THE ANATOMICAL RECORD 242:83-90 (1995) Myelopoiesis in the Thymus of the Sea Bass, Dicentrarchus labrax L. (Teleost) MARCELINO AVILES-TRIGUEROS AND JUAN A. QUESADA Departamento de Biologia Celular, Facultad de Biologia, Uniuersidad de Murcia, Murcia, Spain ABSTRACT Background: In vertebrates the thymus is primarily regarded as a lymphoid organ whose importance lies in its capacity to produce a large number of lymphocytes that enter the circulation as T cells. In higher vertebrates the organ has also been regarded as a site for myelopoiesis, but this capacity has not been observed in fish. In this study we describe morphologically the presence of intrathymic developing myeloid cells in the sea bass. Methods: The thymus samples were morphologically studied by transmission electron microscopy. Results: We describe the coexistence of cells in different stages of erythropoiesis and granulopoiesis that appear to be developing in situ in some thymus lobes. Degenerated thymocytes and epithelial-reticularcells occur simultaneously in the same areas. Conclusions: The coexistence of different cellular components of erythropoiesis and the heterophilic series of granulopoiesis with areas of necrosis suggests a relationship between both processes that is influenced by the microenvironment. Our observations also suggest that the presence of intrathymic developing myeloid cells may imply a nonimmunological role for the thymus. o 1995 Wiley-Liss, Inc. Key words: Thymus, Myelopoiesis, Erythropoiesis, Granulopoiesis, Ultrastructure, Sea bass, Dicentrarchus labrax, Teleostei The thymus is a lymphoid organ whose main function is the generation of T lymphocytes in vertebrates. The presence of myeloid cells in the thymus of vertebrates, including fishes, is generally accepted (Hafter, 1952; Gorgollh, 1983; O’Neill, 1989; Zapata and Cooper, 1990), although whether or not there is any myelopoietic activity is currently receiving much interest. Erythropoietic areas have been described in mammals including humans (Kendall, 1980, 1981; Kendall and Singh, 1980; Bourgeois et al., 19811,and birds (Kendall and Frazier, 1979; Fonfria et al., 1983) but not in the teleosts studied so far (Gorgollh, 1983). The existence of granulopoiesis has been demonstrated in higher vertebrates (Olah et al., 1975; Bourgeois et al., 1981; Kendall, 19811, in holosteans (Hill, 1935), and teleosts (Davina et al., 1980; Gorgollon, 1983). In this report, we demonstrate the existence of myelopoietic areas in the thymus of the sea bass, Dicentrarchus labrax, in which erythrocytes and heterophilic granulocytes are produced. MATERIALS AND METHODS Twenty-five specimens of both sexes of juvenile sea bass (Dicentrarchus labrax),aged 1-2 years, 15-20 cm in size, and healthy, were provided by the Experimental Station of San Pedro del Pinatar (Murcia, Spain) and kept in tanks of running sea water a t 17-20°C, 0 1995 WILEY-LISS, INC. continually aerated and exposed to a natural photoperiod. The specimens were anaesthetised with 0.01% tricaine metanesulphonate (TMS, Crescent Research Chemicals, Phoenix, AZ). The thymus was removed and small pieces of -1 mm3 fixed in ice-cold 2% glutaraldehyde buffered to pH 7.3 with 0.1 M sodium cacodylate buffer and postfixed in 1%osmium tetroxide. The samples were washed again and in block stained with 4.8% uranyl acetate in veronal buffer, pH 7.3, being dehydrated in ethanol and transferred to propylene oxide as transitional fluid. Epon 812 resin was used for embedding. U1trathin sections were contrasted with uranyl acetate and lead citrate and examined in a Zeiss EM-1OC or EM-109 electron microscope, operating at 60 or 80 kV, respectively. RESULTS The thymus of sea bass (Dicentrarchus labrax L.) is a pair organ, located on the dorsal body wall of the gill cavity. It is externally limited by the pharyngeal epithelium and internally bound by a capsule of connec- Received July 25, 1994; accepted November 4, 1994. Address reprint requests to Juan A. Quesada, Departamento de Biologia Celular, Facultad de Biologia, Universidad de Murcia, E-30100 Murcia, Spain. 84 M. AVILES-TRIGUEROS AND J.A. QUESADA tive tissue that is continuous with long trabeculae invaginating the organ, by deeply penetrating the parenchyma, without splitting it into individual thymic areas. In some thymic lobes, in small extensions neighbouring the connective tissue, clearly defined myelopoietic areas appear, replacing the lymphoid tissue. They are formed by several hematopoietic islets or nests of 65 +- 10 pm of diameter, delineated by a unicellular layer of macrophages and with a delicate stroma of dark epithelial-reticular cells, where all cellular elements of the erythropoietic and granulopoietic series are accommodated mixed, without predominate of neither (Fig. 1). Erythropoiesis Proerythroblasts. These represent the first stage of maturation of the erythropoietic series that are morphologically recognizable (Fig. 2a). The earliest forms are difficult to differentiate from myeloblasts or lymphoblasts. Proerythroblasts are the largest cells of the erythrocytic series and have a rounded, slightly lengthened, or polygonal shape. The nuclear membrane has numerous nuclear pores. The nucleus is euchromatinic, large, central, and rounded, or slightly indented. The centrally placed nucleolus reaches its maximum development during this stage. In the cytoplasm there are flattened cisternae of rough endoplasmic reticulum (RER) and small mitochondria, round or oval in shape, that are spread through out the cytoplasm. The proerythroblasts are distinguished from other precursor cells by the presence of microtubules and coated vesicles of moderate electron density in the vicinity of the membrane and sometimes in contact with it, occasionally opening into the extracellular space. These vesicular structures are clearly rhopheocytic vesicles. Basophilic erythroblasfs. These are slightly smaller than the proerythroblasts (Fig. 2b). The nucleus is large, oval, or rounded in shape, with a nucleolus and heterochromatin arranged inside the nuclear membrane or in scarce central clumps. The cytoplasm is characterized by a moderate number of electron light vesicles and an increased number of coated vesicles compared with the previous stage (Fig. 2f), many free ribosomes, some profiles of endoplasmic reticulum, and a poorly developed Golgi apparatus, whereas mitochondria are small and scarce. By this stage the synthesis of hemoglobin can be established by the formation of fine cytoplasmic particles of medium electron density. Polychromafophilic erythroblasfs. These are smaller than their basophilic counterparts (Fig. 2c), and their nucleus is generally rounded, with clumps of heterochromatin, and occupies a greater area than in the basophilic erythrocyte. The nucleolus is observed with difficulty. Bands of microtubules appear in the marginal cytoplasm, and there is a decrease in the number of organelles matched by an increase of their electron density due to the presence of greater amounts of hemoglobin. Free ribosomes are fewer than in the previous stage, but numerous polysomes appear. Small rounded vesicles of low electron density and vesicles of rhopheocytosis are also present, and occasionally siderosomes (Fig. 2g). Acidophilic erythroblasts. These are slightly smaller than the polychromatophilic erythroblasts (Fig. 2d). The nucleus is central, rounded, or oval, and is considerably smaller than in the previous stage. The heterochromatin is arranged marginally and in large central clumps. The number of nuclear pores decrease. The cytoplasmic matrix appears finely granulated because of the increased concentration of hemoglobin. It contains marginal bands of microtubules and vesicles of rhopheocytosis (Fig. 2h). A decreased number of organelles is observed, but there are still groups of polysomes dispersed through the cytoplasm, a poorly developed Golgi apparatus, and scarce small mitochondria, elongated in shape and a matrix of moderate electron-density . Immature erythrocytes. These are elongated in shape (Fig. 2e). Generally, the nucleus, which is ovoid, is smaller and more condensed than in previous stages. Few nuclear pores can be distinguished, and the heterochromatin is arranged in the periphery and in dense central clumps. In this stage the cytoplasm is more homogeneous due to the prevalence of hemoglobin. Some mitochondria remain, along with a small Golgi apparatus, ribosomes, vesicles, and profiles of membranes that seem to be the remains of membraneous organelles. The phenomenon of rhopheocytosis persists. Mature erythrocytes. These are not observed in the hematopoietic areas, although they appear dispersed through the rest of the thymic parenchyma and are occasionally phagocytized by macrophages. They have no cytoplasmic organelles. Granulopoiesis The different stages of differentiation of the heterophilic series are established in accordance with nuclear features, and according to the kinds and densities of the cytoplasmic granules, as well as the differential development of the cytoplasmic organelles in the different phases of cellular differentiation. Blast cell. It was possible to identify only a few undifferentiated cells, presumably myeloblasts (Fig. 3a). These cells are characterized by their large euchromatic nucleus, clearly visible nucleolus, and a cytoplasm of low electron density with abundant free ribosomes. Promyelocytes. These cells have a large rounded or oval nucleus that is weakly indented and eccentrically placed. The heterochromatin is found at the periphery or in central clumps and in most cases, a nucleolus can be distinguished (Fig. 3b). The cytoplasm possesses numerous free ribosomes, frequent flattened cisternae of evenly distributed RER, sometimes forming small stacks, some mitochondria of scarce cristae, and a well-developed Golgi apparatus near the nucleus. The distinguishing feature of the promyelocytes is the appearance for the first time of type I filamentous granules of low electron density that are homogeneously distributed through out the cytoplasm. MYELOPOIESIS I N THE THYMUS 85 Fig. 1. Myelopoieticarea in the inner zone of the sea bass thymus. Macrophages (M+), dark epithelialreticular cells (DER), x 2,500. Heterophilic myelocytes. These cells (Fig. 3c) are rounded and are characterized by having a second type of granule (type 11) in their cytoplasm with an oval or elongated shape and a fibrillar content more electrondense than that of the type I granule (Fig. 3d). The nucleus is more indented and has more condensed chro- matin than the previous stage. The nucleolus, when found, is eccentric. In the cytoplasm, the RER is not so stacked as in the previous stage and contains few ribosomes. The Golgi apparatus is more prominent during this stage. The mitochondria are longer and less numerous than in the 86 M. AVILES-TRIGUEROS AND J.A. QUESADA Fig. 2. Erythropoietic series: (a)proerythroblast, x 5,200, (b)basophilic erythroblast, x 7,800, ( c ) polychromatophilic erythroblast, x 9,400, (d) Acidophilic erythroblast, x 7,200, (el immature erythrocyte, x 9,500, (0 detail of a basophilic erythroblast, X 19,000, (g) de- tail of a polychromatophilic erythroblast, x 19,000, (h)detail of an acidophilic erythroblast, x 28,500. Microtubules (arrows), rhopheocytic vesicles (arrowheads}, siderosomes (s). MYELOPOIESIS IN THE THYMUS Fig. 3.Heterophilic granulocytic series: (a) blast cell, X 12,000, (b) promyelocyte, X 9,600, (c) heterophilic myelocyte, X 9,600, (d) detail of a heterophilic myelocyte, x 14,400, ( e )heterophilic metamyelocyte, 87 X 9,600, (0 young heterophilic granulocyte, x 7,800. Type I granules (arrows), type I1 granules (arrowheads), type p glycogen (double arrowheads). 88 M. AVILES-TRIGUEROS AND J.A. QUESADA Fig, 4. (a)Area of necrosis (remarked area), x 2,500, (b)necrotic cells isolated, x 5,000, (c) necrosis with syncytial structure, x 5,000. Macrophages (M+), dark epithelial-reticular cells (DER), mitosis (m), pyknotic nucleus (P), trabeculae (TI,connective capsule (C), tonofilaments (arrows). promyelocytes and multivesicular bodies are also observed that contain small slightly electron-dense vesicles. In this stage, the number of type I1 granules increases progressively as the myelocytes mature, until they are as numerous as or more numerous than the type I granules. Type p glycogen is progressively being accumulated (Fig. 3d). Heterophilic metamyelocytes. These cells (Fig. 3e) are characterized by their horseshoe shape or deeply in 89 MYELOPOIESIS IN THE THYMUS dented nucleus and their greater content of heterochromatin than in previous stages. In the cytoplasm a decreased number of ribosomes and scarce cisternae of RER can be observed. The Golgi apparatus also decreases in size and the elongated mitochondria are scarcer. Myelin figures can be seen in mature metamyelocytes. There are more type I1 granules than type I and their electron density also increases during this stage. Young heterophilic granulocytes. These cells (Fig. 3f) are larger than their precursors. They are oval with an eccentric bilobed or deeply indented nucleus with abundant heterochromatin. The cytoplasm contains the two types of granules previously described, with a predominance of type 11, a poorly developed Golgi apparatus, very little RER, some free ribosomes, and scarce small mitochondria. Mature heterophilic granulocytes. These cells are not found in the myelopoietic areas, although they appear through the rest of the thymic parenchyma. Areas of Necrosis In some zones close to the connective capsule, we observed isolated involuted cells and areas of varying extension formed by the fusion of numerous cells with signs of regression (Fig. 4a,b). These syncitial regions are characterized by the presence of pyknotic irregular nuclei, with very condensed clumps of chromatin that vary in appearance. Sincitial cytoplasm is segmented in vacuolated structures by membranes that sometimes rupture, releasing their contents. The membraneous organelles appear intact or show symptoms of degeneration, and a substantial vesicular component is observed. In the interior of these syncytial structures, there are normally lymphoid cells and epithelial-reticular cells (ERC) characterizated by tonofilaments remanents (Fig. 4c). Both cell type have lost their membrane and the chromatin begins to condense, suggesting that this is the process that gives rise to these necrotic areas. Macrophages are present in these areas, although necrotic cells do not appear phagocitized by them (Fig. 4a). This suggests a case of nonlysosomal cellular death. DISCUSSION In vertebrates, erythropoietic areas have been described in the thymus of birds (Kendall and Frazier, 1979; Fonfria et al., 1983) and mammals (Olah et al., 1975; Bourgeois et al., 1981; Kendall, 1981). Heterophilic granulopoietic areas have also been observed in the thymus of the teleost Syciases sanguineus (Gorgol16n, 1983). However, in the thymus of sea bass (Dicentrarchus labrax L.), we have observed the presence of definite myelopoietic areas, where heterophilic granulocytes and erythrocytes originate. These myelopoietic areas only appear in some thymic lobes in a similar way to the erythropoietic areas of the thymus in birds (Kendall and Frazier, 1979; Fonfria et al., 1983). In sea bass as in other animals (Olah et al., 1975; Bourgeois et al., 1981; Kendall, 1981), the thymic hematopoietic areas are located next to the connective capsule and are constituted by islets bounded by macrophages and occupied by a network of ERC, in which hematopoietic cells and some lymphocytes can be found. The nomenclature and morphology of the several cellular components of erythropoiesis and of the heterophilic series of granulopoiesis in the thymus agree with the descriptions of other haematopoietic areas of sea bass (Esteban et al., 1989; Meseguer et al., 1990; Quesada et al., 1994). The coexistence of myelopoietic areas and of necrosis in the same thymic lobe suggests a relationship between both processes that is probably influenced by microenvironmental changes, in accordance with the observations of Fonfria et al. (19831, who describe a degeneration of ERC, pyknotic lymphocytes, and an increased number of macrophages in the erythropoietic areas in development of the thymus of birds. The microenvironmental changes that regulate the erythropoietic processes might be controlled by T cells as has been demonstrated in mammals by Lipton and Nathan (1981). Intrathymic erythropoiesis could result in an increased demand of erythrocytes in circulating blood, because of a fall in erythrocyte production in other haematopoietic organs (Ward and Kendall, 1975; Kendall and Frazier, 1979; Fonfria et al., 1983). Erythropoietic activity involves a nonimmunological response of the thymus. We must therefore await future explanations concerning the behavior and capacity of the stem cells before it can be determined whether the presence of erythropoiesis is accessory to the normal functions of this organ. According to Kendall (19811, the thymus always has the potential t o develop erythropoiesis but is usually inhibited or is not stimulated or, in contrast, the factors that induce an increase of erythropoiesis stimulate all the haemato-poietic tissues to do it. The existence of myelopoiesis in the thymus of sea bass necessarily involves the arrival of circulating stem cells capable of developing in erythrocytes and granulocytes, although there is also a selective entrance of prothymocytes, and it will be the microenvironment created by the neuroendocric interactions that will favor one activity or the other, as occurs in the bursa of Fabricius of chicken embryo (Quesada et al., 1985). According to the above and in accordance with Kendall (19801, it would be convenient to refer to the thymus as a hematopoietic organ, where the cellular differentiation of the lymphoid series is favored. Further research into the microenvironmental factors that influence myelopoietic activity in the thymus should be carried out. LITERATURE CITED Bourgeois, N., G. Bergmans, and N. Buyssens 1981 The thymus as haemopoietic tissue of non-lymphoid cells. Virchows Arch., 391: 81-89. Davina J.H.M., G.T. Rijkers, J.H.W.M. Rombout, L.P.M. Timmermans, and W.B. van Muiswinkel 1980 Lymphoid and non-lymphoid cells in the intestine of cyprinid fish. In: Development and differentiation of vertebrate lymphocytes, J.D. Horton ed., Elsevier North Holland, Amsterdam, pp. 129-140. Esteban M.A., J. Meseguer, A. Garcia-Ayala, and B. Agulleiro 1989 Erythropoiesis and thrombopoiesis in the head-kidney of the sea bass (Dicentrurchus Zabrax L.): An ultrastructural study. Arch. Histol. Cytol., 52:407-419. 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