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PubMed1994

Nuclear and cytoplasmic maturation of pig oocytes cultured in the amniotic fluid of developing chick embryos.

Studies were conducted to determine if the amniotic fluid of a developing chick embryo (CEAm) could be used as a co-culture system for the maturation of pig oocytes. In Exp. 1, the pig oocytes were cultured in mTCM-199 (control) or day 3, 4 and 5 CEAm. The maturation rate of pig oocytes in the control vs CEAm was significantly higher (P < 0.001). The maturation rate of pig oocytes cultured in day 3 vs day 4 and 5 CEAm was significantly higher (P < 0.001). In Exp. 2, the pig oocytes were cultured in day 3 CEAm maintained either in a conventional incubator with 40-70% relative humidity or incubator with 5% CO2 in air atmosphere at 37 degrees C or 39 degrees C. A significantly lower maturation rate (P < 0.001) was obtained when CEAm were maintained in an incubator with 5% CO2 gas atmosphere regardless of the temperature. In Exp. 3, pig oocytes cultured in day 3 CEAm and mTCM-199 (control) were fertilized in vitro (IVF). The penetration and male pronucleus (MPN) formation of pig oocytes cultured in day 3 CEAm was significantly lower (P < 0.01) than those cultured in mTCM-199 medium. The results presented here demonstrate that CEAm could be used to induce maturation of pigs oocytes, however its efficacy is influenced by factors such as developmental stages of chick embryos, temperature and gas atmosphere.

Amniotic Fluid↗

PubMed1994

Interaction between glycine and glutamate in the development of spontaneous motility in chick embryos.

In this study we investigated whether also glycine fulfils the function as co-activator in glutamatergic activation of NMDA receptors in the neuronal apparatus of spontaneous motility in chick embryos. The successive application of glycine (5 or 10 mg/kg egg weight (e.w.) and glutamate (15 mg/kg e.w.) in a 10 min interval significantly increased the activation of spontaneous motility of 17-day-old chick embryos in comparison with the effect of glutamate alone. This effect did not depend on the order of application of the drugs. In 13-day-old embryos, glycine was ineffective in both doses. It is concluded from these results that the modulatory effect of glycine is evidently a later developmental acquisition (after day 15 of incubation) in the embryogenesis of NMDA-ergic activation of spontaneous motility in chick embryos similarly as glycinergic inhibition.

Animals↗

PubMed1990

Estrogen-independent and estrogen-induced progesterone receptors, and their regulation by progestins in the hypothalamus and pituitary of the chick embryo: an immunohistochemical study.

The effects of an anti-estrogen and of progestins on the progesterone receptor (PR) in the hypothalamus and pituitary of 10-day-old chick embryos were studied by immunohistochemistry with an antibody to the receptor. In a first experiment, to determine if endogenous estrogens are responsible for the early appearance of PR in the chick embryo, a continuous treatment with the anti-estrogen. Tamoxifen, was applied from day 0 of incubation. In the hypothalamus and pars distalis of the pituitary a Tamoxifen treatment (10 micrograms every other day from day 0) did not modify the distribution of PR-positive cells or the intensity of PR immunoreactivity (PR-IR), compared to oil-injected embryos. In contrast, the same treatment totally blocked the increase of PR-IR in embryos administered estradiol (10 micrograms on day 7). Thus, the estradiol-induced PR-IR is inhibited by Tamoxifen, whereas the natural appearance of PR is not. We conclude that, in the chick embryo, the basal expression of PR is estradiol independent. In a second experiment, the regulation of PR by its own ligands (progesterone and the synthetic progestin, R5020) was studied. Progesterone (150 micrograms), administered to embryos 16 or 48 h before sacrifice on day 10, induced a slight increase in PR-IR in hypothalamus and anterior pituitary, whereas R5020 had no effect. In embryos treated with estradiol on day 7, R5020 decreased the PR-IR to a level comparable to that of control embryos. Thus R5020, a non metabolizable progestin, down-regulates the estradiol-induced PR-IR, but has no effect on the estradiol-independent, naturally expressed PR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Identification of insulin in chick embryo retina during development and its inhibitory effect on DNA synthesis.

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.

Animals↗

PubMed1988

Thyrotropic and peripheral activities of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and adult chicken.

The influence of an intravenous injection of thyrotrophin-releasing hormone (TRH) and bovine thyrotrophin (TSH) on circulating levels of thyroid hormones and the liver 5'-monodeiodination (5'-D) activity is studied in the chick embryo and the adult chicken. In the 18-day-old chick embryo, an injection of 1 microgram TRH and 0.01 I.U. TSH increase plasma concentrations of triiodothyronine (T3) and of thyroxine (T4). TRH, however, preferentially raises plasma levels of T3, resulting in an increased T3 to T4 ratio, whereas TSH preferentially increases T4, resulting in a decreased T3 to T4 ratio. The 5'-D-activity is also stimulated following TRH but not following TSH administration. The increase of reverse T3 (rT3) is much more pronounced following the administration of TSH. In adult chicken an injection of up to 20 micrograms of TRH never increased plasma concentrations of T4, but increases T3 at every dose used together with 5'-D at the 20 micrograms dose. TSH on the other hand never increased T3 or 5'-D, but elevates T4 consistently. It is concluded that TSH is mainly thyrotropic in the chick embryo or adult chicken whereas TRH is responsible for the peripheral conversion of T4 into T3 by stimulating the 5'-D-activity. The involvement of a TRH induced GH release in this peripheral activity is discussed.

Animals↗

PubMed1989

Is extraembryonic angiogenesis in the chick embryo controlled by the endoderm? A morphology study.

The area vasculosa of the chick embryo is subdivided into two concentric zones: the inner transparent area pellucida vasculosa (AVP) and the less transparent surrounding area opaca vasculosa (AOV). The different optical properties of these zones are caused by the different morphology of the endoderm, which consists of flat cells in the APV and of high-prismatic cells containing large yolk vacuoles in the AOV. The present study describes how this endodermal subdivision of the area vasculosa is related to the development of the extraembryonic vascular pattern. By injection of ink into the vascular system of chick embryos at stages 12 to 20 (Hamburger and Hamilton 1951 "HH"), it has been demonstrated that the vascular net of the area vasculosa from stage 14 (HH) onwards develops into different patterns in APV and AOV. The small loops of uniform capillary vessels of stage 13 (HH) are widened due to the rapid expansion of the extraembryonic mesoderm. In the AOV from stage 14 (HH) onwards numerous small blood vessels sprout into the enlarged intervascular spaces. This process is maximal at stage 17 (HH). In contrast, the blood vessels of the APV remain largely unbranched. These findings suggest that the development of the extraembryonic vascular pattern is controlled by the endodermal pattern. To test this hypothesis, both zones (APV and AOV) were examined by light microscopy, transmission and scanning electron microscopy, in vivo observations and by treatment with bromodeoxyuridine (BrdU). TEM examinations show that the ultrastructural organization of the APV mesoderm is different from that of the AOV: The splanchnopleuric cells of the APV form a continuous cover around the endothelial cells connected by numerous desmosomes, whereas the splanchnopleuric cells of the AOV are frequently separated by gaps. The largest gaps are seen in the small blood vessels at stage 17 (HH). These results should be considered in relation to the dynamic changes in the vascular pattern of the AOV. The endodermal cells of APV and AOV are two different populations. In vivo observation of the endodermal transition from APV to AOV detected no transformations of APV cells into AOV cells or vice versa. The borderline between the zones is stable. The AOV endoderm, having been overgrown by the expanding mesoderm, stops proliferating almost completely, whereas the proliferation of the APV endoderm is unaffected by contact with the mesoderm. The rate of its proliferation is approximately as high as that of the AOV prior to contact with the expanding mesoderm (results after treatment with BrdU).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2001

Dorsal aortic flow velocity in chick embryos of stage 16 to 28.

The objective of this study was to evaluate two Doppler frequency-detection methods to measure blood flow velocity in the developing chick embryo. We compared the commonly used directional zero-crossing counter and a customized digital bidirectional spectrum analyzer. At development stages 16 up to 28 (2.5 to 6 days incubation), a reversed flow component in the dorsal aorta was demonstrated using the bidirectional spectrum analyzer. Dorsal aortic velocities obtained with the directional zero-crossing counter were significantly lower than with the bidirectional spectrum analyzer in stages 16, 20 and 28. In addition to the differences in the absolute velocity values, there was also a remarkable discrepancy in the velocity waveform shape using the two Doppler frequency processors. The calculated heart rate using the two Doppler frequency processors was identical. It is concluded that a Doppler velocity detector based on spectral analysis is superior to the hitherto used zero-crossing counter in the chick embryo. With the customized digital bidirectional spectrum analyzer, we can accurately measure the hemodynamics of the developing chick embryo.

Animals↗

PubMed1989

Study of possible teratogenic effects of the fungicide maneb on chick embryos.

The teratogenicity of the fungicide maneb (80% active ingredient and 20% inert ingredients) in chick embryos was evaluated. Eggs, before incubation, were dipped in 0.2% or 1.2% aqueous dispersions of maneb for 30 seconds. An untreated group, a water group and a formulation ingredient group, at the concentration present in the 1.2% maneb group, ensured control in the study. Eggs were incubated for 18 days then transferred to hatching racks and allowed to hatch normally. Viable chicks and chicks "dead in shell" were assessed for external deformities. There was no evidence that maneb or its formulation ingredients were teratogenic or embryotoxic to developing chick embryos.

Animals↗

PubMed2002

Brain expansion in the chick embryo initiated by experimentally produced occlusion of the spinal neurocoel.

The brain expands in the early chick embryo from pressure generated by accumulation of cerebrospinal fluid (CSF) in a closed neural tube. The sealing of the neural tube occurs as the result of occlusion of the spinal neurocoel rostral to and before closure of the posterior neuropore. We have previously demonstrated the dependence of normal brain expansion upon intraluminal pressure. We had yet to demonstrate, however, that brain expansion actually depends upon natural occlusion of the spinal neurocoel. To demonstrate such dependence, we experimentally occluded the spinal neurocoels of embryos 5 hr younger than stage 11 embryos (in which occlusion of the neurocoel occurs naturally). The stage 10 chick embryos were cultured ex ovo and critically staged, and their spinal neurocoels were occluded using microcautery. All embryos were photographed immediately and at 5, 12, and 24 hr after cautery. Serial sections were made of selected embryos, in which the areas of both the brain and the head were measured. Wilcoxon-Mann-Whitney rank-sum nonparametric tests, Hodges-Lehmann estimators, bootstrapping techniques, and resampling randomization tests were used to determine whether the increases in the brain and head areas for the experimental embryos were significantly different from those of the control embryos during three distinct intervals of expansion: 0-5, 5-12, and 0-12 hr. From 0 to 5 hr, the brains of the precociously occluded embryos expanded significantly more than the brains of the non-occluded controls. From 5 to 12 hr, the brains of the embryos with naturally occluded neurocoels grew significantly larger than the brains of the embryos with precociously occluded neurocoels. At 12 hr, there appeared to be no difference in brain size for these two groups. We conclude that the data support the hypothesis that brain expansion is directly dependent upon occlusion of the spinal neurocoel.

Animals↗

PubMed1989

[Quantitative and qualitative variations in the glycoproteins in the chick embryo brain].

Sugar content was examined in soluble and insoluble glycoproteins extracted from the chick embryo brain at different developmental stages. The content of hexosamines and uronic acids in the soluble fraction is higher during the whole period examined. The difference between the two fractions reaches a maximum at the 15th day. The insoluble fraction shows the highest content of sialic acid and fucose in comparison with the soluble one, especially toward hatching. The sialic acid/fucose ratio shows a different pattern in the two fractions examined, particularly in the soluble glycoproteins. The patterns of sialic acid and fucose indicate that quantitative and qualitative developmental changes occur in the soluble and insoluble glycoproteins. All sugars examined show significant changes on the 15th day, suggesting that this stage may represent a critical period in the development of the chick embryo brain.

Animals↗

PubMed2003

Chick embryo back skin organ and fibroblast cultures. Extracellular matrix changes induced by dialysate fluid and uraemic toxins in relation to proliferation and differentiation processes.

AIM: During uraemia, an increase of middle molecules and acetylpolyamines occurs. In vitro the middle molecules produce cell toxicity, while the acetylpolyamines stimulate cell proliferation and differentiation. These phenomena are related to protein and extracellular glycosaminoglycan production. The aim of the present study was to evaluate the activity of dialysate, dialysate fluid and the chromatographic peaks of dialysate fractionated by G-15 Sephadex column on chick embryo skin development. METHODS: We evaluated the effects of protein and glycosaminoglycan synthesis using monolayer and organotypic cultures. RESULTS: Our data show that dialysate, chromatographic peak II, and 2 x 10(-8)M N1-acetylspermine cause inhibition of chick embryo skin culture development. On the contrary, 10(-8)M N-acetylornithine and dialysate fluid increase protein and extracellular glycosaminoglycan synthesis, whereas chromatographic peak I does not reveal differences when compared to controls. CONCLUSIONS: These extracelluar changes are related to cell proliferation and feather formation in chick embryo organotypic culture. Moreover, the pH changes of culture medium do not influence the biological action of acetylpolyamines and dialysate fluid on protein and extracellular glycosaminoglycan synthesis. Cell death in the presence of N1-acetylspermine, dialysate and peak II appears unrelated to the apoptotic process. The data show that acetylpolyamines, dialysis fluid, dialysate and chromatographic peaks act on fibroblasts, and are able to modify glycosaminoglycan synthesis. The organotypic cultures of chick embryo back skin could represent a model for studying the modifications of the extracellular matrix induced by these substances.

Animals↗

PubMed1985

Biosynthesis of ascorbic acid in chick embryos.

Biosynthesis of ascorbic acid was found in the kidneys (mesonephros and metanephros) of the chick embryo as well as in the yolk sac membrane. The activity of L-gulonolactone oxidase in the yolk sac membrane suggested that it was the major source of ascorbic acid in the chick embryo.

Age Factors↗

PubMed1986

Chromium (VI)-induced DNA damage in chick embryo liver and blood cells in vivo.

Uptake of chromium (VI) and subsequent induction of DNA damage was examined in liver and blood cells of 14-day chick embryos after injection of sodium dichromate onto the inner shell membrane. Maximal loss of chromium from the inner shell membrane and distribution of chromium in liver, lung and blood was observed 2 h after injection. DNA strand breaks, interstrand cross-links and DNA--protein cross-links were measured using the alkaline elution technique. In chick embryo liver, chromium (VI) induced DNA cross-links in the absence of strand breaks. Maximal DNA cross-linking was detected in the liver 8 h after injection. Little or no DNA damage remained in the liver 10-24 h after injection. In contrast, chromium (VI) induced DNA strand breaks in the absence of cross-links in chick embryo blood cells. Maximal DNA strand breakage was observed in blood cells 8 h after injection. High levels of DNA strand breaks were present in blood cells even 24 h after treatment. These intra-embryonic tissue differences in chromium (VI)-induced DNA damage may be a result of the differences in glutathione, cytochrome P-450, other pathways of chromium (VI) metabolism or chromatin organization which exist in liver and blood cells.

Animals↗

PubMed1977

Evidence for the presence of adrenergic receptors in 3-day-old chick embryo.

Effects of sympathomimetic amines without and with alpha and beta-adrenergic blocking agents on the heart rate and arterial and venous blood pressures in the 3-day-old chick embryo were studied. No chronotropic effect was observed. Norepinephrine caused a biphasic change in systolic and diastolic arterial pressures, the lower doses effecting a fall, and the higher doses a rise in these pressures. With phenylephrine a sharp rise in systolic, diastolic, and pulse pressures was seen. Isoproterenol caused a dramatic fall in systolic, diastolic, and pulse pressures. In the presence of phenoxybenzamine, the pressor effect of high doses of norepinephrine was reversed, the pressor effect of phenylephrine was abolished, and the hypotension with isoproterenol was enhanced. After propranolol, the hypotensive effect of low doses of norepinephrine was reversed, the pressor response to phenylephrine was unchanged, and the depressor effect of isoproterenol was abolished. These findings suggest the presence of functioning alpha- and beta-receptors in the 3-day-old chick embryo. Additionally, they suggest that the alpha-receptors develop more slowly in the chick embryo.

Animals↗

PubMed1996

Interaction between quercetin and heat shock. A preliminary study on the chick embryo.

A hyperthermic shock (43 degrees C/30 min) enhances in somite stages of the chick embryo development the activity of the caudal morphogenetic system, manifested by intensive growth of the embryonic trunk. The exposure also induced the synthesis of heat shock proteins (HSP 70). A single administration of a bioflavonoid quercetin dissolved in DMSO to chick embryos in stages HH 10-11 (10-14 somites) prior to heat exposure inhibited both the growth acceleration and the HSP induction.

Animals↗

PubMed1996

Immunocytochemical and biochemical characterization of glial phenotypes in normal and immortalized cultures derived from 3-day-old chick embryo encephalon.

We examined properties of glia derived from very early neurogenesis in the chick embryo, as well as their behavior in response to extended cell passages and at various periods in culture. Primary cultures derived from the telencephalic region of 3-day-old chick embryo (stage 19) exhibited intense staining for vimentin (Vim; indicative of immature glial phenotypes) throughout the 17-day culture period, transitioning to Vim-positive/glial fibrillary acidic protein (GFAP)-positive astroblasts after a single cell passage at 13 days in vitro (DIV). With subsequent passage (P; through P6), cell continued to coexpress Vim/GFAP with the occasional appearance of fibronectin (Fib). By P11, Vim staining was faint, whereas GFAP staining gained in intensity, indicating the presence of mature astrocytes. These cultures also featured significant activity of glutamine synthetase (GS), which increased slightly with both cell passage and days in culture, correlating well with immunocytochemical findings. The activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP) remained low, indicating a low percentage of mature oligodendrocytes. Thus, embryonic day (E)3H glia cultures mature into astrocytes given sufficient time in culture. To obtain a stable population of glia at various stages of astrocytic differentiation, we immortalized and subcloned homogenous colonies of E3H glia precursors at specific stages of development. A cell suspension was electroporetically transfected with the pSV40neo gene for large T antigen (E3H.SV5). After 5 passages, the parent colonies consisted of a heterogeneous population of cells, most of which were vim and GFAP positive. Following subcloning, one line (colony 7) displayed the staining pattern of mature astrocytes. However, with advancing age in culture, staining for both vim and GFAP became increasingly faint. Compared with control (normal) cultures, transfected cells exhibited a significantly lower activity of GS that did not fluctuate with days in culture but decreased with advancing cell passage. Furthermore, CNP activity in E3H.SV5 colony 7 was approximately double that observed in normal cultures at the same cell passage. This observation suggests that the phenotype of transfected glia was less stable than that observed in normal glial cultures.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1975

The role of beta-adrenergic activity in the production of cardiac and aortic arch anomalies in chick embryos.

The sympathomimetic amines isoproterenol, epinephrine, norepinephrine, and phenylephrine are structural derivatives of beta-phenylethylamine and have proportionately different effects on alpha- and beta-adrenergic receptors. Chick embryos in ovo were each administered a single dose of one of these compounds at concentrations ranging from 0.4 times 10(-9) to 20 times 10(-9) mol/5 mul saline during Hamburger and Hamilton stages 20-27. In other experiments embryos were pretreated with the beta-antagonist propranolol and subsequently administered isoproterenol. 743 cardiovascular anomalies were produced. The production of cardiovascular anomalies was proportional to the degree of beta-adrenergic activity of each drug. The frequency of anomalies was significantly reduced by pretreatment with propranolol. At all concentrations tested the anomaly rate was greater in chick embryos receiving an experimental compound than in controls. The general types of anomalies included aortic arch defects, ventricular septal defect, double outlet right ventricle, aortic hypoplasia, and truncus arteriosus. These results demonstrate that activation of the beta-adrenergic receptor mechanism is directly related to the cardiovascular anomalies produced in the chick embryos.

Abnormalities, Drug-Induced↗

PubMed1993

Teratogenic effects of a mixture of scopolamine and hyoscyamine in chick embryos.

The effect of a mixture of scopolamine and hyoscyamine (98:2) on developing chick embryos was investigated. The embryos were exposed at 2 stages of development to various doses of this mixture via yolk sac injection. This resulted in deaths at different stages of prenatal development, as well as various malformations and reduction deformities. These findings suggest that the studied mixture has a teratogenic effect when injected into the yolk sac of developing chick embryos.

Abnormalities, Drug-Induced↗