Haemoglobins of the foetal and adult rat: sites of synthesis and the effects of erythropoietin

By J. A. HUNTER 0 1 0 Authors' address: The Beatson Institute for Cancer Research, Royal Beatson Memorial Hospital , 132 Hill Street, Glasgow, C.3, Scotland 1 From the Beatson Institute for Cancer Research , Glasgow Early studies on rat haemoglobins indicated the existence of several components (Gratzer & Allison, 1960). Seven components were found in the blood of adult random-bred Wistar rats by CM-cellulose chromatography; their rates of synthesis appeared unaltered during erythroid cell maturation (Brada & Tobiska, 1964). A 5-component electrophoretic pattern found in some randombred Wistar rats was considered to be a combination of two 4-component patterns observed in two inbred strains (Marinkovic, Martinovic & Kanazir, 1967). A 4-component electrophoretic pattern was also reported by Cole, Hunter & Paul (1968). During gestation the number of components resolved in this buffer system increased from 1 at 13 days to 4 at 15 days; the pattern appeared identical to that of the adult from 18 days onward. Erythropoietin stimulates stem cells to mature to erythrocytes (Filmanowicz & Gurney, 1961; Orlic, Gordon & Rhodin, 1965; Nakao, Miura & Takaku, 1966). It appears to have no direct effect on haemoglobin synthesis (Erslev, 1962, 1964). In preliminary studies it was found that although erythropoietin increases the rate of haemoglobin synthesis by foetal rat liver cells in the early stages of organogenesis, it does not alter the nature of the haemoglobin produced (Cole et al. 1968). This paper presents a more complete study of haemoglobin synthesis during rat foetal development and the effects exerted by erythropoietin. - 362 J. A. HUNTER & J. PAUL replicate tubes. Haemoglobin synthesized in culture was labelled with 59Fe for fixed periods and was released from the cells by lysis in distilled water and extraction with chloroform. The haemoglobins were separated by starch gel electrophoresis using the discontinuous buffer system of Poulik (1957). The distribution of radioactivity after electrophoresis was determined as previously described (Cole et al. 1968). Gels were stained for protein with naphthalene black (Smithies, 1955) and for haemoglobin with o-dianisidine (O'Brien, 1961). Five components were separated in this buffer system; the fastest was termed haemoglobin ' a ' . The relative mobilities of the other four components, termed haemoglobins ' b ' , V , ' d ' and ' e \ were respectively 0-87, 0-68, 0-45 and 0-17 of that of haemoglobin a. In adult blood these were present in the following ratios, taking the value for haemoglobin a as 1; haemoglobin b, 1; haemo I I I I I I I I I i i 1 i r L _ _ l -I I I I l~l I I I I I lOrigin Foetal age (days) Fig. 1. Haemoglobin components from rat peripheral blood at different developmental ages. The diagram represents positions on starch gel electrophoretograms, run in the conditions described in the text. globin c, 0-0-06; haemoglobin d, 0-2; haemoglobin e, 0-17. As shown in Fig. 1, only haemoglobins b and c were found in 12- and 13-day foetal blood, haemoglobin b being the fainter of the two. Haemoglobin a appeared early on day 14, haemoglobin d later in the same day; haemoglobin e appeared on day 15. From day 18 the proportions of haemoglobins a, d and e appeared constant and similar to the adult pattern; the proportion of haemoglobin b increased before and after birth while that of haemoglobin c decreased. Cultures of 10-day yolk-sac cells, prepared from embryos after removal of the ectoplacental cone, synthesized only haemoglobins b and c, and were unaffected by erythropoietin treatment (Table 1). Cultures of 12- and 13-day foetal liver cells synthesized haemoglobins a, b and c; the relative rates of synthesis of the components were the same during periods of incubation some 24 h apart (Table 1). A small amount of haemoglobin d was made by 14-day foetal liver cell cultures, in addition to haemoglobins a, b and c. Regardless of erythropoietin treatment, the relative rates of synthesis of the components were unaltered during incubation (Table I). Tissue of origin 13-day foetal liver 14-day foetal liver 15-day foetal liver 18-day foetal liver 20-day foetal liver Haemoglobin components as % of total haemoglobin Untreated cultures Cultures of 15-day foetal liver cells synthesized all five components and were sensitive to erythropoietin treatment. The proportion of each component was unchanged during incubation, irrespective of erythropoietin stimulation (Fig. 2). Eighteen-day foetal liver cells were unaffected by erythropoietin, quantitatively as well as qualitatively; the relative amounts of each component were unchanged during incubation (Table 1). Cultures of adult marrow cells also synthesized all five components, in proportions differing from those observed in cultures of foetal cells (Fig. 3). These cells responded to erythropoietin with an 364 J. A. HUNTER & J. PAUL over-all increase in the synthesis but no change in the proportions of the components; the latter was also unaltered during 30 h incubation (Table 1). These findings show that treatment with erythropoietin in vitro does not alter the nature of the haemoglobins made by rat erythroid cells. To determine whether this were the case in vivo, studies were made of the pattern of haemoglobin synthesis by marrow cell cultures from normal and anaemic adult rats. A group of animals was made anaemic by bleeding, another by injection of Fig. 2. Rates of synthesis of different haemoglobin compounds from 15-day foetal rat livers. The cells were incubated with 59Fe and the incorporation into haemoglobin determined as described in the text. No erythropoietin added, # ; 0-5u/ml step 2 sheep erythropoietin added, OO. (a) 59Fe added from 0 to 6 h; (b) 59Fe added from 24 to 30 h. phenylhydrazine; an untreated third group of animals provided cultures of normal marrow cells. As shown in Table 2, the patterns of haemoglobin synthesis were virtually identical in cultures of normal and anaemic marrow. It can be seen that the proportions of the different haemoglobins in the circulating blood is reflected in the proportions synthesized by the livers of different ages. Erythropoiesis also occurs in the rat spleen during the last third of gestation; the pattern of haemoglobin synthesis in this organ is similar to that in livers of the same age (Table 1). It can be seen from Table 1 that in culture the haemoglobin components were synthesized in the same ratios during 30 h incubation. To determine whether this pattern persisted in the last stages of cell maturation in vivo the haemoglobin synthesized by circulating cells was examined. The results are shown in Table 3. Haemoglobin synthesis by 12-day foetal blood cells resembled that of 10-day yolk-sac cells, some 42 h earlier. The pattern observed in cultures of 16-day blood cells resembled that of 15-day foetal liver cells some 28 h earlier in Fig. 3. Rates of synthesis of different haemoglobin compounds from adult rat (...truncated)