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PubMed1996

Development and maintenance of outer segments by isolated chick embryo photoreceptor cells in culture.

PURPOSE: To investigate the capacity of isolated chick embryo photoreceptors to develop and maintain outer-segment processes in dissociated cell cultures, in the absence of pigment epithelial and glial cells. METHODS: Cells were obtained from the retinas of embryonic day (ED) 17 chick embryos, after the onset of outer-segment formation in vivo. After 5 to 12-minute incubation in Ca++ and Mg++-free Hank's balanced salt solution, neural retinas were freed from other optical tissues, including the pigment epithelium. Retinal cell suspensions were prepared by repeated pipetting after mild trypsinization and were grown in serum-containing medium on a polyornithine-coated substratum. Cell differentiation was evaluated using phase-contrast and transmission electron microscopes and by autoradiographic analysis of the uptake of putative amino acid neurotransmitters, lectin cytochemical analysis, and immunocytochemical analysis with rod and cone-specific antibodies. Cells isolated from ED 8 retinas, before the onset of outer-segment formation in vivo, were also studied. RESULTS: At culture onset, ED 17 cells appeared morphologically undifferentiated and devoid of processes; differentiated features could be detected after 24 to 48 hours in vitro. Photoreceptor cells were the most abundant cell type after 6 days in vitro, followed by nonphotoreceptor multipolar neurons and morphologically undifferentiated cells. Autoradiographic analysis showed extensive Na+ -dependent uptake of (2,3,4-(3)H)gamma- aminobutyric acid in nonphotoreceptor neurons, whereas photoreceptors were labeled predominantly with 3H-glutamate. Most of the photoreceptors were labeled with fluorescent peanut lectin and with a sheep polyclonal antibody against bovine rhodopsin. Subsets of photoreceptors, on the other hand, were immunoreactive with cone- or rod-specific monoclonal antibodies COS-1, OS-2, 50-1B11, or Rho-4D2. Approximately 50% to 65% of the photoreceptors positive with these monoclonal antibodies showed a remarkable polarization of immunoreactive materials, which accumulated predominantly, or even exclusively, in an outer-segment-like apical process. When viewed on the transmission electron microscope, these outer-segment-like processes appeared as distal expansions of the photoreceptor cilium and contained disc-like membranous profiles. Outer-segment-like processes also could be detected using the electron microscope and by immunocytochemical analysis of cultures of ED 8 retinal cells. CONCLUSIONS: After undergoing morphologic dedifferentiation as a result of tissue dissociation, isolated retinal photoreceptors, grown in the absence of contact-mediated cell interactions and of pigment epithelial and glial cells, can regenerate and maintain a highly polarized pattern of structural and molecular organization, including the formation of outer-segment-like processes. The cultures provide an experimental system for the investigation of cellular and molecular mechanisms regulating further development and maturation of these photoreceptor structures.

Animals↗

PubMed1995

Effect of atrial natriuretic peptide on diastolic filling in the stage 21 chick embryo.

Atrial natriuretic peptide (ANP) exerts hemodynamic effects by direct venodilation in the chick embryo. We hypothesized that ANP-induced venodilation affects ventricular diastolic filling resulting in reduced ventricular preload. Chick ANP (0.1 microgram in 10 microL of normal saline) was suffused onto the vitelline vascular bed in stage 21 (3 1/2 d) chick embryos. Equivalent aliquots of normal saline were suffused as sham controls, and normal embryos received no suffusion. We measured simultaneously dorsal aortic blood velocity and atrioventricular blood velocity with a 20-MHz pulsed-Doppler velocity meter. Analog wave forms were digitally sampled at 500 Hz, and the dorsal aortic cross-sectional area was used to calculate dorsal aortic blood flow. Passive ventricular filling volume equaled dorsal aortic stroke volume multiplied by the fraction of passive area; active filling volume equaled dorsal aortic stroke volume multiplied by the fraction of active area. Data were summarized as mean +/- SEM (n > or = 7 per group) and analyzed by analysis of variance. Cycle lengths were similar in ANP-suffused, sham control, and normal embryos. Dorsal aortic blood flow decreased from 0.49 +/- 0.04 mm3/S at baseline to 0.27 +/- 0.05 mm3/S at 4 min post-ANP suffusion (p < 0.05) and was unchanged in sham control and normal embryos (p > 0.05). Passive ventricular filling was reduced by ANP suffusion, whereas active filling was unaffected, resulting in a decreased passive/active filling ratio from 0.64 +/- 0.07 at baseline to 0.32 +/- 0.08 at 4 min in ANP-suffused embryos (p < 0.05). Passive/active ratio was unchanged in sham control and normal embryos. Thus, ANP-mediated vasodilation reduces cardiac output via decreased passive ventricular filling in the embryonic heart.

Analysis of Variance↗

PubMedDate not supplied

The development of the caeca in the chick embryo. Morphometric and morphological remarks.

Quantitative aspects of the organogenesis of the chick embryo caecum (proximal and distal tracts) were investigated from the 8th to the 15th day of incubation. Histological sections were studied by means of a computerized morphometric system and a semiautomatic digital system (Videoplan 2). The total area of sections, of the lumen, the caecal wall and its components (subserous stratum, muscle layer, lamina propria, epithelium) and the thickness of the epithelium and muscle layer were measured. The mean +/- S.E.M. of the obtained values was calculated. The percentage of shrinkage, due to histological procedures, was calculated for each day considered. The mean values for each day of incubation were modified on the basis of the shrinkage percentage. The differences between the mean values of the areas of the proximal and distal tract were statistically evaluated. Exponential curves and the r coefficient were determined in order to evaluate the general growing pattern of the mean area of the caecal wall components as a function of age. The obtained results demonstrate that there are some differences between the proximal and distal tract of the caecal anlage in the growth pattern of the wall components. In the proximal caecal tract, the lamina propria showed the highest growth rate, while in the distal tract, the caecal wall component the subserous stratum did. Moreover, the caecal anlage showed different developmental behaviour from the ileum, duodenum and colon in the same chick embryo.

Animals↗

PubMed1979

The role of temperature in the crystallization of ribosomes in chick embryos.

The relationship between ribosome crystallization and cell degeneration has been studied in chick embryos at various temperatures, and new methods of inducing ribosome microcrystals are described. A model is discussed that reinterprets the role of low temperatures in these phenomena and provides a unitary explanation of the various cases in which the occurrence of ribosome crystallization in chick embryos has been reported.

Animals↗

PubMed1979

Effect of aflatoxin B1 on the ultrastructural and biochemical development of chick embryo liver during organ culture.

When liver fragments from eleven-day chick embryos were maintained on Eagle's minimal essential medium by the established method of organ culture, they developed ultrastructural features similar to liver cells in vivo, except that they had small amounts of smooth endoplasmic reticulum and little glycogen. The cultured liver cells synthesized DNA, RNA and protein. The addition of aflatoxin B1 to the medium inhibited the synthesis of nucleic acid. Aflatoxin B1 also produced the segregation of granular and fibrillar components in nucleoli and the disarrangement of ribosomes attached to endoplasmic reticulum. Since these results were consistent with the known effects of the toxin in animals, we concluded that organ culture of chick embryo liver could be a useful technique for other studies.

Aflatoxins↗

PubMed1984

The spontaneous occurrence of aortic arch and cardiac malformations in the white Leghorn chick embryo (Gallus domesticus).

Spontaneous aortic arch and cardiac malformations occur in White Leghorn chick embryos at a relatively high rate. Although this breed of Gallus domesticus is widely used for biomedical and biological research, no previous study has recorded the incidence of these defects. We found aortic arch malformations in 7.1% (14 of 196) and ventricular septal defects in 11.7% (23 of 196) of living embryos. Defects occurred alone or as a combined pattern. Our findings suggest that the cardiovascular defects in the chick embryo documented in past studies may, in some cases, have been part of normal spontaneous occurrence, rather than the major result of experimental manipulation.

Animals↗

PubMed1977

Uptake of L-DOPA by the cells of organotypic culture from chick-embryo brain hemispheres.

With the use of the histofluorescence method investigations were performed on L-DOPA uptake by the cells of an organotypic culture from chick-embryo brain hemispheres and newborn rats. It has been found that in chick-embryos between the 8th to 11th day of development and 5 to 14 days in culture in vitro, with a concentration of 0.1 mg/10ml of L-DOPA applied to the culture, intense fluorescence in cell membranes, perinuclear region and sometimes in the whole cell body and the initial parts of the cell processes can be observed. It is supposed that in the stage of development of chick-embryos and newborn rats under study neural elements and glial cells take up the amino-acid applied. The authors suggest that the uptake of L-DOPA proceeds rather through the uptake II mechanism.

Animals↗

PubMed1982

Changes in specific activities of 3',5'-cyclic adenosine monophosphate phosphodiesterase in chick embryo cells grown under different conditions.

The relationship between the density of chick embryo cells in stationary cultures and their specific 3',5'-cAMP phosphodiesterase (PDE) activities and calmodulin (CaM) contents were investigated. An indirect relationship between them was found. The specific CaM-PDE activities were the highest in the starting logarithmic phase of growth and the lowest in grown (72 hr old) monolayer cultures. Changes in the specific activities induced by two serum concentrations were also related to the cell culture density. The results suggested that the density or any metabolic change due to it is the primary factor regulating the level of specific PDE activities in chick embryo cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1967

Development of uridine diphosphate-glucuronyltransferase activity in cultures of chick-embryo liver.

1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.

Aging↗

PubMed1986

Demonstration of cellular retinoic acid binding protein (CRABP) in chick embryo tendon cells and effects of retinoids on collagen synthesis in tendon and sterna.

The binding of all-trans-retinoic acid (all-trans RA) to specific cytosol proteins and the effects of retinoids on procollagen synthesis were studied in chick-embryo tendon cells. For the receptor assay, tendon cytosols were incubated with [3H]all-trans-RA in the presence or absence of 100-fold excess of nonlabeled all-trans-RA up to 20 hr at +4 degrees and unbound retinoid was removed by charcoal-dextran treatment or by gel filtration chromatography. The results indicated that chick-embryo tendon cells contained cellular retinoic acid binding protein (CRABP). The binding of [3H]all-trans-RA could be displaced by 13-cis-retinoic acid, but not by retinol or etretinate. In contrast no CRABP could be found in cartilage cells isolated from sterna or in whole sterna. The treatment of tendon cytosol with proteases (pronase, trypsin, chymotrypsin) abolished the specific binding of [3H]all-trans-RA. Gel filtration studies on Sephadex G-100 indicated an apparent molecular weight of 14,500-15,000 daltons for the all-trans-retinoic acid binding protein. All-trans-RA markedly decreased procollagen synthesis in isolated chick-embryo tendon cells, the inhibition being concentration dependent; the decrease was about 58% of the control in the presence of 10(-5) M all-trans-RA. Similar decrease was noted with 13-cis-RA and etretinate, while retinol was less effective. In isolated cartilage cells the dose of 10(-5) M of all-trans-retinoic acid decreased drastically total protein and collagen synthesis. The mannosylation of procollagen, the conversion of procollagen to collagen and the size of procollagen chains were not significantly affected. The results of the present study indicate that CRABP is not expressed in sterna of chick-embryos, and in contrast high levels of CRABP could be found in tendons. However, retinoids modulated collagen synthesis in both tissues. Thus it is possible that retinoids can affect the metabolism of different collagen types also in clinical use.

Animals↗

PubMed2000

The hemodynamic effects of halothane and isoflurane in chick embryo.

UNLABELLED: The cardiovascular effects of volatile anesthetics in prenatal hearts are not well investigated. The purpose of this study was to determine whether the embryonic cardiovascular system is sensitive to an exposure to clinically relevant, equipotent concentrations of halothane and isoflurane. Stage 24 (4-day-old) chick embryos were exposed to 0.09 and 0.16 mM of halothane and 0.17 and 0.29 mM of isoflurane. Dorsal aortic blood velocity was measured with a pulsed-Doppler velocity meter. Halothane, but not isoflurane, caused a significant decrease in cardiac stroke volume and maximum acceleration of blood (dV/dt(max)), an index of cardiac performance. This effect was reversible, and during washout, stroke volume and dV/dt(max) increased above control levels. Embryonic heart rate was not affected by either drug. Chick and human embryos are similar during early stages of development; therefore, chick embryo may be a useful model to study the cardiovascular effects of anesthetics. IMPLICATIONS: In equipotent, clinically relevant concentrations, halothane, but not isoflurane, markedly decreased aortic blood flow and cardiac performance measured with ultrasound techniques in chick embryos. Chick and human embryos are similar during early stages of development; therefore, chick embryo may be a useful model to study the cardiovascular effects of anesthetics.

Anesthetics, Inhalation↗

PubMed1994

Development regulation of basal and hormone-inducible phosphoenolpyruvate carboxykinase gene expression in chick embryo liver in vivo.

We have examined cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) gene transcription and steady-state mRNA expression in chick embryo liver in vivo both constitutively and in response to dexamethasone, cAMP, retinoic acid, and the protein synthesis inhibitors, cycloheximide and pactamycin. We report here that PEPCK mRNA is constitutively expressed between 12 and 16 days of development. PEPCK expression was also transiently but highly inducible on Day 14 by dexamethasone, cAMP, cycloheximide, and pactamycin, but not by retinoic acid. Cycloheximide pretreatment had an additive or synergistic effect on the induction by dexamethasone and/or cAMP. Dexamethasone- and cycloheximide-induced increases in mRNA expression were principally due to increases in the rate of PEPCK gene transcription. Following an initial induction by dexamethasone, PEPCK expression was no longer responsive to a second administration of dexamethasone but was still responsive to cycloheximide and cAMP. PEPCK induction by dexamethasone or cycloheximide progressively diminished between 12 and 15 days of development. By Day 16, PEPCK expression was no longer responsive to dexamethasone, but was still inducible by cAMP and this induction was increased by cycloheximide. These results indicate that PEPCK is transiently inducible by glucocorticoids in chick embryo liver and that there are two developmental switches to the adult phenotype between Days 14 and 16 of development and between Day 16 and 4 days posthatching. Our results also suggest the presence of a developmentally regulated repressor of PEPCK gene expression in chick embryo liver.

Animals↗

PubMed1996

Comparison of lens biochemistry and structure between BSO-treated and glucocorticoid-treated developing chick embryos.

In this paper various changes in glutathione level, which were influenced by balance of its synthesis, degradation, transport and utilization, were analysed in chick embryos administered with glucocorticoid (GC) or buthionine sulfoximine (BSO; an inhibitor of glutathione synthesis). When BSO (30 mumol egg-1) was administered twice to chick embryos on day 14 and 15, the GSH in both the lens and the liver decreased to 15-20% and 30-40% of the age-matched control level, respectively, between 24 and 48 hr after the second treatment, then began to recover. Although this decline in the GSH level in these tissues was greater and more prolonged in embryos treated with BSO than with GC, the former embryos maintained lens transparency even up to 144 hr by a visual examination. However, histological changes in the lens occurred after 96 hr and more significantly 144 hr after second administration of BSO. The changes mainly consisted of pale epithelial cells on the anterior peripheral surface of the lens, irregular height of the epithelial cells at the equator, clefts between the epithelium and the cortex and swelling of almost all the cortical fibers. These observations may suggest that BSO treatment could produce the beginning of a cataract. Embryos with GC-cataract revealed the following changes at 48 hr: loss of transparency, elevation of LPO (TBA-reacting substance) in the lens, the blood and the liver. These were not observed in BSO-treated embryos during the experimental period. The GC-cataract may well depend on the generation of LPO. BSO cataract, having a distinct mechanism compared to that caused by GC, develops more slowly in GSH-depleted lenses. The BSO-treated chick embryos will be a useful model to screen the risk factors which accelerate cataract formation.

Animals↗

PubMed1976

Developmental changes in plasma membrane fluidity in chick embryo heart.

1. Decreases in the rate of transport of sugars (facilitated transport), amino acids (active transport), and urea (simple diffusion) occur in chick embryo heart during development. This work considers the possibility that changes in the plasma membrane fluidity during development contribute to the observed changes in transport activities. 2. Technics were developed for subcellar fractionation of chick embryos and adult chickens. 3. The depolarization of the fluorescence of 1,6-diphenylhexatriene was used to estimate the fluidity of the lipid portion of plasma membrane enriched fractions of hearts from chick embryos at various stages of development and from adult hearts. 4. There is a pattern of decreasing membrane viscosity as development proceeds. Between 5-6 days and 10 days of embryonic life a 20% decrease in viscosity of the plasma membrane-enriched fraction occurs. Between 10 and 20 days of embryonic life there is no significant change in viscosity. Between 20 days of development (1 day before hatching) and adulthood there is a further 55% decrease in plasma membrane viscosity. 5. It is proposed that the changes in membrane fluidity observed may contribute to developmental changes in membrane transport activities, but other factors must also be involved.

Amino Acids↗

PubMed1991

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo--II. In culture studies.

The effects of muscle-derived factors on the cholinergic and GABAergic neuronal phenotypes were studied in cultures derived from 3-day-old whole chick embryo (E3WE) consisting of proliferating neuroblasts and 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons. The effects of limb muscle extract (LME) were examined either when added to the culture medium or to the polylysine coating substratum cultures. We also compared the effects of LME derived from 8- to 15-day-old chick embryos. We found that LME added to the medium not only increased choline acetyltransferase activity, a marker for cholinergic neurons throughout the development of neurons in E3WE or E8CH culture, but also delayed the decline in the activity observed in untreated cultures. The marked increase in choline acetyltransferase activity in E8CH cultures grown on substratum-bound LME as compared to those grown in medium-containing LME, suggests that LME factors may be adhesion-promoting substances stimulating neuronal growth. These findings provide evidence that muscle-derived factors may be important in early cholinergic phenotypic expression and support our previous in ovo studies indicating that target-derived factors in limb muscle extract have general cholinotrophic effect.

Animals↗

PubMed1989

[Responses of tumor cell lines and surgical specimens transplanted into chorioallantoic membrane of chick embryo to anticancer agents in combination with/without hyperthermia].

The chorioallantoic membrane of chick embryo was used to examine the responses of tumor cell lines and surgically resected specimens to anticancer agents in combination with or without hyperthermia. B16-F10 melanoma, KK-47 cell line derived from a transitional cell carcinoma of the bladder and surgical specimens from urological malignancies were transplanted onto the chorioallantoic membrane of chick embryo 10 days after fertilization. The effects of intravascularly injected cyclophosphamide, cisplatin, adriamycin, mitomycin C, vincristine and vinblastine, in combination with or without hyperthermia, on the growth of the grafts were investigated. A 15-minute 42.5 degrees C hyperthermia exhibited most favorable anticancer effects in B16-F10 grafts (P less than 0.01) of all heating conditions tested. A combined use of hyperthermia and cisplatin, cyclophosphamide or adriamycin demonstrated a significantly high tumor regression rate in B16-F10 grafts (P less than 0.01). In KK-47 grafts, a single use of cyclophosphamide and a combined use of hyperthermia and cyclophosphamide exhibited a highly significant antitumor regression (P less than 0.05). Surgical specimens from 14 urological malignancies were subjected to this assay. Growth which was adequate for sensitivity test was obtained in 11 of the 14 malignancies. The results obtained suggest that this assay system may be a useful chemohyperthermia sensitivity test.

Allantois↗

PubMed1994

Pattern of polyamines and related monoacetyl derivatives in chick embryo retina during development.

Polyamines and related monoacetyl derivatives were studied in chick embryo retina during development (6th-19th day). Putrescine, which is high in the first phase of retinogenesis, is necessary to sustain both tissue proliferation and via N-acetylputrescine, gamma-aminobutyric acid synthesis. A later increase in spermidine and particularly spermine may play a role in the last phase of development when the retina reaches maturation. The presence of N1-acetylspermidine already at the 8th day indicates that in chick embryo retina, putrescine synthesis can depend on two separate pathways. The first involves ornithine decarboxylase activity; the second, spermidine/spermine N1-acetyltransferase and probably polyamine oxidase that converts spermidine to putrescine via N1-acetylspermidine.

Animals↗

PubMed1981

Conversion of 5-aminolaevulinate into haem by liver homogenates. Comparison of rat and chick embryo.

1. We have studied the kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of livers of rats and chick embryos. Homogenates of fresh liver from both species efficiently convert 5-aminolaevulinate into haem. After frozen storage for 1 year, homogenates of rat, but not chick, liver have decreased rates of formation of haem with accumulation of more protoporphyrin. The rate of haem formation after storage is restored by addition of Fe2+ and menadione. 2. At all initial concentrations of 5-aminolaevulinate tested (2 microM-1 mM), homogenates of rat liver accumulate less protoporphyrin than haem. In contrast, homogenates of chick embryo liver accumulate more protoporphyrin than haem at concentration of 5-aminolaevulinate greater than 10 microM. Conversion of protoporphyrin into haem by homogenates of fresh or frozen chick embryo liver is not increased by addition of Fe2+. 3. Homogenates of liver from both species accumulate porphobilinogen; the kinetic parameters for this process reflect those of 5-aminolaevulinate dehydratase. 4. The results show that the rate-limiting enzyme for the hepatic conversion of 5-aminolaevulinate into protoporphyrin is porphobilinogen deaminase. In addition, chick liver, compared with rat liver, has only about one-fifth the activity of ferrochelatase, the final enzyme of the haem biosynthetic pathway, which inserts Fe2+ into protoporphyrin to form haem. 5. Comparison of these results with previous studies indicates that the homogenate system described here provides physiologically and clinically relevant information for study of hepatic haem synthesis and its control.

Aminolevulinic Acid↗