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Myelopoiesis in the thymus of the sea bass Dicentrarchus labrax L. (teleost)

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THE ANATOMICAL RECORD 242:83-90 (1995)
Myelopoiesis in the Thymus of the Sea Bass,
Dicentrarchus labrax L. (Teleost)
Departamento de Biologia Celular, Facultad de Biologia, Uniuersidad de Murcia,
Murcia, Spain
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,
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.
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,
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,
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.
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).
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
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
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.
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.
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
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).
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,
X 9,600, (0 young heterophilic granulocyte, x 7,800. Type I granules
(arrows), type I1 granules (arrowheads), type p glycogen (double arrowheads).
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
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
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.
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
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.
Bourgeois, N., G. Bergmans, and N. Buyssens 1981 The thymus as
haemopoietic tissue of non-lymphoid cells. Virchows Arch., 391:
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.
Fonfria J., M.G. Barrutia, E. Garrido, C.F. Ardavin, A. Villena, and
A. Zapata 1983 Erythropoiesis in the thymus of the spotless starling Sturnus unicolor. Cell Tissue Res., 232:445-455.
Gorgoll6n P. 1983 Fine structure of the thymus in the adult cling fish
Sicyases sanguineus (Pisces, Gobiesocidae). J . Morphol., 177:2540.
Hafter E. 1952 Histological age changes in the thymus of the teleost
Astyanax mexicanus. J . Morphol., 90:555-581.
Hill B.H. 1935 The early development of the thymus glands of Amiu
culva. J . Morphol., 57.61-89.
Kendall M.D. 1980 Avian thymus glands: A review. Dev. Comp. Immunol., 4:191-209.
Kendall M.D. 1981 Cells of the thymus. In: The Thymus Gland, M.D.
Kendall ed., Academis Press, London, pp. 63-83.
Kendall M.D., and J.A. Frazier 1979 Ultrastructural studies on erythropoiesis in the avian thymus. I. Description of cell types. Cell
Tissue Res., 199:37-61.
Kendall M.D., and J . Singh 1980 The presence of erythroid cells in the
thymus gland of man. J . Anat., 130:183-189.
Lipton J.M., and D.G. Nathan 1981 The role of T lymphocytes in
human erythropoiesis. In: The lymphocyte, K.W. Sell and W.V.
Miller eds., Alan R. Liss, New York, p. 57.
Meseguer J., M.A. Esteban MA, A. Garcia-Ayala, A. Lopez-Ruiz, and
B. Agulleiro 1990 Granulopoiesis in the head-kidney of the sea
bass (Dicentrarchus lubrax L.): An ultrastructural study. Arch.
Histol. Cytol., 53:287-296.
Olah I., P. Rohlich, and I. Toro 1975 Ultrastructure of Lymphoid
Or. gans. Masson et Cie, Paris.
O”eil1 J.G. 1989 Lymphoid organ development in Antarctic teleosts.
Antarct. Spec. Top., 77-86.
Quesada J., B. Agulleiro, and M.T. Lozano 1985 Ultrastructure of the
granulopoietic microenvironment in the tunica propia of the
bursa of Fabricius of white leghorn chicken embryo. J . Submicrosc. Cytol., 17537-540.
Quesada J., M.I. Villena and V. Navarro 1994 Ontogeny of the sea
bass spleen (Dicentrarchus labrax):A light and electron microscopy study. J . Morphol., 221t161-176.
Ward P., and M.D. Kendall 1975 Morphological changes in the thymus of young and adult red-billed queleas Quelea quelea (Aves).
Phil. Trans. R. SOC.
B., 273:55-64.
Zapata A.G., and E.L. Cooper 1990 The thymus. In: The Immune
System: Comparative Histophysiology, John Wiley & Sons, New
York, pp. 104-149.
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sea, labrab, dicentrarchus, teleost, myelopoiesis, bass, thymus
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