FOETAL ERYTHROPOIESIS IN GENETICALLY ANAEMIC, FLEXED-TAILED (f/f) MICE

This paper presents measurements of foetal weights, haemoglobin concentrations, red cell counts, blood volumes, erythroblast thymidine labelling indices and cell cycle parameters in genetically anaemic, flexed-tailed ( f/f ) and haematologically normal ( f/+ ) foetuses.
The production of red blood cells from the livers of ( f/f ) foetal mice lags behind that of heterozygote ( f/+ ) foetuses, and the cells that are produced are small and poorly haemoglobinized. The anaemia is not due to an abnormal cell cycle in f/f erythroblasts, which appear to be capable of responding to the anaemia by a small increase in their rates of proliferation. The number of red cells in the foetal circulation can be calculated from the cell cycle time and number of hepatic erythroblasts; the calculated number agrees quite well with the measured number in both f/f and f/+ foetuses. Many characteristics of the anaemia of the f/f foetus can be accounted for if it is assumed that there is a deficiency in the production or activity of an enzyme involved early in the haem synthetic pathway.     

Effect of blood transfusion on erythropoiesis of homozygotic beta-thalassemia patients

The influence of blood transfusion on erythropoiesis (bone marrow erythroblasts, peripheral blood erythroblasts and reticulocytes) has been studied in 20 non splenectomized homozygous beta thalassaemia patients aged 3 to 16 years and in 10 splenectomized patients aged 8 to 24 years affected with the same disease. The number of reticulocytes was the same in the two groups but the number of erythroblasts in the splenectomized group was higher than in the other group. There was no correlation between the erythroblasts and the reticulocytes of the peripheral blood on one hand and the haemoglobin level proceeding from the same sample on the other hand. In the non splenectomized group of patients, an inverse relationship was found between the percentage of bone marrow erythroblasts and the mean annual haemoglobin level (r = -0.71; p less than 0.01). These results demonstrate the effect of blood transfusion on the erythroid cell line in homozygous beta thalassaemia and the delay between the transfusions and the medullary erythroblastic response.     

Haematological and haematopoietic studies in pyridoxine deficient fish, Channa punctatus Bloch

Pyridoxine deficiency was induced in a population of Channa punctatus. The deficiency effects were increased by adding 4-deoxypyridoxine as an antagonist. Haematological and haematopoietic changes were studied. The significant changes in peripheral blood appeared after 150 days in the antagonist treated group and 180 days in the group on the deficient diet. After 240 days when morphological changes and an increased mortality also appeared, the changes in RBC and related values (haemoglobin, haematocrit, MCV, MCH & MCHC) were highly significant and indicated hypochromic microcytic anaemia; this was accompanied by leukopenia. The haematopoietic studies revealed significant increase in the earlier stages of erythrocytic development (basophilic erythroblasts and polychromatophilic eryth-roblasts) with a simultaneous decrease in the later stages (young and mature reticulocytes). A series of pathological disorders in the various developing cells were recorded in the imprints of haematopoietic tissues. The pyridoxine specificity of these changes were confirmed by remedial treatment for a month.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro

Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for practical use in research have been difficult to produce from this system. Here, we report an improved method of hematopoietic stem cell culture for P. vivax infection, which requires less time and produces higher or equivalent percentage of reticulocytes than previously reported systems. Reticulocytes were cultured from cryopreserved erythroblasts that were frozen after 8 day-cultivation of purified CD34 + cells from human umbilical cord blood. This method of production allowed the recovery of reticulocytes in a shorter time than with continuous stem cell culture. We obtained a relatively high percentage of peak reticulocyte production by using co-cultivation with a mouse stromal cell line. Using P. vivax mature stage parasites obtained from infected Aotus monkeys, we observed substantial numbers (up to 0.8% of the total number of the cells) of newly invaded reticulocytes 24 h after initial cultivation. The addition of fresh reticulocytes after 48 h culture, however, did not result in significant increase of second cycle reticulocyte invasion. Assays of invasion inhibition with specific antibodies were successful with this system, demonstrating potential for study of biological processes as well as the conditions necessary for long-term maintenance of P. vivax in vitro.     

Relation between haemoglobin degradation and maturity of the red blood cell infected by P. berghei

The action on haemoglobin of P. berghei growing in mature red cells, P. berghei growing in reticulocytes and P. berghei R.C. (which grows almost exclusively in reticulocytes) was compared. P. berghei growing in reticulocytes had a much higher level of proteolytic activity on haemoglobin than that of P. berghei growing in mature red cells. The amount of residual hematin was considerably reduced. In P. berghei R.C. and P. berghei growing in reticulocytes, the pigment seems to be exocyted as it is forming. The mechanism of haemoglobin degradation seemed therefore to be linked to the nature of the host red cell.     

Penetration of maturating red blood cells by Toxoplasma gondii

Penetration of Toxoplasma gondii tachyzoites was studied in vitro using murine erythroid cells at different stages of development. Toxoplasma gondii penetrated nucleated erythroblasts and macroreticulocytes from foetal mouse liver and the circulating erythrocytes of foetal, neonatal or severely anaemic adult mice. Immature reticulocytes were more susceptible to penetration than mature ones, indicating that some change in their membrane properties occurred during maturation. The present results confirmed our previous finding that the major erythrocyte membrane-specific proteins do not prevent erythrocyte penetration since these proteins are known to be present in the reticulocyte membrane.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.     

Effects of erythrocyte lysate and erythrocyte-conditioned medium on erythroid cells in vitro.

A component of erythrocyte-conditioned medium has been shown to inhibit the incorporation of tritiated thymidine into erythroblasts from fetal mouse liver, proliferating in vitro. This component, however, has no detectable effect on the growth of colonies of erythroid cells stimulated to grow in viscous culture media by the hormone erythropoietin. Erythrocyte lysate and preparations of haemoglobin derived from the lysate increase the number and size of the colonies growing in vitro. Results are discussed in terms of possible control mechanisms in erythropoiesis.     

Erythropoietin control of terminal erythroid differentiation: maintenance of cell viability, production of hemoglobin, and development of the erythrocyte membrane

Using the spleen cells of mice infected with the anemia-inducing strain of Friend leukemia virus, an in vitro model system of erythropoiesis has been developed in which a homogeneous population of murine proerythroblasts terminally differentiates in response to erythropoietin (EP). The biochemical events involved in EP's capacity to maintain viability, induce hemoglobin production, and promote the development of the specialized erythrocyte membrane were studied during the 48-72 hour period required for proerythroblasts to differentiate into reticulocytes. The results show that EP increases glucose uptake and the syntheses of RNA and protein in the first few hours after exposure of the erythroblasts to the hormone. A coordinated production of heme, alpha and beta globin occurs later and peaks at about 48 hours. This peak corresponds to the time at which the majority of cells are undergoing enucleation and becoming reticulocytes. The syntheses of the erythrocyte membrane and membrane skeletal proteins are not coordinated, and multiple patterns of synthesis are found with respect to the time of EP exposure. A number of proteins are lost from the membrane fraction while the characteristic proteins of the mature erythrocyte become prominent in the membrane fraction of erythroid cells as they develop from reticulocytes into erythrocytes.     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

Transformation of erythrocytes and their ultrastructure with respect to aging

Transformation and ultrastructure of erythrocytes have been investigated in 58 white non-inbred rats on the 1st, 2nd, 3d days after birth and at the age of 1.5, 3, 8, 20 months. The transformation index of the erythrocytes drops by the 8th month of life and further it rises in old animals. In newborn rats echino-poikiloid forms predominate; they are mainly presented as reticulocytes with remnants of nuclei, mitochondria and other organells. During first three days of life cells of the erythroid line actively free themselves from the nucleus by means of its pyknosis and chromatinolysis. In newborn rats erythrocytes have folds of plasmolemma of linear and spotted form on their surface; their number sharply decreases in mature animals. The least changes in form and ultrastructure of erythrocytes are noticed in 3-8-month-old animals.     

AN ULTRASTRUCTURAL STUDY OF EARLY MORPHOGENETIC EVENTS DURING THE ESTABLISHMENT OF FETAL HEPATIC ERYTHROPOIESIS

Morphogenetic events are described which characterize early stages of the interaction between mesenchyme and expanding epithelial cell cords derived from the hepatic endodermal diverticulum in the C57BL/6J mouse. This interaction culminates in the differentiation of hepatic epithelial and hematopoietic tissues. No basement membrane separates the presumptive hepatic epithelial cells from the adjacent mesenchyme, while intercellular attachments, both adherent junctions and desmosomes, are established transiently between heterologous cell types across this epithelio-mesenchymal interface. Yolk sac-derived erythroblasts found in the primitive liver are distinguished morphologically from endogenous hepatic erythroid cells; they are confined to the vascular compartment and are not, apparently, precursors for hepatic erythropoiesis. The earliest recognizable endogenous hepatic hematopoietic cells appear, extravascularly, among those mesenchymal cells in intimate contact with the endodermal epithelium between the 10¼ and 10½ gestational day. Definitive erythropoiesis commences between the 10½ and 11th fetal days. The ultrastructure of these primitive hepatic erythroid cells (proerythroblasts) and their transition to more mature forms (basophilic and polychromatophilic erythroblasts) are described.     

五唇兰雌配子体发育和胚胎发生的研究

五唇兰的胚珠倒生型,具薄珠心,两层珠被。胚囊发育为双孢子葱型,成熟胚囊８核。从传粉到受精约５０ｄ,正常双受精。胚具５－６细胞的胚柄,种子成熟时胚柄及胚乳核消失,成熟种子只具单层细胞的种皮和一个未分化的珠珠形胚。     

The red blood cells in growing guinea pigs. (Cavia cobaya). II. Communication: erythropoiesis and red blood cells in the postnatal development period

Bone-marrow smears of 175 guinea pigs aged 1-27 days and venous blood samples of 351 animals aged 1-25 days were prepared for cell counting. A significant increase of erythroblasts were found between life day 1 and 2; normoblasts increased in number synchronously with a decrease of erythroblasts after the 5th day. The percentage of the erythroid bone marrow increased from 10 to 14 during the developmental period. Beyond the perinatal period the red blood picture is characterized by the following changes: a decrease of erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin; a constant mean corpuscular hemoglobin concentration; an increase of the reticulocyte count. The decrease of the red cell count is compensated by a decreasing oxygen affinity attained by an important increase of 2,3-DPG. Nevertheless, the stimulus for a raising erythropoiesis remains constant which can be shown by the growing percentage of erythroid cells and reticulocytes. The difference between the human postnatal development and that of the guinea pig becomes obvious. Cell counts in dependence of body masses in postnatally growing guinea pigs, veil the perinatal finding of the increase in erythrocytes up to the 5th day and the decrease of the mean corpuscular volume after the 3rd day.     

Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co-signaling effects

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.     

Phosphate incorporation into "nuclear" residual proteins of goose erythrocytes

Minor fractions of “residual proteins” from erythroblast-enriched avian erythroid cells were found to incorporate significantly larger amounts of radiophosphate than similar fractions from mature erythrocytes. This higher level of incorporation could not be detected in the bulk, unfractionated residual proteins from cell populations, respectively, enriched in erythroblasts, reticulocytes, or mature erythrocytes, probably because of variable amounts of phosphate-free protein. It was confirmed that when avian erythroid cells incorporated radiophosphate, specific activities of chromosomal “residual proteins” were orders of magnitude greater than those of histones, although levels of alkali-labile phosphate were only a fewfold higher.     

Scanning cytophotometry of carp, Cyprinm carpio L., erythrocyte populations: the influence of short-term hypoxic environment and the recovery period following severe bleeding

Scanning stage absorbance cytophotometry was used to examine haemoglobin contents in individual cells or carp erythrocyte populations. Changes in frequency distributional profiles in response to respiratory stress caused experimentally by hypoxia and/or bleeding were studied at intervals.
Histograms were drawn of haemoglobin contents of individual red blood cells in populations obtained from the same carp on four consecutive days after the fish was temporarily exposed to hypoxic environment. The modes and means of the haemoglobin values increased during the 3 days following to the stimulus, whereas a decline was measured on the 4th day. Erythrocyte counts showed the total number of red blood cells to be approximately constant during the period of investigation. On the basis of these findings, it is concluded that the mature, nucleated red blood cells of carp are capable of resuming haemoglobin synthesis after stimuli such as reducing ambient oxygen concentration.
Frequency distributional profiles covering a period of 4 weeks following severe loss of blood showed that it took 10–12 days after bleeding until masses of immature erythrocytes appeared in the peripheral blood and that there were then two distinct populations of red blood cells. In the course of about 2 weeks the precursor cells developed into mature erythrocytes.