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Fine structural study of the spermatogenic cycle in Pitar rudis and Chamelea gallina (Mollusca, Bivalvia, Veneridae)
Tissue & Cell, 2002 34 (4) 262–272 © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-8166(02)00016-2, available online at http://www.idealibrary.com
Tissue&Cell
Fine structural study of the spermatogenic cycle in Pitar rudis and Chamelea gallina (Mollusca, Bivalvia, Veneridae) M. Erkan, 1 M. Sousa 2
Abstract. A comparative ultrastructural study of spermatogenesis was performed in the bivalve molluscs Pitar rudis and Chamelea gallina (Veneridae) from Turkey. Sertoli cells appeared to be rich in glycogen, lipid droplets and germ-cell phagolysosomes. Premeiotic cells exhibited nuage and a flagellum, with the Golgi complex and the rough endoplasmic reticulum originating proacrosomal vesicles during the pachytene stage. In round spermatids, the acrosomal vesicle migrated linked to the plasma membrane. In P. rudis , the acrosomal vesicle base formed a thin expansion that attached to the nuclear apex and was associated with development of the perforatorium. The cap-shaped acrosomal vesicle then differentiated into external and internal regions, and also into a small apical light region, although some cells exhibited an apical extension of the external component. On the contrary, two lateroapical light pouches developed in C. gallina . During spermiogenesis, chromatin became fibrillar and then condensed while the nucleus turned conical shaped in P. rudis or slightly curved in C. gallina . In P. rudis , the midpiece contained glycogen and four mitochondria, although five mitochondria were sometimes observed, whereas in C. gallina the midpiece contained four mitochondria. Comparison with other members of Veneroida shows a common ectaquasperm type, but novel findings in acrosome biogenesis. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Mollusca, Bivalvia, Heterodonta, Veneroida, spermatogenesis
Introduction The ultrastructural and morphometric characteristics of mature spermatozoa in the Mollusca have been correlated with the different reproductive strategies of organisms, 1 Department of Biology, Faculty of Sciences, University of Istanbul, Istanbul, Turkey, 2 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Professor Abel Salazar 2, 4099-003 Porto, Portugal
Received 1 August 2001 Accepted 14 February 2002 Correspondence to: M ário Sousa, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Professor Abel Salazar 2, 4099-003 Porto, Portugal. Fax: +351 22 206 22 32; E-mail: [email protected]
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including the adaptation to egg structure of the species, and were also shown to be of great value for tracing phylogenetic and taxonomic relationships between species (Popham, 1979; Franzén, 1983; Healy, 1995; Hodgson, 1986, 1995; Morse & Zardus, 1997; Kafanov & Drozdov, 1998). The ultrastructural characteristics of spermatogenesis have also revealed important findings in relation to the individual species under study, and shown that different organisms generally present specific morphogenetic traits, including: (1) the cell stage at which proacrosomal vesicles first appear; (2) the mechanism by which the acrosomal vesicle migrates towards the apical pole of the cell; (3) the morphological appearance and chemical constitution of the acrosome subcomponents; (4) the origin of the subacrosomal
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Fig. 1 P. rudis. Sertoli cells (S), showing glycogen deposits (g) and phagolysosomes with degenerated germ cells (*). Note the presence of germ-cell flagella released in the cytoplasm of Sertoli cells (white arrowheads). SG, spermatogonium; ST, primary spermatocyte; BL, basal lamina. ×8200. Fig. 2 P. rudis. Spermatogonia (SG), with mitochondria (m), nuage (n), distal centriole (C) and intracellular axoneme (Ax). Pachytene spermatocytes (ST) show synaptonemal complexes (SC), mitochondria (m) and proacrosomal vesicles (white arrowheads). Inset: extrusion of the flagellum is due to linkage of the distal centriole (DC) to the plasma membrane and encirclement of the axoneme (Ax) by Golgian (G) vesicles. (∗ ) Sertoli cell phagolysosome with degenerated germ-cells. ×8400; Inset: ×23 100.
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Fig. 3 P. rudis. Spermatogonium (SG), with mitochondria (m), rough endoplasmic reticulum cisternae (rer), nuage (n) and Golgi complexes (G). Note shedding of annulate lamellae (black arrowhead) and rough endoplasmic reticulum cisternae (white arrowheads) by the nuclear envelope. (∗ ) Sertoli cell phagolysosome with degenerated germ-cells. ×16 000. Fig. 4 P. rudis. At pachytene, proacrosomal vesicles (white arrowheads) are released from the Golgi complex (G). Note interaction of small Golgian vesicles (v) with the rough endoplasmic reticulum (rer). m, Mitochondria. Inset: fusion (white arrowheads) between proacrosomal vesicles. ×63 100; Inset: ×34 700. Fig. 5 P. rudis. Metaphase I of meiosis. Note the dense contents (black arrowhead) accumulated inside rough endoplasmic reticulum cisternae (rer) and the close association with proacrosomal vesicles (white arrowheads). Chro, metaphase I chromosomes; m, mitochondria. ×26 000.
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Fig. 6 P. rudis. Round spermatid (Sa) with large dense patches of heterochromatin. In the basal pole there are proacrosomal vesicles (white arrowheads), mitochondria (m) and an intracellular axoneme (Ax). ×14 500. Figs 7 & 8 P. rudis. Round spermatid (Sa) with fine fibrillar cord-like arranged chromatin. Fig. 7 At the basal pole, note chromatin condensation at the contact regions (white arrowheads) with mitochondria (m), the acrosomal vesicle (av), the Golgi complex (G) and the intracellular axoneme (Ax). Fig. 8 The acrosomal vesicle (av) is linked to the plasma membrane by its apex (black arrowhead) and shows a fine dense basal external coat (white arrowhead). ×22 200 and ×41 300. Figs 9 & 10 P. rudis. Round spermatid, with coarse fibrillar cord-like arranged chromatin. At the apical pole, the apex of the flattened acrosomal vesicle (av) remains attached to the plasma membrane (black arrowhead) while its base evaginates to form a basal process (white arrowhead) that attaches to the nucleus (N). At the basal pole, note chromatin condensation at the contact region (white arrowhead) with the proximal centriole. A dense material (*) links the proximal centriole (pC) to the centriolar fossa and to the distal centriole (dC). The latter is connected to the plasma membrane by the pericentriolar complex (pCc) and gives origin to the axoneme (Ax) of the flagellum. m, Mitochondria. ×47 700 and ×23 500. Figs 11–13 P. rudis. Early (Sb) and late elongating (Sc) and elongated (Sd) spermatids. As the nucleus elongates and condenses, chromatin turns coarse filamentous and develops a longitudinal pattern, while the nucleus forms mitochondrial (m) and centriolar (C) fossae (white arrowheads). Note development of the perforatorium (*) as the base of the acrosomal vesicle (av) invaginates. ×21 900, ×21 000 and ×17 400.
material; (5) the pattern of chromatin remodeling during condensation; (6) and the composition and structure of the midpiece (Sousa & Azevedo, 1988; Sousa et al., 1989, 1995, 1998, 2000; Sousa & Oliveira, 1994a–c). In the Veneroida, the specific study of the complete steps of spermatogenesis has been very limited and includes the
Lucinacea (Johnson et al., 1996), Leptonacea (Eckelbarger et al., 1990), Mactracea (Longo & Anderson, 1969), Solenacea (Reunov & Hodgson, 1994), Tellinacea (Sousa et al., 1989; Hodgson et al., 1990; Reunov & Hodgson, 1994; Sousa & Oliveira, 1994a), Corbiculacea (Konishi et al., 1998) and Veneracea (Reunov & Hodgson, 1994).
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Fig. 14 P. rudis. Mature spermatozoon showing a dense nucleus (N) with apical and mitochondrial fossae (white arrowheads), an acrosome with acrosomal vesicle (av) and perfuratorium (*), and a midpiece with mitochondria (m) and glycogen particles (g). Ax, flagellar axoneme. ×29 700. Figs 15 & 16 P. rudis. Acrosomal vesicle and perforatorium (*) of mature spermatozoa. The acrosomal vesicle displays outer dense (o) and inner intermediate-dense (i) regions. Fig. 15 In most sperm, the acrosomal vesicle also exhibits a light apical tip (white arrowhead). Fig. 16 In a few sperm, the apical protrusion of the acrosomal vesicle is longer and filled with the outer dense component (white arrowhead). N, nucleus. ×61 400 and ×40 100. Figs 17–19 P. rudis. Midpiece of mature spermatozoa. Fig. 17 The proximal centriole is linked to the nucleus (N) at the centriolar fossa (white arrowhead), while the distal centriole originates the axoneme (Ax) of the flagellum. Fig. 18 In the majority of the cases, four mitochondria (m) encircle the centrioles (c). Fig. 19 In about 10% of the cases, sperm exhibit five mitochondria. ×21 200, ×22 600 and ×19 600.
The present study describes the ultrastructural features of the germinal epithelium and mature primitive ectaquasperm of two species, Pitar rudis and Chamelea gallina (Veneroidea). Data show that despite very similar structural morphologies and developmental patterns, each can be distinguished by clear traits observed during the spermatogenic cycle.
cut in a Leica ultratome with a Diatome knife, collected in 300 mesh copper grids (Taab), double-stained with alcoholic concentrated uranyl acetate for 20 min followed by lead citrate (Reynolds) for 10 min, and studied at 60 kV in a transmission electron microscope JEOL 100 CX II (Sousa et al., 2000). All chemicals were of analytical grade and purchased from Merck.
Materials and methods
Results
P. rudis and C. gallina (Mollusca, Bivalvia, Heterodonta, Veneroida, Veneroidea, Veneridae) specimens were collected from the intertidal zone of the Turkish coast. Small pieces of testis (<1 mm) were fixed with 2.5% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4 for 2 h at 4 ◦ C and then rinsed in the same buffer. Specimens were post-fixed in 2% osmium tetroxide in the same buffer for 2 h at 4 ◦ C, serially dehydrated in an ethanol series followed by propylene oxide, and finally embedded in Epon. Ultrathin sections were
P. rudis Sertoli cells were irregularly shaped, as to enclose germ cells, extending from the basal lamina towards the lumen. They displayed an ovoid indented nucleus with 2–3 nucleoli. Besides mitochondria, Golgi complexes, endoplasmic reticulum cisternae, ribosomes and lipid droplets, the cytoplasm was rich in microtubules, glycogen, and large phagolysosomes with degenerated germ cells (Fig. 1). Spermatogonia were adjacent to the basal lamina and presented a large, round
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Fig. 20 C. gallina. Sertoli cell (S) with glycogen (g), lipid droplets (L), microtubules (mt), endoplasmic reticulum (rer), dictyosomes (G), lysosomes (Ly) and mitochondria (m). Note that intracellular axonemes (Ax) are derived (inset) from phagocytic vesicles containing germ-cell flagella (white arrowhead). SG< spermatogonia; BL, basal lamina. ×9100; Inset: ×15 200. Fig. 21 C. gallina. Spermatogonia (SG) show intercellular bridges (*), mitochondria (m), nuage (n) and a flagellum (F). They are linked (inset a) to Sertoli cells (S) by desmosome-like junctions (white arrowheads). They also exhibit (inset b) proximal (pC) and distal (dC) centrioles, with the latter being linked to the plasma membrane by a pericentriolar complex (pCc) and gives rise to the axoneme (Ax) of the extruded flagellum. Note their close association with leptotene (Lep) and pachytene (Paq) spermatocytes. Leptotene spermatocytes (inset c) evidence mitochondria (m), rough endoplasmic reticulum cisternae (rer), an active dictyosome (G) and desmosome-like junctions (white arrowhead) with Sertoli cells. ×6600; inset a: ×11 100; inset b: ×16 700; inset c: ×12 800.
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Fig. 22 C. gallina. Pachytene spermatocyte, with synaptonemal complexes (SC), proacrosomal vesicles (v) and an active dictyosome (G). ×9000. Fig. 23 C. gallina. Note meiotic metaphase I chromosomes (Chro), the aster centriole (C), mitochondria (m), rough endoplasmic reticulum cisternae (rer) and proacrosomal vesicles (v). ×7700. Figs 24 & 25 C. gallina. Pachytene spermatocytes. Fig. 24 Shedding of proacrosomal vesicles (white arrowheads) from dictyosomes (G). Fig. 25 Close association (white arrowheads) of proacrosomal vesicles with the rough endoplasmic reticulum (rer). N, nucleus; m, mitochondria; c, centriole. ×26 000 and ×37 600. Fig. 26 C. gallina. Round spermatid. During migration, the acrosomal vesicle (av) is linked by its apex to the plasma membrane (black arrowhead) and coated by a fine dense basal material (white arrowhead). Inset: at the apical pole, the acrosomal vesicle base becomes invaginated to form the subacrosomal space (*). N, nucleus. ×20 200; inset: ×26 000. Fig. 27 C. gallina. In the elongated spermatid nucleus (N), chromatin becomes coarse fibrillar with a longitudinal arrangement. In the acrosome, note the subacrosomal space (*) and the acrosomal vesicle with outer dense (o) and inner intermediately dense (i) regions. In the midpiece, the proximal centriole (pC) is linked to the nuclear base, while the distal centriole (dC) anchors to the plasma membrane by the pericentriolar complex (pCc). m, Mitochondria. ×16 500.
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nucleus with 1–2 nucleoli, mitochondria, small nuage aggregates (granulofibrillar materials containing exported ribonucleoproteins), dictyosomes, endoplasmic reticulum cisternae actively shed from the nuclear envelope, ribosomes, and an intracellular axoneme (Figs 2 & 3). Primary spermatocytes displayed similar organelles and structures as spermatogonia, but from the pachytene stage they extruded the flagellum (Fig. 2) and the Golgi complex began the synthesis of small, dense proacrosomal vesicles (Fig. 4). The latter also seemed to receive components of the rough endoplasmic reticulum cisternae (Figs 4 & 5). During spermiogenesis, the round spermatid nucleus became progressively elongated, with chromatin turning longitudinally filamentous before beginning a homogeneous condensation process, which left small-uncondensed spaces (Figs 7, 11–13). At the nuclear base, chromatin was condensed at the sites of attachment to mitochondria (Figs 7 & 8) and the proximal centriole (Fig. 10). Later, as the cytoplasmic axoneme was extruded to form the flagellum, the nucleus developed mitochondrial and centriolar fossae (Figs 6, 10–13). In the round spermatid, proacrosomal vesicles fused to form a round acrosomal vesicle that exhibited a central ring-shaped dense component, a central and apical intermediate-dense material, and a basal light region (Figs 6–8). The acrosomal vesicle then migrated from the basal pole to the cell apex, linked apically to the plasma membrane, while developing a dense basal coat (Figs 7 & 8). Once at the nuclear apex, the acrosomal vesicle turned so that it was horizontally elongated and its contents became segregated into a central dense material completely surrounded by an intermediate dense component. The acrosomal vesicle base then formed a thin expansion that attached to the nuclear envelope (Figs 9 & 10), being associated with development of acrosomal and nuclear indentations and the subacrosomal material, then filled with a perforatorium (Figs 11–13). The mature spermatozoon showed a conical elongated nucleus (2.6 m) and a cap-shaped elongated acrosomal vesicle (0.6 m) (Fig. 14). The acrosomal vesicle displayed external dense and internal intermediately dense regions, and a small apical light tip (Fig. 15). However, in a few cases the apical protrusion was longer and filled with the outer dense component (Fig. 16). In the midpiece (0.7 m), which contained glycogen, there were two centrioles and four mitochondria, although about <10% of sperm exhibited five mitochondria. The proximal centriole was tightly linked to the nuclear envelope, whereas the distal centriole was connected to the plasma membrane by the pericentriolar complex and gave origin to the flagellum, which contained a 9d + 2s axoneme (Figs 17–19). C. gallina Sertoli cells were very similar to those found in P. rudis, but showed higher numbers of lipid droplets, only a few phagolysosomes containing degenerated germ cells (Fig. 20), and evident desmosome-like junctions with spermatogonia and spermatocytes (Fig. 21). Spermatogonia were adjacent to
the basal lamina and presented intercellular bridges, a large ovoid nucleus with 1–2 nucleoli, mitochondria, small nuage materials, a small dictyosome, a few endoplasmic reticulum cisternae, ribosomes and a flagellum (Fig. 21). Primary spermatocytes displayed similar organelles and structures as spermatogonia, with small dense proacrosomal vesicles being produced by the Golgi complex and the rough endoplasmic reticulum since the pachytene stage (Figs 22–25). During spermiogenesis, the nucleus elongated and chromatin condensed as shown for P. rudis (Figs 26 & 27). In early spermatids, the acrosomal vesicle also migrated linked apically to the plasma membrane, while developing an outer dense basal coat. Their contents became differentiated into a thin peripheral layer of intermediate density, a floccular matrix, and a central ring-shaped dense component (Fig. 26). Once at the nuclear apex, it developed a basal invagination to originate the subacrosomal space (Fig. 26, inset) that developed a perforatorium in later stages (Fig. 27). The mature spermatozoon showed a conical elongated and slightly curved nucleus (3.5 m) and a cap-shaped elongated acrosomal vesicle (0.6 m) with external dense and internal intermediately dense regions, and two small lateroapical light tips (Figs 28 & 29). In the midpiece (0.6 m), there were two centrioles and four mitochondria (Figs 28 & 30).
Fig. 28 C. gallina. Mature sperm, showing an elongated and condensed nucleus (N) with mitochondrial (m) and centriolar (c) fossae (white arrowheads). Note the acrosomal vesicle (av) and the subacrosomal space (*). Ax, flagellar axoneme. ×18 700. Fig. 29 C. gallina. Acrosome of mature sperm. The acrosomal vesicle displays outer dense (o) and inner intermediately dense (i) regions, as also two apicolateral light pouches (white arrowheads). Note the perforatorium (*) and the apical nuclear (N) protrusion. ×40 000. Fig. 30 C. gallina. Midpiece of mature sperm, with four mitochondria (m) encircling the proximal centriole (c). ×20 000.
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Discussion Invertebrate spermatozoa are divided into three main types: aquasperm (ectaquasperm and entaquasperm), introsperm and dimorphic or polymorphic sperm (Jamieson, 1987; Rouse & Jamieson, 1987). Ectaquasperm are primitive and shed in water, where they fertilize. The ‘primitive’ sperm type is characterized by a small head, containing a rounded or conical nucleus, surmounted by a cup-shaped acrosomal vesicle, a midpiece with a ring of 4–5 rounded mitochondria encircling two centrioles, and a tail flagellum about 50 m long (axoneme with a 9d + 2s microtubule pattern) (Franzén, 1983). Within the Veneroida (Bivalvia, Heterodonta) the spermatozoon is of the ‘primitive’ type in almost all species studied, although this definition includes some modifications of the sperm nucleus and midpiece that deviate from the most common type (Lucinacea: Healy, 1995; Johnson et al., 1996; Leptonacea: Galeommatidae: Eckelbarger et al., 1990; Cardiacea: Sousa & Azevedo, 1988; Healy, 1995; Keys & Healy, 1999, 2000; Tellinacea: Sousa et al., 1989; Healy, 1995; Dreissenacea: Denson & Wang, 1994, 1998; Walker et al., 1996; Veneracea: Turtoniidae: Ockelmann, 1964), as also a few examples of modified and dimorphic sperm (Lucinacea: Healy, 1995; Moueza & Frankiel, 1995; Leptonacea: Lasaeidae: Ó’Foighil, 1985; Leptonacea: Montacutidae: Ockelmann, 1965; Corbiculacea: Komaru & Konishi, 1996; Konishi et al., 1998). The spermatogenesis of P. rudis and C. gallina as studied here represents the first detailed description of the seminiferous epithelium in the Veneroidea. Comparisons between members of this superfamily show only slight differences between the specific steps of spermatogenesis, which nevertheless confer individual characteristics to each different species. Sertoli cell structure has only been described in other Veneroida superfamilies. Similar to P. rudis and C. gallina, Sertoli cells rich in glycogen and lipid droplets were described in Loripes lucinalis (Lucinacea) (Johnson et al., 1996), Divariscintilla sp. (Leptonacea) (Eckelbarger et al., 1990) and Scrobicularia plana (Tellinacea) (Sousa et al., 1989), whereas germ-cell phagocytosis was observed only in Divariscintilla sp. In the Veneroidea, the presence of a flagellum in premeiotic germ cells was described in primary spermatocytes of Tivela polita (Reunov & Hodgson, 1994). In the present study, it is shown that whereas spermatogonia of C. gallina already have a flagellum, P. rudis exhibited an intracellular axoneme in spermatogonia that became extruded only in primary spermatocytes. The presence of a flagellum in premeiotic germ cells was also found in other Veneroida species, such as Divariscintilla sp. (Eckelbarger et al., 1990), Solen sp. (Hodgson et al., 1987; Reunov & Hodgson, 1994) and S. plana (Sousa et al., 1989). In the Veneroidea, proacrosomal vesicles derived from Golgian activity have been described during the pachytene stage in T. polita (Reunov & Hodgson, 1994), or during spermiogenesis in Venerupis sp. and Callista chione
(Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971; Nicotra & Zappata, 1991). On the contrary, in P. rudis and C. gallina, proacrosomal vesicles, although produced during the pachytene stage by dictyosomes, also seemed to incorporate components of the endoplasmic reticulum. Comparisons with other Veneroida members show that proacrosomal vesicles may form either during the leptotene stage, as in Divariscintilla sp. (Eckelbarger et al., 1990), or the zygotene/pachytene stage (similar to P. rudis and C. gallina) as in Solen sp. (Solenacea) (Hodgson et al., 1987; Reunov & Hodgson, 1994), Donax sp. (Tellinacea) (Hodgson et al., 1990; Reunov & Hodgson, 1994; Sousa & Oliveira, 1994a) and Corbicula fluminea (Corbiculacea) (Konishi et al., 1998). However, in all these species, proacrosomal vesicles also originated from dictyosomes, and thus the active participation of the rough endoplasmic reticulum in this process here is firstly described in the Veneroida for P. rudis and C. gallina. Description of the mechanism by which the acrosomal vesicle migrates during spermiogenesis has also not been studied in most of the species. In P. rudis and C. gallina, the apex of the acrosomal vesicle first links to the plasma membrane and then migrates to the apical pole of the cell. During this process, it acquires an outer dense material that defines the basal pole of the acrosomal vesicle and corresponds to the precursors of the subacrosomal material. A similar situation has only been found previously for D. trunculus and S. plana (Sousa et al., 1989; Sousa & Oliveira, 1994a). Formation of the subacrosomal space is supposed to be driven by deposition of the subacrosomal material. However, in P. rudis the acrosomal vesicle base formed an evaginated process that was associated with formation of the subacrosomal region. This type of activity of the acrosomal vesicle has never been described before and thus constitutes a novel description in Mollusca. In relation to acrosomal vesicle contents, whereas all members of the Veneroidea show a dual differentiation of the acrosomal contents, here it is shown that P. rudis and C. gallina have a tripartite composition due to the presence of apical acrosomal protrusions. Regarding the presence of a perforatorium, which is also present in P. rudis and C. gallina, in the Veneroidea only T. polita shows an absence of such subacrosomal specialization (Reunov & Hodgson, 1994). In the Veneroidea, the sperm nucleus also shows wide variations, with Tivela minuta exhibiting a very long conical and spiral nucleus (Reunov & Hodgson, 1994), and Venerupis sp. a long conical and slightly curved nucleus (Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971). On the contrary, P. rudis and C. gallina (in the current study), C. chione (Nicotra & Zappata, 1991) and T. polita (Reunov & Hodgson, 1994) all show a short conical and slightly curved nucleus. The process of nuclear chromatin condensation during spermiogenesis has not been described in the Veneroidea. In comparison with other Veneroida, a similar situation to P. rudis and C. gallina, where chromatin condensation progresses through a filamentous stage, was found in species with an elongated nucleus, such as L. lucinalis (Johnson et al., 1996), Divariscintilla
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sp. (Eckelbarger et al., 1990) and S. plana (Sousa et al., 1989). Finally, and in relation to the midpiece, P. rudis and C. gallina (in the current study), T. minuta (Reunov & Hodgson, 1994) and Venerupis sp. (Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971) show four mitochondria, whereas other members of the Veneroidea possess five mitochondria. In this regard, P. rudis constitutes an exception and shows a polymorphic trait, as about 10% of sperm also exhibited five mitochondria. On the contrary, the presence of glycogen in the midpiece, as found in P. rudis, was only observed in other Veneroida, such as Divariscintilla sp. (Eckelbarger et al., 1990) and Donax sp. (Hodgson et al., 1990).
ACKNOWLEDGEMENTS We acknowledge E. Oliveira for technical assistance, J. Carvalheiro and J. Gonçalves for iconography, Professor Dr Dinçer Gulen for institutional support (to M.E.) and S. Albayrak for assistance in specimen collection. This study was partially supported by FCT (UMIB; Sapiens 36363/99, 35231/99).
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Johnson, M.J., Casse, N. and Le Pennec, M. 1996. Spermatogenesis in the endosymbiont-bearing bivalve Loripes lucinalis (Veneroida: Lucinidae). Mol. Reprod. Dev., 45, 476–484. Kafanov, A.I. and Drozdov, A.L. 1998. Comparative sperm morphology and phylogenetic classification of recent mytiloidea (Bivalvia). Malacologia, 39, 129–139. Keys, J.L. and Healy, J.M. 1999. Sperm ultrastructure of the giant clam Tridacna maxima (Tridacnidae: Bivalvia: Mollusca) from the Great Barrier Reef. Mar. Biol., 135, 41–46. Keys, J.L. and Healy, J.M. 2000. Relevance of sperm ultrastructure to the classification of giant clams (Mollusca, Cardioidea, Cardiidae, Tridacninae). In: Harper, E.M., Taylor, J.D. and Crame, J.A. (eds) The Evolutionary Biology of the Bivalvia, Vol. 177. Geological Society, Special Publications, London, pp 191–205. Komaru, A. and Konishi, K. 1996. Ultrastructure of biflagellate spermatozoa in the freshwater clam, Corbicula leana (Prime). Invert. Reprod. Dev., 29, 193–197. Konishi, K., Kawamura, K., Furuita, H. and Komaru, A. 1998. Spermatogenesis of the freshwater clam Corbicula AFF. fluminea Müller (Bivalvia: Corbiculidae). J. Shellfish Res., 17, 185–189. Longo, F.J. and Anderson, E. 1969. Spermiogenesis in the surf clam Spisula solidissima with special reference to the formation of the acrosomal vesicle. J. Ultrastruct. Res., 27, 435–443. Morse, M.P. and Zardus, J.D. 1997. Bivalvia. In: Harrison, F.W. and Kohn, A.J. (eds) Microscopic Anatomy of Invertebrates. 2: Mollusca II, Vol. 6A. Wiley-Liss, Inc., New York, pp 7–118. Moueza, M. and Frenkiel, L. 1995. Ultrastructural study of the spermatozoon in a tropical lucinid bivalve: Codakia orbicularis L. Invert. Reprod. Dev., 27, 205–211. Nicotra, A. and Zappata, S. 1991. Ultrastructure of the mature sperm and spermiogenesis in Callista chione (Mollusca, Bivalvia). Invert. Reprod. Dev., 20, 213–218. Ó’Foighil, D. 1985. Fine structure of Lasaea subviridis and Mysella tumida sperm (Bivalvia, Galeommatacea. Zoomorphology, 105, 125–132. Ockelmann, K.W. 1964. Turtonia minuta (Fabricius), a neotenous veneracean bivalve. Ophelia, 1, 121–146. Ockelmann, K.W. 1965. Redescription, distribution, biology, and dimorphous sperm of Montacuta tenella Lovén (Mollusca, Leptonacea). Ophelia, 2, 211–221. Pochon-Masson, J. and Gharagozlou, I.-D. 1970. Particularité morphologique de l’acrosome dans le spermatozo¨ıde de Tapes decussatus L. (Mollusque lamellibranche). Annales Sci. Naturelles Zoologie Paris, 12, 171–180. Popham, J.D. 1979. Comparative spermatozoon morphology and bivalve phylogeny. Malacological Rev., 12, 1–20. Reunov, A.A. and Hodgson, A.N. 1994. Ultrastructure of the spermatozoa of five species of South African bivalves (Mollusca), and an examination of early spermatogenesis. J. Morphol., 219, 275–283. Rouse, G.W. and Jamieson, B.G.M. 1987. An ultrastructural study of the spermatozoa of the polychaetes Eurythoe complanata (Amphinomidae), Clymenella sp. and Micromaldane sp. (Maldanidae), with definition of sperm types in relation to reproductive biology. J. Submicrosc. Cytol. Pathol., 19, 573–584. Sousa, M. and Azevedo, C. 1988. Comparative silver staining analysis on spermatozoa of various invertebrate species. Int. J. Invert. Reprod. Dev., 13, 1–8. Sousa, M. and Oliveira, E. 1994a. Ultrastructural and cytochemical study of spermatogenesis in Donax trunculus (Mollusca, Bivalvia). J. Submicrosc. Cytol. Pathol., 26, 305–311. Sousa, M. and Oliveira, E. 1994b. An ultrastructural study of spermatogenesis in Helcion pellucidus (Gastropoda, Prosobranchia). Invert. Reprod. Dev., 26, 119–126. Sousa, M. and Oliveira, E. 1994c. An ultrastructural study of Crassostrea angulata (Mollusca, Bivalvia) spermatogenesis. Mar. Biol., 120, 545–551. Sousa, M., Corral, L. and Azevedo, C. 1989. Ultrastructural and cytochemical study of spermatogenesis in Scrobicularia plana (Mollusca, Bivalvia). Gamete Res., 24, 393–401. Sousa, M., Oliveira, E., Carvalheiro, J. and Oliveira, V. 1995. Comparative silver staining of molluscan spermatozoa. In: Jamieson, B.G.M., Ausio, J. and Justine, J.-L. (eds) Advances in Spermatozoal Phylogeny and Taxonomy, Vol. 166. Mém. Mus. Natl. Hist. Nat., Paris, pp 179–187.
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Sousa, M., Guerra, R., Oliveira, E. and Torres, A. 1998. Comparative PTA staining of molluscan spermatozoa. J. Submicrosc. Cytol. Pathol., 30, 183–187. Sousa, M., Cunha, C., Erkan, M., Guerra, R., Oliveira, E. and Baldaia, L. 2000. Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and
comparative enzymatic ultracytochemical study. Tissue Cell, 32, 88–94. Walker, G.K., Black, M.G. and Edwards, C.A. 1996. Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: light and electron microscopy. Can. J. Zool., 74, 809–815.
Tissue&Cell
Fine structural study of the spermatogenic cycle in Pitar rudis and Chamelea gallina (Mollusca, Bivalvia, Veneridae) M. Erkan, 1 M. Sousa 2
Abstract. A comparative ultrastructural study of spermatogenesis was performed in the bivalve molluscs Pitar rudis and Chamelea gallina (Veneridae) from Turkey. Sertoli cells appeared to be rich in glycogen, lipid droplets and germ-cell phagolysosomes. Premeiotic cells exhibited nuage and a flagellum, with the Golgi complex and the rough endoplasmic reticulum originating proacrosomal vesicles during the pachytene stage. In round spermatids, the acrosomal vesicle migrated linked to the plasma membrane. In P. rudis , the acrosomal vesicle base formed a thin expansion that attached to the nuclear apex and was associated with development of the perforatorium. The cap-shaped acrosomal vesicle then differentiated into external and internal regions, and also into a small apical light region, although some cells exhibited an apical extension of the external component. On the contrary, two lateroapical light pouches developed in C. gallina . During spermiogenesis, chromatin became fibrillar and then condensed while the nucleus turned conical shaped in P. rudis or slightly curved in C. gallina . In P. rudis , the midpiece contained glycogen and four mitochondria, although five mitochondria were sometimes observed, whereas in C. gallina the midpiece contained four mitochondria. Comparison with other members of Veneroida shows a common ectaquasperm type, but novel findings in acrosome biogenesis. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Mollusca, Bivalvia, Heterodonta, Veneroida, spermatogenesis
Introduction The ultrastructural and morphometric characteristics of mature spermatozoa in the Mollusca have been correlated with the different reproductive strategies of organisms, 1 Department of Biology, Faculty of Sciences, University of Istanbul, Istanbul, Turkey, 2 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Professor Abel Salazar 2, 4099-003 Porto, Portugal
Received 1 August 2001 Accepted 14 February 2002 Correspondence to: M ário Sousa, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Professor Abel Salazar 2, 4099-003 Porto, Portugal. Fax: +351 22 206 22 32; E-mail: [email protected]
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including the adaptation to egg structure of the species, and were also shown to be of great value for tracing phylogenetic and taxonomic relationships between species (Popham, 1979; Franzén, 1983; Healy, 1995; Hodgson, 1986, 1995; Morse & Zardus, 1997; Kafanov & Drozdov, 1998). The ultrastructural characteristics of spermatogenesis have also revealed important findings in relation to the individual species under study, and shown that different organisms generally present specific morphogenetic traits, including: (1) the cell stage at which proacrosomal vesicles first appear; (2) the mechanism by which the acrosomal vesicle migrates towards the apical pole of the cell; (3) the morphological appearance and chemical constitution of the acrosome subcomponents; (4) the origin of the subacrosomal
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Fig. 1 P. rudis. Sertoli cells (S), showing glycogen deposits (g) and phagolysosomes with degenerated germ cells (*). Note the presence of germ-cell flagella released in the cytoplasm of Sertoli cells (white arrowheads). SG, spermatogonium; ST, primary spermatocyte; BL, basal lamina. ×8200. Fig. 2 P. rudis. Spermatogonia (SG), with mitochondria (m), nuage (n), distal centriole (C) and intracellular axoneme (Ax). Pachytene spermatocytes (ST) show synaptonemal complexes (SC), mitochondria (m) and proacrosomal vesicles (white arrowheads). Inset: extrusion of the flagellum is due to linkage of the distal centriole (DC) to the plasma membrane and encirclement of the axoneme (Ax) by Golgian (G) vesicles. (∗ ) Sertoli cell phagolysosome with degenerated germ-cells. ×8400; Inset: ×23 100.
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Fig. 3 P. rudis. Spermatogonium (SG), with mitochondria (m), rough endoplasmic reticulum cisternae (rer), nuage (n) and Golgi complexes (G). Note shedding of annulate lamellae (black arrowhead) and rough endoplasmic reticulum cisternae (white arrowheads) by the nuclear envelope. (∗ ) Sertoli cell phagolysosome with degenerated germ-cells. ×16 000. Fig. 4 P. rudis. At pachytene, proacrosomal vesicles (white arrowheads) are released from the Golgi complex (G). Note interaction of small Golgian vesicles (v) with the rough endoplasmic reticulum (rer). m, Mitochondria. Inset: fusion (white arrowheads) between proacrosomal vesicles. ×63 100; Inset: ×34 700. Fig. 5 P. rudis. Metaphase I of meiosis. Note the dense contents (black arrowhead) accumulated inside rough endoplasmic reticulum cisternae (rer) and the close association with proacrosomal vesicles (white arrowheads). Chro, metaphase I chromosomes; m, mitochondria. ×26 000.
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Fig. 6 P. rudis. Round spermatid (Sa) with large dense patches of heterochromatin. In the basal pole there are proacrosomal vesicles (white arrowheads), mitochondria (m) and an intracellular axoneme (Ax). ×14 500. Figs 7 & 8 P. rudis. Round spermatid (Sa) with fine fibrillar cord-like arranged chromatin. Fig. 7 At the basal pole, note chromatin condensation at the contact regions (white arrowheads) with mitochondria (m), the acrosomal vesicle (av), the Golgi complex (G) and the intracellular axoneme (Ax). Fig. 8 The acrosomal vesicle (av) is linked to the plasma membrane by its apex (black arrowhead) and shows a fine dense basal external coat (white arrowhead). ×22 200 and ×41 300. Figs 9 & 10 P. rudis. Round spermatid, with coarse fibrillar cord-like arranged chromatin. At the apical pole, the apex of the flattened acrosomal vesicle (av) remains attached to the plasma membrane (black arrowhead) while its base evaginates to form a basal process (white arrowhead) that attaches to the nucleus (N). At the basal pole, note chromatin condensation at the contact region (white arrowhead) with the proximal centriole. A dense material (*) links the proximal centriole (pC) to the centriolar fossa and to the distal centriole (dC). The latter is connected to the plasma membrane by the pericentriolar complex (pCc) and gives origin to the axoneme (Ax) of the flagellum. m, Mitochondria. ×47 700 and ×23 500. Figs 11–13 P. rudis. Early (Sb) and late elongating (Sc) and elongated (Sd) spermatids. As the nucleus elongates and condenses, chromatin turns coarse filamentous and develops a longitudinal pattern, while the nucleus forms mitochondrial (m) and centriolar (C) fossae (white arrowheads). Note development of the perforatorium (*) as the base of the acrosomal vesicle (av) invaginates. ×21 900, ×21 000 and ×17 400.
material; (5) the pattern of chromatin remodeling during condensation; (6) and the composition and structure of the midpiece (Sousa & Azevedo, 1988; Sousa et al., 1989, 1995, 1998, 2000; Sousa & Oliveira, 1994a–c). In the Veneroida, the specific study of the complete steps of spermatogenesis has been very limited and includes the
Lucinacea (Johnson et al., 1996), Leptonacea (Eckelbarger et al., 1990), Mactracea (Longo & Anderson, 1969), Solenacea (Reunov & Hodgson, 1994), Tellinacea (Sousa et al., 1989; Hodgson et al., 1990; Reunov & Hodgson, 1994; Sousa & Oliveira, 1994a), Corbiculacea (Konishi et al., 1998) and Veneracea (Reunov & Hodgson, 1994).
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Fig. 14 P. rudis. Mature spermatozoon showing a dense nucleus (N) with apical and mitochondrial fossae (white arrowheads), an acrosome with acrosomal vesicle (av) and perfuratorium (*), and a midpiece with mitochondria (m) and glycogen particles (g). Ax, flagellar axoneme. ×29 700. Figs 15 & 16 P. rudis. Acrosomal vesicle and perforatorium (*) of mature spermatozoa. The acrosomal vesicle displays outer dense (o) and inner intermediate-dense (i) regions. Fig. 15 In most sperm, the acrosomal vesicle also exhibits a light apical tip (white arrowhead). Fig. 16 In a few sperm, the apical protrusion of the acrosomal vesicle is longer and filled with the outer dense component (white arrowhead). N, nucleus. ×61 400 and ×40 100. Figs 17–19 P. rudis. Midpiece of mature spermatozoa. Fig. 17 The proximal centriole is linked to the nucleus (N) at the centriolar fossa (white arrowhead), while the distal centriole originates the axoneme (Ax) of the flagellum. Fig. 18 In the majority of the cases, four mitochondria (m) encircle the centrioles (c). Fig. 19 In about 10% of the cases, sperm exhibit five mitochondria. ×21 200, ×22 600 and ×19 600.
The present study describes the ultrastructural features of the germinal epithelium and mature primitive ectaquasperm of two species, Pitar rudis and Chamelea gallina (Veneroidea). Data show that despite very similar structural morphologies and developmental patterns, each can be distinguished by clear traits observed during the spermatogenic cycle.
cut in a Leica ultratome with a Diatome knife, collected in 300 mesh copper grids (Taab), double-stained with alcoholic concentrated uranyl acetate for 20 min followed by lead citrate (Reynolds) for 10 min, and studied at 60 kV in a transmission electron microscope JEOL 100 CX II (Sousa et al., 2000). All chemicals were of analytical grade and purchased from Merck.
Materials and methods
Results
P. rudis and C. gallina (Mollusca, Bivalvia, Heterodonta, Veneroida, Veneroidea, Veneridae) specimens were collected from the intertidal zone of the Turkish coast. Small pieces of testis (<1 mm) were fixed with 2.5% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4 for 2 h at 4 ◦ C and then rinsed in the same buffer. Specimens were post-fixed in 2% osmium tetroxide in the same buffer for 2 h at 4 ◦ C, serially dehydrated in an ethanol series followed by propylene oxide, and finally embedded in Epon. Ultrathin sections were
P. rudis Sertoli cells were irregularly shaped, as to enclose germ cells, extending from the basal lamina towards the lumen. They displayed an ovoid indented nucleus with 2–3 nucleoli. Besides mitochondria, Golgi complexes, endoplasmic reticulum cisternae, ribosomes and lipid droplets, the cytoplasm was rich in microtubules, glycogen, and large phagolysosomes with degenerated germ cells (Fig. 1). Spermatogonia were adjacent to the basal lamina and presented a large, round
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Fig. 20 C. gallina. Sertoli cell (S) with glycogen (g), lipid droplets (L), microtubules (mt), endoplasmic reticulum (rer), dictyosomes (G), lysosomes (Ly) and mitochondria (m). Note that intracellular axonemes (Ax) are derived (inset) from phagocytic vesicles containing germ-cell flagella (white arrowhead). SG< spermatogonia; BL, basal lamina. ×9100; Inset: ×15 200. Fig. 21 C. gallina. Spermatogonia (SG) show intercellular bridges (*), mitochondria (m), nuage (n) and a flagellum (F). They are linked (inset a) to Sertoli cells (S) by desmosome-like junctions (white arrowheads). They also exhibit (inset b) proximal (pC) and distal (dC) centrioles, with the latter being linked to the plasma membrane by a pericentriolar complex (pCc) and gives rise to the axoneme (Ax) of the extruded flagellum. Note their close association with leptotene (Lep) and pachytene (Paq) spermatocytes. Leptotene spermatocytes (inset c) evidence mitochondria (m), rough endoplasmic reticulum cisternae (rer), an active dictyosome (G) and desmosome-like junctions (white arrowhead) with Sertoli cells. ×6600; inset a: ×11 100; inset b: ×16 700; inset c: ×12 800.
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Fig. 22 C. gallina. Pachytene spermatocyte, with synaptonemal complexes (SC), proacrosomal vesicles (v) and an active dictyosome (G). ×9000. Fig. 23 C. gallina. Note meiotic metaphase I chromosomes (Chro), the aster centriole (C), mitochondria (m), rough endoplasmic reticulum cisternae (rer) and proacrosomal vesicles (v). ×7700. Figs 24 & 25 C. gallina. Pachytene spermatocytes. Fig. 24 Shedding of proacrosomal vesicles (white arrowheads) from dictyosomes (G). Fig. 25 Close association (white arrowheads) of proacrosomal vesicles with the rough endoplasmic reticulum (rer). N, nucleus; m, mitochondria; c, centriole. ×26 000 and ×37 600. Fig. 26 C. gallina. Round spermatid. During migration, the acrosomal vesicle (av) is linked by its apex to the plasma membrane (black arrowhead) and coated by a fine dense basal material (white arrowhead). Inset: at the apical pole, the acrosomal vesicle base becomes invaginated to form the subacrosomal space (*). N, nucleus. ×20 200; inset: ×26 000. Fig. 27 C. gallina. In the elongated spermatid nucleus (N), chromatin becomes coarse fibrillar with a longitudinal arrangement. In the acrosome, note the subacrosomal space (*) and the acrosomal vesicle with outer dense (o) and inner intermediately dense (i) regions. In the midpiece, the proximal centriole (pC) is linked to the nuclear base, while the distal centriole (dC) anchors to the plasma membrane by the pericentriolar complex (pCc). m, Mitochondria. ×16 500.
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nucleus with 1–2 nucleoli, mitochondria, small nuage aggregates (granulofibrillar materials containing exported ribonucleoproteins), dictyosomes, endoplasmic reticulum cisternae actively shed from the nuclear envelope, ribosomes, and an intracellular axoneme (Figs 2 & 3). Primary spermatocytes displayed similar organelles and structures as spermatogonia, but from the pachytene stage they extruded the flagellum (Fig. 2) and the Golgi complex began the synthesis of small, dense proacrosomal vesicles (Fig. 4). The latter also seemed to receive components of the rough endoplasmic reticulum cisternae (Figs 4 & 5). During spermiogenesis, the round spermatid nucleus became progressively elongated, with chromatin turning longitudinally filamentous before beginning a homogeneous condensation process, which left small-uncondensed spaces (Figs 7, 11–13). At the nuclear base, chromatin was condensed at the sites of attachment to mitochondria (Figs 7 & 8) and the proximal centriole (Fig. 10). Later, as the cytoplasmic axoneme was extruded to form the flagellum, the nucleus developed mitochondrial and centriolar fossae (Figs 6, 10–13). In the round spermatid, proacrosomal vesicles fused to form a round acrosomal vesicle that exhibited a central ring-shaped dense component, a central and apical intermediate-dense material, and a basal light region (Figs 6–8). The acrosomal vesicle then migrated from the basal pole to the cell apex, linked apically to the plasma membrane, while developing a dense basal coat (Figs 7 & 8). Once at the nuclear apex, the acrosomal vesicle turned so that it was horizontally elongated and its contents became segregated into a central dense material completely surrounded by an intermediate dense component. The acrosomal vesicle base then formed a thin expansion that attached to the nuclear envelope (Figs 9 & 10), being associated with development of acrosomal and nuclear indentations and the subacrosomal material, then filled with a perforatorium (Figs 11–13). The mature spermatozoon showed a conical elongated nucleus (2.6 m) and a cap-shaped elongated acrosomal vesicle (0.6 m) (Fig. 14). The acrosomal vesicle displayed external dense and internal intermediately dense regions, and a small apical light tip (Fig. 15). However, in a few cases the apical protrusion was longer and filled with the outer dense component (Fig. 16). In the midpiece (0.7 m), which contained glycogen, there were two centrioles and four mitochondria, although about <10% of sperm exhibited five mitochondria. The proximal centriole was tightly linked to the nuclear envelope, whereas the distal centriole was connected to the plasma membrane by the pericentriolar complex and gave origin to the flagellum, which contained a 9d + 2s axoneme (Figs 17–19). C. gallina Sertoli cells were very similar to those found in P. rudis, but showed higher numbers of lipid droplets, only a few phagolysosomes containing degenerated germ cells (Fig. 20), and evident desmosome-like junctions with spermatogonia and spermatocytes (Fig. 21). Spermatogonia were adjacent to
the basal lamina and presented intercellular bridges, a large ovoid nucleus with 1–2 nucleoli, mitochondria, small nuage materials, a small dictyosome, a few endoplasmic reticulum cisternae, ribosomes and a flagellum (Fig. 21). Primary spermatocytes displayed similar organelles and structures as spermatogonia, with small dense proacrosomal vesicles being produced by the Golgi complex and the rough endoplasmic reticulum since the pachytene stage (Figs 22–25). During spermiogenesis, the nucleus elongated and chromatin condensed as shown for P. rudis (Figs 26 & 27). In early spermatids, the acrosomal vesicle also migrated linked apically to the plasma membrane, while developing an outer dense basal coat. Their contents became differentiated into a thin peripheral layer of intermediate density, a floccular matrix, and a central ring-shaped dense component (Fig. 26). Once at the nuclear apex, it developed a basal invagination to originate the subacrosomal space (Fig. 26, inset) that developed a perforatorium in later stages (Fig. 27). The mature spermatozoon showed a conical elongated and slightly curved nucleus (3.5 m) and a cap-shaped elongated acrosomal vesicle (0.6 m) with external dense and internal intermediately dense regions, and two small lateroapical light tips (Figs 28 & 29). In the midpiece (0.6 m), there were two centrioles and four mitochondria (Figs 28 & 30).
Fig. 28 C. gallina. Mature sperm, showing an elongated and condensed nucleus (N) with mitochondrial (m) and centriolar (c) fossae (white arrowheads). Note the acrosomal vesicle (av) and the subacrosomal space (*). Ax, flagellar axoneme. ×18 700. Fig. 29 C. gallina. Acrosome of mature sperm. The acrosomal vesicle displays outer dense (o) and inner intermediately dense (i) regions, as also two apicolateral light pouches (white arrowheads). Note the perforatorium (*) and the apical nuclear (N) protrusion. ×40 000. Fig. 30 C. gallina. Midpiece of mature sperm, with four mitochondria (m) encircling the proximal centriole (c). ×20 000.
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Discussion Invertebrate spermatozoa are divided into three main types: aquasperm (ectaquasperm and entaquasperm), introsperm and dimorphic or polymorphic sperm (Jamieson, 1987; Rouse & Jamieson, 1987). Ectaquasperm are primitive and shed in water, where they fertilize. The ‘primitive’ sperm type is characterized by a small head, containing a rounded or conical nucleus, surmounted by a cup-shaped acrosomal vesicle, a midpiece with a ring of 4–5 rounded mitochondria encircling two centrioles, and a tail flagellum about 50 m long (axoneme with a 9d + 2s microtubule pattern) (Franzén, 1983). Within the Veneroida (Bivalvia, Heterodonta) the spermatozoon is of the ‘primitive’ type in almost all species studied, although this definition includes some modifications of the sperm nucleus and midpiece that deviate from the most common type (Lucinacea: Healy, 1995; Johnson et al., 1996; Leptonacea: Galeommatidae: Eckelbarger et al., 1990; Cardiacea: Sousa & Azevedo, 1988; Healy, 1995; Keys & Healy, 1999, 2000; Tellinacea: Sousa et al., 1989; Healy, 1995; Dreissenacea: Denson & Wang, 1994, 1998; Walker et al., 1996; Veneracea: Turtoniidae: Ockelmann, 1964), as also a few examples of modified and dimorphic sperm (Lucinacea: Healy, 1995; Moueza & Frankiel, 1995; Leptonacea: Lasaeidae: Ó’Foighil, 1985; Leptonacea: Montacutidae: Ockelmann, 1965; Corbiculacea: Komaru & Konishi, 1996; Konishi et al., 1998). The spermatogenesis of P. rudis and C. gallina as studied here represents the first detailed description of the seminiferous epithelium in the Veneroidea. Comparisons between members of this superfamily show only slight differences between the specific steps of spermatogenesis, which nevertheless confer individual characteristics to each different species. Sertoli cell structure has only been described in other Veneroida superfamilies. Similar to P. rudis and C. gallina, Sertoli cells rich in glycogen and lipid droplets were described in Loripes lucinalis (Lucinacea) (Johnson et al., 1996), Divariscintilla sp. (Leptonacea) (Eckelbarger et al., 1990) and Scrobicularia plana (Tellinacea) (Sousa et al., 1989), whereas germ-cell phagocytosis was observed only in Divariscintilla sp. In the Veneroidea, the presence of a flagellum in premeiotic germ cells was described in primary spermatocytes of Tivela polita (Reunov & Hodgson, 1994). In the present study, it is shown that whereas spermatogonia of C. gallina already have a flagellum, P. rudis exhibited an intracellular axoneme in spermatogonia that became extruded only in primary spermatocytes. The presence of a flagellum in premeiotic germ cells was also found in other Veneroida species, such as Divariscintilla sp. (Eckelbarger et al., 1990), Solen sp. (Hodgson et al., 1987; Reunov & Hodgson, 1994) and S. plana (Sousa et al., 1989). In the Veneroidea, proacrosomal vesicles derived from Golgian activity have been described during the pachytene stage in T. polita (Reunov & Hodgson, 1994), or during spermiogenesis in Venerupis sp. and Callista chione
(Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971; Nicotra & Zappata, 1991). On the contrary, in P. rudis and C. gallina, proacrosomal vesicles, although produced during the pachytene stage by dictyosomes, also seemed to incorporate components of the endoplasmic reticulum. Comparisons with other Veneroida members show that proacrosomal vesicles may form either during the leptotene stage, as in Divariscintilla sp. (Eckelbarger et al., 1990), or the zygotene/pachytene stage (similar to P. rudis and C. gallina) as in Solen sp. (Solenacea) (Hodgson et al., 1987; Reunov & Hodgson, 1994), Donax sp. (Tellinacea) (Hodgson et al., 1990; Reunov & Hodgson, 1994; Sousa & Oliveira, 1994a) and Corbicula fluminea (Corbiculacea) (Konishi et al., 1998). However, in all these species, proacrosomal vesicles also originated from dictyosomes, and thus the active participation of the rough endoplasmic reticulum in this process here is firstly described in the Veneroida for P. rudis and C. gallina. Description of the mechanism by which the acrosomal vesicle migrates during spermiogenesis has also not been studied in most of the species. In P. rudis and C. gallina, the apex of the acrosomal vesicle first links to the plasma membrane and then migrates to the apical pole of the cell. During this process, it acquires an outer dense material that defines the basal pole of the acrosomal vesicle and corresponds to the precursors of the subacrosomal material. A similar situation has only been found previously for D. trunculus and S. plana (Sousa et al., 1989; Sousa & Oliveira, 1994a). Formation of the subacrosomal space is supposed to be driven by deposition of the subacrosomal material. However, in P. rudis the acrosomal vesicle base formed an evaginated process that was associated with formation of the subacrosomal region. This type of activity of the acrosomal vesicle has never been described before and thus constitutes a novel description in Mollusca. In relation to acrosomal vesicle contents, whereas all members of the Veneroidea show a dual differentiation of the acrosomal contents, here it is shown that P. rudis and C. gallina have a tripartite composition due to the presence of apical acrosomal protrusions. Regarding the presence of a perforatorium, which is also present in P. rudis and C. gallina, in the Veneroidea only T. polita shows an absence of such subacrosomal specialization (Reunov & Hodgson, 1994). In the Veneroidea, the sperm nucleus also shows wide variations, with Tivela minuta exhibiting a very long conical and spiral nucleus (Reunov & Hodgson, 1994), and Venerupis sp. a long conical and slightly curved nucleus (Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971). On the contrary, P. rudis and C. gallina (in the current study), C. chione (Nicotra & Zappata, 1991) and T. polita (Reunov & Hodgson, 1994) all show a short conical and slightly curved nucleus. The process of nuclear chromatin condensation during spermiogenesis has not been described in the Veneroidea. In comparison with other Veneroida, a similar situation to P. rudis and C. gallina, where chromatin condensation progresses through a filamentous stage, was found in species with an elongated nucleus, such as L. lucinalis (Johnson et al., 1996), Divariscintilla
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sp. (Eckelbarger et al., 1990) and S. plana (Sousa et al., 1989). Finally, and in relation to the midpiece, P. rudis and C. gallina (in the current study), T. minuta (Reunov & Hodgson, 1994) and Venerupis sp. (Pochon-Masson & Gharagozlou, 1970; Gharagozlou & Pochon-Masson, 1971) show four mitochondria, whereas other members of the Veneroidea possess five mitochondria. In this regard, P. rudis constitutes an exception and shows a polymorphic trait, as about 10% of sperm also exhibited five mitochondria. On the contrary, the presence of glycogen in the midpiece, as found in P. rudis, was only observed in other Veneroida, such as Divariscintilla sp. (Eckelbarger et al., 1990) and Donax sp. (Hodgson et al., 1990).
ACKNOWLEDGEMENTS We acknowledge E. Oliveira for technical assistance, J. Carvalheiro and J. Gonçalves for iconography, Professor Dr Dinçer Gulen for institutional support (to M.E.) and S. Albayrak for assistance in specimen collection. This study was partially supported by FCT (UMIB; Sapiens 36363/99, 35231/99).
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