Differentiation in the early chick embryo: effects of bromodeoxyuridine on erythropoiesis

Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the tissue becomes resistant to the inhibitor when addition is delayed for approximately 10 hr. The inhibitory effect of BrdU is canceled or reversed if thymidine replaces BrdU at the beginning of culture or later. Transfer to thymidine containing medium even after 20 hr permits hemoglobin formation to occur at almost the normal time, thus making it unlikely that a complete cell cycle or DNA replication during a complete S period is required for the reversal of inhibition.Even when the appearance of cytologically detectable hemoglobin is inhibited by BrdU, some globin is synthesized and RNA sequences specific for globin are present, but in decreased amount, unless the inhibitor is given very early. BrdU does not affect the synthesis of any particular RNA species or of polyadenylic acid, but it does lower the rate of uptake of adenosine into the ATP pool. While not affecting cell cycle times in cell cultures, BrdU greatly reduces cell numbers.     

Enzymes of glycogen metabolism in chick embryo liver]

At all stages of ontogenesis glycogen phosphorylase (EC 2.4.1.1) from liver chick embryos in represented by an isoenzyme whose properties are close to those of isoenzyme IL or F. Total enzyme activity (a+b forms) from the 8th day of development up to hatching gradually increases 1.5-fold, a practically complete activation of enzyme being observed by the end of embryogenesis. Phosphorylase b possesses high catalytic activity in the presence of 1 mM AMP and it activated by protamine and 0.2 M Na2SO4. Glycogen synthetase (EC 2.4.1.11) has a constant Km(UDFG) value during ontogenesis. This value is about 5.10(-4) M in the presence of 10 mM glucose-6-phosphate, both for I- and D-forms of enzyme. The total enzyme activity reaches its maximum on the 17th postembryonic day and is decreased more than 6-fold thereafter. In the course of embryogenesis the I/D ratio is increased from 0.2 on the 8th day of development up to 0,45 during extensive accumulation of glycogen and falls down to 0.33 before hatching. Glycogen biosynthesis in embryonic liver is wellcorrelated with the increase in the I/D ratio, i.e. the increase of the active form of enzyme. The proportion of granular glycogen in embryonic liver is increased from 15% up to 90% of total glycogen content between the 8th and 14th days of development. The activity of glycogen synthetase contained in granular glycogen is increased from 40% in the 8-day-old embryos up to 90% in the 18-day-old ones. The activity of phosphorylase is found in granular glycogen only on the 12th day of embryogenesis and reaches its maximum (80% of total enzyme activity) only on the 19th days of development. It is concluded that in the adult chicken liver the embronic enzymes--glycogen phosphorylase and glycogen synthetase--are retained.     

Effect of insulin on glycogen metabolism in isolated catfish hepatocytes

Insulin effect on carbohydrate metabolism in catfish hepatocytes consisted of a significant decrease of cell glycogen concentration both in the absence and in the presence of glucose in the medium. The hormone did not influence either the output of glucose from the cell or the intracellular glucose level. Experiments with radioactive glucose showed a very low uptake of the sugar by the hepatocytes; correspondingly the incorporation of radioactivity into glycogen was very low and not influenced by insulin. The glycogen content in catfish liver cells was influenced by the hormone in the opposite way to rat liver cells.     

Primitive erythropoiesis in chick embryogenesis. 3. Effect of FUdR on Hb synthesis

A single hematocytoblast in the yolk sac of the chick embryo has been shown previously to give rise on the average to a clone of 128 erythrocytes. Furthermore, in any given generation the erythroid cell synthesizes a characteristic amount of hemoglobin (Hb). In these experiments day 4 embryos were treated with FUdR for 12 hours, and then reversed with thymidine. We have monitored both the passage of these erythroblasts through the cell cycle, and the effect of this perturbation on the Hb content of single cells. As a result of this disruption the amount of Hb synthesized in a given generation can be varied, but the final amount of Hb/cell in the mature erythrocyte is the same as in the untreated controls. Apparently the total amount of the Hb/cell does not in itself influence the passage of the cell through the cycle. The coefficients of variation of the Hb values in the mature erythrocytes from both normal an perturbed embryos are similar.