Interaction of the anti-oestrogen, nafoxidine hydrochloride, with the soluble nuclear oestradiol-binding protein in chick liver.

Nafoxidine hydrochloride (Upjohn, 11100A)injected with oestradiol into immature chicks inhibits the hormone-induced increase in [3H]oestradiol-binding activity in salt extracts of liver nuclei as well as the subsequent production by liver of egg-yolk phosphoprotein. Substantial inhibition of both oestradiol-induced responses is seen when nafoxidine is given in a dose approximately equimolar with that of oestradiol. In vitro nafoxidine competitively inhibits binding of [3H]oestradiol in nuclear extracts. The Ki for the inhibition is 43 nM, which indicates an affinity of nafoxidine for the binding protein about 4% of that of oestradiol. The inhibitory action of nafoxidine in vivo thus is more potent than the relative binding affinity determined in vitro might indicate. One possible explanation is that the primary site of nafoxidine action is at a point proximal to nuclear receptor interaction. Nafoxidine injected alone into the chick does not induce phosphoprotein synthesis, but it does increase [3H]oestradiol-binding activity in extracts of liver nuclei to a limited extent. No differences in the properties of the oestradiol-binding activity in extracts from nafoxidine-treated chicks or from oestradiol-treated chicks were detected. Chick liver cytosol does not contain detectable high-affinity oestradiol-binding activity. A low-affinity oestradiol-binding component with a sedimentation coefficient of 3.5S was found, but it was unaffected by treatment of chicks with earlier nafoxidine or oestradiol. The results suggest a difference in the mechanism of oestradiol action in the chick liver and in the widely studied rat uterus, on which the usual model for oestradiol action is largely based.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.     

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.     

Metabolism of cholesterol in the tissues and blood of the chick embryo

Three artificially inseminated laying White Leghorn hens were given 35-50 micro c of cholesterol-4-(14)C intravenously. Their subsequently produced eggs contained cholesterol-(14)C-labeled yolks. Some of the fertilized eggs were analyzed for cholesterol content and radioactivity. Other eggs were incubated until hatching. The specific activity of the cholesterol contained in the serum and tissues of newly hatched chicks was determined and compared with that of yolk sac, which was taken as representative of egg yolk cholesterol before its metabolic transfer into the chick embryo. The specific activities of cholesterol in intestine, liver, serum, heart, and skeletal muscle and the whole chick were 95-98% of that in yolk sac, but that of brain cholesterol was only 11% of this value. These results indicate that whereas most of the cholesterol in the chick originated from the egg yolk, cholesterol biosynthesis was active in the brain and provided about 90% of its cholestero content. Newly hatched chicks were found to be hyperlipemic compared with older chicks and had fatty livers with a high cholesterol content. Desmosterol was found in 9- and 15-day old chick embryos but not in the newly hatched chicks, in which the only sterol was cholesterol.     

Inhibition of the estrogen-induced synthesis of vitellogenin mRNA in chick liver by tamoxifen

Cloned vitellogenin cDNA (labelled with ³²P) was used as a probe for measuring vitellogenin mRNA sequences in RNA preparations from the liver of chicks treated with estradiol and/or tamoxifen. For the first time it was shown that the antiestrogen tamoxifen inhibits the estradiol-induced synthesis of vitellogenin mRNA in chick liver. This inhibition correlates very well with a reduced capacity of the liver to synthesize vitellogenin. Furthermore, evidence is presented that tamoxifen lacks any agonistic activity in chick liver. Vitellogenin mRNA is not measurable after tamoxifen alone.     

Lack of effect of exogenous insulin-like growth factor-I (IGF-I) on chick embryo growth rate

Direct evidence that IGF-I has any significant effect on embryo growth is lacking. We therefore studied the effect of administration of IGF-I on the chick embryo in ovo. Five hundred ng pure IGF-I (purified from human plasma) were given to chick embryos on 2 occasions (7 and 14 d) by injection directly into the allantoic sac. Treated and control (saline injected) chicks hatched on the same day and were killed. IGF-I appeared to reach the tissues as the [35S]-sulphate uptake of treated sternal cartilage was significantly greater than that of control (P less than 0.02). However, there were no significant effects of treatment on total body weight, bone length measurements, organ (lung, liver, heart) weights, muscle DNA, RNA or protein levels. From these results we conclude that administration of exogenous IGF-I to the chick embryo at 7 and 14 d does not stimulate further growth of the chick embryo.     

Induction of microsomal stearyl coenzyme A desaturase in the newly hatched chicks.

The development of the stearyl-CoA desaturase system was studied in newly hatched chicks. The desaturation activity was very low in hepatic microsomes from chick embryos, less than 0.05 nmol of oleate formed min^?1 (mg of protein)^?1. After hatching and feeding, the desaturation activity gradually increased to 4–5 nmol of oleate formed min^?1 (mg of protein)^?1 in 6-day-old chicks. This increase could be prevented by administration of cycloheximide or actinomycin D. Measurement of the microsomal electron transfer components throughout the induction period showed no significant changes in the NADH- or NADPH-specific reductases or in the concentrations of cytochromes b₅ and P-450. However, the activity of the terminal component of the desaturase system (the desaturase enzyme) increased in parallel with the desaturation activity. Supplementing the liver microsomes from chick embryos with isolated desaturase enzyme resulted in the formation of an active desaturation system. It is proposed that the induction of the stearyl-CoA desaturase system during development of newly hatched chicks is dependent on the synthesis of the terminal desaturase enzyme.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

The dual regulation by glucagon of the adenylate cyclase system in the embryonic chick heart]

Taken in physiological concentrations, glucagon increases the activity of adenylate cyclase from the heart of 11-day chick embryos, i.e. at the earliest investigated stage. High glucagon concentrations inhibit the enzyme from cardiac membranes at all ontogenetic stages except mature chicks in which glucagon produces stimulating effect. Guanine nucleotides potentiate this effect up to the 16th day of incubation, this effect being absent at later periods. Reconstruction of adenylate cyclase system from the heart of 16-day embryos with N-proteins from mature liver tissue of chicks results in the recovery of potentiating effect. However, at later developmental stages, potentiation was absent even in the presence of N-proteins.     

Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ～0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.     

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks

1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (-40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.     

Hormonal regulation of fatty acid synthetase in chick embryo liver.

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.     

Studies on Polyadenylic Acid-Containing RNA from Developing Nervous System of the Chicken the

Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)⁺ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T₁ ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)⁺ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)⁺ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)⁺ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)⁻ RNA was larger than 18s. The proportion of poly(A)⁺ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults.     

Maturation of the homeothermic response of heart rate to altered ambient temperature in developing chick hatchlings (Gallus gallus domesticus)

On the basis of evidence showing that instantaneous heart rate (IHR) of chick hatchlings responds to exposure to altered ambient temperature (Ta; Tazawa H, Moriya K, Tamura A, and Akiyama R. Comp Biochem Physiol A 131A: 797-803, 2002), we elucidate here the developmental timeline for the homeothermic response of HR in newly hatched chicks (days 0-7) maintained at room temperature ( approximately 24-27 degrees C). Hatchlings were exposed to Ta of 25, 35, and 25 degrees C for 1-h periods, respectively, and IHR was measured together with skin temperature (Ts) during this warming and cooling bout. Early 0-day-old (0 day) chicks responded to warming and cooling exposures with various changes in HR baseline. In newly hatched chicks (0-7 h old), HR baseline was elevated during warming (Delta126 beats/min, n = 13) and declined during cooling (-Delta94 beats/min). With progress of development on day 0, the elevation of HR baseline during warming decreased and advanced 0-day chicks tended to decrease HR baseline during warming rather than increase HR. The more developed 1- to 7-day-old chicks exhibited the expected homeothermic decrease in HR during warming. The diurnal variations of HR responses during warming and cooling on the first day of post-egg life indicate that pronounced development of thermoregulatory competence occurs during the day of hatching (day 0). The response of IHR fluctuations to altered Ta was observed in the form of low- and high-frequency oscillations. High-frequency oscillations corresponding to respiratory sinus arrhythmia developed as the hatchlings aged. There was a significant increase in the number of chicks exhibiting both low- and high-frequency oscillations that depended on age and the development of thermoregulatory competence of hatchlings.     

Regulation of sugar content in the blood during artificial hyperglycaemia in chick embryos]

Intravenous injections of glucose (8.6 mg/ml of blood) have been made 9-18 day chick embryos, 1-day chicks and adult hens. Glucose content of the blood was measured 15 and 30 min., 1,4 and 8 hours after the injection. It was shown that already 9-day embryos possess a regulatory system which restores the initial glycaemic level. During the development of the organism, the efficiency of this system increases. The data obtained may be used in theoretical studies on the sugar system of the blood.