THE FORM OF HAEMOGLOBIN IN THE ERYTHROCYTES OF THE COD, GADUS CALLARIAS

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1 J. Cell Set. 8, (1971) 407 Printed in Great Britain THE FORM OF HAEMOGLOBIN IN THE ERYTHROCYTES OF THE COD, GADUS CALLARIAS N.W.THOMAS Department of Anatomy, Marischal College, Aberdeen, Scotland SUMMARY The erythrocytes of the cod, Gadiu callarias have a flattened discoid shape; each contains a peripheral marginal band of approximately 10 microtubules and a centrally placed nucleus. The cytoplasmic and nuclear haemoglobins have a paracrystalline organization; individual filaments, presumably polymers of haemoglobin, are grouped into bundles which course in all directions through the cell. This organization shows a remarkable similarity to that of deoxygenated haemoglobin in sickled human red cells. However, paracrystalline haemoglobin in the cod is characteristic of the normal red cells and is not associated with a crenated shape. INTRODUCTION The detailed organization of functioning haemoglobin within the cytoplasm of the erythrocyte has yet to be elucidated. In previous fine-structure studies of nucleated erythrocytes particular attention has been given to the morphology of the erythrocyte nucleus (Davies, 1961, 1968; Davies & Spencer, 1962; Davies & Small, 1968; Tooze & Davies, 1963; Everid, Small & Davies, 1970; Small & Davies, 1970), and the nature and significance of the marginal band of microtubules (Fawcett & Witebsky, 1964; Maser & Philpott, 1964; Barclay, 1966; Sekhon & Beams, 1969; Behnke, 1970). In the electron micrographs from these studies haemoglobin appeared as a homogeneous electron-dense material. This communication presents observations made on the fine structure of the erythrocytes of the cod in which haemoglobin appears to have a paracrystalline organization; the findings are discussed with reference to similar studies on sickled human red cells (Dobler & Bertles, 1968; White, 1968) in which haemoglobin has a paracrystalline form. MATERIAL AND METHODS Specimens of cod (Gadus callarias) which had been collected by netting in Aberdeen Bay were killed with a blow on the head, and tissues and 2-4 ml of cardiac blood removed. No special steps were taken to ensure the complete deoxygenation of the cardiac blood samples while the tissues were assumed to contain erythrocytes at different states of deoxygenation. The tissues were fixed immediately in 3 % glutaraldehyde buffered at ph 7-3 with 01 M phosphate buffer, at 0-4 C. (Sabatini, Bensch & Barrnett, 1963). Blood samples were reduced to a pellet by centrifugation (30 x io 4 g av min. in a M.S.E. Mistral 6L centrifuge, angle head 62302) and the buffy layer removed before treatment with the same fixative. After fixation for 1 h the material was washed in buffer and post-fixed in 2 % osmium tetroxide in the same buffer

2 408 N. W. Thomas system, dehydrated through graded acetones and embedded in Epon (Luft, 1961). Sections were cut on a Reichert Om 2 ultramicrotome, stained with uranyl acetate and/or lead citrate (Reynolds, 1963) and viewed in a Jem 7 electron microscope. OBSERVATIONS In these specimens the erythrocytes had a flattened discoid shape and a diameter between 7-5 and 9-5 /im (Fig. 1). The nucleus was centrally placed and flattened in the main axis of the cell. At low magnifications the cytoplasm appeared homogeneous and finely granular while at higher magnifications fine filaments were apparent. These filaments were of indeterminate length and of about 13 nm diameter; when observed in cross-section the filaments appeared tubular with an electron-lucid core, and had an angular outline (Fig. 4). In some micrographs the filaments appeared to be made up of subunits and to have fine lateral arms projecting towards adjacent filaments. Large numbers of filaments in parallel array and equidistant from each other were grouped into bundles which coursed through the cytoplasm (Fig. 2) and showed no preferred orientation. The bundles did not distort the discoid shape of the cell. Bundles of similar filaments were observed in the nucleoplasm and were easily distinguished from the granular chromatin distributed around the periphery of the nucleoplasm (Fig. 3). The nuclear membrane was pierced by nuclear pores but no continuity between cytoplasmic and nuclear bundles was observed. Free ribosomes, mitochondria and myelin bodies were present in the cytoplasm surrounding the nucleus, especially at the poles of the nucleus (Fig. 2). A group of microtubules which made up the marginal band was located beneath the plasma membrane at the region of the cells' greatest circumference (Fig. 4). Up to 12 microtubules were present in the marginal band and each measured approximately 27 nm in diameter. DISCUSSION The erythrocyte of the cod appears to have a similar gross appearance to nucleated red cells from other species, with a marginal band of microtubules and haemoglobin rilling the cytoplasm and some of the nucleoplasm. However, the organization of normal haemoglobin in situ into filaments or tubules, smaller than typical microtubules (Slautterback, 1963), and different from tubular units associated with the nuclear membrane in some species (Davies & Small, 1968; Everid et al. 1970), does not seem to have been described previously. R. Bulger (unpublished observations) has found similar fibrillar haemoglobin in the erythrocytes of the large sea horse (Hippocampus punctulatus) and the midshipman (Porichthys notatus), but in these species the filaments do not appear to have an electron-lucid core and the cells contain fewer bundles. Fawcett & Witebsky (1964) observed crystalline haemoglobin in the nucleus of the toadfish; this consisted of parallel electron-dense striations 6 nm wide separated by interspaces of 6 nm. They pointed out the difficulty of assessing whether crystalline haemoglobin existed in vivo, and the same must also be said of the paracrystalline organization of the cod, in which each filament presumably represents a polymer of

3 Haemoglobin in cod erythrocytes 409 haemoglobin. Ponder (1945) observed that crystallization of haemoglobin in rat erythrocyte occurred after prolonged storage (72 h) in 3 % sodium citrate at 4 C. The present observations were made on tissues fixed immediately on death of the fish and on blood samples fixed within 17 min of death, and it seems likely, therefore, that the paracrystalline organization of cod haemoglobin was not induced by cold treatment. The organization was not preserved when osmium fixation was used alone, but was present in cells in situ and in blood pellets when primary fixation was with glutaraldehyde. The organization showed a remarkable similarity to that observed in sickled cells from patients with sickle cell anaemia. Oxygenated red cells from patients with sickle cell anaemia have a biconcave discoid shape and resemble red cells from normal patients: in the deoxygenated state the cell outlines become irregular and they take on a prickled or holly-leaf shape. Murayama (1966) observed hollow filaments 17 nm in diameter in whole mount preparations of haemoglobin (HbS) from these cells and postulated that each filament was composed of 6 strands of a hypothetical monofilament. White (1968) described filaments 6-7 nm in diameter, which he presumed corresponded to Murayama's monofilament, and also rods nm m diameter in the cytoplasm of sickled erythrocytes. Dobler & Bertles (1968) observed 16-nm diameter filaments arranged in a paracrystalline form in erthrocytes in vivo. The filaments of the cod are slightly smaller than the filaments described in sickled cells but the subunit of the cod tubule may correspond to the monofilament postulated by Murayama (1966). It has been proposed that the change in cell shape in sickling involves the conformation of the erythrocyte membrane to the filaments or rods of deoxygenated HbS (Stetson, 1966; Murayama, 1966; Dobler & Bertles, 1968; White, 1968). However, Bertles & Dobler (1969), observed some sickled cells which showed a typical crenated outline but lacked haemoglobin filaments, and therefore suggested that cell distortion may be another factor dictatingfilament arrangement. The flattened discoid shape of the cod erythrocyte is unaffected by the paracrystalline bundles of haemoglobin and these observations would therefore support this latter hypothesis. The functional significance of the paracrystalline haemoglobin in the cod erythrocyte is as yet unknown. Normal human haemoglobin (HbA) and HbS differ only in the single replacement of the number 6 glutamic acid residue for valine in the B chain (Ingram, 1957) and it is tempting to postulate that cod haemoglobin may show similar substitutions. Unlike the other species offish studied by R. Bulger (unpublished observations), the paracrystalline haemoglobin in the cod completely fills the cell cytoplasm and presumably reflects a highly effective packing system. Preliminary results (N. W. Thomas & T. L. Coombs, unpublished) indicate that treatment with different gases will induce a change in this organization and it seems likely that the cod erythrocyte will prove a model system for the investigation of fine-structural changes of haemoglobin under different gaseous conditions. I am grateful to Dr P. T. Grant, Director of the Fisheries Biochemical Research Station, Aberdeen, for the provision of fish, Dr T. L. Coombs of the Station for valuable discussion and Professor D. C. Sinclair who read this manuscript. I am also grateful to the Science Research Council for financial support.

4 410 N. W. Thomas REFERENCES BARCLAY, N. (1966). Marginal bands in duck and camel erythrocytes. Anat. Rec. 154, 313. BEHNKE, O. (1970). A comparative study of microtubules of disk-shaped blood cells. J. Ultrastruct. Res. 31, BERTLES, J. F. & DOBLER, J. (1969). Reversible and irreversible sickling: a distinction by electron microscopy. Blood 33, DAVIES, H. G. (1961). Structure in nucleated erythrocytes. J. biophys. biochem. Cytol. 9, DAVIES, H. G. (1968). Electron-microscope observations of the organization of heterochromatin in certain cells. J. Cell Sci. 3, DAVIES, H. G. & SMALL, J. V. (1968). Structural units in chromatin and their orientation on membranes. Nature, Lond. 217, DAVIES, H. G. & SPENCER, M. (1962). The variation in the structure of the erythrocyte nuclei with fixation. J. Cell Biol. 14, DOBLER, J. & BERTLES, J. F. (1968). The physical state of hemoglobin in sickle cell anemia erythrocytes in vivo. J. exp. Med. 127, EVERID, A. C, SMALL, J. V. & DAVIES, H. G. (1970). Electron-microscope observations on the structure of condensed chromatin: Evidence for orderly arrays of unit threads on the surface of chicken erythrocyte nuclei. J. Cell Sci. 7, FAWCETT, D. W. & WITEBSKY, F. (1964). Observations on the ultrastructure of nucleated erythrocytes and thrombocytes with particular reference to the structural basis of their discoidal shape. Z. Zellforsch. mikrosk. Anat. 62, INGRAM, V. M. (1957). Gene mutation in human haemoglobin: The chemical difference between normal and sickle cell haemoglobin. Nature, Lond. 180, LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, MASER, M. & PHILPOTT, C. (1964). Marginal bands in nucleated erythrocytes. Anat. Rec. 150, MURAYAMA, M. (1966). Molecular mechanism of red cell sickling. Science, N.Y. 153, PONDER, E. (1945). The paracrystalline state of the rat red cell. J. gen. Physiol. 29, REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SABATINI, D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, SEKHON, S. E. & BEAMS, H. W. (1969). Fine structure of the developing trout erythrocytes with special reference to the marginal band and the cytoplasmic organelles. Am. J. Anat. 125, SLAUTTERBACK, D. B. (1963). Cytoplasmic microtubules, I. Hydra. J. Cell Biol. 18, SMALL, J. V. & DAVIES, H. G. (1970). The haemoglobin in the condensed chromatin of mature amphibian erythrocytes: a further study. J. Cell Sci. 7, STETSON, C. A. (1966). The state of hemoglobin in sickled erythrocytes. J. exp. Med. 123, TOOZE, J. & DAVIES, H. G. (1963). The occurrence and possible significance of hemoglobin in the chromosomal regions of mature erythrocyte nuclei of the newt, Triturus cristatus cristatns. J. Cell Biol. 16, WHITE, J. G. (1968). The fine structure of sickled hemoglobin in situ. Blood 31, {Received 29 August 1970) Fig. 1. Electron micrograph of cod pancreas tissue showing 2 nucleated erythrocytes (e) within a small venule (v). x Fig. 2. Electron micrograph of an erythrocyte in situ showing bundles of haemoglobin tubules running in different directions through the cytoplasm. The tubular nature of the individual units of haemoglobin is clearly seen (arrow). («;, mitochondrion ; my, myelin body; n, nucleus; r, free ribosomes.) x

5 Haemoglobin in cod erythrocytes

6 412 N. W. Thomas Fig. 3. Electron micrograph of an erythrocyte from a pellet showing nuclear haemoglobin (nh) with the same electron-microscopic appearance as that of the cytoplasm, (p, nuclear pore.) x Fig. 4. Electron micrograph of an erythrocyte from a pellet showing the tubular nature of the haemoglobin units. Some have an angular outline with short lateral arms (arrows), (mb, marginal band.) x