitored for growth retardation and congenital anomalies. The oxidant/antioxidant status was examined by oxygen radical absorbance capacity and lipid peroxidation assays, whereas the yolk sac function was evaluated by endocytosis assay. RESULTS: Diabetic culture medium inhibited embryonic and yolk sac growth, induced a high rate of NTDs, reduced yolk sac endocytosis and embryonic antioxidant capacity, and increased lipid peroxidation. These effects were more prominent in the embryos with NTD compared to those without NTD. TPL added to diabetic teratogenic medium improved embryonic and yolk sac growth, reduced the rate of NTDs, and improved yolk sac function. The oxidant/antioxidant status of embryos was also improved. TPL at 1 mM did not damage the embryos cultured in control medium. CONCLUSIONS: In diabetic culture medium, oxidative damage is higher in the malformed rat embryos compared to those without anomalies; the nitroxide provides protection against diabetes-induced teratogenicity in a dose-dependent manner. The yolk sac damage, apparently caused by the same mechanism, might be an additional contributor to the embryonic damage observed in diabetes.     

Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions

Diabetes mellitus in pregnancy is associated with an increased incidence of various congenital anomalies that occur during organogenesis. Because a well functioning yolk sac is crucial to embryonic growth and development during this period, we performed an ultrastructural study of the effects of excess glucose (total glucose 750 mg/dl, osmolality 305 mOsm/kg) on pregnancy day 10 (Witschi stage 13) rat conceptuses cultured for 48 hr in heat-inactivated male rat serum with and without added d- or l-glucose. Embryos exposed to excess d-glucose demonstrated decreased conceptus size (P less than 0.001), and gross malformations in a dose-related fashion. The visceral yolk sac capillaries and vitelline vessels of conceptuses in excess d-glucose were sparse, patchy, and nonuniformly located. Ultrastructurally, the visceral yolk sac endodermal cells had reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These obvious defects in yolk sac structure suggest that hyperglycemia during organogenesis has a primary deleterious effect on yolk sac function with resultant embryopathy.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

胚胎神经管缺陷大鼠模型差异基因的表达

通过构建妊娠合并糖尿病诱发先天性神经管缺陷的SD大鼠模型, 与胚胎不伴有先天性神经管缺陷组大鼠和正常对照组大鼠胚胎进行研究, 提取卵黄囊细胞的mRNA, cDNA 基因芯片技术对表达差异基因进行检测, 应用特异性抗磷酸化抗体进行免疫共沉淀及Western blotting, 对卵黄囊细胞MAP Kinase信号途径蛋白激酶活性进行分析。在神经管缺陷大鼠胚胎卵黄囊细胞和对照组1 200个基因中, 共筛选出表达差异基因79个, 其中42个基因表达上调、37个基因表达下调。同时发现神经管缺陷胚胎卵黄囊细胞出现细胞凋亡特征性的DNA ladder(梯状电泳), 凋亡相关基因 caspase-3、Bax 高表达, 凋亡抑制基因 AKT活性明显受抑; 与正常对照组相比ERK1/2蛋白激酶活性显著下降、JNK1/2活性明显升高。因此, 认为妊娠合并糖尿病诱发胚胎先天性神经管缺陷的发生存在多种差异基因表达, 以及MAP Kinase、凋亡信号传导机制的共同作用。     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

An in vivo/in vitro evaluation of the teratogenic action of excess vitamin A

Pregnant rats of CFHB strain were injected 81/2 days postcoitum with a 1% suspension of retinoic acid (RA) in arachis oil to give 20 mg RA per kg body weight. Control rats were injected with arachis oil only. After 26 hours, one uterine horn was removed from each rat and the embryos cultured in serum from untreated rats. The embryos in the other horn were allowed to continue development in vivo. After a further 48 hours the cultures were terminated and the second uterine horn removed from each rat. This provided four groups of embryos for comparison: (1) embryos from RA-treated rats, (2) cultured embryos from RA-treated rats, (3) embryos from control rats, and (4) cultured embryos from control rats. The results showed that the effects of the teratogen on the cultured embryos were similar to those on the embryos allowed to continue development for the same period in the mother. In both groups RA reduced protein synthesis, inhibited somite and limb bud formation, and caused various neural tube defects, particularly microcephaly and abnormalities in the closure of the anterior and posterior neuropores.     

WHOLE-EMBRYO CULTURE AND THE STUDY OF MAMMALIAN EMBRYOS DURING ORGANOGENESIS

1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation. 2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta. 3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice. 4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac. 5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate. 6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac. 7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds. 8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.     

妊娠合并糖尿病诱发胚胎先天性神经管缺陷 动物模型的MAP 激酶信号传导机制MAP

为了揭示妊娠合并糖尿病诱发胚胎先天性神经管缺陷的分子机制,并探讨其有效的防治方法。 实验选用6个实验组的 Sprague-Dawley 大鼠：第1组为常规饲养的正常对照组;第2组尾静脉注射65mg/kg Streptozotocin (STZ) 构建妊娠合并糖尿病、且诱发先天性神经管缺陷的实验组大鼠;第3组为STZ构建的糖尿病、但胚胎不伴有先天性神经管缺陷的大鼠模型;第4、5、6 组为 STZ 构建的糖尿病治疗组大鼠,每日分别给予80mg/mL花生四烯酸 (arachidonic acid,AA)、400mg 维生素E、抗氧化剂(维生素 E) 和不饱和脂肪酸 (saflower oil) 混合物 cocktail 治疗。于妊娠第12天取出各组胚胎,解剖显微镜下进行形态学分析;提取卵黄囊细胞蛋白质,应用特异性抗磷酸化抗体进行免疫共沉淀及 Western 印迹,对MAP 激酶 信号途径上各蛋白激酶ERK1/2、JNK1/2、RAF-1活性进行分析。 与正常对照组相比,妊娠合并糖尿病诱发的先天性神经管缺陷胚胎中(第2组),ERK1/2蛋白激酶活性显著下降,其上游 RAF-1活性相应降低;与此相反,JNK1/2活性明显升高。在给予花生四烯酸、维生素E 补充物治疗后,通过调节MAP 激酶 信号通路蛋白激酶活性,逆转了胚胎神经管缺陷的发生。妊娠合并糖尿病诱发的胚胎先天性神经管缺陷的发生,与MAP 激酶信号传导机制异常密切相关。不饱和脂肪酸和抗氧化剂补充物的治疗作用通过对MAP 激酶信号途径的调控实现的。 AbstractThe aim of the present study was to determine molecular mechanism in hyperglycemia-induced congenital neural tube defects and the its potential pharmacologic rescuing agents. In order to explore these questions, six study groups of Sprague-Dawley rats were employed: Group 1 was normal control rats with normal diet; group 2 represented streptozotocin (STZ) -induced diabetic rats with congenital neural tube defects in offspring; group 3 included STZ-induced diabetic rats with normal offspring; groups 4,5 and 6 included rats exposed to the same STZ-induced diabetic condition, but receiving daily oral supplementation of 80mg/mL of the sodium salt of arachidonic acid (AA), 400mg of vitamin E and a cocktail of a polyunsaturated fatty acid (saflower oil) plus an antioxidant ( vitamin E) respectively. Yolk sac cells were harvested at gestational day 12 from each rat group. Changes in MAPK signaling pathways were detected by western blot analysis using special antibodies directed against phosphorylated forms of extracellular signal regulated kinase (ERK), Jun N-terminal/stress-activated protein kinase (JNK/SAPK). Furthermore, activity of RAF-1, an upstream kinase in ERK1/2 signaling cascade, was evaluated by immunoprecipitation assay. The results showed that in yolk sac cells in embryopathic offspring from experimentally-induced diabetic rats, activities of ERK1/2 were dramatically decreased (group 2). Consisted with these observation, reduction in RAF-1 kinase activity could be discerned in these diabetic yolk sac cells. In contrast, activities of JNK1/2 were significantly increased in yolk sac cells of group 2. Under rescuing circumstance,activations of ERK1/2 and RAF-1 were increased, and JNK1/2 were decreased. MAP kinase signal pathway plays a very important role in hyperglycemia induced neural tube defects. The supplementation of polyunsaturated fatty acid arachidonic acid, and antioxident vitamin E rescued conceptuses from diabetic embryopathy by triggering a restoration of normal membrane signaling pathways.     

Role of reactive oxygen species (ROS) in the diabetes-induced anomalies in rat embryos in vitro: reduction in antioxidant enzymes and low-molecular-weight antioxidants (LMWA) may be the causative factor for increased anomalies

A disturbed embryonic antioxidant defense mechanism may play a major role in diabetes-induced teratogenesis. We therefore studied the antioxidant capacity of 10.5-day-old rat embryos and their yolk sacs after culture for 28 hr in vitro under diabetic conditions (3 mg/ml glucose, 2 mg/ml beta-hydroxybutyrate (BHOB) and 10 microg/ml of acetoacetate), as compared with control embryos in vitro. We found a high rate of congenital anomalies, decreased growth and protein content, and a decrease in the activity of both superoxide dismutase (SOD) and catalase (CAT) under diabetic conditions, as compared with controls. The reducing power, which reflects the concentration and type of water-soluble and of lipid-soluble low-molecular-weight antioxidants (LMWA), was measured by cyclic voltammetry. Generally, LMWA were reduced in the embryos and yolk sacs under diabetic conditions. In the water-soluble fraction of control embryos and yolk sacs, two peak potentials were found, indicating two major groups of LMWA, while only one peak potential was found under diabetic conditions, indicating that an entire group of LMWA is missing. HPLC studies have demonstrated a decrease in vitamin C (water-soluble fraction) and in vitamin E (lipid-soluble fraction) under diabetic culture conditions, and an increase in uric acid. Generally, the concentration of LMWA was higher in the embryos than in the yolk sac. LMWA concentration, protein content, and antioxidant enzyme activity were lower in the malformed experimental embryos than in experimental embryos without anomalies. The addition of vitamins C and E to the diabetic culture medium abolished the deleterious effects of the diabetic serum on the embryos. The disturbed antioxidant defense mechanism under diabetic conditions may be explained, at least in part, by a direct effect of diabetic metabolic factors on the activity of antioxidant enzymes and on the concentration of reducing equivalents. This, in turn, may be embryotoxic.     

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Evidence that suramin and aurothiomalate are teratogenic in rat by disturbing yolk sac-mediated embryonic protein nutrition

Suramin (250 mg/kg) and sodium aurothiomalate (100 mg/kg) both induced congenital malformations in the offspring following treatment of pregnant rats at either 8.5 or 9.5 days of gestation. Conceptuses from 9.5-day pregnant rats were cultured for 48 h in homologous serum to which either suramin or sodium aurothiomalate was added for the final 6 h. The presence of suramin up to 5 mg/ml had no effect on the protein content of yolk sacs at harvesting, but at 10 mg/ml caused a significant decrease. In contrast sodium aurothiomalate increased the protein content of yolk sacs at harvesting, in a concentration-dependent manner up to 100 micrograms/ml. Neither suramin nor sodium aurothiomalate significantly affected embryo protein content. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. When suramin (2-10 mg/ml) was present for the final 6 h of culture, the quantity of radioactivity measured in the yolk sac at harvesting was significantly decreased in a concentration-dependent manner. No radioactivity was detected in the embryos. Sodium aurothiomalate had no effect on the uptake of 125I-labelled polyvinylpyrrolidone. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in the conceptus (both yolk sac and embryo) at harvesting. Suramin (5 mg/ml), present for the final or penultimate 6 h, significantly decreased the uptake of radioactivity into conceptuses and caused a significant increase in the proportion of the captured radiolabel that was associated with the yolk sac. Sodium aurothiomalate (25 or 500 micrograms/ml) had no effect on the total uptake of radio-label but caused a significant increase in the proportion of total radioactivity captured that was associated with the yolk sac. These data indicate that suramin, by interfering with both the uptake and intralysosomal digestion of protein, and sodium aurothiomalate, by inhibiting digestion of captured protein, disturb the normal pathway of yolk sac-mediated protein utilization with a consequent diminution of the supply of amino acids to the conceptus. The effects of suramin are seen only at high concentration, those of sodium aurothiomalate at much lower concentrations. It is likely that the two drugs exert their teratogenic action by their effects on the yolk sac nutritional pathway with resultant amino acid deprivation of the conceptus at a critical stage of development.     

Vitamins C and E improve rat embryonic antioxidant defense mechanism in diabetic culture medium.

BACKGROUND: Diabetes teratogenicity seems to be related to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We have studied the mechanism by which antioxidants, such as vitamins C and E, reduce diabetes-induced embryonic damage. We therefore compared the antioxidant capacity of 10.5-day-old rat embryos and their yolk sacs cultured for 28h in diabetic culture medium with or without vitamins C and E. METHODS: The embryos were cultured in 90% rat serum to which 2mg/ml glucose, 2mg/ml beta hydroxy butyrate (BHOB) and 10 microg/ml of acetoacetate were added. Rat embryos were also cultured in a diabetic medium with 25 microg/ml of vitamin E and 50 microg/ml of vitamin C. Control embryos were cultured in normal rat serum with or without vitamins C and E. RESULTS: Decreased activity of Cu/Zn superoxide dismutase (SOD) and of catalase (CAT) in the "diabetic" embryos and their yolk sacs, and reduced concentrations of low molecular weight antioxidant (LMWA) were found. Under these conditions we also found a decrease in vitamin C and vitamin E concentrations in the embryos, as measured by HPLC. In situ hybridization for SOD mRNA showed a marked reduction of SOD mRNA in the brain, spinal cord, heart and liver of embryos cultured in diabetic medium in comparison to controls. Following the addition of vitamins C and E to the diabetic culture medium, SOD and CAT activity, the concentrations of LMWA, the levels of vitamin C and E and the expression of SOD mRNA in the embryos and yolk sacs returned to normal. CONCLUSIONS: Diabetic metabolic factors seem to have a direct effect on embryonic SOD gene and perhaps genes of other antioxidant enzymes, reducing embryonic endogenous antioxidant defense mechanism. This in turn may cause a depletion of the LMWA, such as vitamins C and E. The addition of these vitamins normalizes the embryonic antioxidant defense mechanism, reducing the damage caused by the diabetic environment.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum.

In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy. However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium. This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate. The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats. Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos. Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations. At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation. When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis. Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures. There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs. 50%). Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations. By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of ceruloplasmin synthesis in embryogenesis and in the early postnatal period of laboratory white rats

The dynamics of ceruloplasmin content was studied by immunochemical methods in the postimplantation rat embryos and postnatal animals. Ten to twenty two day old embryos contained ceruloplasmin (CP) in yolk sac, serum, and amniotic fluid. The highest CP levels were found in yolk sac. CP concentration profiles were almost identical in the serum and amniotic fluid being the highest on the 12th day (0.26 mg%) and the lowest (0.04) on the 16th day of gestation. CP concentration in the serum increased rapidly up to 3.5 mg% from the 17th day of gestation till the term (22nd day) while remaining at a constant and rather low level in the amniotic fluid. Within 16-18 days after birth, CP concentration in the serum remained at the level of 11 +/- 0.3 mg%. Later on it gradually increased and attained plateau (46-48 mg%) by the time of sex maturity. The maternal serum CP does not penetrate, in the embryo, as can be inferred from the experiments with 125I-CP injected into pregnant rats. Differences in the CP degradation rate and modes were found between the embryos and postnatal rats. It is suggested that CP is initially synthesized by the yolk sac endoderm during organogenesis (10-16 days of gestation) and predominantly by the liver during the foetal period (17-22 days).     

In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos

The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture. Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture. No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose. From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture. Embryonic growth of 9.5-day explants was similar to that previously observed in vivo. Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM). The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h. The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.     

Maternal transferrin uptake by and transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro

Uptake and transfer of maternal transferrin by rat embryos during organogenesis in vitro was investigated using radiolabelled rat transferrin and rocket immunoelectrophoresis. Colloidal gold to which rat transferrin was adsorbed was used as an electron microscopical marker in order to follow the route taken by internalised transferrin across the visceral yolk sac. Culture of rat conceptuses from 9.5 to 11.5 days of gestation in rat or human sera resulted in the passage of rat or human transferrin from the culture medium into the extraembryonic coelom as determined by quantitative immunoelectrophoretic analysis of exo-coelomic fluid. The concentration of human transferrin which was transferred to the exo-coelomic fluid of conceptuses cultured in whole human serum at 10.5 days and 11.5 days of gestation was similar to the concentration of rat transferrin in the fluid of conceptuses cultured in rat serum which had been diluted with Hanks' saline to 50% in order to match the levels of transferrin found in human serum. Growth of rat embryos in 50% rat serum was identical to embryonic growth in 100% rat serum. Uptake of radiolabelled rat transferrin by the visceral yolk sac at 11.5 days of gestation, following culture for 60 min in radiolabelled medium, was much greater than nonspecific uptake of radiolabelled bovine serum albumin. Accumulation of radiolabelled transferrin by the embryo was reduced by the inclusion of unlabelled transferrin into the culture medium. Uptake of transferrin adsorbed 18 nm gold particles was mediated by attachment to coated pits on the apical cell surface of the extraembryonic endoderm. Transferrin-adsorbed gold colloid was internalised via coated vesicles and found in cisternal structures of the peripheral and juxtanuclear areas, as well as in smooth and coated vesicles deep within the cell. The intercellular presence of gold particles in the endodermal layer of the visceral yolk sac and their presence in the mesoderm after 60 min of incubation suggested that passage of transferrin was rapid and mediated by vesicular evagination from the extraembryonic endoderm. These findings suggest that maternal transferrin is the primary source of transferrin for the early rat embryo and its passage to the exo-coelom and embryo is mediated by specific receptors on the apical surface of the extraembryonic endoderm.