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Freeze-etching observation on the development of intercellular junctions of the duodenal epithelial cells in the chick embryo.

Development of intercellular junctions in duodenal epithelium in the chick embryo was studied by electron microscopy using the thin-section and freeze-fracture techniques. Incomplete tight junctions are already seen in 6 and 7 day old embryos at the apical portion of the lateral plasma membrance, and consist of 1-7 strands, their mean depth measuring 0.2 micron. This corresponds to a "very leaky type" (CLAUDE and GOODENOUGH) of tight junction. Ridges on the PF are discontinuous and rarely cross or link. The tight junctions extend basally at the place where more than three epithelial cells are in contact. On the lateral plasma membrane, particle-aggregates suggesting primitive gap junctions are already recognized. Some are dense aggregations of 3-5 membrane-particles with a halo free of the particles and others are rather loose aggregations of 5-10 particles with an indistinct halo. In 9 day old embryos, the ridges of the tight junction become more discontinuous, although the frequency of the linkage of the neighboring ridges increases. The compartments bounded by the tight junctional strands are angular. These strands become continuous and the facets surrounded by them are roundish in 12 day old embryos. The presumptive immature gap junctions show a characteristic polygonal pattern in 9 day old embryos and gradually increase in size. Mature tight junctions and typical gap junctions of 0.3-0.4 micron diameter are seen after 18 days of incubation. The strands number 6-8 and the depth of the tight junction measures about 0.4 micron in 18 day old embryos. In the chick embryo duodenal epithelium, the tight and gap junctions develop independently from each other without any direct interaction between them.

Animals↗

Ameliorating effects of thyroxine and atropine in phosphamidon intoxicated chick embryos.

The effects of thyroxine and atropine in ameliorating phosphamidon intoxication in chick embryos was studied. Treatment of phosphamidon significantly enhanced the mortality and abnormality rates, decreased the average body weights, and cholinesterase activity in chick embryos. When thyroxine was administered to the phosphamidon intoxicated embryos, the above parameters changed significantly, indicating an ameliorating effect of thyroxine against phosphamidon intoxication in chick embryos. The combined thyroxine and atropine therapy did not further improve the ameliorating effect. Since in many respects chick embryo development parallels that of mammalian embryos, a short-term use of thyroxine as a protective agent against organophosphate toxicity might be useful.

Animals↗

[Effect of ethyl alcohol, nicotine, and both combined on certain parameters of chick embryo development].

Experiments on 220 chick embryos were made to study the embryotoxic action of ethyl alcohol and nicotine administered alone and combined, depending on the drug dose, route and frequency of administration. It has been disclosed that the toxic effect of the drugs manifests primarily at the cellular and subcellular levels and mainly affects the cardiovascular system and liver. The drug side effects increased on repeated administration. Combined use of alcohol and nicotine does not lead to potentiation of their toxic effect on the developing embryo.

Animals↗

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos.

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.

Animals↗

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development.

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.

Animals↗

The effect of isoproterenol on cardiovascular function in the stage 24 chick embryo.

The developing cardiovascular system of the chick embryo is susceptible to teratogenic effects of catecholamines. Yet the mechanism for the teratogenetic action is unclear. Since catecholamines affect cardiovascular physiology, we studied the acute effect of the beta-agonist isoproterenol on mean atrial pressure, heart rate, mean dorsal aortic blood flow, mean arterial pressure and vascular resistance in stage 24 chick embryos. Dorsal aortic blood velocity was measured with a 20-MHz pulsed-Doppler velocity meter and intravascular pressure was measured with a servo-null pressure system. Isoproterenol in doses of 2 X 10(-4) micrograms (2.5 micrograms/kg), 8 X 10(-4) micrograms (10 micrograms/kg), and 1.2 X 10(-3) micrograms (15 micrograms/kg) was injected intravenously in 5-microliters aliquots of chick Ringer's solution. Additional groups of embryos were treated with the beta-antagonist propranolol, and isoproterenol plus propranolol. Control embryos received 5 microliters chick Ringer's solution to assess the hemodynamic effects of a volume injection. We found that isoproterenol caused no change in mean atrial pressure, heart rate, or mean arterial pressure. However, isoproterenol caused a dose-related decrease in dorsal aortic blood flow and a 2.5-fold increase in vascular resistance. The effects of isoproterenol were blocked by propranolol, which suggested that the increase in vascular resistance was mediated by beta-receptor stimulation.

Animals↗

Proliferative and steroidogenic effects of follicle-stimulating hormone during chick embryo gonadal development.

The aim of this study was to evaluate the in vitro effect of human follicle-stimulating hormone (hFSH) on cellular proliferation and steroid hormone secretion in the left ovary, the right ovary, and the testis of the chick embryo. Gonads from 8- to 18-day-old chick embryo were cultured in a defined medium during 60 h under basal and hFSH-stimulated conditions (0.5 IU/ml of culture medium). At the end of the culture, the incorporation of ¿(3)Hthymidine and the total number of cells were measured to estimate gonadal cell proliferation. The secretion of 17beta-estradiol and testosterone in the culture medium was radioimmunologically assayed in order to evaluate the steroidogenic function of the cultured gonadal cells. The response to hFSH stimulation was observed in the left ovary, the right ovary, and the testis from the 8-day-old chick embryo. In the left ovary, cellular proliferation was not augmented by hFSH in the 8-, 10-, and 13-day-old chick embryo; meanwhile, the proliferative stimulus of hFSH was observed in the 15- and 18-day-old embryos. In the same ovary, 17beta-estradiol and testosterone secretion were stimulated after hFSH treatment at all evaluated stages (8-18 days of chick embryo development). In the right ovary, an increment in proliferation and steroidogenesis was induced by hFSH in the 8-, 10-, and 13-day-old chick embryo. Afterward, the right gonad did not respond to hFSH. Testis cells displayed hFSH response as an increment in cell proliferation at all embryonic ages (8-18 days of chick embryo development). There was a transient lack of response to hFSH in testosterone secretion at 10 and 13 days of development. The in vitro effect of hFSH on cell proliferation and steroid hormone secretion changed in the ovary and the testis according to the age of the embryo. These changes could be attributed to the growth of the left ovary and the testis and the regression of the right ovary. Probably, paracrine factors modulated the gonadotropin effect on the target cells during embryonic development of chick embryo gonads.

Animals↗

Factors influencing premature induction of UDP-glucuronyltransferase activity in cultured chick embryo liver cells.

Very young (5-day-old) chick embryo livers during organ culture on rafts over a chemically defined nutrient medium precociously develop, from zero, adult levels of UDP-glucuronyltransferase activity. Induction of enzyme does not require DNA synthesis or mitosis, or exposure to hormones, serum, or tissue extracts. Thus, this functional differentiation of embryo liver is apparently not dependent on cell proliferation or on extrinsic substances other than simple nutrients.UDP-glucuronyltransferase activity develops rapidly also in embryo liver cells cultured as monolayers or aggregates; specific activity of enzyme on a protein basis increases at the same rate in cells undergoing widely different rates of cell division, and net protein synthesis. These findings suggest that precocious development of UDP-glucuronyltransferase activity in chick embryo liver is due, not to incidents of culture, but to removal from the embryonic environment. Supporting this, chick embryo liver grafted to the chorioallantoic membrane does not develop UDP-glucuronyltransferase activity until subsequently transferred to an in vitro cell culture system.

Animals↗

Alteration of hepatic lipidperoxide levels during cataract formation caused by glucocorticoids in developing chick embryos.

The level of hepatic lipidperoxides in chick embryos was determined during cataract formation resulting from glucocorticoid treatment. When 15 day old chick embryos were administered 0.25 mumol of hydrocortisone acetate their hepatic lipidperoxide level, determined by thio-barbituric acid, increased after a lag time of 20 hr and reached approximately 8-fold of control at 48 hr after the treatment. These studies indicate that the peroxidation of lipid in tissues should be considered in elucidating mechanisms of action or adverse effects of glucocorticoids.

Animals↗

Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis.

Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties.

Animals↗

Immunohistochemical localisation of monoclonal antibody R 24-recognized ganglioside Glac2 in early chick embryos.

The spatio-temporal cellular expression and biosynthesis of ganglioside Glac2 was investigated in early chick embryogenesis. For demonstration of embryonic Glac2-biosynthesis, chick embryos of stage 0 and of stages 4-5 were incubated in vitro in the presence of radioactive sugar precursors. It was found that chick embryos synthesize Glac2 as early as at the blastula stage as well as at the gastrula stage, both within the area pellucida and the area opaca. In contrast to the biosynthetical findings immunohistochemical staining of the chick embryos at various stages by aid of the mouse monoclonal antibody (mAb) R 24, specific for the immunoepitope NeuAc alpha, 8NeuAc alpha, 3Gal beta less than, as present on the ganglioside Glac2, revealed a spatio-temporal cellular pattern of expression of this ganglioside in early chick embryos. Immunohistochemical staining of the chick embryo at stage 0 shows that all cells of the embryo, the extraembryonic epiblast and the yolk endoderm included, are mAb R 24-positive. At the intermediate streak stage (stage 3), the cranial part of the deep layer, the so-called endophyll, is strongly mAb R 24-positive, whereas at the end of gastrulation (stage 5), mAb R 24-recognized epitopes appear to be restricted to a narrow band of deep-layer cells in the endophyllic crescent and to the yolk endoderm of the area opaca. At this stage, no labelling by the antibody is observed in cell layers of the future embryo. The beginning of neurulation (stage 7) is characterized by the expression of the mAb R 24-recognized epitope in the notochord, whilst the deep layer in the cranial part of the neural fold still expresses this epitope. No ecto- or mesodermal structures are stained by the antibody at this developmental stage. During further development (stage 12 and 13), mAb R 24-reactivity is restricted to the cranial part of the embryo with a preferential staining of cells of endodermal origin. At these stages, the notochord expresses mAb R 24 binding sites only in its cranial region. The spatial and temporal correlation between the presence of mAb R 24-recognized epitopes and the morphogenetic positioning of tissues may be indicative for a possible role of the ganglioside Glac2 in corresponding cellular interactions.

Animals↗

The carcinogen chromate causes DNA damage and inhibits drug-mediated induction of porphyrin accumulation and glucuronidation in chick embryo hepatocytes.

DNA damage by chromate in chick embryo hepatocytes has been correlated with the effect of chromate on inducible cell functions. Treatment of chick embryo hepatocytes with chromium(VI) in the form of sodium chromate resulted in the rapid uptake of chromate and the induction of DNA lesions in a time- and concentration-dependent manner. DNA interstrand cross-links, strand breaks and DNA-protein crosslinks, as measured by the alkaline elution technique, were observed after treatment of the hepatocytes with chromate concentrations (2.5- 0 microM) which did not affect cell viability. The effect of chromate on inducible cell functions was measured by assaying propylisopropylacetamide-induced accumulation of porphyrin and glucuronidation of phenol red by intact cells. Chromate inhibited propylisopropylacetamide-induction of porphyrin accumulation and phenol red glucuronidation in a time- and concentration-dependent manner which paralleled DNA damage. DNA damage was removed and inducibility of porphyrin accumulation by propylisopropylacetamide plus deferoxamine methanesulfonate was restored 21 h following a 2 h pretreatment with chromate. Chromium(III) in the form of chromic nitrate at concentrations up to 25 times those used with chromate had no effect on DNA damage or the induction of porphyrin accumulation and phenol red glucuronidation by propylisopropylacetamide in the cultured chick hepatocytes.

Animals↗

Protective role of thyroxine in methylparathion intoxicated chick embryos.

The efficacy of thyroxine against methylparathion poisoning in chick embryos was studied. The mortality rate and survival rate, frequency of abnormalities, growth rate and size of embryos, and also the change in cholinesterase activity were determined to evaluate the protective effect of thyroxine and atropine. It was observed that the survival rate, growth rate and size, and the cholinesterase activity significantly declined in the methylparathion treated group while the mortality rate and the frequency of abnormalities increased. When thyroxine was given, a significant reversal in these parameters was seen, indicating an effective protective action of thyroxine against methylparathion intoxication in chick embryos. The results also showed that the therapeutic treatment of the combination of thyroxine and atropine did not further improve the effects. Since in many respects, chick embryo development parallels that of mammalian embryos, a short term use of thyroxine as an effective protective agent against organophosphate methylparathion (perhaps other compounds) poisoning may have important implications.

Animals↗

Homocysteine interference in neurulation: a chick embryo model.

BACKGROUND: Periconceptional folic acid supplementation reduces the occurrence and recurrence risk of neural tube defects (NTD). Mothers of children with NTD have elevated plasma homocysteine levels. Administering homocysteine to chick embryos is reported to cause 27% NTD. Therefore, elevated plasma homocysteine levels per se or a disturbed homocysteine metabolism may be teratogenic to the embryo and may interfere with neural tube closure. Our aim was to obtain a chick embryo model to explore the interference of homocysteine in neural tube closure. METHODS: Homocysteine or saline was administered to chick embryos in ovo at 3 hr, 30 hr, and 60 hr of incubation and harvested at 74 hr. Homocysteine was then applied to chick embryos in vitro at a defined time window of four to six somites and followed for 6 hr. RESULTS: Homocysteine administration to chick embryos in ovo resulted in several malformations but not in an increased number of NTDs. Homocysteine administration to chick embryos in vitro resulted in a transient, dose-dependent widening of the anterior neuropore and closure delay of the rhombencephalic neuropore. After 16 hr of incubation the neural tube was closed. CONCLUSIONS: The in vitro chick embryo model appears a good model to explore the interference of a disturbed homocysteine metabolism in neurulation.

Abnormalities, Multiple↗

Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma.

The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.

Animals↗

The overlapping effects of thyrotropin and gonadotropins on chick embryo gonads in vitro.

Pieces of 12- and 15-day-old chick embryo testes and ovaries were cultured in vitro in the presence of thyrotropin (TSH), gonadotropins (FSH + LH) and adrenocorticotropin (ACTH) for different periods. All the explants of treated gonads differentiated into typical testes or ovaries according to their genetic sex. The gonads of 12-and 15-day-old chick embryos showed a good response to both thyrotropic and gonadotropic stimulation. On the other hand, they did not respond to adrenocorticotropic stimulation. Fifteen-day-old chick embryo testes were grown in tissue culture in the presence of the said hormones. Gonadotropins and TSH enhanced the growth and migration of testicular cells as compared with the control or ACTH treated group. In addition, they maintained the germ cells on the upper surface of epithelial cells. These results have confirmed our previous results in vivo in that gonadotropins and thyrotropin hormones accelerated the development of 12- or 15-day-old chick embryo gonads.

Adrenocorticotropic Hormone↗

A new technique for studying potential changes in nephrons of chick embryo kidneys in vivo.

To make the chick embryo accessible to electrophysiological measurements in its mesonephric kidney during the period between embryonic days (e.d.) 5 and 10, a special "chick-embryo-incubation bath" was constructed. It consists of an aerated chamber covering the egg and maintaining the gas exchange across the shell, and of a warmed reservoir of the incubation medium, into which the embryo is pulled out of the egg through a window in the shell. The two compartments are separated with a rubber membrane tightly fitting to the edges of the shell-window. The incubation medium contains a modified Krebs-Henseleit-Ringer solution and anesthetic Tricaine (Sigma). Access to the mesonephric nephrons is achieved by surgical excision of the body wall on the right side performed at e.d. 5. On average only about 35 percent of the operated embryos survive till the third day after surgery but during the next two days a mortality rate recedes to zero. The tolerance of short-term survival of embryos placed in the incubation bath was tested for up to 4 1/2 h. It was very good in embryos of age 5 to 7 e.d. as assessed by a steady heart rate and the presence of arterio-venous differences. A modified differential amplifier containing circuits for frequency compensation of the two channels was used for high-fidelity registration of voltage changes in the embryonic nephron with a single double-barrel microelectrode.

Animals↗

Ontogeny of spontaneous antigen-binding cells in developing chick embryos.

Bursal and splenic lymphocytes in developing chick embryos were examined for the number of antigen binding cells (ABC) which could form rosettes with red blood cells from sheep, guinea-pig, rabbit or dinitrophenylated sheep red blood cells (DNP-SRBC). The kinetics of the development of bursal ABC to rabbit red blood cells (RRBC) did not vary significantly with age of embryo examined, and its frequency was constantly high. The number of cells which bind to SRBC, guinea-pig red blood cells (GPRBC) or DNP-SRBC was low in the bursa and increased in an approximately linear fashion with the age of embryo. With the exception of RRBC-ABC, similar results were obtained with splenic ABC. Furthermore, the frequencies of ABC were higher in the spleen than in the bursa. These events suggest the antigen-independent generation of clonal diversity during the developmental stage of chick embryos.

Animals↗