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Histones were prepared and purified from chick embryo, chick and chicken liver nuclei. The comparative analysis of these histone preparations, fractionated by polyacrylamide gel electrophoresis, indicates that histone fractions of chick embryo, chick and chicken livers are respectively identical and they comigrate with calf thymus histones.
A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.
After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.
Chick embryos at stage 8, prior to neural crest cell migration, were explanted on whole egg medium with or without vitamin A and cultured for 3 days. Sections through the head regions showed that the cranial neural crest cells had migrated into the first visceral arch in the controls but were absent from this structure in the treated embryos. These observations suggest that vitamin A inhibits neural crest cell development or migration, an effect which may in part account for the facial malformations produced by excess vitamin A.
Chick embryos ranging from the stage of first cleavage to that of about 700 cells were removed from the oviduct and examined by transmission electron microscopy. Beneath the cell membrane is yolk-free cortical region containing microfilaments. Beneath this lies cytoplasm which contains yolk spheres which are graded in size, the dorsal ones being smaller than the ventral ones. The subgerminal periblast possesses a greater proportion of yolk to cytoplasm than do the cells proper, but it merges with the cytoplasm at the incomplete borders of the 'open' cells. Specialized accumulations of membranes lie in the marginal periblast, and it is suggested that they play a role in cell membrane formation.
Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.
A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.
The level of UDPG-pyrophosphorylase activity has been determined spectrophotometrically. The enzymic activity in skeletal muscles gradually increases during the development of chick embryos and chick. Adrenalin does not affect the activity of the enzyme neither in embryos, nor in chick.
Fluorescence microscopy (Falck and Hillarp technique) of 11th day old chick embryo shows aminergic neurons and processes in the paraventricular organ area. Administration of tritiated amines permits us to observe uptake and storage of serotonin only as early as the 10th day; this suggests a very early hypothalamic serotoninergic nervous system differentiation.
Treatments at the rate of 3 rad of chick embryos and young birds have led to the stabilizing of the mitochondrial structures and to lowering the access of enzymes to their substrates. The free activity of malate dehydrogenase drops. Electron microscopic studies reveal that the number of mitochondria rises following irradiation with stimulating doses (3 rad). Mitochondrial cristae remain intact and the matrix presents higher electronic density.
Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.
Chick primordial germ cells (PGCs) which separated from the "germinal crescent" entoderm in the period from stages 4 to 8 circulated mostly through the developing blood vessels from stage 10 onward and finally migrated into the gonad. The PGCs making their appearance up to this stage were generally spherical in profile, about 14 mum in diameter. Some of the PGCs in contrast, did not enter the blood vessels but remained in the tissue (mesenchyme) of the embryo proper (tissue PGCs) and possessed pseudopodial processes, suggesting their migration by means of amoeboid movements. The circulating PGCs emerged from blood vessels in the vicinity of developing gonads by three days (gonadal PGCs). The principal mechanism responsible for the subsequent migration of gonadal PGCs is assumed to be amoeboid movements as in the case of tissue PGCs. Notable amounts of PAS-positive glycogen were demonstrated in the cytoplasm of PGCs in all stages obsreved. They also contained yolk and lipids intracytoplasmically, the former dissipating in relatively early stages of development. Electron microscopic observation revealed the electron-opaque, "fragmented nucleolus" in the large nucleus (8 mum in diameter), which represented another prominent feature of chick PGCs. PGCs contained a well-developed Golgi complex and endoplasmic reticulum.
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The chick embryo was used to study the relationship between the onset of tumour neovascularization and tumour growth. Walker 256 carcinosarcoma was implanted on the chrioallantoic membrane (CAM) of about 600 embryos aged 5-16 days. Tumour diameter and changes in the CAM vasculature in response to the implants were recorded daily. Representative tumours were examined by light microscopy of Epon-embedded tissue and autoradiography after injection of [3H]-thymidine. Tumours remained avascular for 72 h, after which they were penetrated by new blood vessels and began a phase of rapid growth. The rate of growth during this vascular phase was greatest for implants on 5- and 6-day-old embryos and decreased the later the day of implantation. The time of onset of tumour angiogenesis appears to be independent of the immunological state of the chick embryo, although the rate of growth after vascularization may be modified by the onset of immunity. This study suggests that the avascular and vascular phases of tumour growth are separable, and that the avascular tumour population lives under the growth constraints which limit the size of a tumour spheroid growing in soft agar or aqueous humour.
The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryo-genesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17-20 cell divisions are required for differentiation of erythrocytes from stem cells.
Studied were the level of ADP and the adenosinetriphosphatase in nuclei, mitochondria, and microsomes taken from the brain and liver of singly gamma-irradiated at 1000 rd chick embryos and birds. As a result of the treatment of ADP content dropped, while the activity ADP rose. These changes were most strongly expressed in the nuclei, then in the mitochondria, and to a lesser extent-in the microsomes. Twelve-day chick embryos showed more markedly expressed radiosensitivity than newly hatched chicks. This embryonal state is characterized by intense growth, differentiation and metabolic processes in the liver, which substantiate not only the higher radiosensitivity of this age group but the more strongly expressed changes in the liver as compared with the brain.