J. Anat. (1975), 120, 2, pp. 239-252 With 16 figures Printed in Great Britain

Megakaryocytopoiesis in the liver of the developing opossum (Didelphis virginiana) D. B. PAONE, J. HARRY CUTTS AND W. J. KRAUSE

Department of Anatomy, University of Missouri, Columbia, Missouri

(Accepted 6 February 1975) INTRODUCTION

The name 'megakaryocyte' was introduced by Howell (1890) to designate a certain type of giant cell in bone marrow. Numerous early investigations soon provided a large body of information concerning the morphology and function of these cells (Wright, 1906, 1910; Bunting, 1909; Downey, 1913; Jordan, 1918; Minot, 1922; Rothermal, 1930) and more recent studies have detailed the ultrastructure of megakaryocytes and the way platelets are formed and released by these cells (Yamada, 1957; Jones, 1960; Sorenson, 1960, 1963; DeBruyn, 1964; Han & Baker, 1964; Zamboni, 1965; Behnke, 1968; Zucker-Franklin, 1970). Although hepatic megakaryocytes have been studied histochemically (Ackerman & Knouff, 1960) and at the fine-structure level (Sorenson, 1960, 1963; Zamboni, 1965), there have been few studies of megakaryocytopoiesis in the liver and these are found only in general surveys of hematopoiesis in this organ (Bloom, 1938; Gilmour, 1941; McKeller, 1949; Block, 1964; Leeson & Cutts, 1972; Cutts, Leeson & Krause, 1973). The present study details features of megakaryocytopoiesis as it occurs in the developing liver of the postnatal opossum. The study encompasses morphological aspects of the megakaryocytes at both the light and electron microscopic levels, quantitative changes that occur during the development of the liver, and indicates some features of the orientation of megakaryocytes within the liver lobule. MATERIALS AND METHODS

Megakaryocytes were studied in the developing livers of 35 young opossums. The livers were obtained at birth and at 9, 20 and 44 days of postnatal development, corresponding to snout-rump lengths of 1!5, 2 5, 4 5 and 7-0 cm respectively. The animals were killed by decapitation and the livers fixed in 3-5 % glutaraldehyde buffered with 0 1 M phosphate to a pH of 7.3. The tissues were washed twice in buffer and osmicated for two hours in 1 0 % osmium tetroxide, following which they were processed for embedding in Epon 812. Alternate thick (2-3 ,tm) and thin sections were cut so that electron microscopic studies could be carried out on cells that already had been identified by light microscopy. The thick sections for light microscopy were stained in' a 1: 1 solution of 0 1 M toluidine blue and 0 1 M sodium borate. Sections for electron microscopy were mounted on coated grids, stained in



2 % uranyl acetate and lead citrate (Reynolds, 1963) and examined in a RCA EMU-3F electron microscope. A total of 435 megakaryocytes was examined for their cytological features. These comprised 112, 125, 147, and 51 cells at the 1 5, 25, 45 and 7 0 cm stages respectively. Quantitative evaluation of hepatic megakaryocytes, and an appraisal of their distribution throughout the liver lobule were carried out by mapping the entire areas of the thick sections at a magnification of x 40. The location of each megakaryocyte, and of the vascular channels within the liver section, was outlined on the maps, the cross sectional areas of which were determined by means of a calibrated grid. Thus the number of megakaryocytes at each stage of development could be determined in the entire section and related to a standard unit area. Additionally, the location of megakaryocytes in the developing liver lobule, and with respect to vascular channels, could be assessed. RESULTS

Qualitative findings Megakaryocytes in the developing liver of the opossum range in size from 12-43 ,um diameter and, within this range, small, medium, large and very large cells can be recognized (Figs. 1-4). Cells with diameters of 12-19,um are classed as small, while the medium, large and very large cells are represented by cells with diameters of 20-26, 27-34 and 35-43 ,um respectively. Figs. 1-4 show the different sizes of megakaryocytes found in developing liver of the opossum. Fig. 1. This small megakaryocyte shows a type I cytoplasm in which there is no evidence of granule formation and only a modest degree of cytoplasmic streaking. 1-5 cm opossum; x 1000. Fig. 2. The nuclei of this medium-sized megakaryocyte show features of type III morphology. There is a more advanced degree of cytoplasmic streaking, indicating a type 1I cytoplasm. 2-5 cm opossum; x 1000. Fig. 3. This large megakaryocyte shows advanced nuclear and cytoplasmic differentiation. The nuclei show a type II morphology in which the generally oval nuclei show some irregularities of the nuclear border and the presence of flakes and blocks of heterochromatin. The type III cytoplasm shows prominent streaking that extends to the cell boundary. 2-5 cm opossum; x 1000. Fig. 4. This cell is representative of the 'very large' type of megakaryocyte. The nucleus shows the more irregular shape and outline characteristic of the type III morphology. The cytoplasm is of the type III variety. 4-5 cm opossum; x 1000. Fig. 5. This small megakaryocyte shows two type I nuclei. The nuclei are rounded, smoothly contoured and show a thin rim of heterochromatin at the periphery. The cytoplasm is somewhat intermediate between type I and type II. 1-5 cm opossum; x 1200. Fig. 6. The nuclei of this medium-sized megakaryocyte show a type II morphology. The cytoplasm shows a moderate degree of streaking which is patchy in distribution and represents a type II cytoplasm. The arrow points to a dense chromatin body lying close to the nucleus. 4 5 cm opossum; x 1000. Fig. 7. Prominent streaking and a zoning of the cytoplasm characterizes the type IV cytoplasm of this megakaryocyte. The nuclear and marginal zones are homogeneous and streaking is confined to the intermediate zone. A bleb of cytoplasm extends from the marginal zone(*). A small chromatin body lies next to the nucleus (arrow). 4-5 cm opossum; x 1000. Fig. 8. The chromosomes in this mitotic megakaryocyte reveal a multipolar arrangement. At the arrow are chromosomes that appear to be separating from the main body of chromosomes. Such chromosomes possibly give rise to the chromatin bodies seen in some interphase cells. 4-5 cm opossum; x 1000.

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Megakaryocytopoiesis in the liver of the developing opossum (Didelphis virginiana).

The development of hepatic megakaryocytes has been examined in the livers of 35 young opossums at four different stages of postnatal development. Mega...
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