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PubMed1994

Kinetics and differentiation of somite cells forming the vertebral column: studies on human and chick embryos.

We have studied the kinetics of somite cells with an antibody against proliferating cell nuclear antigen (PCNA/cyclin) in human and chick embryos, and with the BrdU anti-BrdU method in chick embryos, to investigate whether the metameric pattern of the developing vertebral column can be explained by different proliferation rates. Furthermore we applied antibodies against differentiation markers of chondrogenic and myogenic cells of the somites in order to study the correlation between proliferation and differentiation. There are no principal differences in the proliferation pattern of the vertebral column between human and chick embryos. In all stages examined, the cell density is higher in the caudal sclerotome halves than in the cranial halves. Laterally, the caudal sclerotome halves, which give rise to the neural arches, are characterized by a higher proliferative activity than the cranial halves. Although there is a high variability, the labelling indices show significant differences between the two halves with both proliferation markers. With the onset of chondrogenic differentiation, only the perichondrial cells retain a high proliferation rate. During fetal development, the neural arches and their processes grow appositionally. Even at the earliest stages, there is practically no immunostaining for PCNA or BrdU in the desmin-positive myotome cells of human and chick embryos. Axially, a higher proliferation rate is found in the condensed mesenchyme of the anlagen of the intervertebral discs than in the anlagen of the vertebral bodies. During fetal development, cells at the borders between vertebral bodies and intervertebral discs proliferate, indicating appositional growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

[Study of the synthesis of Rous virus-specific RNA in chick embryo cells].

The presence of virus-specific RNA in commercial chick embryos and its lack in chick embryos of leukemia-free chicken farm of the USSR AMS Oncological Research Center as well as in cell cultures from RIF-free chicken infected with Marek's disease virus was demonstrated by hybridizationof 3H-DNA-product of Rous sarcoma virus synthesized in vitro in the presence of actinomycin D with the total preparation of cellular RNA.

Animals↗

PubMed2005

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo.

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.

Animals↗

PubMed1992

Visual demonstration of the limb-forming zone in the chick embryo lateral plate.

The present study was undertaken to determine whether a visible Wolffian ridge, distinct from the lateral fold, can be identified in chick embryos. Ectoderm thickness was measured in stage 11-17 chick embryos. There was a general trend, from thin ectoderm in the midline, to an ectodermal thickening over the somites, intermediate mesoderm, and lateral plate. Other embryos were cut from the yolk, pinned out, and photographed. The lateral fold was then eliminated, and the embryo was rephotographed. The photographs reveal a definite opaic zone, distinct from the lateral fold, in stage 11-18 chick embryos. Furthermore, there is a direct correlation between the opacity of this cellular band and the limb-forming potential of grafted wing, flank, and leg regions (see Stephens et al., '89). At stages 11-14, the wing, flank, and leg exhibit a uniform opacity, and a uniform capacity for limb formation when grafted to a host celom. From stage 15 to stage 18, the opacity in the flank diminishes, and its limb-forming capability disappears. This study demonstrates the presence of an opaic zone, which we have called the limb-forming zone (LFZ) along the lateral side of early chick embryos, which is independent of the lateral fold, is not as extensive as the lateral plate, and is not simply associated with ectodermal thickening, but which is directly correlated with limb-forming potential in the lateral plate.

Animals↗

PubMed1991

Non-linear dependence of the dielectric properties of chick embryo myoblast membranes exposed to a sinusoidal 50 Hz magnetic field.

Primary chick embryo myoblasts can be a useful tool for studying the developmental events which accompany myoblast differentiation, particularly myoblast membrane fusion. To determine whether the electrical properties and/or fusion in these systems are affected by 50 Hz magnetic fields, chick embryo myoblast cultures were exposed to B-field intensities ranging from 1 to 10 mT. The electrical parameters of the myoblasts, i.e. membrane conductivity, membrane permittivity and the conductivity of the cell interior (cytosol) were determined by the analysis of conductivity dispersion data in the radio frequency range (10 kHz-100 MHz). Preliminary results indicate that the time of fusion (60 h) is not affected by these fields, but that the absolute values of the two membrane electrical parameters are affected. In particular, a B-field intensity-dependent decrease was observed. The maximum effect resulted after a 1 h exposure to a magnetic flux density of about 5 mT. The conductivity of the cytosol remained unchanged. These data seem to indicate that exposure to 50 Hz magnetic fields affects both static and dynamic membrane properties in primary chick embryo myoblasts.

Animals↗

PubMed1976

Nonsuppressible insulin-like activity and thyroid hormones: major pituitary-dependent sulfation factors for chick embryo cartilage.

Serum from hypothyroid hypophysectomized rats did not stimulate sulfation or incorporation of amino acids into chick embryo sterna. When such rats were treated for a short time with growth hormone (somatotropin), their serum stimulated incorporation both of sulfate and of amino acids. The different actions of the two types of sera were not due to changes in thyroid state. The results support the existence in serum of a sulfation factor for chick embryo cartilage that is dependent upon growth hormone. Highly purified preparations of nonsuppressible insulin-like activity from human serum stimulated incorporation of amino acids, and of uridine into RNA, in chick embryo sterna in vitro; chondrocytes prepared from this tissue had specific high-affinity binding sites for this insulin-like activity. However, sulfate incorporation was stimulated very little, unless serum from hypothyroid hypophysectomized rats was also present. When L-3,5,3'-triiodothyronine was added as well, the stimulation was enhanced further. From these and other experiments, we conclude that (i) nonsuppressible insulin-like activity or a closely related peptide is the growth-hormone-dependent growth and sulfation factor for chick embryo cartilage: (ii) a second, unidentified factor must be present for the insulin-like activity to stimulate sulfation; and (iii) stimulation of sulfation by thyroid hormones in vitro is additive to that of nonsuppressible insulin-like activity.

Animals↗

PubMed2006

Transplantation of human embryonic stem cells to the chick embryo.

The traditional methods of studying the differentiation of human embryonic stem cells (hESCs) are to differentiate them in vitro or in immune-deficient mice as teratomas. The chick embryo is a well-studied and accessible experimental system that has been shown to permit the development of mammalian cells, including murine embryonic stem cells. We therefore performed experiments transplanting colonies of hESCs into organogenesis-stage chick embryos, hoping this might provide a novel system for studying the developmental programs and decisions of these important cells. hESCs, constitutively expressing green fluorescent protein or labeled with the dye CFDA, were used to allow the following the hESC in living embryos. As a first step, we chose to transplant hESCs into the trunk of chick embryos, both into and instead of developing somites. Our first results showed that hESCs survive, migrate, and integrate into the tissues of the chick embryo. Some of the hESCs differentiate and the type of embryonic microenvironment that the implanted cells were exposed to modified their differentiation. Therefore, this hESC-chick embryo system has potential for complementing studies in rodents and in vitro, and uniquely, to shed light on early processes in the development of human cells in the embryonic context.

Animals↗

PubMed1987

Evidence for a dual mechanism of chick embryo fibroblast adhesion on fibronectin and laminin substrata.

Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.

Animals↗

PubMed1985

[Influence of the concentration of precursors on the biosynthesis of testosterone by the chick embryo testis].

The aim of this study was to determine whether substrate concentration was of crucial importance in the formation of testosterone by the chick embryo testis. Testes from 15 to 18-day old chick embryos were cultured in medium 199 with dehydroepiandrosterone 1, 2, 6, 7-3H or androstenedione-4-14C added at different concentrations. Testosterone was identified and measured by crystallization to constant specific activity or by gas chromatography-mass spectrometry. The formation of testosterone could not be demonstrated at a substrate concentration of 76 nM. Concentrations in the micromolar range yielded measurable quantities of testosterone which increased to about 10% of the added substrate when the concentration was 70 microM. However, the capacity to form testosterone was shared by other organs such as the ovary or the mesonephros. These results should settle definitively the question concerning the formation of testosterone by the chick embryo testis. When exposed to high substrate concentrations, the testis can form testosterone, but under physiological conditions there is no such testosterone formation. This conclusion is in agreement with the absence of any physiological role of testosterone in the chick embryo.

Androstenedione↗

PubMed1986

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture.

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.

Animals↗

PubMed1977

Developmental changes in collagen glycosyltransferase activities in chick embryos.

The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.

Animals↗

PubMedDate not supplied

Lethality and EROD-inducing potency of chlorinated chrysene in chick embryos.

Chrysene was non-specifically chlorinated and the toxic potency of the mixture formed was studied. The chlorinated mixture was considerably more potent than the parent compound in terms of embryolethality and EROD and AHH induction in a 2-week test in chick embryos. The chlorinated chrysene caused anomalies, including edema and beak defects, similar to those previously found after treatment of chick embryos with coplanar PCBs. Chlorinated chrysene was also much more potent than chrysene as an inducer of EROD in a 72-hour test in chick embryos in ovo and in chick embryo liver in vitro. It seems that the mechanism of toxicity of chlorinated chrysene in chick embryos is similar to that of the coplanar PCBs and other Ah receptor ligands. The effects of the chlorinated mixture were mainly accounted for by 6- chlorochrysene and 6,12-dichlorochrysene.

Animals↗

PubMedDate not supplied

Porphyrinogenic xenobiotic-induced N-alkylprotoporphyrin IX formation: a bioassay utilizing chick embryo hepatic ferrochelatase.

INTRODUCTION: The porphyrinogenicity of some xenobiotics results from mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes accompanied by conversion of prosthetic heme groups to N-alkylprotoporphyrins (N-alkylPPs), some of which inhibit ferrochelatase (FC). Problems have arisen in extrapolating xenobiotic porphyrinogenicity observed in test animals to humans, due in part to differences among CYP enzymes. Our goal was to develop a bioassay to detect N-alkylPPs formed following interaction of porphyrinogenic xenobiotics with rat liver microsomal CYP. METHODS: Seventeen-day-old chick embryo livers were homogenized, and the mitochondrial fraction was isolated. The FC activity of this fraction was determined by means of the pyridine hemochromogen method. Inhibition of FC was used to detect N-alkylPP formation following interaction of porphyrinogenic xenobiotics with rat liver microsomes. RESULTS: The 17-day-old chick embryo hepatic mitochondrial preparation served as a stable source of FC activity, which was linear with respect to time and protein concentration. FC activity was higher than previously reported in a homogenate of 17-day-old chick embryo hepatocytes in culture and in an aqueous extract of 17-day-old chick embryo mitochondria. The EC(50) of N-methylprotoporphyrin IX in the chick embryo liver mitochondrial preparation was similar to that in the homogenate of chick embryo liver cell culture. The FC bioassay could detect N-alkylPPs formed following the interaction of porphyrinogenic xenobiotics with rat liver microsomes containing 2.4-9.0 nmol of CYP. DISCUSSION: In future studies investigating N-alkylPP formation following interaction of xenobiotics with CYP enzymes, we recommend using a combination of a fluorescence technique and the chick embryo hepatic mitochondrial FC assay. This would provide information both on the formation of N-alkylPPs and distinguish between those N-alkylPPs that produced porphyrin accumulation via FC inhibition and those that do not.

Animals↗

PubMed1996

Chick embryo liver microsomal steroid hydroxylations: induction by dexamethasone, phenobarbital, and glutethimide and inactivation following the in ovo administration of porphyrinogenic compounds.

Metabolites of [4-(14)C]androstenedione (AD) and [4-(14)C]progesterone (PG) were separated and quantitated following incubation with hepatic microsomes from chick embryos. PG 2 alpha-hydroxylase and AD 7 alpha-hydroxylase, which are diagnostic markers in rat liver for cytochrome P450 (P450) 2C11 and 2A1/2, respectively, were not identified in chick embryo liver. PG 6 beta-hydroxylase and AD 6 beta-hydroxylase, diagnostic markers for P450 3A1/2 activity in rat liver, were identified in chick embryo liver, and we were able to show that radiolabeled 6 beta-hydroxyprogesterone and 6 beta-hydroxyandrostenedione, the metabolites of PG and AD 6 beta-hydroxylase, respectively, were homogeneous and identical with authentic standards. Dexamethasone, phenobarbital, and glutethimide were found to be significant inducers of PG and AD 6 beta-hydroxylases in chick embryo liver. The in ovo administration of the porphyrinogenic compounds 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) and 3-[2-(2,4,6-trimethylphenyl)-thioethyl]-4-methylsydnone (TTMS) caused inactivation of chick embryo hepatic PG and AD 6 beta-hydroxylases. Therefore, we suggest that PG and AD 6 beta-hydroxylases may serve as diagnostic markers for a P450 3A-like isozyme in the chick embryo, and that this isozyme is one of the targets for inactivation by 4-ethyl DDC and TTMS.

Animals↗

PubMed1985

Behavioral analysis of opiate-mediated inhibition in the early chick embryo.

Morphine (opiate agonist) produced a dose-dependent decrease in the spontaneous motility of 5- and 9-day chick embryos. Naloxone (opiate antagonist) appeared to reverse competitively the inhibition of motility caused by morphine. The effects of morphine on spontaneous motility in 5-day embryos were also reversed stereospecifically by the opiate antagonist pairs WIN 44441-3/WIN 44441-2 and levallorphan/dextrallorphan. Levorphanol (opiate agonist) also produced a dose-dependent decrease in the motility of 5-day embryos while its inactive (+)-isomer, dextrophan, was not effective. Etorphine (opiate agonist) was more than 1000-fold more effective than morphine in inhibiting the motility of 5-day embryos. The effectiveness of several opiate agonists and antagonists on the spontaneous motility of 5-day embryos was similar to their effectiveness in radioligand-binding studies on isolated membrane receptors from either adult mammalian brain or ileum. Levorphanol was more effective than dextrophan and etorphine was substantially more effective than morphine in decreasing the spontaneous motility of 4-day embryos. WIN 44441-3 was more effective than WIN 44441-2 in reversing the inhibition of motility in 4-day embryos caused by morphine. Morphine inhibited spontaneous hind-limb motility in both thoracic spinal and sham-operated 7-day embryos; the inhibition of motility caused by morphine was reversed by WIN 44441-3 in both thoracic spinal and sham-operated 7-day embryos. [Leu5]enkephalin-like immunoreactivity in the lumbar spinal cord was concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of immunoreactive processes occurred in the medial and lateral motor columns where labelled varicosities appeared to contact motoneurons. Opiate receptors appear to be present at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. Opiate receptors appear to be present in the lumbar spinal cord of the chick embryo at least as early as day 7. The structural requirements for ligand binding to opiate receptors in the 5-day chick embryo are similar to the requirements for ligand binding to opiate receptors in the adult.

Age Factors↗

PubMed1979

Responses of the early chick embryo to external cAMP sources.

Early chick embryos were stimulated with local sources of cAMP. Three major effects were observed: bending of the embryonic axis, attraction of cells on the ventral surface of the embryo, and disruption of the blastodisc. Each had a characteristic concentration dependence. These results are compared with those from studies of cells disaggregated from similar embryos.

Animals↗

PubMed1978

The induction of axial blisters in the chick embryo by trypan blue.

The hemorrhage, blister formation, and rumplessness observed in the chick embryo following treatment with trypan blue may be due to (1) increased ventricular blood pressure or (2) to necrosis and edema in the caudal region of the embryo by inhibiting nutrient utilization. To test the role of increased ventricular blood pressure in the induction of caudal blisters, primitive streak to 7-somite chick embryos were cut in half, separating the upper presumptive heart region from the lower presumptive trunk and tail regions. Each half was then explanted on media containing trypan blue for 24 hours. In intact embryos treated with 0.04 mM trypan blue the frequency of blisters in the posterior region was 80.8%. The blisters usually appeared on both sides of the neural tube, below or in the region of the last few somites. In transected embryos treated with trypan blue, the frequency of blisters in the posterior halves which had beating hearts was 2.6%. However, the frequency of blisters in the posterior halves which were not connected to beating hearts was 47.8%. In some cases the blisters were found in posterior halves in which the rump was not well developed or present at all. Thus, we may conclude that: (1) direct connection between the heart and the rump is not necessary for the induction of caudal blisters. (2) The presence of a well-differentiated rump is also not necessary for blister formation. We suggest that trypan blue acts directly on organs or structures found in the caudal region of the chick embryos.

Abnormalities, Drug-Induced↗

PubMed1986

Properties of 17- to 19-day-old chick embryo liver microsomes. Induction of cytochrome P-450, effect of storage at low temperature, and resistance to lipid peroxidation.

Maximal hepatic cytochrome P-450 levels were induced in the 17-day-old chick embryo (four to five times control) with a dose of sodium phenobarbital of 6 mg/egg/day for 2 days. Similar levels of hepatic cytochrome P-450 were induced with a dose of propylisopropylacetamide of 4 mg/egg/day for 1 day. Chick embryo hepatic microsomes from phenobarbital-pretreated or from untreated chick embryos could be stored for periods of 14 days at -70 degrees C without a decrease in cytochrome P-450 levels. Moreover, no significant differences was discerned between the degree of suicidal inactivation of chick embryo hepatic cytochrome P-450 by 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine in fresh and stored microsomes. Unlike rat hepatic microsomes, chick embryo hepatic microsomes do not undergo lipid peroxidative loss of cytochrome P-450 and heme when incubated in the presence of NADPH.

Animals↗