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The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods
Tissue & Cell, 1999 31 (6) 561–567 © 1999 Harcourt Publishers Ltd Article no. tice.1999.0068
Tissue&Cell
The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods I. Quagio-Grassiotto, E. D. Carvalho
Abstract. The ultrastructure of Sorubim lima spermatogenesis during the premeiotic and meiotic periods was studied. Our observations showed that the germ cells in the cysts are connected by cytoplasmic bridges and the mitotic and meiotic divisions are slightly asynchronous. The first and the last spermatogonial generations differ in the cellular and nuclear volume, nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of ‘lamellae anulata’, amount and dimension of the ‘nuages’, and movement of the centrioles. In addition to the nuclear prophase structures, the spermatocyte I shows changes in all other cellular organelles and elongated vesicles appear in the cytoplasm. The accentuated cytoplasmic density and thickened walled vesicles are morphological characteristics that differentiate spermatocytes II from the other germ cells in the cysts of Sorubim lima testis. © 1999 Harcourt Publishers Ltd.
Keywords: S. lima, teleostei, ultrastructure, spermatogenesis, spermatogonia, spermatocytes
Introduction The ultrastuctural descriptions of the premeiotic and meiotic fish male germ cells show certain nuclear and cytoplasmic common characteristics (Billard & Fléchon, 1969; Grier, 1975; Clerot, 1976; Billard, 1984; Selman & Wallace, 1986; Cruz-Landim & Cruz- Hofling, 1986/1987; Silveira et al., 1990; Mattei et al., 1993; Thiaw et al., 1995). In studies on sex differentation, these characteristics have been used to recognize the male and female early gonad development (Satoh, 1974; Bruslé & Bruslé, 1978; Colombo et al., 1984; 1987; Winkoop et al., 1992; Flowers & Burns, 1993; Grandi & Colombo, 1997). This has also been useful in the gonad analysis of sex inversion in hermaphroditic fish (BrusléSicard & Reinboth, 1990; Bruslé-Sicard et al., 1994) and in hybrid or polypoid species (Stoumboudi & Abraham, 1996). UNESP, Instituto de Biociências de Botucatu, Departamento de Morfologia, Cx. Postal 510, 18.618–000, Botucatu, Sao Paulo, Brazil
Received 6 April 1999 Accepted 13 August 1999 Correspondence to: Irani Quagio-Grassiotto. Tel.: +55 14 8206264; Fax: +55 14 8206322; E-mail: [email protected]
Despite this, the knowledge of the ultrastructure of the early fish male germ cells is limited to a few species. South and Central American neotropical freshwater ichthyofauna, estimated at 8000 species, represent up to 25% of all species of both freshwater and marine fish worldwide (Vari & Malabarba, 1998), and very little is known about them (Cruz-Landim & Cruz-Hofling, 1986/1987; Matos & Azevedo, 1989; Matos et al., 1993). To further knowledge on fish spermatogenesis, we have studied the premeiotic and meiotic periods of male germ cell development in Sorubim lima, Teleostei, Siluriformes, Pimelodidae, as this is a neotropical catfish, endemic in the Amazonas River and Paraná River Basins, Brazil. This species has a good reputation in fishing and has good breeding potential.
Materials and methods Adult male S. lima were collected from the Hidrology and Aquiculture Sector of Salto Grande/CESP-SP, Brazil. They were reared under natural photoperiod (yearly range: 561
562
QUAGIO-GRASSIOTTO, CARVALHO
10 h 14 min–13 h 56 min LD) and temperature (yearly range: 15.9–28.8°C) in a tank (100 m long / 10 m wide / 2 m deep). The fish were fed twice daily with dry food pellets (35–46% protein). Each month from April 1995 to March 1996, three fishes were taken at random from the tank, previously anesthetized, and killed. Gonads of 36 specimens were removed, reduced to fragments, and fixed in 4% paraformaldehyde and 2% glutaraldehyde in Sorensen phosphate buffer 0.1 M pH 7.4 (Karnovsky, 1965) overnight. The material was post-fixed for 2 h in the dark in 1% osmium tetroxide in the same buffer, contrasted in block with aqueous solution of 5% uranyl for two hours, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% alcohol and lead citrate (Reynolds, 1963). The specimens were observed and electromicrographs were obtained using a Phillips- CM 100 transmission electron microscope.
Results The Sorubim lima testes are paired and elongated organs, located in the dorsal region of the celomate cavity. They are surrounded by a tunica albuginea and subdivided into lobules by incomplete septae. The lobules converge forming the spermatic ducts. Inside the lobules, the germinal epithelium composed of Sertoli and germ cells is supported by the basal lamina. The germ cells are surrounded by cytoplasmic processes of Sertoli cells, forming cysts (Fig. 1–Inset). Spermatogenesis occurs inside the cysts. The germ cells inside the same cyst are at a similar developmental stage. This organization is characteristic of the testis of the unrestricted spermatogonial (Grier, 1981) or lobular type (Billard, 1986), with a permanent germinal epithelium (Grier, 1992). In the lobules, the primordial germ cells are in contact with the basal lamina and they do not form cysts. They show a pronounced electron density, a voluminous nucleus of irregular outline, and a well developed nucleolus. Their cytoplasm shows ‘nuages’ and innumerable mitochondria. They divide by mitosis, giving rise to the primary spermatogonia. Sertoli cell processes enclosed a single primary spermatogonium, forming the cyst. It is the largest cell of the germ line. Inside the cysts, it undergoes several mitotic
divisions with incomplete cytokinesis, originating the secondary spermatogonia (Figs 1–6). The primary and the secondary spermatogonia resulting from the first mitosis present voluminous nucleus of regular outline with decondensed chromatin and well-developed nucleolus. Their cytoplasm has a large amount of free ribosomes, many mitochondria, and ‘nuages’. The ‘nuages’ initially appear juxtaposed to the nuclear envelope, accumulating near nuclear pores. They move to the cytoplasm forming dense circular masses with contiguous diffuse areas, and they are frequently surrounded by mitochondria. There were also a well-developed Golgi complex and ‘lamellae anulata’. The ‘lamellae anulata’ were linear, formed by only one not so long cistern close to the ‘nuages’ (Figs 1–4). In the secondary spermatogonia, the centrioles are near the cytoplasmic bridges close to the plasma membrane, and with one of the component pairs forming a short cilium (Figs 4 & 7). Inside the same cyst, the cells in division are not necessarily synchronized (Fig. 5). The secondary spermatogonia resulting from the last mitosis are smaller than the initial cells. They have a nucleus with predominantly diffuse chromatin, an eccentric nucleolus, a smaller amount of ‘nuages’ in the cytoplasm with no ‘lamellae anulata’. Their mitochondria are slightly less voluminous, denser, and more elongated (Figs 4 & 6). The intercellular cytoplasmic bridges present a thickened plasma membrane (Figs 4 & 7). The initiation of the germ cell meiosis does not determine a meaningful cell volume increase. At the beginning of prophase, in the nucleus of the leptotenic spermatocyte I, the chromatin is diffuse with areas of peripheral concentration and few dense granules (Fig. 8). In the cytoplasm, the centrioles duplicate, move to the cell periphery close to cytoplasmic bridges. One of the diplosome components anchors to the plasma membrane and elongates, creating a short cilium that projects from the cellular surface (Fig. 15a–d). The mitochondria, now smaller, slightly denser and more elongated, and the Golgi complex tend to concentrate in the proximity of the cytoplasmic bridges, giving a certain polarity to the cell (Figs 8–10; 12 & 13). Elongated vesicles appear in the cytoplasm (Figs 10; 13 & 14). With the progress of prophase, the formation of the synaptonemical complexes begins in the nucleus. First, the
Fig. 1 Sorubim lima primary spermatogonium. CF, collagen fibers; IM, interstitial myoid cell; LL, lobular lumen; MI, mitochondria; N, nucleus; NE, ‘nuages’; SC, Sertoli cell; arrow head, basal lamina. X 9750. Inset X 15 500. Fig. 2 S. lima primary spermatogonium cytoplasm. G, Golgi complex; LA, ‘lamellae anulata’; MI, mitochondria; NE, ‘nuages’. X 56 700. Fig. 3 S. lima early secondary spermatogonium. G, Golgi complex; MI, mitochondria; NE, ‘nuages’; NU, nucleolus; P, plasma membrane; double arrow, nuclear pores. X 11 900. Fig. 4 S. lima secondary spermatogonium. B, cytoplasmic bridges; C, centriole; MI, mitochondria; N, nucleus; NE, ‘nuages’; SC, Sertoli cell. X 14 850. Inset X 18 900. Fig. 5 Mitotic division of the S. lima secondary spermatogonia. CR, chromosome; M, metaphase; PR, prophase; arrow head, mitochondria. X 4350. Fig. 6 S. lima late secondary spermatogonia. N, nucleus; NE, ‘nuages’; NU, nucleolus; arrow head, mitochondria. X 5750. Fig. 7 S. lima secondary spermatogonium cytoplasm. B, cytoplasmic bridges; C, centrioles; G, Golgi complex; MI, mitochondria; N, nucleus; P, plasma membrane; arrow head, mitochondria. X 18 400.
PREMEIOTIC AND MEIOTIC PERIODS OF SPERMATOGENESIS IN FISH
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lateral components appear in the zygotenic spermatocytes I anchored to the nuclear envelope (Fig. 9). This is followed by the progressive compactation of the associated homologous chromosomes. Finally, in the pachytenic spermatocytes I, central component organization is completed (Figs 10 & 11). Later, to facilitate the genic change, the synaptonemical complexes disappear and the chromosomes move to the periphery of the nucleus (Fig. 16). Nuclear envelope fragmentation releases the chromosomes that progress in the meiotic I process (Fig. 16 a–d). The spermatocytes I in diakinesis, metaphase, anaphase, and telophase show a more electron-dense cytoplasm than during the prophase, smaller, elongated, and highly electron dense mitochondria, and numerous thickened walled vesicles (Fig. 16 a–d). In this cellular type, the cytoplasmic bridges show membranous reinforcements (Fig. 16a, b–Insets). The spermatocytes II, resulting from the first division of meiosis, are characterized and differ from the spermatocytes I by their accentuated electronic density and smaller cell volume. During prophase in the nucleus, chromosomes in compactation, centrioles at opposite sides of the cell, and innumerable thickened walled vesicles in the cytoplasm can be seen (Fig. 17). The metaphasic and anaphasic spermatocytes II maintain their accentuated cytoplasmic electron density and present a large quantity of thickened wall vesicles in the cell periphery (Fig. 18a, b). The newly-formed spermatids are smaller than the spermatocytes II. They present accentuated cytoplasmic electron density, eccentric nucleus, and thickened walled vesicles concentrated in the proximity of the cytoplasmic bridges. Dense bodies can be seen in the cytoplasmic bridges (Fig. 19).
Discussion ‘Nuages’, ‘lamellae anulata’, and mitochondrial clusters are common cytoplasmic characteristics of the earliest fish male germ cells (Billard & Fléchon, 1969; Satoh, 1974; Grier, 1975; Clerot, 1976; Bruslé & Bruslé, 1978; Billard, 1984; Colombo et al., 1984; Selman & Wallace, 1986; CruzLandim & Cruz-Hofling, 1986/1987; Silveira et al., 1990; van Winkoop et al., 1992; Flowers & Burns, 1993; Mattei et al., 1993; Thiaw et al., 1995; Grandi & Colombo, 1997). ‘Nuages’ are cytoplasmic electron-dense materials not surrounded by membranes. They are of different sizes and shapes, receive different names, and are present during the whole life cycle of these cells. They are abundant during oogenesis and spermatogenesis, contain RNA, appear isolated in the cytoplasm close to the nuclear envelope, and are associated with mitochondria (Eddy, 1975) or with ‘lamellae anulata’ (Kessel, 1983). The electrondense material, observed between clusters of mitochondria, is known as intermitochondrial cement and presents various types of RNAs, proteins, cytochromes, and lipids (Toury et al.,
1977). ‘Lamellae anulata’ are transient organelles composed of stacks of cisterns with pores similar to nuclear pores. They are found in several cells, especially in animal germinal cells and are well developed in oocytes (Kessel, 1983; 1992). Kessel (1981) proposed that the pores of these ‘lamellae anulata’ are associated with the activation of genic products of long duration, such as rRNAs and mRNAs contained in the dense cytoplasmic masses. As well as the ‘nuages’, ‘lamelae anulata’, and mitochondrial clusters, the primordial germ cells are frequently highly electron dense. The stem cells in the testis of Sorubim lima share these characteristics. They do not form cysts and are easily found in the rest periods, during the reproductive cycle of the animal. On the other hand, the primary spermatogonia originating from the stem cells are the largest cells of the germinative lineage in Sorubim lima. They form cysts and their nuclear and cytoplasmic characteristics do not differ from those described for other teleosts. According to Selman and Wallace (1986), when these cells have incomplete cytokinesis, they are considered secondary spermatogonia. Billard (1984) mentions that this cell type undergoes morphological changes throughout its numerous generations. As expected, the changes observed in the Sorubim lima secondary spermatogonia give rise to morphological differences between the first and the last generation. These changes are related to the decrease of cellular and nuclear volume, position and size of the nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of the ‘lamellae anulata’, amount and dimension of the ‘nuages’, and movement of the centrioles. The Sorubim lima spermatocytes I, in addition to these nuclear alterations characteristic of prophase, undergo changes in the cytoplasm. ‘Nuages’, mitochondria, centrioles, and the endomembrane system are different in spermatocyte I. The thickened walled vesicles, which appear in the cytoplasm of spermatocytes I of Sorubim lima, will be part of the intermediary piece of the future spermatozoon. (Quagio-Grassiotto, personal communication). The formation of a cilium or short flagellum, originating from one of the centrioles, as observed in Sorubim lima, was observed in spermatocytes I of other groups of animals (QuagioGrassiotto & Lello, 1996). In teleosts, these have also been observed in the spermatocytes I and the spermatogonia (Billard & Fléchon, 1969; Billard, 1984; Thiaw et al., 1995). The asynchronous characteristic of the cell divisions of the germ cells in mitosis or meiosis was detected by Selman and Wallace (1986). In Sorubim lima, this asynchrony can lead to the coexistence of spermatocytes II and newlyformed spermatids in the same cyst (Quagio-Grassiotto, personal communication). The high cytoplasmic density, smaller cellular volume, and also the thickened walled vesicles are the morphological characteristics that differentiate spermatocytes II from the other cell types present in the cysts of Sorubim lima testis.
PREMEIOTIC AND MEIOTIC PERIODS OF SPERMATOGENESIS IN FISH
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Fig. 8 Sorubim lima leptotenic spermatocytes I (LS1). B, cytoplasmic bridges; C, centrioles; N, nucleus; arrow head, mitochondria. X 10 000. Fig. 9 S. lima zygotenic spermatocytes I (ZS1). B, cytoplasmic bridges; C, centrioles; LC, lateral component of the synaptonemical complex; MI, mitochondria; N, nucleus; arrow, nuclear envelope. X 6200. Fig. 10 S. lima pachytenic spermatocytes I (PS1). N, nucleus; V, vesicles; arrow head, mitochondria agglomerations. X 5750. Fig. 11 Synaptonemical complex. CC, central component; LC, lateral component. X 64 000. Fig. 12 Cytoplasmic bridges (B) region of the S. lima prophasic spermatocytes I. C, centrioles; MI, mitochondria; N, nucleus. X 17 000. Fig. 13 S. lima spermatocytes I cytoplasm. C, centrioles; G, Golgi complex; MI, mitochondria; V, vesicles. X 40 250. Fig. 14 Thickened walled vesicles (V) on the S. lima prophasic spermatocytes I cytoplasm. X 28 350. Fig. 15 Centriole behaviour during the S. lima spermatocytes I prophase. B, cytoplasmic bridges; C, centrioles; CI, cilium; G, Golgi complex; MI, mitochondria; N, nucleus; P, plasma membrane; T, microtubules. a: X 21 000; b: X 31 500; c: X 16 500; d; X 58 500.
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Fig. 16 Sorubim lima spermatocytes I (S1) in genic material division. A, anaphase; B, cytoplasmic bridges; CR, chromosomes; D, diakinesis; IM, interstitial myoid cell; M, metaphase; SC, Sertoli cell; TL, telophase; V, thickened walled vesicles; arrow head, mitochondria. a: X 13 250; Inset X 9750; b: X 5400; Inset X 31 500; c: X 8500; d: X 10 000. Fig. 17 S. lima spermatocyte II (S2). C, centrioles in opposite sides of the cell; N, nucleus; V, thickened walled vesicles; arrow head, mitochondria. X 8050. Fig. 18 S. lima spermatocytes II (S2) in genic material division. A, anaphase; C, centrioles; M, metaphase; V, thickened walled vesicles in the cell periphery; arrow head, mitochondria. a: X 10 000; b: X 12 900. Fig. 19 S. lima newly formed spermatid (S). N, eccentric nucleus; V, thickened walled vesicles in the cytoplasmic bridges; B, proximity; arrow, dense bodies; arrow head, mitochondria. X 13 250.
PREMEIOTIC AND MEIOTIC PERIODS OF SPERMATOGENESIS IN FISH
The behavior of the cytoplasmic bridges in Sorubim lima, a result of incomplete cytokinesis during mitosis and meiosis, follows the patterns described for teleosts and also for other groups of animals (Fawcett et al., 1959; Fawcett, 1961; Clerot, 1971; Dym & Fawcett, 1971; Gondos, 1973; Billard, 1984; Weber & Russell, 1987; Silveira et al. 1990). The cytoplasmic bridges on the different male germ cell types include lateral thickenings, membranous reinforcements in the spermatocytes, and dense bodies in the newlyformed spermatids. ACKNOWLEDGEMENTS We would like to thank the EM Laboratory of IB, Botucatu for the use of their facilities and Dr Heidi Dolder for revising the manuscript. This research was supported by CAUNESP and CESP. REFERENCES Billard, R. 1984. Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis. Cell Tissue Res., 273, 219–226. Billard, R. 1986. Spermatogenesis and spermatology of some teleost fish species. Repord. Nutr. Dev., 26, 877–1024. Billard, R. and Fléchon, J.E. 1969. Spermatogonies et spermatocytes flagellés chez Poecilia reticulata (Poissons téléostéen Cyprinidontiforme). Ann. Biol. Anim. Biochim. Biophys., 281, 281–286. Bruslé, S. and Bruslé, J. 1978. Early sex differentiation in Mugil (Liza) auratus. Risso, 1810, an ultrastructural study. Ann. Biol. Anim. Biochim. Biophys., 18, 871–875. Bruslé-Sicard, S. and Reinboth, R. 1990. Protandric hermaphrodite peculiarities in Amphiprion frenatus Brevoort (Teleostei, Pomacentridae). J. Fish Biol., 36, 383–390. Bruslé-Sicard, S., Reinboth, R. and Fourcault, B. 1994. Germinal potentialities during sexual state changes in a protandric hermaphrodite, Amphiprion frenatus (Teleostei, Pomacentridae). J. Fish Biol., 45, 597–611. Clerot, J.C. 1971. Les ponts intercellulaires du testicule du Gardon, organisation syncitiale et synchronie de la différenciation des cellules germinales. J. Ultrastruct. Res., 37, 690–703. Clerot, J.C. 1976. Les groupements mitochondriau des cellules germinales des poissons téleostéens cyprinidés. I. Etude ultrastructurale. J. Ultrastruct. Res., 54, 461–475. Colombo, G., Grandi, G. and Rossi, R. 1984. Gonad differentation and body growth in Anguilla anguilla L. J. Fish. Biol., 24, 225–228. Colombo, G., Grandi, G., Romeo, A., Giovannini, G., Pellizzola, D., Catozzi, L. and Piffanelli, A. 1987. Testis cytological structure, plasma sex steroids and gonad cytosol steroid receptors of heterologous gonadotropin (hCG) stimulated silver eel, Anguilla anguilla L. Gen. Comp. Endocrinol., 65, 167–178. Cruz-Landim, C. and Cruz-Hofling, M.A. 1986/1987. Aspectos da espermatogênese de tucunaré Cichla ocellaris Schneider, 1801 (Telostei, Cichlidae). Acta Amazônica, 16/17, 65–72. Dym, M. and Fawcett, D.W. 1971. Further observations on the numbers of spermatogonia, spermatocytes and spermatids connected by intercellular bridges in the mammalian testis. Biol. Reprod., 4, 195–215. Eddy, E. M. 1975. Germ plasm and differentiation of the germ cell line. Int. Rev. Cytol., 43, 229–280. Fawcett, D.W. 1961. Intercellular bridges. Exp. Cell Res., 8 (suppl.), 174–187. Fawcett, D.W. Ito, S. and Slauterback, D.L. 1959. The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation. J. Biophys. Biochem. Cytol., 5, 453–460.
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Flowers, J.A. and Burns, J.R. 1993. Ultrastructural study of embryonic and early adult germ cells and their support cells, in both sexes of Xiphophorus (Teleostei, Poeciliidae). Cell Tissue Res., 271, 263–270. Gondos, B. 1973. Intercellular bridges and mammalian germ cell differentiation. Differentiation, 1, 177–182. Grandi, G. and Colombo, G. 1997. Developmental and early differentiation of gonad in the European eel (Anguilla anguilla [L], Anguilliformes, Teleostei): a cytological and ultrastructural study. J. Morphol., 231, 195–216. Grier, H. J. 1975. Aspects of germinal cyst and sperm development in Poecilia latipinna (Teleostei, Poeciliidae). J. Morphol., 146, 229–250. Grier, H.J. 1981. Cellular organization of the testis and spermatogenesis in fishes. Am. Zool., 21, 345–357. Grier, H.J. 1992. Chordate testis: the extracellular matrix hypothesis. J. Exp. Zool., 261, 151–160. Karnovsky, J.M. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microcopy. J. Cell Biol., 27, 137a. Kessel, R.G. 1981. Origin, differentiation, distribution and possible functional role of annulate lamellae during spermatogenesis in Drosophila melanogaster. J. Ultrastruct. Res., 75, 72–96. Kessel, R. G. 1983. The structure and function of annulate lamellae, porous cytoplasmic and intranuclear membrane. Int. Rev. Cytol., 82, 181–303. Kessel, R.G. 1992. Annulate lamellae, a last frontier in cellular organelles. Int. Rev. Cytol., 133, 43–100. Matos, E. and Azevedo, C. 1989 Estudo ultra-estrutural da espermatogênese de Lepidosiren paradoxa (Pisces, Dipnoi) na amazônia. Rev. bras. Ciênc. Morfol., 6, 67–71. Matos, E., Matos, P., Oliveira, E. and Azevedo, C. 1993. Ultraestrura da espermatogênese do tamoatá, Hoplosternum littorale (Teleostei, Callichgthyidae) do rio Amazonas. Rev. Bras. Zool., 10, 219–227. Mattei, X., Siau, Y. O. T. and Thiam, D. 1993. Peculiarities in the organization of testis of Ophidion sp (Pisces Teleostei). Evidence for two types of spermatogenesis in teleost fish. J. Fish Biol., 43, 931–937. Quagio-Grassiotto, I. and Lello, E. 1996. Centriole behavior during meiosis of male germ cells of Dermatobia hominis (Diptera, Cuterebridae) Cytobios, 85, 73–80. Reynolds, E.S. 1963. The use of lead citrate at high pH as electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208–212. Satoh, N. 1974. An ultrastructural study of sex differentiation in the teleost Oryzias latipes. J. Embryol.. Exp. Morphol., 32, 195–215. Selman, K. and Wallace, R.A. 1986. Gametogenesis in Fundulus heteroclitus. Am. Zool., 26, 173–192. Silveira, H., Rodrigues, P. and Azevedo, C. 1990. Fine structure of the spermatogenesis of Blennius pholis (Pisces, Blenniidae). J. Submicrosc. Cytol. Pathol., 22, 103–109. Stoumboudi, M. Th. and Abraham, M. 1996. The spermatogenetic process in Barbus longiceps, Capoeta damascina and their natural sterile hybrid (Teleostei, Cyprinidae). J. Fish Biol., 49, 458–468. Thiaw, O.T., Mattei, X. and Ndiaye, M. 1995. Unusual syncytial structure of the germ cells in the Cyprinodontidae Aphyosemion cameronense (Pisces, Teleostei). J. Submicrosc. Cytol. Pathol., 27, 375–380. Toury, R., Clérot, J.C. and André, J. 1977. Les groupments mitochondriaux des cellules germinales des poissons teleostéens Cyprinides. IV Analyses biochimique des constituants du “ciment” intermitochondrial isolé. Biol. Cell., 30, 225–232. Van Winkoop, A., Booms, G.H.R., Dulos, G.J. and Timmermans, L.P.M. 1992. Ultrastructural changes in primordial germ cells during early gonadal development of common carp. (Cyprinus carpio L., Teleostei). Cell Tissue Res., 267, 337–346. Vari, R.P. and Malabarba, L.R. 1998. Neotropical Ichthyology: an overview. In: Malabarba, L.R. Reis, R.E., Var Lucena, Z.M.S., Lucena, C.A.S. (eds) Phylogeny and classification of neotropical fishes EDIPUCRS, Porto alegre, 1–12. Weber, J. E. and Russell, L. D. 1987. A study of intercellular bridges during spermatogenesis in the rat. Am. J. Anat., 180, 1–24.
Tissue&Cell
The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods I. Quagio-Grassiotto, E. D. Carvalho
Abstract. The ultrastructure of Sorubim lima spermatogenesis during the premeiotic and meiotic periods was studied. Our observations showed that the germ cells in the cysts are connected by cytoplasmic bridges and the mitotic and meiotic divisions are slightly asynchronous. The first and the last spermatogonial generations differ in the cellular and nuclear volume, nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of ‘lamellae anulata’, amount and dimension of the ‘nuages’, and movement of the centrioles. In addition to the nuclear prophase structures, the spermatocyte I shows changes in all other cellular organelles and elongated vesicles appear in the cytoplasm. The accentuated cytoplasmic density and thickened walled vesicles are morphological characteristics that differentiate spermatocytes II from the other germ cells in the cysts of Sorubim lima testis. © 1999 Harcourt Publishers Ltd.
Keywords: S. lima, teleostei, ultrastructure, spermatogenesis, spermatogonia, spermatocytes
Introduction The ultrastuctural descriptions of the premeiotic and meiotic fish male germ cells show certain nuclear and cytoplasmic common characteristics (Billard & Fléchon, 1969; Grier, 1975; Clerot, 1976; Billard, 1984; Selman & Wallace, 1986; Cruz-Landim & Cruz- Hofling, 1986/1987; Silveira et al., 1990; Mattei et al., 1993; Thiaw et al., 1995). In studies on sex differentation, these characteristics have been used to recognize the male and female early gonad development (Satoh, 1974; Bruslé & Bruslé, 1978; Colombo et al., 1984; 1987; Winkoop et al., 1992; Flowers & Burns, 1993; Grandi & Colombo, 1997). This has also been useful in the gonad analysis of sex inversion in hermaphroditic fish (BrusléSicard & Reinboth, 1990; Bruslé-Sicard et al., 1994) and in hybrid or polypoid species (Stoumboudi & Abraham, 1996). UNESP, Instituto de Biociências de Botucatu, Departamento de Morfologia, Cx. Postal 510, 18.618–000, Botucatu, Sao Paulo, Brazil
Received 6 April 1999 Accepted 13 August 1999 Correspondence to: Irani Quagio-Grassiotto. Tel.: +55 14 8206264; Fax: +55 14 8206322; E-mail: [email protected]
Despite this, the knowledge of the ultrastructure of the early fish male germ cells is limited to a few species. South and Central American neotropical freshwater ichthyofauna, estimated at 8000 species, represent up to 25% of all species of both freshwater and marine fish worldwide (Vari & Malabarba, 1998), and very little is known about them (Cruz-Landim & Cruz-Hofling, 1986/1987; Matos & Azevedo, 1989; Matos et al., 1993). To further knowledge on fish spermatogenesis, we have studied the premeiotic and meiotic periods of male germ cell development in Sorubim lima, Teleostei, Siluriformes, Pimelodidae, as this is a neotropical catfish, endemic in the Amazonas River and Paraná River Basins, Brazil. This species has a good reputation in fishing and has good breeding potential.
Materials and methods Adult male S. lima were collected from the Hidrology and Aquiculture Sector of Salto Grande/CESP-SP, Brazil. They were reared under natural photoperiod (yearly range: 561
562
QUAGIO-GRASSIOTTO, CARVALHO
10 h 14 min–13 h 56 min LD) and temperature (yearly range: 15.9–28.8°C) in a tank (100 m long / 10 m wide / 2 m deep). The fish were fed twice daily with dry food pellets (35–46% protein). Each month from April 1995 to March 1996, three fishes were taken at random from the tank, previously anesthetized, and killed. Gonads of 36 specimens were removed, reduced to fragments, and fixed in 4% paraformaldehyde and 2% glutaraldehyde in Sorensen phosphate buffer 0.1 M pH 7.4 (Karnovsky, 1965) overnight. The material was post-fixed for 2 h in the dark in 1% osmium tetroxide in the same buffer, contrasted in block with aqueous solution of 5% uranyl for two hours, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% alcohol and lead citrate (Reynolds, 1963). The specimens were observed and electromicrographs were obtained using a Phillips- CM 100 transmission electron microscope.
Results The Sorubim lima testes are paired and elongated organs, located in the dorsal region of the celomate cavity. They are surrounded by a tunica albuginea and subdivided into lobules by incomplete septae. The lobules converge forming the spermatic ducts. Inside the lobules, the germinal epithelium composed of Sertoli and germ cells is supported by the basal lamina. The germ cells are surrounded by cytoplasmic processes of Sertoli cells, forming cysts (Fig. 1–Inset). Spermatogenesis occurs inside the cysts. The germ cells inside the same cyst are at a similar developmental stage. This organization is characteristic of the testis of the unrestricted spermatogonial (Grier, 1981) or lobular type (Billard, 1986), with a permanent germinal epithelium (Grier, 1992). In the lobules, the primordial germ cells are in contact with the basal lamina and they do not form cysts. They show a pronounced electron density, a voluminous nucleus of irregular outline, and a well developed nucleolus. Their cytoplasm shows ‘nuages’ and innumerable mitochondria. They divide by mitosis, giving rise to the primary spermatogonia. Sertoli cell processes enclosed a single primary spermatogonium, forming the cyst. It is the largest cell of the germ line. Inside the cysts, it undergoes several mitotic
divisions with incomplete cytokinesis, originating the secondary spermatogonia (Figs 1–6). The primary and the secondary spermatogonia resulting from the first mitosis present voluminous nucleus of regular outline with decondensed chromatin and well-developed nucleolus. Their cytoplasm has a large amount of free ribosomes, many mitochondria, and ‘nuages’. The ‘nuages’ initially appear juxtaposed to the nuclear envelope, accumulating near nuclear pores. They move to the cytoplasm forming dense circular masses with contiguous diffuse areas, and they are frequently surrounded by mitochondria. There were also a well-developed Golgi complex and ‘lamellae anulata’. The ‘lamellae anulata’ were linear, formed by only one not so long cistern close to the ‘nuages’ (Figs 1–4). In the secondary spermatogonia, the centrioles are near the cytoplasmic bridges close to the plasma membrane, and with one of the component pairs forming a short cilium (Figs 4 & 7). Inside the same cyst, the cells in division are not necessarily synchronized (Fig. 5). The secondary spermatogonia resulting from the last mitosis are smaller than the initial cells. They have a nucleus with predominantly diffuse chromatin, an eccentric nucleolus, a smaller amount of ‘nuages’ in the cytoplasm with no ‘lamellae anulata’. Their mitochondria are slightly less voluminous, denser, and more elongated (Figs 4 & 6). The intercellular cytoplasmic bridges present a thickened plasma membrane (Figs 4 & 7). The initiation of the germ cell meiosis does not determine a meaningful cell volume increase. At the beginning of prophase, in the nucleus of the leptotenic spermatocyte I, the chromatin is diffuse with areas of peripheral concentration and few dense granules (Fig. 8). In the cytoplasm, the centrioles duplicate, move to the cell periphery close to cytoplasmic bridges. One of the diplosome components anchors to the plasma membrane and elongates, creating a short cilium that projects from the cellular surface (Fig. 15a–d). The mitochondria, now smaller, slightly denser and more elongated, and the Golgi complex tend to concentrate in the proximity of the cytoplasmic bridges, giving a certain polarity to the cell (Figs 8–10; 12 & 13). Elongated vesicles appear in the cytoplasm (Figs 10; 13 & 14). With the progress of prophase, the formation of the synaptonemical complexes begins in the nucleus. First, the
Fig. 1 Sorubim lima primary spermatogonium. CF, collagen fibers; IM, interstitial myoid cell; LL, lobular lumen; MI, mitochondria; N, nucleus; NE, ‘nuages’; SC, Sertoli cell; arrow head, basal lamina. X 9750. Inset X 15 500. Fig. 2 S. lima primary spermatogonium cytoplasm. G, Golgi complex; LA, ‘lamellae anulata’; MI, mitochondria; NE, ‘nuages’. X 56 700. Fig. 3 S. lima early secondary spermatogonium. G, Golgi complex; MI, mitochondria; NE, ‘nuages’; NU, nucleolus; P, plasma membrane; double arrow, nuclear pores. X 11 900. Fig. 4 S. lima secondary spermatogonium. B, cytoplasmic bridges; C, centriole; MI, mitochondria; N, nucleus; NE, ‘nuages’; SC, Sertoli cell. X 14 850. Inset X 18 900. Fig. 5 Mitotic division of the S. lima secondary spermatogonia. CR, chromosome; M, metaphase; PR, prophase; arrow head, mitochondria. X 4350. Fig. 6 S. lima late secondary spermatogonia. N, nucleus; NE, ‘nuages’; NU, nucleolus; arrow head, mitochondria. X 5750. Fig. 7 S. lima secondary spermatogonium cytoplasm. B, cytoplasmic bridges; C, centrioles; G, Golgi complex; MI, mitochondria; N, nucleus; P, plasma membrane; arrow head, mitochondria. X 18 400.
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lateral components appear in the zygotenic spermatocytes I anchored to the nuclear envelope (Fig. 9). This is followed by the progressive compactation of the associated homologous chromosomes. Finally, in the pachytenic spermatocytes I, central component organization is completed (Figs 10 & 11). Later, to facilitate the genic change, the synaptonemical complexes disappear and the chromosomes move to the periphery of the nucleus (Fig. 16). Nuclear envelope fragmentation releases the chromosomes that progress in the meiotic I process (Fig. 16 a–d). The spermatocytes I in diakinesis, metaphase, anaphase, and telophase show a more electron-dense cytoplasm than during the prophase, smaller, elongated, and highly electron dense mitochondria, and numerous thickened walled vesicles (Fig. 16 a–d). In this cellular type, the cytoplasmic bridges show membranous reinforcements (Fig. 16a, b–Insets). The spermatocytes II, resulting from the first division of meiosis, are characterized and differ from the spermatocytes I by their accentuated electronic density and smaller cell volume. During prophase in the nucleus, chromosomes in compactation, centrioles at opposite sides of the cell, and innumerable thickened walled vesicles in the cytoplasm can be seen (Fig. 17). The metaphasic and anaphasic spermatocytes II maintain their accentuated cytoplasmic electron density and present a large quantity of thickened wall vesicles in the cell periphery (Fig. 18a, b). The newly-formed spermatids are smaller than the spermatocytes II. They present accentuated cytoplasmic electron density, eccentric nucleus, and thickened walled vesicles concentrated in the proximity of the cytoplasmic bridges. Dense bodies can be seen in the cytoplasmic bridges (Fig. 19).
Discussion ‘Nuages’, ‘lamellae anulata’, and mitochondrial clusters are common cytoplasmic characteristics of the earliest fish male germ cells (Billard & Fléchon, 1969; Satoh, 1974; Grier, 1975; Clerot, 1976; Bruslé & Bruslé, 1978; Billard, 1984; Colombo et al., 1984; Selman & Wallace, 1986; CruzLandim & Cruz-Hofling, 1986/1987; Silveira et al., 1990; van Winkoop et al., 1992; Flowers & Burns, 1993; Mattei et al., 1993; Thiaw et al., 1995; Grandi & Colombo, 1997). ‘Nuages’ are cytoplasmic electron-dense materials not surrounded by membranes. They are of different sizes and shapes, receive different names, and are present during the whole life cycle of these cells. They are abundant during oogenesis and spermatogenesis, contain RNA, appear isolated in the cytoplasm close to the nuclear envelope, and are associated with mitochondria (Eddy, 1975) or with ‘lamellae anulata’ (Kessel, 1983). The electrondense material, observed between clusters of mitochondria, is known as intermitochondrial cement and presents various types of RNAs, proteins, cytochromes, and lipids (Toury et al.,
1977). ‘Lamellae anulata’ are transient organelles composed of stacks of cisterns with pores similar to nuclear pores. They are found in several cells, especially in animal germinal cells and are well developed in oocytes (Kessel, 1983; 1992). Kessel (1981) proposed that the pores of these ‘lamellae anulata’ are associated with the activation of genic products of long duration, such as rRNAs and mRNAs contained in the dense cytoplasmic masses. As well as the ‘nuages’, ‘lamelae anulata’, and mitochondrial clusters, the primordial germ cells are frequently highly electron dense. The stem cells in the testis of Sorubim lima share these characteristics. They do not form cysts and are easily found in the rest periods, during the reproductive cycle of the animal. On the other hand, the primary spermatogonia originating from the stem cells are the largest cells of the germinative lineage in Sorubim lima. They form cysts and their nuclear and cytoplasmic characteristics do not differ from those described for other teleosts. According to Selman and Wallace (1986), when these cells have incomplete cytokinesis, they are considered secondary spermatogonia. Billard (1984) mentions that this cell type undergoes morphological changes throughout its numerous generations. As expected, the changes observed in the Sorubim lima secondary spermatogonia give rise to morphological differences between the first and the last generation. These changes are related to the decrease of cellular and nuclear volume, position and size of the nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of the ‘lamellae anulata’, amount and dimension of the ‘nuages’, and movement of the centrioles. The Sorubim lima spermatocytes I, in addition to these nuclear alterations characteristic of prophase, undergo changes in the cytoplasm. ‘Nuages’, mitochondria, centrioles, and the endomembrane system are different in spermatocyte I. The thickened walled vesicles, which appear in the cytoplasm of spermatocytes I of Sorubim lima, will be part of the intermediary piece of the future spermatozoon. (Quagio-Grassiotto, personal communication). The formation of a cilium or short flagellum, originating from one of the centrioles, as observed in Sorubim lima, was observed in spermatocytes I of other groups of animals (QuagioGrassiotto & Lello, 1996). In teleosts, these have also been observed in the spermatocytes I and the spermatogonia (Billard & Fléchon, 1969; Billard, 1984; Thiaw et al., 1995). The asynchronous characteristic of the cell divisions of the germ cells in mitosis or meiosis was detected by Selman and Wallace (1986). In Sorubim lima, this asynchrony can lead to the coexistence of spermatocytes II and newlyformed spermatids in the same cyst (Quagio-Grassiotto, personal communication). The high cytoplasmic density, smaller cellular volume, and also the thickened walled vesicles are the morphological characteristics that differentiate spermatocytes II from the other cell types present in the cysts of Sorubim lima testis.
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Fig. 8 Sorubim lima leptotenic spermatocytes I (LS1). B, cytoplasmic bridges; C, centrioles; N, nucleus; arrow head, mitochondria. X 10 000. Fig. 9 S. lima zygotenic spermatocytes I (ZS1). B, cytoplasmic bridges; C, centrioles; LC, lateral component of the synaptonemical complex; MI, mitochondria; N, nucleus; arrow, nuclear envelope. X 6200. Fig. 10 S. lima pachytenic spermatocytes I (PS1). N, nucleus; V, vesicles; arrow head, mitochondria agglomerations. X 5750. Fig. 11 Synaptonemical complex. CC, central component; LC, lateral component. X 64 000. Fig. 12 Cytoplasmic bridges (B) region of the S. lima prophasic spermatocytes I. C, centrioles; MI, mitochondria; N, nucleus. X 17 000. Fig. 13 S. lima spermatocytes I cytoplasm. C, centrioles; G, Golgi complex; MI, mitochondria; V, vesicles. X 40 250. Fig. 14 Thickened walled vesicles (V) on the S. lima prophasic spermatocytes I cytoplasm. X 28 350. Fig. 15 Centriole behaviour during the S. lima spermatocytes I prophase. B, cytoplasmic bridges; C, centrioles; CI, cilium; G, Golgi complex; MI, mitochondria; N, nucleus; P, plasma membrane; T, microtubules. a: X 21 000; b: X 31 500; c: X 16 500; d; X 58 500.
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Fig. 16 Sorubim lima spermatocytes I (S1) in genic material division. A, anaphase; B, cytoplasmic bridges; CR, chromosomes; D, diakinesis; IM, interstitial myoid cell; M, metaphase; SC, Sertoli cell; TL, telophase; V, thickened walled vesicles; arrow head, mitochondria. a: X 13 250; Inset X 9750; b: X 5400; Inset X 31 500; c: X 8500; d: X 10 000. Fig. 17 S. lima spermatocyte II (S2). C, centrioles in opposite sides of the cell; N, nucleus; V, thickened walled vesicles; arrow head, mitochondria. X 8050. Fig. 18 S. lima spermatocytes II (S2) in genic material division. A, anaphase; C, centrioles; M, metaphase; V, thickened walled vesicles in the cell periphery; arrow head, mitochondria. a: X 10 000; b: X 12 900. Fig. 19 S. lima newly formed spermatid (S). N, eccentric nucleus; V, thickened walled vesicles in the cytoplasmic bridges; B, proximity; arrow, dense bodies; arrow head, mitochondria. X 13 250.
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The behavior of the cytoplasmic bridges in Sorubim lima, a result of incomplete cytokinesis during mitosis and meiosis, follows the patterns described for teleosts and also for other groups of animals (Fawcett et al., 1959; Fawcett, 1961; Clerot, 1971; Dym & Fawcett, 1971; Gondos, 1973; Billard, 1984; Weber & Russell, 1987; Silveira et al. 1990). The cytoplasmic bridges on the different male germ cell types include lateral thickenings, membranous reinforcements in the spermatocytes, and dense bodies in the newlyformed spermatids. ACKNOWLEDGEMENTS We would like to thank the EM Laboratory of IB, Botucatu for the use of their facilities and Dr Heidi Dolder for revising the manuscript. This research was supported by CAUNESP and CESP. REFERENCES Billard, R. 1984. Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis. Cell Tissue Res., 273, 219–226. Billard, R. 1986. Spermatogenesis and spermatology of some teleost fish species. Repord. Nutr. Dev., 26, 877–1024. Billard, R. and Fléchon, J.E. 1969. Spermatogonies et spermatocytes flagellés chez Poecilia reticulata (Poissons téléostéen Cyprinidontiforme). Ann. Biol. Anim. Biochim. Biophys., 281, 281–286. Bruslé, S. and Bruslé, J. 1978. Early sex differentiation in Mugil (Liza) auratus. Risso, 1810, an ultrastructural study. Ann. Biol. Anim. Biochim. Biophys., 18, 871–875. Bruslé-Sicard, S. and Reinboth, R. 1990. Protandric hermaphrodite peculiarities in Amphiprion frenatus Brevoort (Teleostei, Pomacentridae). J. Fish Biol., 36, 383–390. Bruslé-Sicard, S., Reinboth, R. and Fourcault, B. 1994. Germinal potentialities during sexual state changes in a protandric hermaphrodite, Amphiprion frenatus (Teleostei, Pomacentridae). J. Fish Biol., 45, 597–611. Clerot, J.C. 1971. Les ponts intercellulaires du testicule du Gardon, organisation syncitiale et synchronie de la différenciation des cellules germinales. J. Ultrastruct. Res., 37, 690–703. Clerot, J.C. 1976. Les groupements mitochondriau des cellules germinales des poissons téleostéens cyprinidés. I. Etude ultrastructurale. J. Ultrastruct. Res., 54, 461–475. Colombo, G., Grandi, G. and Rossi, R. 1984. Gonad differentation and body growth in Anguilla anguilla L. J. Fish. Biol., 24, 225–228. Colombo, G., Grandi, G., Romeo, A., Giovannini, G., Pellizzola, D., Catozzi, L. and Piffanelli, A. 1987. Testis cytological structure, plasma sex steroids and gonad cytosol steroid receptors of heterologous gonadotropin (hCG) stimulated silver eel, Anguilla anguilla L. Gen. Comp. Endocrinol., 65, 167–178. Cruz-Landim, C. and Cruz-Hofling, M.A. 1986/1987. Aspectos da espermatogênese de tucunaré Cichla ocellaris Schneider, 1801 (Telostei, Cichlidae). Acta Amazônica, 16/17, 65–72. Dym, M. and Fawcett, D.W. 1971. Further observations on the numbers of spermatogonia, spermatocytes and spermatids connected by intercellular bridges in the mammalian testis. Biol. Reprod., 4, 195–215. Eddy, E. M. 1975. Germ plasm and differentiation of the germ cell line. Int. Rev. Cytol., 43, 229–280. Fawcett, D.W. 1961. Intercellular bridges. Exp. Cell Res., 8 (suppl.), 174–187. Fawcett, D.W. Ito, S. and Slauterback, D.L. 1959. The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation. J. Biophys. Biochem. Cytol., 5, 453–460.
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