MORPHOMETRY AND MORPHOLOGY OF NUCLEUS OF THE SERTOLI AND INTERSTITIAL CELLS OF THE TAMBAQUI Colossoma macropomum ( CUVIER , 1881 ) ( PISCES : CHARACIDAE ) DURING THE REPRODUCTIVE CYCLE

This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes.


INTRODUCTION
During gonadal development, the testis presents more discreet variations than do ovaries.These variations relate to weight, volume, shape, and coloration.In that period, the testis presents seminiferous lobules having spherical or oval cysts inside of which the spermatogenic lineage cells develop.In each cyst, all germinative cells are in the same developmental phase and Sertoli cells in variable number envelop them.
During the reproductive cycle the Sertoli cells occur in variable forms mainly relat to the morphology of nucleus and cytoplasm.These cells constitute the somatic envelope of spermatogenic lineage cells, distributed in the cysts' peripheral area.
In spite of the Roosen Runge (1977) and Billard et al. (1982) terminology for teleosts, we consider the term "cyst cells" more appropriate; the expression Sertoli cell is used here due to the homology with mammalian Sertoli cells (Mitsuiki, 2002).
Taking into account the function of Sertoli cells, authors like Grier & Linton (1977) and Cruz-Höfling & Cruz-Landim (1984) have established a relationship between lipid presence in Sertoli cells and steroidogenic activity.On the other hand, Mattei et al. (1982) have suggested that if such cells do not synthesize steroids, at least they are the storage site of these hormones.Another function related to Sertoli cells is gametic phagocytosis, mainly in the testes of the pacu Piaractus mesopotamicus when maintained in captivity.This fact might be related to unnatural elimination of spermatozoids, which consequently degenerate and are reabsorbed (Romagosa, 1991).
Although several studies have analyzed the morphology of both interstitial cells and Sertoli cells as well as their roles, research is still necessary on the structure and morphometrey of these cells in different stages of the reproductive cycle and, particularly, on the interstitial cells of teleost testes.Until now, these have been not well characterized.
Our study therefore aims to analyze these features of the tambaqui Colossoma macropomum.

MATERIAL AND METHODS
Tambaqui Colossoma macropomum males were collected in ponds of the Aquaculture Center, State University of São Paulo (UNESP-CAUNESP, Jaboticabal, SP), and of the Power Station of Minas Gerais (CEMIG, Conceição das Alagoas, MG), at the following reproductive cycle stages of the: immature, resting, maturation I, maturation II, mature, and regression.After sampled, each fish was weighed (total weight) and measured (total length and standard length).The animals were sacrificed by spinal cord destruction and ventrally incised gonad for analysis and extirpation.Gonads were weighed and samples of cranial, medial, and caudal portions taken.They were fixed in Bouin's solution for 6 hours at 4ºC and for 24 hours at ambient temperature.Inclusion of the samples was done in paraffin.Cross-sectioning was 6 µm thickness and staining was by the hematoxilin techniques of Harris-eosin (HE), Periodic Acid Schiff (PAS), and Masson's Tricromic (MT).All these procedures, used to get a histological crosssection of the studied testes, were carried out at the Laboratory of Fish Histology of the Department of Morphology and Animal Physiology (Agronomic and Veterinary Sciences College, Jaboticabal, UNESP).
The area of the nucleus of intralobular Sertoli cells and interstitial tissue, located among three seminiferous lobules of testes in stages of maturation I and II of the reproductive cycle, was calculated using an Image Analyzer System (Videoplan -Image Analysis Kontron Elektronik -Zeiss).Measures were done in 30 nuclei of Sertoli cells, located alongside each type of testicular germinal cell, resulting in a total of 150 Sertoli cells (per animal and per developmental stage), for which the nuclear areas were determined.Comparisons were done among samples of cranial, medial, and caudal regions.The triangular areas, occupied by interstitial tissue, were measured in the same way as that of the cyst cell nucleus, i.e., using a 40 x objective.Basically, 240 interstitial areas and 120 for each maturation stage were measured.Data obtained by measurements done through the Image Analyzer System were analyzed by Test F, and mean values were compared by Tukey test.

RESULTS
In the histological preparations of tambaqui testes no differences among the cephalic, medial and caudal regions were oberved.
A testes in the immature stage of the reproductive cycle consisted of small seminiferous lobules and much interstitial tissue.Primary spermatogonia were found throughout the lobules and characterized as cells having a spherical centrally located nucleus with a single nucleolus (Fig. 1A).
In the resting stage, the seminiferous lobules were bigger and contained several spermatogonia (primary and secondary) (Fig. 1B).In these lobules, interstitial tissue was less abundant.
In testes in maturation I (Fig. 1C) the following structures were identified: isolated primary spermatogonia; secondary spermatogonia cysts; spermatocyte (I and II) cysts; spermatid cysts; and some spermatozoids in seminiferous lobule lumen and interstitial tissue, which were clearly seen in areas where three or more lobules were fitted together.
In testes in maturation II (Fig. 1D), the seminiferous lobules appeared big and had several spermatozoids in their lumen.Interstitial tissue was evident among the lobules, mainly where three of them were joined.Primary spermatogonia and the cysts of the other germ cells were also present in this reproductive cycle.
Mature testes showed lobules full of spermatozoids, with spermatogonia surrounding them, and a thin interstitial tissue layer among them (Fig. 1E).
Testes in regression showed irregular seminiferous lobules, with several spermatogonia and some residual spermatozoids.The interstitial tissue was very abundant among the seminiferous lobules (Fig. 1F).
In several analyzed testes, Sertoli cells were found within the seminiferous lobules, beside the primary spermatogonia and at the periphery of several germ cell cysts.Sertoli cell nuclei located near the primary spermatogonia (Fig. 1C) appeared bigger and wider, and triangular or egg-shaped.Some nuclei appeared more elongated, in accordance with the profile of each spermatogonia.Testis in maturation I and II showed Sertoli cells making up a somatic envelope of several germ cell cysts (Fig. 1C).They showed a flattened nucleus and thin cytoplasmatic extensions.Such nuclei appeared increasingly flat as testes developed further.
Interstitial cells were better evidenced in testes in maturation I and II, mainly in Maturation I.They were identified in interstitial tissue, located among the seminiferous lobules.Interstitial cells were better visualized in triangular areas, containing interstitial tissue and formed by three adjacent lobules, always alongside the blood vessels.
Interstitial cell cytoplasmatic borders were not well defined, thus making it difficult to recognize the eccentric position of the nucleus, which was egg-shaped.
Statistical analyses showed that the Sertoli cell nuclear area, situated beside the primary spermatogonium, was bigger than the nuclear area of the same cell, producing a somatic envelope of diverse germ cell cysts (p < 0.01) (Table 1).
The nuclear Sertoli cell area, located near the primary spermatogonia, was bigger in testes in maturation II when compared with that of maturation I (p < 0.05).In the secondary spermatogonia, the Sertoli cell nuclear areas showed no significant difference in testes in maturation I and II.

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In spermatocytes I, the Sertoli cell nuclear area showed a significant difference in testes in maturation I and II (p < 0.01), so that in maturation I the difference was bigger than in stage II.Spermatocytes II showed a significant difference in the Sertoli cell nuclear area of the testes in maturation I and II (p < 0.01) and in maturation I the difference was bigger than in stage II.At the periphery of the spermatid cysts, the Sertoli cell nuclear area was significantly different in testes in maturation I and II (p < 0.01), and was bigger in maturation I (Table 1).
The Sertoli cell nuclear area related with primary spermatogonium and producing the sheathing of several intralobular cysts of germ cells, showed no significant difference among the sampled testes in the cranial, medial, and caudal regions.
The statistical analysis applied to interstitial tissue area data (Table 2) evidenced a significant difference (p < 0.05) in testes in two maturation stages: maturation I the difference was bigger than in maturation II (p < 0.05).
The area occupied by the interstitial tissue located among three seminiferous lobules of testes in maturation I and II did not show any difference among the regions in which the samples were taken (cranial, medial, and caudal).

DISCUSSION
In the testes of tambaqui no histological differences among the cephalic, medial, and caudal regions were observed.According to Romagosa (1991), the same was true for pacu.Testes of tambaqui fit the first pattern of classification described by Grier et al. (1980) (spermatogonial unrestricted), since the spermatogonia lie alongside the entire seminiferous lobule.The primary spermatogonia of tambaqui were considered the biggest cells of the germinative lineage.They occured individually in seminiferous lobules, distributed throughout the testes, during all reproductive cycle phases.These morphological observations are in accordance with those of Romagosa (1991).
Secondary spermatogonia, observed in tambaqui testes appeared inside the cysts.They were small cells with more condensed chromatin and their morphology is similar to that described by Silva (1987) and Romagosa (1991), for species they studied.These cells result from mitotic division of primary spermatogonia later transformed into spermatocytes I.
Until now, there is no literature regarding Sertoli cell karyometry of teleost fish gonads.Agostinho et al. (1987) and Billard et al. (1972) studied Rhinelepesis aspera and Onchorhinchus mykiss, respectively, and reported that Sertoli cells have their nuclei modified during the spermatogenic process.The nuclei of these cells appeared bigger and almost spherical in testes in resting, and smaller and elongated in testes in maturation.In mature testes, the nucleus of the Sertoli cells can be entangled in the tubular wall.Zaiden (1997Zaiden ( , 2000) ) studied Brycon orbignyanus and Brycon hilari respectively and verified that Sertoli cell nuclei became more and more elongated as spermatogenic lineage developed.
Data presented in Table 1 shows that in tambaqui, the nucleus narrowed during development, in accordance with the results obtained by Zaiden (1997Zaiden ( , 2000)).The size of Sertoli cell nuclei did not differ statistically among the three studied regions (cranial, medial and caudal).
The analyses resulted in the following conclusions: the testes of tambaqui Colossoma macropomum are composed of several types of germinative cells (primary spermatogonia, secondary spermatogonia, spermatocyte I, spermatocyte II, spermatid, and spermatozoid).The primary spermatogonium is the biggest germinative cell and the spermatozoid is the smallest.Primary spermatogonia and spermatozoid did not produce cysts.Primary spermatogonia are distributed throughout the seminiferous lobule and are isolated, while spermatozoid lie in the lobule lumen.Sertoli cells constitute the somatic envelope of germinative cells, from secondary spermatogonia to spermatids (they are also found beside primary spermatogonia and their nucleus decreases in size and changes in shape during spermatogenic cell development).Interstitial cells are clearly in evidence in testes at the beginning of maturation and are always located beside blood vessels, characterizing the beginning of a new reproductive cycle.At this point, the steroids necessary to testes development have been synthetized.Furthermore, the area development have been synthesize.Furthermore, the area occupied by interstitial tissue diminishes with testicular development.
The Sertoli cells found in tambaqui testes presented bigger nuclei in the resting period, becoming flat in the maturation period, due to the modifications undergone in the formation of spermatogenic cysts.Romagosa (1991) found a triangularly-shaped cyst cell nuclei in Piaractus mesopotamicus.Cecílio & Agostinho (1991) showed that the same nuclei in Hypotalmus edentatus are comma-shape, changing to rod-shape with gonadal maturation.Zaiden (2000) showed in piraputanga testes, that this cell has an oval nucleus in the resting period, gradually flattening in the maturation period.
A great variety of information exists on Sertoli cells in fish, describing, e.g., there function and employin differing nomenclature, but controversies are not few, making necessary many more studies to obtain histophysiological information that will show their role in the tambaqui's reproductive cycle and contribute to further understanding the reproductive cycle of these species, which is of great importance in aquaculture.