On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Mouse hemoglobin during gestation. Absence of fetal hemoglobin

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.     

DNA synthesis and content and the accumulation of total protein and hemoglobin during the differentiation of primary erythroid cells in chickens

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in globin messenger RNA content during erythroid cell differentiation.

Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis.     

Alterations in lysine transfer RNA during erythroid differentiation of the Friend cell.

The proportion of lysine tRNA represented by the isoacceptor species lysine tRNA4 has previously been shown to be largest in cells with the greatest ability to proliferate. Using reverse phase chromatography (RPC-5), we have analyzed the changes in the relative quantities of lysine tRNA species which occur in different cellular states of the Friend cell, a transformed murine cell infected with Friend erythroleukemia virus complex. This cell undergoes erythroid differentiation when exposed to various chemicals. Lysine tRNA4 comprises 32% of the total lysine tRNA in rapidly dividing, uninduced Friend cells, but only 16% of the total lysine tRNA in uninducase. Friend cells undergoing erythroid differentiation divide more slowly than uninduced cells, and finally cease proliferation, but lysine tRNA4 becomes the major lysine tRNA species (greater than 50%). This does not appear to reflect erythroid properties of the cell, since the lysine tRNA of the mouse reticulocyte contains very little lysine tRNA4. The non-dividing erythroid Friend cell, therefore, represents an exception to the finding that non-dividing cells usually have little or no lysine tRNA4 present.     

Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell.

The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis.     

Variations of chromosome radiation sensitivity in fetal and adult mice during gestation

Mice were exposed to 2 Gy of γ-rays at various days of pregnancy, and just before and after gestation. Chromosomes were analyzed 4 h after irradiation in spontaneously dividing hematopoietic cells from liver for fetuses and bone marrow for mothers. On average, there was significantly less chromosome damage in fetuses than in mothers. A very strong increase of chromosome breakage was observed in mothers at days 16–19 of gestation. This increase parallels that of gestation hormones, suggesting a direct relationship. The differences between fetuses and mothers in relation to gestation age result from the increase in the rate of chromatid and chromosome breaks but not of chromatid exchanges, which remained stable. This suggests that a DNA repair step involved in joining broken extremities is the cause. More experiments are needed to understand the origin of these variations of radiation sensitivity and the possible extrapolation of these observations to other species.     

Intranuclear and cytoplasmic hemoglobin in human erythroblasts during maturation. Electron microscopic immunocytochemistry

Changes in the hemoglobin level in human bone marrow erythroblasts associated with cell maturation were studied by the electron microscopic immunocytochemical technique using protein A-gold. Intense reaction of gold to hemoglobin was observed diffusely in the cytoplasm, but the reaction was weak in the Golgi zone. No reaction was observed in mitochondria or granules. Cytoplasmic hemoglobin was noted in basophilic erythroblasts and increased with maturation. Hemoglobin was also noted in the nucleus, especially in the euchromatin, though in smaller amounts than in the cytoplasm. Since intranuclear hemoglobin tended to increase in the euchromatin but to decrease in the heterochromatin with erythroblast maturation, the ratio of the amount of hemoglobin in the euchromatin to that in the heterochromatin increased with maturation.     

Carbohydrate metabolism in the fetal pig during late gestation.

In acute experiments on pregnant sows under sodium pentobarbitone anaesthesia, acid base balance, oxygenation and plasma metabolite concentrations were well maintained in the dam and all fetuses which remained undisturbed in utero, irrespective of the duration of the experiment. Fetal liver glycogen concentrations were also unaffected by the time of removal of the fetus. By contrast, intravascular catheterization and withdrawal of blood led to fetal hyperglycaemia and depletion of hepatic glycogen although blood gas and pH values were not changed by these procedures. In the 1 1/2--2 h sampling period following catheterization the normal positive umbilical venous-arterial differences in plasma glucose and lactate generally became reversed. These changes were prevented by the administration of hexamethonium (10--15 mg . kg-1 i.v.) but the drug did not block the fall in hepatic glycogen in catheterized fetuses. Both adrenaline and noradrenaline, which were each infused intravenously at 2.7--3.9 or 0.6--0.9 microgram . kg-1 . min-1, resulted in fetal hyperglycaemia and lacticacidemia together with a fall in arterial blood pH; hepatic glycogen concentrations in these fetuses were also reduced. The apparent sensitivity of the glycogenolytic mechanism to surgical trauma and haemorrhage in the fetal piglet is discussed in relation to findings in other species.     

Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4′,6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50–60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p?<?0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.     

Studies of murine erythroid cell development. Synthesis of heme and hemoglobin

Techniques of cell separation were used to isolate murine erythroid precursors at different states of maturation. Cells were studied before and after short-term incubation in the presence or absence of erythropoietin. Complementary results were obtained by direct examination of the cell fractions and by the short-term culture experiments. Indices of heme synthesis, including incorporation of 59Fe or [2-14C]glycine into heme and activity of delta-aminolevulinic acid synthetase, were already well developed in the least mature cells, chiefly pronormoblasts. Activity then rose moderately in the cell fractions consisting primarily of basophilic and polychromatophilic normoblasts, and fell off with further increases in cell maturity. On short-term culture in the presence of erythropoietin, activity declined with increasing cell maturation except in the least mature fraction where the original level of activity was maintained. By contrast, synthesis of labeled hemoglobin ([3H]leucine) was very low in the least mature cell fractions and rose progressively with increasing cell maturity. The rate of hemoglobin synthesis increase in cells at all stages of maturation when cultured in the presence of erythropoietin. Despite the different patterns observed for heme synthesis and hemoglobin synthesis, both synthetic activities were consistently higher in cells cultured with erythropoietin as compared to controls. These findings suggest that erythropoietin stimulates biochemical differentiation of erythroid precursors at various stages of maturation. They also demonstrate an asynchronism between heme synthesis and hemoglobin syhthesis; heme synthesis is already well developed in the least mature erythroid cells and begins to diminish as the capacity for hemoglobin synthesis continues to rise.     

Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts

Summary The method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).The morphological characteristics of the most primitive erythroblast (EI cells) were described. The proerythroblasts which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of orthochromatic erythroblasts (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.Partly supported by Alexander von Humboldt-Stiftung and Deutsche Forschungsgemeinschaft DFG-Grant Bo 395/3     

The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.