Iron incorporation and haemoglobin synthesis in erythropoietic cells during the ontogenesis of the mouse

0 the Department of Anatomy, Erasmus University , Rotterdam 1 From the Beatson Institute for Cancer Research , Glasgow SUMMARY Iron incorporation (59Fe) into erythropoietic cells from adult and foetal (11- to 15-day) peripheral blood and from foetal (12- to 15-day) livers has been investigated. Ion-exchange chromatography of haemolysates from such cells revealed two groups of 59Fe-containing proteins. The first group (X-fraction) was eluted from CMC-columns in the void volume and was highest in lysates of immature erythropoietic cells. This fraction contained a radiolabelled haemprotein of high molecular weight as well as other 59Fe-containing proteins. The haemprotein does not appear to be related to haemoglobin. The second group consisted of haemoglobins. One major (Ax) and two minor (A2 and A3) haemoglobins were found in adult peripheral blood. In foetal liver lysates two major (Fx and Ax) and two minor (F2 and A3) haemoglobins were present. The relative proportion of the major haemoglobins changed during development. Haemoglobin Fx was highest in the more mature livers. Fx proved to be different from Ax by chromatographic behaviour, in polypeptide chain composition and in fingerprints. A unique foetal polypeptide chain, intermediate in electrophoretic behaviour between the adult a- and /?-chain, was identified. In young foetal peripheral blood (11-day), in which 95 % of the cells are of yolk-sac origin, one major (Ex), two intermediate (E2 and E3) and one minor (Fj) haemoglobin were demonstrable. Haemolysates of the peripheral blood of older embryos contain haemoglobins from erythroid cells of both yolk-sac and foetal liver origin. The haemoglobin pattern of such lysates is explicable in terms of the decreasing amount of embryonic haemoglobins (Ei, E2 and E3) and the increasing amount of foetal haemoglobins (Fx and Ax). Since Ax and Ex are the most prominent haemoglobins of livers from young embryos and yolk-sac erythrocytes respectively, and since they are very similar in chromatographic behaviour, foetal peripheral blood at all stages contains one dominant haemoglobin peak in the Ax-Ex region. Most authors have neglected the relatively slight elevation of the foetal haemoglobin peak (Fx) in front of Ax-Ex, the more because the Fx-Ai region has been suspected sometimes to contain artificial haemoglobin components (Riggs, 1965). This probably explains why no foetal haemoglobin (Fx) has been reported previously in the peripheral blood of foetal mice. - Erythroid cell differentiation represents the process whereby a multipotential precursor cell becomes committed to devoting most of its synthetic machinery to the production of a specific protein, haemoglobin. This process involves the co-ordinated control of many biochemical and morphological events, some of which have been elucidated using in vitro cultures of erythropoietic cells stimulated to differentiate by the hormone erythropoietin (for review, see Harrison, Conkie & Paul, 1973). A relatively early event in such cultures is the stimulation of iron incorporation and the synthesis of haem (Krantz & Fried, 1968; Hrinda & Goldwasser, 1969; Gross & Goldwasser, 1970; Shepp, Toff, Yamada & Gabuzda, 1972). This is then followed at a later stage by the synthesis of haemoglobin. However, the mechanism whereby iron is transported from the cell surface, where it is delivered by serum transferrin (Katz & Jandl, 1964), to the mitochondrion, where it is utilized for haem synthesis, has not yet been elucidated. Furthermore, very little is known about the factors involved in transport of haem to the globin chains. Thus more information would be valuable concerning the role of iron and haem-iron-containing proteins, including the haemoglobins, during erythroid cell development. This paper describes experiments concerning the relative proportions and characteristics of iron- and haemproteins in erythroid cells of yolk-sac and foetal liver origin in the mouse. It is likely that the relative proportions of the iron- and haemproteins differ greatly in mature and immature cells. Information about this may be useful, not only as information about embryonic development, but also to explain discrepancies which may arise when calculations of radioactive iron or haem-iron incorporation into haemopoietic cells are used to quantitate haemoglobin synthesis (Harrison et al. 1973). MATERIALS AND METHODS Collection and lysis of cells. Adult Porton white Swiss mice were bled by decapitation. Foetuses of the same strain aged 11-15 days (the day on which vaginal plugs were observed being taken as day zero) were washed thoroughly and bled from the umbilical vessels for peripheral blood cells. The livers were excised, washed thoroughly and disaggregated by pipetting. Isotonic ice-cold saline was used for washings and cell collection. The blood cell suspensions were washed three times in at least a tenfold volume of saline. Following the final washing the cells were resuspended in a buffer containing 0-81 % NaCl, 0-12 % Tris and 0-03 % Mg acetate, pH 7-0. When all clumps were dissociated, NP40 (Nonidet P40, Shell Chemicals) was added to 0-5 %. Lysis was allowed for about 30 min. In some experiments the cells were lysed by adding glass-distilled water, but, especially with foetal liver cells, the NP40 method gave better results. The haemoglobins were converted to the CO-form, centrifuged and dialysed overnight against the starting buffer. All manipulations were done at 4 C. No Fe incorporation in erythropoietic freezing-and-thawing, ammonium sulphate precipitation, conversion to cyanmethaemoglobin or ageing of the haemolysate (more than 24 h) was allowed, as all this is known to promote extra haemoglobin peaks (Riggs, 1965; Manwell, Baker & Betz, 1966). An occasional experiment was done with Friend cells (clone M2) grown in bulk cultures as described by Conkie, Affara, Harrison & Paul (1974). Erythroid differentiation in Friend cells was induced by culturing for 5 days in the presence of 1-5 % dimethyl sulphoxide. Preparation and estimation of radiolabelled proteins. Ten /*Ci [59Fe]chloride or citrate (spec. act. 2-10 ^Ci/^g Fe) was injected intraperitoneally into adult and pregnant animals, 24 h before death. In one experiment [3H]leucine (250/tCi) was added to a cell culture of mouse erythropoietic cells, 5 days before cell harvest. The radioactivity of the 59Fe-labelled fractions was measured by adding aliquots of the separated fractions to Triton X-100/toluene-based scintillator (1:2, v/v) and counting them in a liquid scintillation counter (Beckman LS-100). [3H]leucine-labelled fractions were acid-precipitated on to glass-fibre discs and counted in a toluene-based scintillator. Chromatography ofproteins. Anion-exchange chromatography was performed over carboxymethyl cellulose (Whatman CM 52) columns using 0-01 M sodium phosphate buffer in a linear gradient from pH 6-7 to 8-2 for elution. Cationexchange chromatography was performed over (...truncated)