Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.     

Magnetic resonance microscopy of chick embryos in ovo

Magnetic resonance imaging (MRI) of the live 11-day chick embryo with special radiofrequency coils and 3-D imaging methods has produced contiguous 1.25-mm-thick slices with 200-microns pixel resolution, permitting definition of cardiac chambers, cerebral ventricles, spinal cord, liver, and lungs. It was the objective of this study to image younger chick embryos in ovo with higher spatial resolution through the application of implanted radiofrequency coils. Fertilized Arbor Acre eggs were windowed at 9, 6, and 4 days. Circular coils 18 mm in diameter tuned to 85.5 MHz were suspended around the developing embryo. The eggs were sealed with tape and maintained at 37 degrees C during the imaging procedure. MRI was performed in a 2.0-Tesla GE system utilizing a 3-D Fourier transform acquisition in sagittal and axial planes with a partial saturation sequence (TR = 400 ms, TE = 27 ms). Approximately 1 hour of imaging time was required to obtain 16 contiguous 600-microns-thick slices with 50-microns pixel resolution. Embryos remained viable through the imaging procedure. Embryos were photographed, fixed, and cleared for correlative anatomical study. Vitelline vessels, dorsal aorta, aortic arches, cardinal veins, and cardiac chambers were identified as areas of decreased signal intensity. Cerebral ventricles and the vitreous portion of the eye have signal intensities that are less than adjacent neural, scleral, and lens tissue. Further refinements in MR instrumentation and imaging sequences promise improvements in resolution and offer the potential for sequential observations of the intact embryo.     

The teratogenic effects of 6-mercaptopurine on chick embryos in ovo.

Fertilized eggs of single-combed White Leghorn hens were each injected through a shell window directly beneath the embryo at 70 or 96 h of incubation with 2 mg of the sodium salt of 6-MP dissolved in 0.1 M phosphate buffer, and at 11 days of incubation the embryos were examined for gross morphological abnormalities. Various gross malformations and growth retardation were found, the most frequently and severely affected structures being limbs, beak, and eyes. Treatment at 70 h caused more severe abnormalities than at 96 h, but the spectrum of defects was not very different.     

Fine structure of chick embryonic parathyroid glands cultured on media with different concentrations of calcium.

An electron microscopical study of parathyroid glands from 13-day old chick embryos cultured for 2 days on media with different concentrations of calcium was conducted. The same as in non-cultured embryonic glands, the cells in all cultures contained a moderate amount of rough endoplasmic reticulum and a large Golgi complex with many cisternae, vacuoles, vesicles, coated vesicles and small prosecretory granules. In addition, large secretory granules, which are very rare in the non-cultured controls appeared frequently in the cultures and were especially numerous in the glands cultured on high-calcium medium. The fact that the amount of secretory granules varied according to the concentration of calcium in the medium is interpreted as indicating that the rate of parathyroid hormone secretion in the embryo is already responsive to variations in the concentration of calcium. To the extent that in vitro results may be accepted as representative of what happens in vivo these results support the idea that the embryonic gland may be controlled by variations in the calcemia as the adult gland does.     

Introduction of DNA into chick embryos by in ovo electroporation

Gene transfer by in ovo electroporation has been applied to the study of developmental biology, especially to central nervous system (CNS) development. Plasmids are injected into the neural tube of stage 10 chick embryos, and a 25-V 25-msec square pulse is applied five times. Since DNA moves toward the anode, the cathode side of the neural tube is transfected, and the cathode side is used as the control. Expression of translation product of the introduced DNA is observed 2 h after electroporation, peaks around 20 h after electroporation and then weakens. Expression is transient when plasmids are used as expression vectors, but they are very suitable for studying early developmental events (e.g., gene expression cascades or interactions). Misexpression of Pax-5 is shown as an example.     

Abnormal characteristics of the blood from chick embryos maintained in "shell-less" culture

Chick embryos were maintained in shell-less culture up to a total age of 15 days. The composition of their blood was analyzed together with the blood from coontrol embryos of comparable degree of differentiation. The blood from cultured embryos had lower hematocrit values; their serum contained a reduced concentration of proteins, phospholipids and total calcium and an increased concentration of inorganic phosphorus. In view of the reduced concentration of proteins and phospholipids, the concomitant hypocalcemia must represent, at least in part, a reduction in the binding capacity of the serum and not only a decrease in the ionic fraction of serum calcium.     

Embryotoxicity of 1,2-dibromoethane in chick embryos in ovo: early and late effects.

Embryotoxic effects of 1,2-dibromoethane (DBE), a compound still widely used in industry, have been analyzed using chick embryos in ovo. Administration on embryonic days (ED) 3,4 or 5 induced dose-dependent embryotoxicity, manifested namely as the early embryonic death. A serious disturbance of the vascular system represented probably the main cause of strong embryolethality and growth retardation in the group of survivors. Amniotic bands in the parietal region and defects of brain and aorta prevailed in the malformation spectrum registered on ED 10. The local character of early induced changes suggests a direct effect of DBE itself in the embryotoxic action. This process is probably accomplished through interaction with lipids in cell membranes owing to the hydrophobic character of DBE molecules. The results, however, did not exclude an involvement of reactive metabolites in final embryotoxicity via the formation of DNA-adducts. In any case, a decreasing embryotoxicity of DBE with the age of treated embryos documented that the onset of liver function, assumed to occur on ED 5, did not increase the efficacy of DBE bioactivation. Our results confirmed the short-term embryotoxic properties of DBE reported in rat embryonic cultures. In addition, the in ovo system enabled us to reveal also long-term consequences represented namely by the formation of amniotic bands, not detectable in studies in vitro. The results obtained with the chick embryo in ovo confirmed the suitability of this system for embryotoxicity testing.     

Ultrastructure of isoproterenol-induced myocardial damage in chick embryos

Isoproterenol is known to cause severe myocardial lesions when given in toxic doses to adult homoiotherms. Previous studies on chick embryos revealed myocardial damage with scattered necroses in the outer layer of ventricular myocardium. The present ultrastructural study, performed on embryos 6 to 20 days old, has shown various types of cellular lesions; mainly cellular oedema, mitochondrial swelling, necroses of isolated cardiac muscle cells, fatty degeneration, accumulation of glycogen, and signs of increased proteosynthesis in the surviving muscle cells. Morphological features of the lesions differed from those which are known to be induced by isoproterenol in adult animals and seemed to depend on the stage of embryonic development.     

Early changes in parathyroid hormone response and proteoglycan synthesis of chick embryonic femur produced by exposure to 6-aminonicotinamide in ovo

The mechanism of induction of micromelia in 6-day-old chick embryo by 6-aminonicotinamide (6-AN) was investigated. Six-day-old chick embryo exposed to 6-AN did not show micromelia when tenfold excess of nicotinamide over 6-AN was co-administered. The ability of nicotinamide to prevent the induction of micromelia was partially offset after 4 hr of exposure to 6-AN and completely disappeared after 6 hr. The length of time necessary for the induction of micromelia was not affected by the concentration of 6-AN. These results indicate that exposure to 6-AN for only a short period of 6 hr is sufficient to commit the limb to micromelia and that cellular components involved in the induction of micromelia alter during this period. During this period, newly synthesized proteoglycan monomers typical of cartilage decreased in average molecular size, and isolated femora did not respond to parathyroid hormone (PTH) but to dibutyryl cyclic AMP to stimulate growth of cartilage in organ culture.     

Development of calcium reabsorption by the allantoic epithelium in chick embryos grown in shell-less culture

The allantoic sac of the chick embryo functions as a primitive urinary bladder, storing and modifying the excretory fluid produced by the embryo. We have used chick embryos grown in shell-less culture to study the in situ handling of Ca2+ by the allantoic epithelium. Between Days 8 and 13 of incubation (38 degrees C, 5% CO2), the [Ca2+] of the allantoic sac fluid declines from about 1.5 mM to less than 0.3 mM, with most of this Ca2+ reabsorption occurring between Days 10 and 11. In 13-day-old embryos, the allantoic epithelium reabsorbs within 24 hr 85-92% of 45Ca2+ injected into the allantoic sac, while in 9-day-old embryos 45Ca2+ reabsorption is less than 40% by 24 hr. This is evidence for the developmental onset of a Ca2+ reabsorption process in the allantoic epithelium. The allantoic fluid Ca2+ is reabsorbed into the embryo's blood in which the serum [Ca2+] is about 1.5 mM. Also, electrical potential profiles reveal that the serosal (mesenchymal) side of the allantoic epithelium is 15-30 mV positive compared to the mucosal (luminal) side. Thus, by electrochemical criteria this reabsorption process appears to be active.     

The proteins of the embryonic chick epidermis : I. During the normal development in ovo

As a preliminary to a study of the proteins of the embryonic chick epidermis when grown in vitro under various culture conditions, the proteins of the anterior metatarsal epidermis, from 11 days of embryonic life up to 9 days posthatching, have been studied. Carboxymethylated derivatives of the proteins extracted by a thiol reduction procedure have been analyzed by polyacrylamide gel electrophoresis. The results have shown that the differentiation of the epidermis is characterized by the appearance between days 14 and 17 of at least 11 major protein bands in the electrophoretic pattern. Two of these bands are of relatively high molecular weight protein and appear earlier than the remaining bands which form a group of closely related, low molecular weight protein species. The differentiation of the tissue also involves the disappearance from the electrophoretic pattern of all but one of the five major bands present in extracts of the 11/12-day epidermis. A study of the proteins derived from the isolated periderm of the 14-day chick embryo beak has suggested that one of the major bands in the 11/12-day metatarsal epidermal extracts may be a peridermal protein.     

Calcium paradox of aldosteronism and the role of the parathyroid glands

The hypercalciuria and hypermagnesuria that accompany aldosteronism contribute to a fall in plasma ionized extracellular Ca2+ and Mg2+ concentrations ([Ca2+]o and [Mg2+]o). Despite these losses and the decline in extracellular levels of these cations, total intracellular and cytosolic free Ca2+ concentration ([Ca2+]i) is increased and oxidative stress is induced. This involves diverse tissues, including peripheral blood mononuclear cells (PBMC) and plasma. The accompanying elevation in plasma parathyroid hormone (PTH) and reduction in bone mineral density caused by aldosterone (Aldo)-1% NaCl treatment (AldoST) led us to hypothesize that Ca2+ loading and altered redox state are due to secondary hyperparathyroidism (SHPT). Therefore, we studied the effects of total parathyroidectomy (PTx). In rats receiving AldoST, without or with a Ca2+-supplemented diet and/or PTx, we monitored urinary Ca2+ and Mg2+ excretion; plasma [Ca2+]o, [Mg2+]o, and PTH; PBMC [Ca2+]i and H2O2 production; plasma alpha1-antiproteinase activity; total Ca2+ and Mg2+ in bone, myocardium, and rectus femoris; and gp91(phox) labeling in the heart. We found that 1) the hypercalciuria and hypermagnesuria and decline (P < 0.05) in plasma [Ca2+]o and [Mg2+]o that occur with AldoST were not altered by the Ca2+-supplemented diet alone or with PTx; 2) the rise (P < 0.05) in plasma PTH with AldoST, with or without the Ca2+-supplemented diet, was prevented by PTx; 3) increased (P < 0.05) PBMC [Ca2+]i and H2O2 production, increased total Ca2+ in heart and skeletal muscle, and fall in bone Ca2+ and Mg2+ and plasma alpha1-antiproteinase activity with AldoST were abrogated (P < 0.05) by PTx; and 4) gp91(phox) activation in right and left ventricles at 4 wk of AldoST was attenuated by PTx. AldoST is accompanied by SHPT, with parathyroid gland-derived calcitropic hormones being responsible for Ca2+ overload in diverse tissues and induction of oxidative stress. SHPT plays a permissive role in the proinflammatory vascular phenotype.     

Ultrastructure of the ring-shaped nucleoli in the hepatocytes of chick embryos]

The fine structure of ring-shaped nucleoli in hepatocytes of chick embryos at different terms of development was studied. Structural differences were shown between annular nucleoli and nucleoli with typical nucleolonemal organization. Ring-shaped nucleoli, as a rule, are devoid of nucleolonemal structure and their fibrillar component is reduced. The material which fills the central cavity always appears similar to the nucleoplasm in its structure. On the basis of serial sections, we propose that central cavity is isolated from the nucleoplasm. From the hypotonical treatment and EDTA staining application it is concluded that the central cavity of ring-shaped nucleoli contains DNP associated with the intranucleolar chromatin. The number of these nucleoli increased after the injection of a liver homogenate cytoplasmic fraction extracted from adult hen. The physiological significance of such nucleoli is discussed.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Isolation and characterization of proteoglycans synthesized in ovo by embryonic chick cartilage and new bone

Cartilage proteoglycans have been well characterized in a number of developing systems, both in vitro and in vivo, but the newly synthesized molecules have been analyzed only from culture material. Because of potential culture artifacts, an attempt was made to characterize the proteoglycans newly synthesized in ovo in chick embryo sternum, tibial epiphysis, and tibial shaft. These in ovo synthesized proteoglycans share many structural features with chick proteoglycans synthesized by chondrocytes in culture including average monomer size, chondroitin sulfate chain size, keratan sulfate chain size, and the ability to aggregate with hyaluronic acid. Moreover, the newly synthesized in ovo proteoglycans, notably those of the tibial epiphysis, display reproducible changes in their structure as a function of embryonic age. These changes correlate with similar changes documented for chick cartilage proteoglycans synthesized in culture. Finally, the proteoglycans synthesized in ovo in the day 17 tibial shaft include, in addition to cartilage proteoglycans, one proteoglycan which seems to be characteristic of bone.