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.     

Expression of calcium transport proteins in the extraembryonic membranes of a viviparous snake, Virginia striatula

Yolk is the primary source of calcium for embryonic growth and development for most squamates, irrespective of mode of parity. The calcified eggshell is a secondary source for embryonic calcium in all oviparous eggs, but this structure is lost in viviparous lineages. Virginia striatula is a viviparous snake in which embryos obtain calcium from both yolk and placental transport of uterine calcium secretions. The developmental pattern of embryonic calcium acquisition in V. striatula is similar to that for oviparous snakes. Calbindin-D(28K) is a marker for epithelial calcium transport activity and plasma membrane Ca(2+)-ATPase (PMCA) provides the energy to catalyze the final step in calcium transport. Expression of calbindin-D(28K) and PMCA was measured by immunoblotting in yolk sac splanchnopleure and chorioallantois of a developmental series of V. striatula to test the hypothesis that these proteins mediate calcium transport to embryos. In addition, we compared the expression of calbindin-D(28K) in extraembryonic membranes of V. striatula throughout development to a previously published expression pattern in an oviparous snake to test the hypothesis that the ontogeny of calcium transport function is independent of reproductive mode. Expression of calbindin-D(28K) increased in yolk sac splanchnopleure and chorioallantois coincident with calcium mobilization from yolk and uterine sources and with embryonic growth. The amount of PMCA in the chorioallantois did not change through development suggesting its expression is not rate limiting for calcium transport. The pattern of expression of calbindin-D(28K) and PMCA confirms our initial hypothesis that these proteins mediate embryonic calcium uptake. In addition, the developmental pattern of calbindin-D(28K) expression in V. striatula is similar to that of an oviparous snake, which suggests that calcium transport mechanisms and their regulation are independent of reproductive mode.     

Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.     

Loss of yolk platelets and yolk glycoproteins during larval development of the sea urchin embryo

The fate of the yolk platelets and their constituent yolk glycoproteins was studied in Strongylocentrotus purpuratus eggs and embryos cultured through the larval stage. Previous studies have shown that the yolk glycoproteins undergo limited proteolysis during early embryonic development. We present evidence that the yolk glycoproteins stored in the yolk platelets exist as large, disulfide-linked complexes that are maintained even after limited proteolysis have occurred. We provide additional evidence that acidification of the yolk platelet may activate a latent thiol protease in the yolk platelet that is capable of correctly processing the major yolk glycoprotein into the smaller yolk glycoproteins. Because we previously showed that these yolk glycoproteins are not catabolized during early embryonic development, it was of interest to study their fate during larval development. Using a specific polyclonal antibody to a yolk glycoprotein, we found that both yolk glycoproteins and the yolk platelets disappeared in feeding, Day 7, larval stage embryos, but that starvation did not significantly affect the levels of the yolk glycoproteins. We also found that the yolk glycoproteins reappeared in 30-day-old premetamorphosis larvae.     

Expression of endogenous avian myeloblastosis virus information in different chicken cells.

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.     

Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis

In silkworms, yolk proteins comprise vitellin, egg-specific protein and 30K proteins, which are sequentially degraded by endogenous proteases strictly regulated during embryogenesis. Although the process has been extensively investigated, there is still a gap in the knowledge about the degradation of silkworm yolk proteins on the last two days of embryonic development. In the present study, we isolated and purified a gut serine protease P-IIc, which demonstrated optimal activity at 25 °C and pH 11. Semi-quantitative RT-PCR combined with western blotting showed that P-IIc was actively expressed and significantly accumulated in the gut on the last two days of embryogenesis. When natural yolk proteins were incubated with P-IIc in vitro, vitellin and ESP were selectively degraded. P-IIc also demonstrated activity towards 30K proteins as evidenced by rapid and complete digestion of BmLP1 and partial digestion of BmLP2 and BmLP3. Furthermore, RNAi knockdown of P-IIc in silkworm embryos significantly reduced the degradation rate of residual yolk proteins on embryonic day 10. Taken together, our results indicate that P-IIc represents an embryonic gut protease with a relatively broad substrate specificity, which plays an important role in the degradation of yolk proteins at the late stage of silkworm embryogenesis.     

Embryotoxicity of T-2 toxin and secalonic acid in embryonic chicks varies with the site of administration.

A crucial role of the site of administration in the sensitivity of the alternative system using chick embryo for testing embryotoxicity was demonstrated by morphological evaluation of the effects of T-2 toxin and secalonic acid D, and by incorporation of [14C]sodium acetate radioactivity. Secalonic acid D, administered to 2-, 3-, and 4-day-old embryos in doses higher than 1 microgram produced mostly malformations of the face (bilateral cleft beak, microphthalmia) while the teratogenic effects of T-2 toxin were being limited to the embryonic trunk of 2-day-old embryos (rumplessness) after administering doses higher than 0.001 microgram. In case of subgerminal and intraamniotic injections, the doses of both mycotoxins needed for producing embryotoxic effects comparable to those obtained with the more commonly used yolk sac injections appeared to be lower by one and two orders of magnitude, respectively. The results stress the need of using the shortest transport channel of test substances from the site of application to the target tissues of the embryo, when the maximum sensitivity and reproducibility of the test system are to be expected.     

Effects on properties of a thiol protease from Xenopus embryos of changes in substrate and assay conditions.

A protease was purified from Xenopus embryos. Proteolytic activity of the protease against BSA had an optimum pH of 3.8 in acetate buffer and was not detectable at neutral pH. However, when embryonic proteins were used as substrates and digested in phosphate buffer, proteolysis of embryonic proteins was enhanced and was detectable from pH 5.0 to pH 7.0. Digestion of three proteins were mainly detected in digestion of total embryonic proteins. The proteins digested had the same mobilities (on SDS polyacrylamide gel) as yolk proteins. The protease was present in the cytoplasm and around yolk granules. We propose that this protease mainly cleaves a certain yolk proteins in the cytoplasm of Xenopus embryos.     

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.     

Mouse placenta is a major hematopoietic organ

Placenta and yolk sac from 8- to 17-day-old (E8-E17) mouse embryos/fetuses were investigated for the presence of in vitro clonogenic progenitors. At E8-E9, the embryonic body from the umbilicus caudalwards was also analysed. Fetal liver was analysed beginning on E10. At E8, between five and nine somite pairs (sp), placenta, yolk sac and embryonic body yielded no progenitors. The first progenitors appeared at E8.5 at the stage of 15 sp in the yolk sac, 18 sp in the embryonic body, 20 sp in the placenta and only at E12 in the fetal liver (absent at E10, at E11 not determined). Progenitors with a high proliferation potential that could be replated for two months, as well as the whole range of myeloid progenitors, were found at all stages in all organs. However, the earliest of these progenitors (these yielding large, multilineage colonies) were 2-4 times more frequent in the placenta than in the yolk sac or fetal liver. In the fetal liver, late progenitors were more frequent and the cellularity increased steeply with developmental age. Thus, the fetal liver, which is a recognized site for amplification and commitment, has a very different hematopoietic developmental profile from placenta or yolk sac. Placentas were obtained from GFP transgenic embryos in which only the embryonic contribution expressed the transgene. 80% of the colonies derived from these placental cells were GFP+, and so originated from the fetal component of the placenta. These data point to the placenta as a major hematopoietic organ that is active during most of pregnancy.     

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.     

Expression of calbindin-D28K by yolk sac and chorioallantoic membranes of the corn snake, Elaphe guttata

The yolk splanchnopleure and chorioallantoic membrane of oviparous reptiles transport calcium from the yolk and eggshell to the developing embryo. Among oviparous amniotes, the mechanism of calcium mobilization to embryos has been studied only in domestic fowl, in which the mechanism of calcium transport of the yolk splanchnopleure differs from the chorioallantoic membrane. Transport of calcium is facilitated by calbindin-D(28K) in endodermal cells of the yolk splanchnopleure of chickens but the chorioallantoic membrane does not express calbindin-D(28K). We used immunoblotting to assay for calbindin-D(28K) expression in yolk splanchnopleure and chorioallantoic membrane of the corn snake, Elaphe guttata, to test the hypothesis that the mechanism of calcium transport by extraembryonic membranes of snakes is similar to birds. High calbindin-D(28K) expression was detected in samples of yolk splanchnopleure and chorioallantoic membrane during late embryonic stages. We conclude that calbindin-D(28K) is expressed in these extraembryonic membranes to facilitate transport of calcium and that the mechanism of calcium transport of the chorioallantoic membrane of the corn snake differs from that of the chicken. Further, we conclude that calbindin-D(28K) expression is developmentally regulated and increases during later embryonic stages in the corn snake.     

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.     

The composition of liver chromatin proteins from embryos, chicks and adult chickens

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

Antioxidant systems of the developing quail embryo are compromised by mycotoxin aurofusarin

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails (Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.     

Growth and uptake of mineral by embryos of the direct-developing frog Eleutherodactylus coqui

Embryos of the direct-developing frog Eleutherodactylus coqui take up small quantities of yolk and yolk mineral early in incubation but increase their uptake of yolk reserves at later stages of development. Growth and accumulation of calcium and magnesium by embryos also occur slowly at first and at a higher rate later. Accumulation of calcium and magnesium by embryos is largely a function of variation in size of embryos, but uptake of phosphorus is unrelated to size. Althrough patterns of growth and uptake of mineral by embryonic coquis resemble those for embryos of oviparous amniotes, embryonic coquis do not deplete the yolk of its nutrients to the same degree. Thus, residual yolk of coqui hatchlings contains a high percentage of the nutrient reserves originally present in the egg. This difference between embryonic coquis and embryos of oviparous amniotes may indicate that transfer of nutrients from yolk to embryo becomes limiting during the grwoth phase. Alternatively, some aspects of the neurologic system are so poorly developed at hatching that coqui may not be able to find prey effectively. A large nutrient reserve could sustain hatchling while the neurologic system continues to mature.     

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.     

Development of the interaction of GABA and oxazepam on spontaneous motility in chick embryos

The author studied the development of the interaction of GABA and oxazepam on embryonic spontaneous motility in chick embryos during the second half of incubation. In 13-day-old embryos the two substances already potentiated each other's action, despite the fact that GABA, by itself, did not yet have an inhibitory effect. In older embryos this potentiation increased until spontaneous motor activity was almost completely depressed.