THE NATURE OF IMMATURE AVIAN ERYTHROCYTES IN SEVERE ANAEMIA INDUCED BY PHENYLHYDRAZINE

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1 J. Cell Sci. ii, 77>-776 (1972) 771 Printed in Great Britain THE NATURE OF IMMATURE AVIAN ERYTHROCYTES IN SEVERE ANAEMIA INDUCED BY PHENYLHYDRAZINE A. F. WILLIAMS* Department of Biochemistry, Adelaide University, Adelaide, Australia SUMMARY Hens were rendered severely anaemic by the use of phenylhydrazine and the characteristics of the polychromatic erythrocytes appearing in the blood examined. Even the least-dense polychromatic erythrocytes obtained after density fractionation of anaemic blood on BSA density gradients had a volume mode only 1-5 times that of mature erythrocytes. Also the immature cells always contained less haemoglobin than mature erythrocytes. It was concluded that in severe anaemia avian erythropoiesis still consists of only one cell line, and that cells do not miss divisions as is believed to be the case in mammalian erythropoiesis. INTRODUCTION It is well known that the severe anaemia produced in mammals after phenylhydrazine treatment is characterized by the production of very large reticulocytes with a high haemoglobin content per cell (Brecher & Stohlman, 1961; Borsook, Lingrel, Scaro & Millette, 1962; Glowacki & Millette, 1965). In rats the modal cell volume of reticulocytes in severe anaemia was found to be almost 3 times that of normal erythrocytes (Brecher & Stohlman, 1961), and Glowacki & Millette (1965) have reported the identification of reticulocytes up to 4 times larger than normal in low-density fractions of anaemic rabbit cells separated on a BSA density gradient. Borsook et al. (1962) also showed that the large reticulocytes had the lowest density of anaemic blood cells, and that they had a haemoglobin content of up to 40 % more than the normal amount on a per cell basis. From the viewpoint of one wishing to study the erythropoietic cell series as a model of differentiation, the production of atypical reticulocytes gives rise to added technical and conceptual difficulties. For instance, Borsook and co-workers (Borsook et al. 1968; Borsook, Ratner & Tattrie, 1969) define 2 erythroid cell lines which differ in predominance in the anaemic rabbit compared with the normal animal. As mentioned in an earlier paper (Williams, 1972) avian erythropoiesis is a useful system in which to study differentiation since the cell nucleus is retained at all cell stages and progressively expresses less of the genome. For such an experimental system induction of anaemia by phenylhydrazine treatment is essential and therefore it is of importance to know whether it provokes the formation of atypical cells in birds as well as mammals. Present address: Department of Biochemistry, University of Oxford, England.

2 772 A. F. Williams In this paper the size, haemoglobin content, and density of the cells present in anaemic hen blood are described. Cells analogous to the atypical reticulocytes of anaemic mammals were not found and it appeared that even in severe anaemia there is only one cell series in avian erythropoiesis. MATERIALS AND METHODS The materials, methods and nomenclature used to describe avian cell types were as described in Williams (1972). RESULTS In a previous paper (Williams, 1972) details of the cell populations of anaemic and normal hens were described. Cell volume distributions were reported, and the modal cell volume of polychromatic erythrocytes from anaemic blood was 50 % greater than that of mature erythrocytes in normal blood. This volume increment was much less than that found between reticulocytes and erythrocytes of anaemic and normal mammalian blood. One can further examine the avian situation by studying anaemic blood cells centrifuged on BSA density gradients. If atypical cells were produced similar to those in mammalian erythropoiesis the least dense polychromatic erythrocytes should be much larger than normal erythrocytes and have a higher haemoglobin content. The results of such an experiment are seen in Fig. 1 which shows cell distribution, modal cell volume and haemoglobin content (Fig. 1 A) and distribution of histological cell types (Fig. IB). It will be noted that the lowest-density cells had a low haemoglobin content (one-third of the normal amount of 3 x io~ 8 mg/cell) and were larger than the more dense ones only in the fractions containing erythroblasts which would probably divide again (Williams, 1972). Cell volume distributions of the fractions shown in Fig. 1 are plotted in Fig. 2 and reinforce the above conclusions. More than 10 experiments similar to that shown in Fig. 1 have been carried out, and in no case have the low-density cells had a higher haemoglobin content than the denser ones. The results are completely typical with the exception that clear-cut separation of erythroblasts was not always obtained from anaemic blood. Thus, it seemed clear that in severe anaemia avian erythropoiesis does not respond in the same bizarre manner typical of the mammalian process. However, the results *n Fig. 1 do not clearly show that erythropoiesis is precisely the same as normal in the anaemic hen. Are the polychromatic erythrocytes from anaemic blood larger than mature erythrocytes simply because they are immature or for some other reason? To clarify this point the cell volume distribution of a preparation of polychromatic erythrocytes obtained from normal bone marrow cells was determined. After centrifugation of normal marrow cells on a BSA density gradient, cell distribution, haemoglobin content and modal cell volume were determined (Fig. 3 A) as was the distribution of histological cell types'(fig. 3B).

3 Anaemia and avion erythrocytes 773 E 200 H u Density, g cm" Fig. i. Centrifugation of anaemic blood cells on a BSA density gradient. Blood cells from a highly anaemic hen were centrifuged on a BSA density gradient. The following parameters were determined on the fractions obtained, A: A, cell distribution (cells x io~* per fraction);, haemoglobin content (mg xio 8 per erythroid cell); O. median cell volume Gum 3 ), B, cell-type distribution as a percentage of cell population: #, erythroblasts; A, early and mid-polychromatic erythrocytes; O, late polychromatic erythrocytes;, non-erythroid cells.

4 774 A. F. Williams 200 Volume, /tm* Fig. 2. Volume distribution of anaemic blood cells of different densities. Cell volumes were determined on samples from the fractions obtained in the experiment shown in Fig. i A, B. D, Density fraction gem- 1 ;, 1-064; A, 1-072; A., 1-082; O The arrow indicates the modal cell volume of normal blood cells. 400 The fraction at density 1-07 gcm~ 3 contained 65% polychromatic erythrocytes of low haemoglobin content. Contamination with non-erythroid cells was high as was the case with all low-density fractions from normal marrow;* however, the modal volume of polychromatic erythrocytes could be identified in the cell volume distributions determined with the Coulter Counter. As shown in Fig. 3 c this was 22 % greater than that for mature erythrocytes from the blood of the same animal. From these results it seems unlikely that all of the volume increment between the polychromatic erythrocytes of anaemic blood, and mature erythrocytes can be attributed to the immaturity of the former cells. DISCUSSION Results reported in this communication suggest that in severely anaemic hens the polychromatic erythrocytes appearing in the blood were slightly larger than normal The contamination of low-density fractions with non-erythroid cells was much worse in experiments where normal bone marrow was separated on BSA density gradients than in those where anaemic bone marrow was used (Williams, 1972). The great proliferation of low-density erythroid cells in the anaemic bone marrow probably explains the difference.

5 Anaemia and avian erythrocytes S 20 - o E Density, g cm" 3 n in Is per <J \ c D 1 m /\ / \ / ) K \ / a i 100, Cell volume, Fig. 3. Centrifugation of normal bone marrow on a BSA density gradient. Normal bone marrow cells were centrifuged on a BSA density gradient. The following parameters were determined on the fractions, A: T, cell distribution (cells x io" 8 per fraction);, haemoglobin content (mg Hb x io 8 /erythroid cell); O, median cell size (ftm 3 ). B, cell-type distribution as a percentage of the cell population: #, erythroblasts; A, early and mid-polychromatic erythrocytes; O. l ate polychromatic erythrocytes; Q, mature erythrocytes;, non-erythroid cells, c, cell volume distributions:, normal blood;, fraction of density i'o7 g cm"' from density gradient.

6 776 A. F. Williams polychromatic erythrocytes. However, they had a normal or reduced haemoglobin content and the volume increment was not such as to suggest that cells had missed divisions as seems to occur in mammalian erythropoiesis. One might suggest that the failure to observe very large cells was due to the fact that anaemic stress was not great enough. However, this seems unlikely since the phenylhydrazine dose could not be increased without causing death. Also as has been previously reported (Williams, 1972), the cell populations used have been characterized as immature by standards of density, brilliant cresyl blue staining and ability to synthesize RNA. Thus, it seems likely that in the anaemic hen a basically normal erythropoietic cell series is maintained. This idea is in accord with the results presented here and is also consistent with the fact that clear-cut separation of cells at various stages of differentiation could be made from anaemic bone marrow on the basis of density (Williams, 1972). Such a simple situation does not seem to hold in the separation of erythroid cells from mammalian bone marrow (Borsook et al. 1969). Thus, it seems that in mammals the additional process of enucleation is superimposed upon the more primitive avian erythropoietic series. In severe anaemic stress this may be an advantage as cells can be recruited from the marrow at much earlier stages than normal. During the course of this work the author was the holder of a C.S.I.R.O. post-graduate studentship. REFERENCES BORSOOK, H., LINGREL, J. B., SCARO, J. L. & MILLETTE, R. L. (1962). Synthesis of haemoglobin in relation to the maturation of erythroid cells. Nature, Land. 196, BORSOOK, H., RATNER, K., TATTRIE, B., TIEGLER, D. & LAJTHA, L. G. (1968). Erythropoietin and the development of erythrocytes. Nature, Land. 217, BORSOOK, H., RATNER, K. & TATTRIE, B. (1969). Studies on erythropoietin. II. A method of segregating immature from mature adult rabbit erythroblasts. Blood 34, BRECHER, G. & STOHLMAN, F. (1961). Reticulocyte size and erythropoietic stimulation. Proc. Soc. exp. Biol. Med. 107, GLOWACKI, E. R. & MILLETTE, R. L. (1965). Polyribosomes and the loss of haemoglobin synthesis in the maturing reticulocyte. J. molec. Biol. 11, WILLIAMS, A. F. (1972). DNA synthesis in purified populations of avian erythroid cells. J. Cell Sci. io, {Received 21 February 1972)