Revista chilena de anatomía
versión impresa ISSN 0716-9868
Rev. chil. anat. v.20 n.1 Temuco 2002
THE GRANULOPOIETIC ORGANS OF THE NARROW NOSE SMOOTH
HOUND Mustelus schmitti (CHONDRICHTHYES, TRIAKIDAE).
A LIGHT AND ELECTRON MICROSCOPIC STUDY
ÓRGANOS GRANULOPOYÉTICOS DEL GATUSO (Mustelus schmitti)
(CHONDRICHTHYES, TRIAKIDAE). ESTUDIOS A TRAVÉS
DE LOS MICROSCOPIOS DE LUZ Y ELECTRÓNICO
* Laboratorio de Histología Animal, Universidad Nacional del Sur, Bahía Blanca, Argentina.
** Cátedra de. Fisiología. Humana. Dpto. Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Argentina. This work was supported by a grant of CONICET (PIA n° 512/92).
SUMMARY: The elasmobranchs are important natural models of hemopoiesis since they possess naturally dissected the inductive microenvironment. In this study we describe the general structure and cellular morphology of the two principal granulopoietic foci of Mustelus schmitti (Springer, 1936): the Leydig and the epigonal organs, likewise we semiquantify the hemopoietic compartments. Myeloid cells pertain to three different lineages. The principal nonmyeloid cells are lymphocytes, macrophages and reticular cells. No erythropoietic or thrombopoietic elements were found. The general structure of both organs is similar to those of other selachians, but their size and cellular diversity is typical for this species. Better knowledge of the stromatic environment of these specific hemopoietic organs may give further insight on the nature of their specific inductive functions.
KEY WORDS: 1. Elasmobranchs; 2. Granulopoiesis; 3. Epigonal organ; 4. Leydig organ; 5. Mustelus schmitti.
Basic hemopoietic structures and mechanisms in fishes are similar to those operating in other vertebrates (Galíndez & Aggio, 1997 and 1998). However, while in teleosts the pronephros is the main hemopoietic organ (Willett et al., 1999) and in higher vertebrates all myeloid cells proliferate and mature in the bone marrow, in cartilaginous fishes the inductive hemopoietic microenvironment seems to be naturally dissected, since they show different loci for erythro, thrombo and granulopoiesis (Zapata et al., 1995). As for granulopoiesis, it takes place in two particular sites: the Leydig and the epigonal organs, structures observed only in this class (Zapata & Cooper, 1990) and whose configuration is known in a few species. Their different cell types are variable between orders, as yet on the same genus (Galíndez & Aggio, 1995). Differentiation of blood cells in cartilaginous fishes was studied by cytochemical as well as by ultrastructural methods (Honma et al., 1984; Chiba et al., 1988). Long-term cell cultures studies were also done but only in teleosts (Siegl et al., 1993).
The histological and cytological features of granulopoietic tissues of M. schimitti are described in this work , as a contribution for a better understanding of the cellular and tisular interactions in the differentiation process.
MATERIAL AND METHOD
Twenty specimens from both sexes weighing between 200 and 600 g were caught in the fishing zone of the Bahía Blanca Bay (38 45'-39 30'S and 61 30'- 62 30'W) from February to December (1992) and brought alive to the laboratory. The animals were provided from the artisanal fisheries boats and were caught with "tapa-canal" nets.
They were killed by pithing and the epigonal and Leydig organs immediately dissected out. Stamp preparations were made from the cut surface of these organs and stained with May Grünwald-Giemsa, Lepehne and peroxidase reaction (Dacie & Lewis, 1995). Thin sections of paraffin embedded pieces (4-6 mm) were made and stained with hematoxylin-eosin, Masson's trichromic stain and PAS reaction. For electron microscopy, small pieces of tissue were first fixed in 2.5% glutaraldehyde in 0.5M cacodylate buffer (pH 7.4) with 12% of sucrose (Hyder et al., 1983) at 4ºC, for 12 hours, and then postfixed in osmium tetroxide in the same buffer, for 1 hour at 4ºC. They were then dehydrated in graded ethanol and infiltrated in low-density resin (Spurr). The grids were counterstained with uranyl acetate and lead citrate. Specimens were examined in a Jeol CX II electron microscope. The differential counts of myeloid cells were made in the stamp slides used for morphology, by random counting 100 cells of each type in 10 different slides from 10 different specimens.
Although the studied animals were not caught at the same season of the year, we did not find differences among the structure of both organs.
Epigonal Organ: The epigonal organ of M. schimitti is a whitish structure running ventrally from the last branchial arch to the rectal gland. The organ weight:body weight ratio averages 0.4% for males and 0.33% for females. Significant differences (p<<0.01) were found between sexes using a Covariance Analysis (Galíndez & Aggio, 1996).
Histology: Females have one lobe of myeloid tissue adjacent to the right ovary (Fig. 1). In males, the organ is restricted to the mesorchio and separated from testicular ampoules by a thin layer of well-vascularized glandular-like cells (Fig. 2). In both sexes, a thin connective capsule, strongly PAS positive and lacking trabeculae and muscular cells surrounds the organ. Many thin blood vessels irrigate the stroma and the vascular sinus shows a thick basal membrane (120-200 nm), (Fig. 2).
Fig. 1. General view of male and female epigonal organs. t: testis; o: ovary; ep: epigonal organ; arrows: cranial portion of the epigonal organ; *: rectal gland. Millimetric paper = 2 cm.
Fig. 2. Glandular-like tissue interspersed between testis and granulopoietic tissue. t: seminiferous ampoule; ep: epigonal parenchyma; s: sinus, arrows: cellular (glanaaaadular-like) trabeculae. Bar = 20 µm.
Cytology: Nonmyeloid cells: Two types of reticular cells were seen. One is a uniformly distributed star-like cell ultrastructurally similar to fibroblasts, with packages of collagen fibbers in the cytoplasmic process, extending between myeloid cells (Fig. 3). The other type is more electrodense, with a heterochromatic nucleus and bears short cytoplasmic projections (Fig.4). These cells are scarce and located close to thin vessels. Melanomacrophages were not found, and the structure of the macrophages was typical.
Fig. 3. Reticular "light"cell. Arrow heads: fibrilar packages; *: cytoplasmic projections. Bar = 2 µm.
Lymphocytes are the most abundant nonmyeloid cells. They account for 45 % to 55 % of the total cellularity and are interspersed among the granulopoietic elements, forming isolated groups or nodular-like structures without germinal centres. Plasmocytes and plasmoblasts are scarce (< 2% of the total cellularity).
Myeloid cells: These cells include different developmental stages of the granulocytic lineage, representing the remaining 45-50% of the total cellularity. It was possible to group the most differentiated steps into discrete "hemopoietic compartments". The committed compartment includes myeloblasts; they are small irregular-shaped cells (10 µm diameter), with a nucleus:cytoplasm ratio close to 80%, 3 or 4 nucleoli, and undifferentiated cytoplasm. The maturative-proliferative compartment encompasses semi-mature cells (promyelocytes and myelocytes) bearing the specific characteristics of each developmental stage. Promyelocytes are medium sized cells (12-14 µm) with a heterochromatic horseshoe-shaped nucleus (n:c=60-70% ). Their cytoplasm has scarce eosinophilic round granules (0.2-0.8 µm) covering less than the 10% of cell surface (Fig. 5). Myelocytes are large eosinophilic or colourless cells (12-18 µm) with a lobbed heterochromatic nucleus and specific granules filling the cytoplasm. With transmission electron microscopy, it is possible to differentiate three diverse types. The first (Figs. 6-7) has round inclusions of various sizes covered by a single membrane unit, and a clear lateralized granular endoplasmic reticulum. Interactions between these cells and lymphocytes are common. The second type has a horseshoe-shaped, euchromatic nucleus and two types of cytoplasmic granules, one of them homogeneous, indented, roundish, with a dense core and the other large, elongated and fibrillar (Figs. 8-9). The third class of myelocytes have lobed, electrodense, eccentric nuclei, and their cytoplasm is filled by coalescent vacuoles (Fig. 10). Finally, the functional compartment includes cells similar to those circulating in peripheral blood. No peroxidase activity was found in any developmental stage of granulocytic cells.
Fig. 7: Early myelocyte with clear lateralized endoplasmic reticulum. In the inset, a developed Golgi complex is detailed. Bar = 2.5 µm.
Fig. 8: Myelocyte with heterogeneous granules. Arrow indicates the amplified granule of fig. 3-4. Bar = 2.5 µm.
Leydig Organ: In M. schmitti this organ is a small pinkish structure included in the oesophagus submucosa, representing only 0.0075-0.043% of the total weight of the animal (N=44, Galíndez & Aggio, 1996). Its dissection is difficult due to its small size and location, although observation has been possible using light microscopy in sections of the whole oesophagus. It consists of two lobes that run dorsally and ventrally from the last branchial arch to the beginning of the cardiac stomach. Cranially only the dorsal mass is present. The parenchyma consists in an aggregation of lymphocytes and granulocytic cells separated by vascular sinuses. A fine reticular network supports each "lobe". The cells are similar to those described in the epigonal organ.
The epigonal organ of M. schmitti is located more caudal than in other selachian species, so the name "epigonal" should be changed to "subgonal", as proposed by Honma et. al. The Leydig organ is immersed into the sub-mucosa of the oesophagus as in other species of the class.
The granulopoietic parenchyma of both organs is similar to the myeloid cranial tissue of ancestral fishes (Mattisson et al., 1982), the cordoidal myeloid tissue of lampreys (Kélényi & Larsen, 1976), the intestinal myeloid tissue of mixins (Zapata & Cooper) and the mammalian bone marrow (Tavassoli, 1986). The stroma is devoid of groups of smooth muscle fibres, which represents a difference from batoids (Chiba & Honma, 1986). The glandular-like cells present in the epigonal organ of males (closely related with myeloid tissue) were not found in other selachians. Cells described as homologous to adrenal medulla are seen between kidneys in Scyliorhinus canicula (Oguri, 1978).
As in mammals, a great number of eosinophilic cells is secondary to parasitism or bacterial infection. Although we did not observe parasites in our specimens, has found high prevalence of intestinal and hepatic parasites in specimens from the same population.
It is known that there are no uniform criteria for classification of fish granulocytes, and no standardised methods for their manipulation are available (Parish et al., 1986). Morphological criteria were used to define the hemopoietic lines. By looking to the structure of the granules, we found three cellular types. As to the genus Mustelus, references exist for two (Kanesada 1956; Barnes et al., 1967) and three types of granules (Hine & Wain, 1987). In M. schmitti there are three classes of granulocytic cells: cells with round granules in which the most interesting finding is the lateralization of the granular endoplasmic reticulum reported for chondrichthyans (Hine & Wain, 1988); cells with a mixed granular population, common in fishes (Parish et al., and vacuolated cells with evident degranulation, also seen in fish granulocytes (Fänge & Pulsford, 1983) and associated to secretory functions. Evidence of cellular interactions between granulocytes and lymphoid cells and the presence of plasmocytes and plasmoblasts agree with the immuno-competent activity of these organs.
The relative size of the myelopoietic compartments suggest that proliferation is important up to an advanced stage of differentiation, resulting in a similar model to that observed in mammals. Likewise, the functional compartment may be interpreted as a "reserve pool" of mature cells, similar to that existing in the mammalian bone marrow. These similarities indicate a conservative mechanism for the hemopoietic process along the vertebrate phylogeny.
No erythropoietic tissue was seen in these organs, although the epigonal organ carries erythropoietic and thrombopoietic functions in splenectomized chondrichthyans (Fänge & Johansson-Sjöbeck, 1975). A strong association may exist, in cartilaginous fishes, between the fibroblastic stroma for the granulopoiesis and the reticular stroma for erythropoiesis (Pulsford & Zapata, 1989). The reticular cell types, essentially fibroblastic, present in M. schmitti would support the former interpretation.
Scarce information is available on the Leydig organ (Fänge, 1968; Oguri, 1983 and Mattisson & Fänge, 1982). It is likely that its concealed location inside another organ and its very variable size between species (Galíndez & Aggio, 1996) makes it an overlooked object of study. Likewise, the lowest size shows in this species, in contrast with the relevance of the epigonal organ, suggests that the Leydig parenchyma plays a secondary role in hemopoietic and immunological functions of M. schmitti.
RESUMEN: Los elasmobranquios son modelos naturales espontáneos importantes para el estudio de la hemopoyesis, dado que tienen disecados naturalmente los distintos microambientes inductores.
Describimos la microarquitectura y la morfología celular de las dos estructuras granulopoyéticas principales de Mustelus schmitti (Springer, 1936): los órganos epigonal y de Leydig; además semicuantificamos los distintos compartimientos hemopoyéticos. Las células mieloides pertenecen a tres líneas diferentes y las principales células no mieloides son linfocitos, macrófagos y células reticulares. No se encontraron focos eritroides ni trombocitoides en ninguno de los parénquimas. La morfología general de ambos órganos fue concordante con la reportada para otros selacios, pero sus tamaños y la diversidad celular es típica para la especie.
Un conocimiento más detallado del ambiente estromático de estas estructuras tan específicas y únicas puede brindar información importante para la comprensión de sus funciones inductivas.
PALABRAS CLAVE: 1. Elasmobranquios; 2. Granulopoyesis; 3. Órgano epigonal; 4. Órgano de Leydig; 5. Mustelus schmitti.
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Dra. Elena J. Galíndez
Lab. Histología Animal
Dpto. Biol., Bioqca. y Farmacia,
UNS, San Juan 670, 8000
Recibido : 02-01-2002