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ISSN: 1817-3098

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Corresponding Author: Dr. Adel A.B. Shahin, Department of Zoology, Faculty of Science, Minia University, 61519 El-Minia, Egypt1

Volume 1 Number(1) : 01-16, Jan-Jun, 2006

Spermatogenesis and Spermatozoon Ultrastructure in the Nile Pebblyfish Alestes dentex (Teleostei: Characiformes: Alestidae) in Egypt

Adel A.B. ShahinDepartment of Zoology, Faculty of Science, Minia University, 61519 El-Minia, Egypt

Abstract: The ultrastructure of spermatogenesis and spermatozoon of Alestes dentex is described by using scanning andtransmission electron microscopy. The testis is lobular in shape and spermatogenesis is of the unrestricted type. The germcells are found in clusters within the seminiferous tubules and surrounded by cytoplasmic processes of Sertoli cells.Spermiogenesis is characterized by chromatin condensation, flagellum development, nuclear rotation, migration ofdiplosome and mitochondria, nuclear indentation, nuclear fossa formation and loss of excess cytoplasm. The maturespermatozoon is of the primitive type; type I aquasperm. The spermatozoon has no acrosome and has a roundedheterogeneously electron-dense nucleus with a deep axial nuclear fossa and a nuclear notch. The nuclear fossa contains thecentriolar complex and part of the basal body of the axoneme. The short midpiece contains a mitochondrial ring, whichconsists of several unequal-sized and unevenly distributed mitochondria. The flagellum has the classical axoneme pattern9+2 and has no lateral fins or membranous compartment. In addition, the spermatozoon has some peculiar features, whichare not previously described in Characiformes and include the presence of two fibrous bodies anchoring the proximalcentriole to the nucleus, a thick outer membrane of dense fibers separating the flagellum from the cytoplasmic canal anda basal foot and alar sheets attaching the basal body to the nucleus and plasma membrane, respectively. These findingssuggest that the ultrastructural features of spermiogenesis and spermatozoa of A. dentex are synapomorphy ofOstariophysi, Perciformes and Salmoniformes. Spermiogenesis and spermatozoan features of many fish taxa includingCharaciformes are discussed.

Key words: Spermatogenesis sperm Alestes dentex ultrastructure morphology characiformes

INTRODUCTION congeneric species [10, 15, 18-21], important morphological

Spermatogenesis, in general, exhibits only limited fine [2, 22-24]. These differences can, therefore, be used forstructural variations in animal species; however, spermiogenesis inferring both the taxonomic and phylogenetic relationshipsshows a wide variety of patterns [1]. In teleosts, two types among taxa [7, 11, 15, 17, 22, 24-35].(I and II) of spermiogenesis have been recognized [2]. In type I, Despite the immense studies on fish spermatozoa, therotation of the nucleus occurs and the diplosome enters the majority of teleost spermatozoa remain unexamined. Moreover,nuclear fossa and the flagellum is symmetrically located, while there appear to be no published data describing the finein type II, there is no nuclear rotation, the diplosome remains structure of spermatozoa of the Nile characid fishes. The goalsoutside the fossa and the flagellum is asymmetrically located. of this study are to examine the ultrastructure ofDepending upon variation in these features, spermiogenesis has spermatogenesis and spermatozoon of the pebblyfish Alestesbeen used to provide worth insights into the relationships of dentex belonging to one of the endemic families of Characoideimany fish groups, especially at or above the family level [3-10]. in Africa, the family Alestidae and to compare the present dataAccordingly, teleost spermatozoa are widely different in on A. dentex with those available on other fish groups in generalmorphology [11] and their structure is influenced by both the and Characiformes in particular to assess its phylogeneticreproductive mode and the systematic position [12, 13]. relationship with other teleosts.Generally, they vary from aflagellate to biflagellate and have anenormous range of shapes, sizes and structures; the number and MATERIALS AND METHODSlocation of organelles also vary [11, 14-17]. Both light andelectron microscopy of a wide spectrum of teleost spermatozoa In June, during the breeding season (April-September),have shown that although quite similarities are found among mature males of Alestes dentex (Linnaeus, 1758) measuring

differences can also be found between different species

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Fig. 1a-d: Transmission electron micrographs in the spermatogenic cells of Alestes dentex. a) Primary spermatogonia with aprominent nucleus (nu), well-defined nucleolus (nc) and electron-dense chromatin (ch). Mitochondria (m) are unevenlydistributed in the cytoplasm and granular materials including nuage (ng) are distributed either free in the cytoplasm orassociated with mitochondria and referred as cement (ce). b) Secondary spermatogonia have a large nucleus and electron-dense granular chromatin. Note the cytoplasm contains several spherical mitochondria, Golgi vesicles (G) and endoplasmicreticulum (er). c) A primary spermatocyte displays a clumped or slightly mottled chromatin within the large nucleus. Thecytoplasm contains mitochondria, cement and Golgi vesicles. d) A secondary spermatocyte shows a small nucleus withgranular and electron-dense mottled chromatin and a synaptonemal complex (sc). Mitochondria, Golgi vesicles andendoplasmic reticulum are clearly visible in the cytoplasm

15-22 cm in total length were collected from the Nile at El-Minia JEOL JEM-100CX II TEM operated at 80 kV accelerating(Middle Egypt). Samples of testis and semen were fixed in 3% voltage.glutaraldehyde made in 0.12 M phosphate buffer (pH 7.4) for On the other hand, dehydrated samples of semen were1 h at 4°C and postfixed in 1% osmium tetroxide for 1 h. After critical point-dried, sputter-coated with gold and carbon andfixation, samples were dehydrated in a graded ethyl alcohols images were recorded with a JEOL JSM-5400LV SEM operatedseries. Dehydrated samples of testis were cleared in propylene at 15 kV. Germ cells and spermatogenic stages were identifiedoxide and embedded in low viscosity Epoxy resin. Afterwards, according to definite criteria [29].thick plastic sections were cut using a LKB ultramicrotome witha glass knife and stained with toluidine blue. When appropriate RESULTSregions were found, ultrathin sections were subsequently madeand stained with drops of 2% uranyl acetate followed by lead The testis of A. dentex is lobular in shape and the germcitrate for 30 min. Sections were examined and photographed in cells are arranged in cysts or clusters within the seminiferous

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tubules. Spermatogenesis occurs in several places along the Spermiogenesis: The polarization of germ cells starts at thislength of each tubule and the testis structure could be stage. The nucleus moves to an eccentric position, while the celldescribed as the unrestricted spermatogonial testicular type. organelles migrate and concentrate at the opposite pole ofSpermatogonia are found near the periphery along the length of the cell. Early spermatids produced by second meiotic divisionthe tubule, while spermatocytes, spermatids and spermatozoa of secondary spermatocytes have a small round nucleusare found toward the interior. Sertoli cells are found surrounding (about 2.1 µm in diameter) with granular chromatin distributedthe germ cells. Within the lobules, spermatogonia undergo in small electron-dense patches of heterogeneous density,numerous mitotic divisions producing cysts containing several reduced cytoplasm and inconspicuous ribosomes (Fig. 2a). Thespermatogonial cells. Depending upon the morphology and size spherical spermatids remain interconnected by cytoplasmicof the nucleus, presence of organelles and centriolar bridges, which result from incomplete cytokinesis of mitoticmorphology, particularly the pericentriolar structures, germ cells and meiotic divisions. The centriolar complex appears lateral toare classified into: the nucleus and close to the plasma membrane to form the

Spermatogonia: Primary spermatogonia are the smallest germ shape are located near the centrioles. At subsequent stage, thecells (Fig. 1a). They measure about 2.4 µm in diameter and centrioles migrate to the basal pole of the nucleus (Fig. 2c). Thepossess a prominent dark rounded nucleus and an extensive process of spermiogenesis could be divided into the followingcytoplasm. The cytoplasmic processes of the Sertoli cell stages based on formation of the flagellum, rotation andsurround either one or two spermatogonia. The nucleus is small condensation of the nucleus and loss of the superfluous(about 1.0 µm in diameter), nearly central in position and cytoplasm (exocytosis).contains darkly stained granular chromatin that exhibits irregularcondensed patches. The nucleus contains one distinct nucleolus Formation of the flagellum: In this stage, the two centrioles,with fibrilar and granular chromatin. The cytoplasm contains each with nine triplet microtubules, are arranged at right anglesgranular materials that are either free (nuage) or associated with to each other and appear to be interconnected by osmiophilicmitochondria (cement). Mitochondria are unevenly distributed filaments. Both centrioles lie close to the plasma membrane andthroughout the cytoplasm, while the endoplasmic reticulum is the distal centriole (or its basal body) starts to form theconcentrically organized in the peripheral cytoplasm. flagellum (Fig. 2b). The flagellum reveals a typical axonemal

Secondary spermatogonia produced by mitotic division of configuration with two single central and nine double peripheralthe primary spermatogonia are comparatively larger cells, about microtubules. The axoneme then detaches itself from the3.7 µm in diameter, with less well-defined nucleoli, electron- perinuclear region and extends backward parallel to the nucleus,dense material (nuage) and cement (Fig. 1b). The nucleus is bringing with it the plasma membrane and leaving a space, theabout 2.6 µm in diameter and contains irregular patches of cytoplasmic canal, between the plasma membrane and thegranular chromatin. Within the cysts, spermatogonial cells flagellum (Fig. 2b). The diplosome-flagellar axis is tangential toremain connected together by cytoplasmic bridges during mitotic the nucleus and the mitochondria aggregate around the base ofdivision. the flagellum. Golgi apparatus appears as vesicular cisternae.

Spermatocyte: Primary spermatocytes are the largest germ and coarse granular heterochromatin. The cell outline appearscells (Fig. 1c). They measure about 6.3 µm in diameter and very irregular in shape and cytoplasmic bridges becomedistinguished by having a very large nucleus (about 5.7 µm in narrower than in previous stages.diameter) and a dense granular chromatin. The nucleus appearsclumped or slightly mottled in shape and without nucleolus. Rotation and condensation of the nucleus: The cells in thisSecondary spermatocytes produced by first meiotic division of stage display a finely granular appearance because of thethe primary spermatocytes are comparatively smaller cells homogeneity of chromatin (Figs. 2c, d). The nucleus becomes(Fig. 1d). They have nearly about half of the size of the primary indented and a nuclear fossa is formed. Due to the rotation ofcell, about 3.7 µm in diameter, while the nucleus is relatively the nucleus (90°) the diplosome-flagellar axis becomessmaller, about 2.6 µm in diameter. Pachytene stage perpendicular to the base of the nucleus. The proximal centriolespermatocytes have clumped nuclear chromatin and is then located within the nuclear fossa (Fig. 2c). A nuclearsynaptonemal complexes. The cytoplasm forms a narrow notch appears in the region between the proximal and distalstrand with irregular-shaped and electron dense-matrix centriols (Fig. 2d). In addition, two fibrous bodies, each ofmitochondria. which consists of osmiophilic disks alternating with lighter

flagellum (Figs. 2a, b). Some mitochondria of spherical or ovoid

The nucleus is electron dense with prominent patches of fine

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Fig. 2a-d: Transmission electron micrographs through the early stages of spermiogenesis in A. dentex. a) Transverse section showingthe proximal centriole (pc) and the distal centriole (dc) located at right angle to each other lateral to the nucleus (nu) andclose to the plasma membrane and interconnected by osmiophilic filaments (of). Chromatin (ch) is condensed andmitochondria (m) and Golgi vesicles (G) appear close to the centriolar complex. b) Longitudinal section showing the twocentrioles interconnected by osmiophilic filaments and lie close to the plasma membrane (pm) and the distal centriole orits basal body (bb) starts to form the flagellum (f), which extends parallel to the nucleus. A cytoplasmic canal (cc) isformed between the plasma membrane and the flagellum and extends longitudinally around the midpiece (mp). Themitochondria appear around the base of the flagellum and Golgi apparatus appears as vesicular cisternae around thecentrioles. The nucleus is electron dense with prominent patches of fine and coarse granular chromatin (ch). c) Longitudinalsection showing the finely granular homogeneous chromatin. The nucleus becomes indented and a nuclear fossa (nf)(arrowhead) is formed. Note the diplosome-flagellar axis becomes perpendicular to the base of the nucleus due to therotation of the nucleus. The proximal centriole is located within the nuclear fossa and the distal centriole gives rise to thebasal body, which forms the flagellum. The basal body is traversed by the basal plate (bp) at its connection with themidpiece. Mitochondria, Golgi flattened cisternae and endoplasmic reticulum (er) are distributed within the residualcytoplasm. d) Oblique transverse section (left) showing that the nucleus becomes more compact with increasing dense andthick chromatin and the 9+2 microtubular pattern of the axoneme in the midpiece region that is surrounded by thecytoplasmic canal. Longitudinal section (right) showing the rounded nucleus with increasing dense and thick chromatinfilaments and the nuclear fossa is further expanded into the nucleus as far as one-fourth of the nucleus length and containsthe centriolar complex and part of the basal body, the base of which is traversed by the basal plate. Note in the midpiece,the cytoplasmic canal separates the mitochondria from the flagellum. Most of the residual cytoplasm (cy) surrounds themidpiece

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Fig. 3a-d: Transmission electron micrographs in the late spermatids of A. dentex. a) Longitudinal section showing that the nucleus(nu) is more compact and chromatin (ch) is more condensed with dense and thick filaments. The fibrous body (fb) liesabove the proximal centriole (pc) and connects with the nucleus. The nuclear fossa (nf) completely surrounds the centriolarcomplex and part of the basal body (bb). The flagellum (f) consists of peripheral (pt) and central (ct) microtubularfilaments. The transitional region (small arrow) between the basal body (bb) and the midpiece (mp) lacks the centralmicrotubules. b) Oblique longitudinal section at the level of the head showing the nucleus with heterogeneous chromatin,the fibrous body above the proximal centriole and the nuclear fossa contains the proximal and the distal (dc) centrioles andpart of the basal body. c) Oblique transverse section through the head region showing the nucleus with heterogeneouschromatin and the upper fibrous body anchors to the nucleus by two bands of electron-dense filaments (arrowheads).Double arrowheads refer to the location of the lower fibrous body. d) Transverse section through the head region at thelevel of the lower fibrous body showing that the latter lies above the proximal centriole and interconnected withosmiophilic filaments and the nuclear fossa is circular. bp basal plate, er endoplasmic reticulum, G Golgi apparatus, mmitochondria, nn nuclear notch

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Fig. 4a-d: Transmission electron micrographs in the late spermatids of A. dentex. a) Oblique longitudinal section (upper) showingthe nuclear notch (nn) that anchors the proximal centriole (pc) to the nucleus (nu) and the basal foot (bf, white arrow)extending from the lateral side of the basal body (bb). Transverse sections in the head region at the level of the extensionof the nuclear notch from the proximal centriole (lower left) and the basal body (lower right) showing their microtubularstructure pattern. b) Transverse section in the head region at the level of the proximal centriole showing the nucleus withcircular nuclear fossa (nf), the nuclear notch and the basal foot extending from the basal body and anchors to the nucleus.Note the location of the distal centriole (dc). c) Oblique longitudinal section (left) showing the nucleus with heterogenouschromatin (ch), nuclear notch and nuclear fossa that contains the centriolar complex and the basal body with its basal foot.Oblique transverse section (right) at the level of the proximal centriole showing the nucleus with the circular nuclear fossaand nuclear notch. d) Longitudinal section in the early spermatozoon showing the spherical nucleus with heterogeneouslygranular and electron-dense chromatin, which contains tightly packed fibers and irregular small clear lacunae and coveredby a double-layered undulating nuclear envelope (ne) that applies tightly to the plasma membrane (pm). Note that themitochondria (m) lie close to the basal pole of the nucleus and surround the midpiece (mp) and the peripheral microtubular(pt) doublets of the axoneme are connected to the plasma membrane by a Y-shaped electron-dense body (y). bp basal plate,cc cytoplasmic canal, ct central microtubules, dc distal centriole, f flagellum, of osmiophilic filaments

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Fig. 5a-d: Electron micrographs of the mature spermatozoon in A. dentex. a) Scanning electron micrograph showing that the spermhas a spherical head (h), a short midpiece (mp) and a tail or flagellum (f), but there is no acrosome. b-d Transmissionelectron micrographs; b) Longitudinal section showing the nucleus (nu) with heterogeneous electron-dense chromatin (ch)that contains tightly packed fibers and irregular small lacunae and covered by a double-layered undulating nuclear envelope(ne) that is and the plasma membrane (pm) are applied tightly to the anterior part of the nucleus. The posterior part ofthe nucleus is indented by a nuclear fossa (nf), which has a length of about more than one half of the nucleus diameter. Thefossa contains the proximal (pc) and distal centriole (dc) and part of basal body (bb). Note the basal foot (bf) extendingfrom the basal body, the cytoplasmic canal (cc) surrounding the midpiece and separates it from the mitochondria (m) andthe central (ct) and peripheral (pt) microtubules of the midpiece region. c) Enlarged part of the posterior part of the nucleusand midpiece showing the basal foot and alar sheets (as) extending on both sides of the basal body and attach it to thenucleus and the plasma membrane. Note that mitochondria are unevenly distributed on both sides of the midpiece and twoclosely apposed membranes run along between the cytoplasmic canal and the outer layer of mitochondria ring, in additionto another membrane of dense fibrous layer (fl) extends from the posterior end of the midpiece collar to the anterior endof the cytoplasmic canal in close vicinity to the flagellar plasma membrane (fm) and separates the midpiece from thecytoplasmic canal. Arrowhead refers to the transitional region free of central microtubules between the basal body andthe midpiece.The peripheral microtubules are connected to the plasma membrane by a Y-shaped electron-dense link (y).d) Longitudinal section showing clearly the two fibrous bodies (fb), upper and lower, interconnected with osmiophilicfilaments (of) and anchor the proximal centriole to the nucleus. Note the nuclear notch (nn) connecting the lower fibrousbody with the nucleus, the osmiophilic filaments interconnecting the proximal centriole with distal centriole, the lengthof the nuclear fossa, which is about more than one half the length of the nucleus and the basal part of the basal body istraversed by the basal plate (bp)

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Fig. 6a-d: Transmission electron micrographs in the head and midpiece of the mature spermatozoon of A. dentex. a) Two transversesections at the level of the proximal centriole (pc) (upper) showing the nucleus (nu) with tightly packed fibers and irregularsmall lacunae in the chromatin (ch) and at the level of location of the lower fibrous body (fb) above the proximal centriole(lower). b) Transverse section at the level of the basal region of the distal centriole (dc) showing the typical 9+2construction pattern, which is surrounded by osmiophilic ring (or) of electron-dense material filling the nuclear fossa (nf).All nine outermost microtubules of the triplets of the distal centriole disappear proceeding backwards. c) Two transversesections at the level of the distal centriole and the basal body (bb) (upper) and at the level of the basal plate (bp) of thebasal body (lower) showing their microtubular construction pattern (mt). d) Transverse section through the midpiece (mp)showing the cytoplasmic canal (cc) separating the mitochondria (m) from the midpiece. Note that the mitochondria arebounded by an outer and an inner membrane (arrow) and exhibit transverse cristae and each of the peripheral microtubulardoublets of the midpiece is connected to the plasma membrane by a Y-shaped link (y)

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Fig. 7a, b: Transmission electron micrographs in the midpiece of the mature spermatozoon of A. dentex. a) Transverse sectionshowing the 9+2 microtubular pattern of the axoneme in this region, each of the peripheral microtubular doublets (pt) isconnected to the plasma membrane (pm) by a Y-shaped link (y). Note the double-layered flagellar membrane (fm) and itsouter fibrous layer (fl) surrounding the plasma membrane and the mitochondrial envelope (me) in close vicinity to theplasma membrane separating the mitochondria (m) from the cytoplasmic canal (cc). b) Transverse section showing severalmitochondria (rarely 6) around the midpiece (mp) region. Note the double-layered flagellar membrane and its outer fibrouslayer, which surround the plasma membrane. Arrowhead refers to the Y-shaped link that connects each of the peripheralmicrotubular doublets with the plasma membrane

and coarse granular heterochromatin. The cell outline appears is further expanded into the nucleus as far as one-fourth of thevery irregular in shape and cytoplasmic bridges become nucleus length and completely surrounds the centriolar complexnarrower than in previous stages. (Figs. 4a-c). As the nuclear fossa develops, the nucleus

Rotation and condensation of the nucleus: The cells in this proximal centriole, which gradually increases and becomes filledstage display a finely granular appearance because of the with electron-dense material (Figs. 4a-c). The cell begins tohomogeneity of chromatin (Figs. 2c, d). The nucleus becomes discard excess cytoplasm, leaving the so-called residualindented and a nuclear fossa is formed. Due to rotation of the bodies of various sizes and shapes in the intercellular spacesnucleus to 90°, the diplosome-flagellar axis becomes (Fig. 4d).perpendicular to the base of the nucleus. The proximal centrioleis then located within the nuclear fossa (Fig. 2c). A nuclear Spermatozoon: The mature spermatozoon is a relativelynotch appears in the region between the proximal and distal simple elongated cell composed of a head, a short medpiece andcentriols (Fig. 2d). In addition, two fibrous bodies, each of a relatively long tail or flagellum (Fig. 5a).which consists of osmiophilic disks alternating with lighter The head is spherical in shape, about 1.9 µm in diametermaterial, appear perpendicular to each other above the proximal and has no acrosome. The nucleus is spherical, about 1.7 µm incentriole within the nuclear fossa (Figs. 3a-d). The two bodies diameter and covered by a typical double-layered undulatingare interconnected with osmiophilic filaments and the upper one nuclear envelope. The nuclear envelope and the plasmaattaches to the nucleus with two bands of osmiophilic filaments membrane are applied tightly to the anterior part of the nucleus(Fig. 3c); similarly, the lower body connects with the proximal (Fig. 5b). However, the posterior part is indented by a nuclearcentriole (Fig. 3d). Moreover, a basal foot appears laterally in fossa, the length of which is about more than one half of thethe basal part of the distal centriole and anchors it to the nucleus nucleus diameter (Figs. 5b-d). The fossa is bell-shaped in(Figs. 4a-c). The axoneme appears surrounded by mitochondria, longitudinal sections and circular in transverse sections andwhich are separated from the flagellum by the cytoplasmic canal contains the centriolar complex, part of basal body and(Fig. 4a). As development proceeds, the size of the spermatids cytoplasm. A peculiar nuclear notch with some electron-densedecreases and the intercellular spaces enlarge to form a lumen in material connects the lower fibrous body to the nucleusthe cyst. (Fig. 5d). The chromatin is heterogeneously granular, highly

Exocytosis: The nucleus becomes more compact with increasing irregular small clear lacunae (Figs. 5b-d). The proximal and distaldense and thick chromatin filaments (Fig. 4b). The nuclear fossa centrioles occupy only the distal part of the nuclear fossa and

condenses and a small nuclear notch appears at the level of the

electron-dense and contains tightly packed fibers and

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are perpendicular to each other and lie at right angle to the base DISCUSSIONof the head (Fig. 5d). Both centrioles display a characteristicnine-microtubular triplet pattern. An osmiophilic ring embedded Earlier stages of spermatogenesis in A. dentex do notin an electron-dense material surrounds the anterior end of the show any peculiarities; spermatogonia and spermatocytesdistal centriole. The triplets of the proximal and distal centrioles are generally similar to those described in other teleostsare connected together by means of osmiophilic filaments [18, 29, 36-40]. Nevertheless, its spermiogenic characters are(Figs. 6b, c). Two fibrous bodies, upper and lower, consisting consistent with those reported for type I aquasperm [2, 11].of osmiophilic disks alternating with lighter material lie Rotation of the nucleus is 90°, the diplosome enters the nuclearperpendicularly above the proximal centriole (Fig. 6a). These fossa and the flagellum is symmetrically located. Similar findingstwo dense bodies give rise to short electron-dense fibers, which have been found in Lepomis macrochirus [41], Acanthopagrusconnect them together with the proximal centriole and schlegeli [29, 31], poeciliid and zenarchopterid species [6],anchor the proximal centriole to the nucleus (Fig. 5d). However, Diplomystes mesembrinus [8], Hoplias malabaricus [9] andthe distal centriole, which forms the basal body of the curimatid species [10]. On the contrary, rotation of the nucleusaxoneme, extends from the level of the anterior end of the either occurs only to a slight degree as in Cyprinus carpio andcytoplasmic canal to the basal nuclear fossa and its base is Esox lucius [42], Oreochromus niloticus [43] andtraversed by a prominent basal plate (Figs. 5b-d). A striated Paracheirodon innesi [11] or entirely does not occur as in Lizabasal foot attaches laterally in the midregion of the basal body aurata [18], Diapoma speculiferum and Diapoma sp. [22],and anchors it to the nucleus (Figs. 5b, c). Alar sheets radiate Mimagoniates barberi [39], Acestrorhynchus falcatus [44],from the basal body triplets and attach it to the plasma Merluccius merluccius [7] and Chrysichthys auratus [45]. Inmembrane (Fig. 5c). these cases, except in C. auratus [45], the diplosome remains

The midpiece is short, about 0.5 µm in length and 0.9 µm outside the nuclear fossa and the flagellum is asymmetricallyin diameter and contains a mitochondrial ring (Figs. 5b, c). The located, i.e. it inserts laterally to the head (type II) [2]. Inlatter consists of several (rarely four to five) unequal-sized C. auratus, it has been found that the nuclear rotation is absent,spherical or ovoid mitochondria, which measure about the diplosome enters the medial nuclear fossa and the flagellum0.6-0.8 µm in diameter and are completely separated from is symmetrically located [45]. Additionally, it has been pointedthe midpiece by the cytoplasmic canal (Figs. 6d, 7a). The out that the position of the nuclear fossa and accordingly thedistribution of mitochondria is irregular on both sides of the attachment of the flagellum to the nucleus depend upon themidpiece (Figs. 5b, c, 7b). The mitochondria have cristae and rotation of the nucleus [10]. So, when the nuclear rotation iselectron-dense matrix and are separated from the cytoplasmic incomplete, the nuclear fossa is eccentric and so is the flagellum,canal by two closely apposed membranes (Figs. 5b, c, 6d, 7a). which is perpendicular to the nucleus [10, 11, 19, 22, 46, 47].Another membrane of dense fibrous layer extends from the If rotation is complete (90°), as in A. dentex examined hereinposterior end of the midpiece collar to the anterior end of the and some other characids [48-52], the nuclear fossa is medialcytoplasmic canal in close vicinity to the flagellar plasma and the flagellum is medial and perpendicular to the nucleus. Butmembrane and separates the midpiece from the cytoplasmic when the nucleus does not rotate, the nuclear fossa is lateralcanal (Figs. 5b, c, 6d, 7a, b). in position or may be also absent and thus the flagellum is

The flagellum is about 28.5 µm in length and 0.3 µm in parallel to the nucleus [22, 44] or the nuclear fossa is medial anddiameter and is surrounded by the flagellar plasma shallow and the initial segment of the flagellum arises directly inmembrane. In the transitional region between the base of the a perpendicular position to the basal pole of the nucleus [45].axoneme and the basal plate, the axoneme consists of nine Moreover, it has been described that the position of centriolardouble outer tubules and lacks the central tubules (Fig. 5c). complex is related to the shape of nuclear fossa [10]. When theDistal to these regions, the flagellum has the classic nine nuclear fossa is deep, the centriolar complex is located insidedouble outer and two single central microtubular constructions it. If the nuclear fossa is of the moderate type, it may contain(Figs. 6d, 7a, b). The doublets are connected to the flagellar the entire centriolar complex or part of it, or only one of theplasma membrane by short Y-shaped bridges. The central centrioles, while the other one lies outside, but if it istubules are surrounded by a thin sheath and appear to be completely absent, the centriolar complex usually lies close tointerconnected by a single strand. Each of the nine outer the nucleus.doublets consists of subfibers A and B. Two dynein arms arise Accordingly, the nuclear fossa in A. dentex is of the deepfrom subtubule A of each doublet and extend toward the next type, since it has more than one half of the nuclear diameter intubule (Figs. 6d, 7a, b). length and contains the centriolar complex and part of the basal

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body of the axoneme. The presence of a shallow to deep nuclear some Characiformes [9-11, 19, 22, 46, 49, 59]. In the otherfossa containing the centriolar complex and proximal portion of situation, the midpiece is located laterally to the nucleus as inthe flagellum is also common in the spermatozoa of many some members of Characiformes [22, 39, 44].viviparous and some oviparous taxa, e.g. Jenynsia lineata [53], In A. dentex, the midpiece is short and contains a shortPoecilia reticulata [26], Poecilia latipinna [3], Gambusia affinis cytoplasmic canal enveloped by the cytoplasmic sheath[4], Oncorhynchus tshawtscha [54], Salvelinus fontinalis [55], that is formed by as a thin cytoplasmic projection [11, 17].Cymatogaster aggregata [12], Characoden lateralis [13], Similar cases have been recorded in the majority ofOncorhynchus mykiss [36], Platichthys flesus [16], O. niloticus Characiformes [10, 19, 44, 46, 47] and in many teleosts [8, 10,[43], Labidesthes sicculus [56], Thymallus thymallus [57], 11, 17, 24, 27, 29, 31, 32, 34]. However, long midpiece andA. schlegeli [29, 31], Plecoglossus altivelis [58], Acanthopagrus long cytoplasmic canal have been observed in some characidslatus [27], Chanos chanos [33], Oncorhynchus masou [11, 45, 48, 50-52, 59]. Moreover, long midpiece and longformosanus [24], Brycon orbignyanus [48, 50], Brycon hilarii cytoplasmic sheath attached to one side of the nucleus have[52], D. mesembrinus [8], Salminus maxillosus [51] and C. been found in the species of Glandulocaudinae [22] exceptauratus [45]. Nevertheless, the nuclear fossa is moderately species of Mimagoniates [22, 39] that have a short cytoplasmicdeveloped in the curimatid species [10, 19], H. malabaricus [9] sheath. However, a long midpiece with no cytoplasmic sheathand in the subfamilies Aphyocharacinae [22] and Myleinae [59], has been found in the inseminating cheirodontine M.while it is poorly developed in Epinephelus malabaricus and uruguayanae [22].Plectropomus leopardus [32]. On the contrary, it is completely In the spermatozoa of A. dentex, two closely apposedabsent in most oviparous and some viviparous species, e.g. membranes run in the vicinity of the plasma membraneC. carpio [26], Carassius auratus [60], eels [20, 35], L. aurata between the outer mitochondrial envelope and the cytoplasmic[18], Micropongius undulatus [28, 61], Argyropelecus gigas canal membrane. A similar structure has been described in[62], Rhodeus sericeus sinensis [63], Macropsobrycon T. thymallus [57], A. schlegeli [29, 31], A. latus [27] anduruguayanae [22], M. barberi [39], A. falcatus [44] and M. merluccius [7]. Nonetheless, one membrane has beenM. merluccius [7]. found in L. aurata [18], O. mykiss [36], M. undulatus [28, 61],

On the other hand, the arrangement of the centriolar E. malabaricus and P. leopardus [32], C. chanos [33], O. m.complex is variable in many cypriniform species; therefore, formosanus [24], M. barberi [22, 39], D. speculiferum,it is considered species-specific feature [64]. The proximal Diapoma sp., Corynopoma riisei, Pseudocorynopoma doriae,centriole may be anterior or lateral to the distal centriole. In Mimagoniates microlepis, M. uruguayanae and Aphyocharaxeither case, it may be coaxial, parallel, oblique or perpendicular anisitsi [22], D. mesembrinus [8], Steindachnerina insculpta,to the distal centriole. In A. dentex spermatozoa, the two Cyphocharax gillii, C. modestus, C. spilotus and Potamorhinacentrioles are arranged perpendicularly to each other and the altamazonica [10] and C. auratus [45]. Conversely, theproximal one lies anterior to the distal centriole. Similar cytoplasmic canal is absent in P. altivelis [32], Citharinus sp.conditions have been described in A. schlegeli [29, 31], A. latus [49] and H. malabaricus [9]. The cytoplasmic canal is formed[27], C. chanos [33], D. mesembrinus [8], M. merluccius [7] and during spermiogenesis by movement of the centriolar complexC. auratus [45]. Nonetheless, the two centrioles are oriented towards the nucleus, taking with it the plasma membrane andat right angle to each other in Anguilla japonica [30], the initial segment of the flagellum [2].P. altivelis [58], E. malabaricus and P. leopardus [32] and Situation of mitochondria shows considerable variationO. m. formosanus [24]. Among Characiformes variable among teleosts. In the spermatozoa of most taxa [2, 7-11, 17,orientation of the centrioles has been reported. For example, 19, 24, 27, 29, 31-33, 45, 65, 66] including A. dentex examinedthe proximal centriole is either antero-lateral and slightly oblique herein, mitochondria are located adjacent to the caudal poleto the basal body [11], lateral, oblique and distant from the of the nucleus and surround the initial segment of the axonemedistal centriole [49], anterior, coaxial and slightly oblique to the and separated from it by the cytoplasmic canal. Similar locationbasal body [9], anterior, lateral and perpendicular to the basal of mitochondria has been reported also among subfamilies ofbody [44], anterior, medial and perpendicular to the basal Characidae, particularly that have a long midpiece, such asbody [22, 39, 48, 50, 52], or anterior, medial to slightly lateral Myleinae [59], Bryconinae [48, 50, 52], Hyphessobrycon innesiand at a right or slightly oblique angle relative to the distal of the Tetragonopterinae [11] and Salmininae [51], where theycentriole [10]. are grouped in the anterior third of the midpiece around the

Furthermore, the midpiece exhibits two situations among initial region of the axoneme and separated from it by theteleosts, one of which is at the posterior end of the nucleus as cytoplasmic canal. The same arrangement has been observedin A. dentex examined herein, many teleosts [11, 17, 45] and also in an inseminating species of the Cheirodontinae [22].

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In species of the Glandulocaudinae, the elongate mitochondria Moreover, spermatozoan head nuclear chromatin isare grouped and located close to the posterior end of the widely different in appearance among fishes. It is dense,nucleus [22, 39]. The Acestrorhynchidae have few elongate homogeneous and compact in C. carpio [26], C. auratusmitochondria located around the nucleus and around the [15, 60], R. s. sinensis [63], Goodeidae [13], P. latipinna [3],initial region of the axoneme and separated from the axoneme by G. affinis [4], Blenniidae [34, 67], A. gigas [62], C. aggregatathe cytoplasmic canal. However, mitochondria are found in the [12], C. lateralis [13], J. lineata [53], L. sicculus [56],nuclear indentation in the flounder [16], many blenniid species A. schlegeli [29, 31], A. latus [27], D. mesembrinus [8],[34, 67] and several eels [20, 30, 35]. In the citharinid species, A. japonica [30] and M. merluccius [7]. However, it is granularmitochondria are located close to the nucleus near the centriolar and heterogeneous in L. aurata [18], O. niloticus [43],complex [49]. In P. altivelis [58] and H. malabaricus [9], due to salmonids, e.g. O. tshawtscha [54], S. fontinalis [55], Coregonusthe absence of the cytoplasmic canal, a single mitochondrion in wartmanni [69] and O. mykiss [36], M. undulatus [28, 61],the former species or several mitochondria in the latter species C. chanos [33], O. masou formosanus [24], E. malabaricusare situated close to the nucleus and laterally in relation to the and P. leopardus [32] and P. altivelis [58]. In A. dentex, theflagellum. In addition, the number and distribution of spermatozoon chromatin appears heterogeneously granular,mitochondria are frequently variable among teleosts; they highly electron-dense and contains tightly packed fibers andrange from a single mitochondrion to several (up to ten) irregular small clear lacunae. Similar appearance of chromatinmitochondria. For example, one mitochondrion has been found has been found in C. auratus [45] and some Characiformeslying either in a concavity in the anterior end of the nucleus as [22, 48-52, 59]. On the contrary, among other members of thein A. japonica [30], lateral to the flagellum as in P. altivelis [58] Characiformes, spermatozoa exhibit thick fibers ofor encircling the base of the flagellum as in C. chanos [33], homogeneously condensed chromatin [9-11, 19, 22, 39, 44,O. m. formosanus [24] and D. mesembrinus [8]. Nevertheless, 47, 49].it has been reported that there are three mitochondria in In A. dentex, the nucleus is spherical. Similar appearance ofA. latus [27], four in A. schlegeli [29, 31], five (rarely six) in the nucleus has been recorded in some characids [9-11, 17, 19]E. malabaricus and P. leopardus [32] and C. auratus [45] and and in some teleosts [11, 17, 27, 29, 31, 32]. Nevertheless,several, up to ten, mitochondria in M. merluccius [7], which conical and elongate form of the nucleus has been described insurround the base of the flagellum. Among Characiformes, C. auratus [45] and several characids [22, 39, 49]. In addition,however, it has been reported that there is either one neither nuclear elongation in the spermatozoon nor structuresmitochondrion that initially extends laterally to the flagellum used to attach the flagellum to the nucleus duringto one side of the nucleus or several mitochondria, as the case spermiogenesis are found in A. dentex. In other teleosts,in A. dentex examined herein, which surround the base of the however, nuclear elongation has been described in manyflagellum at the posterior part of the nucleus [9, 10, 22, 39]. teleosts [3, 16, 20, 22, 34, 35, 39]. Moreover, several

Development of an acrosomal complex (the acrosomal structures that connect the flagellum with the nucleus arevesicle and the subacrosomal material) from the functions of reported. In O. mykiss [36] and eels [20, 30, 35], there is asmaller Golgi-derived proacrosomal vesicles is not apparent in structure called flagellar rootlet. In Oryzias laptipes [5],A. dentex. The Golgi apparatus appears at the posterior region T. thymallus [57] and P. flesus [16], there is an electron-denseof the diplosome during spermiogenesis and disappears at the material. Nonetheless, there are bundles of microtubules inend of spermiogenesis. The absence of acrosome, although it is G. affinis [4].very common in the majority of teleosts, an acrosomal vesicle In spermatozoa of A. dentex, there are intercentriolarhas been observed, however, in O. mykiss [36]. connections in the form of osmiophilic filaments arise from

In A. dentex, progressive condensation of chromatin occurs the triplets of the proximal centriole and connect it with theduring spermiogenesis. In early stages, the chromatin appears as distal one. A similar structure has been described also inirregular aggregations of coarse and dense granules scattered O. mykiss [36], P. flesus [16], T. thymallus [57], M. undulatusthroughout the nucleus. This is followed with gradual [28, 61], A. schlegeli [29, 31], P. altivelis [58], E. malabaricuscoalescence of the thick chromatin strands into denser and P. leopardus [32], A. latus [27] and C. chanos [33].aggregations. Changes in the nuclear chromatin condensation Moreover, in A. dentex, there are two fibrous bodies consistpattern during spermiogenesis have previously been reported of osmiophilic disks alternating with lighter material andin O. mykiss [36], O. niloticus [43], Blennius pholis [67], lie perpendicularly above the proximal centriole in theA. schlegeli [29], M. barberi [39], D. mesembrinus [8], upper part of nuclear fossa. These two dense bodies giveH. malabaricus [9] and C. auratus [45]. The progressive rise to two short electron-dense fibers, which connect themalterations of chromatin packing have been interpreted as the together with the proximal centriole and anchor the latter toresult of the transformation of nuclear basic proteins [68]. the nucleus. Similar findings have been described in the

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salmon O. m. formosanus [24], where there is a fibrous body, role in the formation of these structures [29]. The otherconsisting of osmiophilic disks alternating with lighter structures are the presence of two fibrous bodies, which occupymaterial, adheres to the two centrioles laterally and links them the upper part of the nuclear fossa and connect the proximaltogether and also links the proximal centriole with dense centriole with the nucleus and the basal foot and alar sheets thatbodies in the nuclear fossa. However, in the gadiform attach the basal body to the nucleus and plasma membrane,M. merluccius [7], the proximal centriole is linked to the respectively.nuclear surface by granular material. In addition, nine radial As regards, the general features of spermiogenesis offibers project from the basal body of the distal centriole A. dentex are considerably similar to those of Ostariophysitriplets and contact the plasma membrane at the caudal part with external fertilization, which includes Cypriniformesof the centriole. Nevertheless, a structure called the [14], Characiformes [9-11, 22, 48, 50, 59, 72], Siluriformesintercentriolar lamellate body has been observed in the [8, 11, 17, 73-75] and Gonorynchiformes [33]. Moreover,poeciliids P. latipinna [3] and G. affinis [4]. This structure A. dentex spermatozoon has some of the characteristics ofappears during the development of spermatids to form an Perciformes [27, 29, 31, 32] and Salmoniformes [24].electron-dense cap on the proximal end of the basal body Therefore, it could be concluded that the ultrastructuraland then disappears during the final stage of spermiogenesis. features of spermiogenesis and spermatozoa of A. dentexFurthermore, a connecting piece has been described in are synapomorphy of Ostariophysi, Perciformes andA. japonica [30]. The morphology of the intercentriolar Salmoniformes. Nevertheless, it is difficult to understand theconnection is suggested to have a phylogenetic importance significance of the ultrastructural characteristics of A. dentexamong fishes [5]. spermatozoa in relation to other species of Characiformes

Further, in A. dentex there is a striated basal foot because of the lack of data about spermatozoa from otherattaches the basal body to the nucleus and alar sheets radiate families of this order. On the other hand, the similarity of somefrom the basal body triplets and joins it to the plasma morphological features of A. dentex spermatozoa to those ofmembrane. The same structures have been observed in Cypriniformes supports the hypothesis that the CypriniformesE. malabaricus and P. leopardus [32] and A. latus [27]. are the sister-group of the Otophysi, Characiformes andHowever, alar sheets have been described in O. m. formosanus Siluriformes [76-78].[24]. As has been described, it is probable that both the alarsheets and basal foot function in the attachment and ACKNOWLEDGEMENTSstabilization of the tail, thereby enabling the centriole towithstand the torque generated by the movement of the The author would like to thank Prof. Dr. Emeritusflagellum [27]. M.A. Ramadan for reading the manuscript, Dr. H.A. El Azm,

Vesicles, which are commonly shown with more or less Electron Microscopy Unit in Minia University Lab forregularity in the midpiece of Characiformes spermatozoa [10] Microanalysis, for technical assistance and the Fishermanand in C. auratus [45], are entirely absent in A. dentex. In Mr. M. Hussein for providing the fish.addition, the “lattice tubule” described in Citharinus sp. [49]or the membranous compartment found in the initial region REFERENCESof the flagellum of some Characiformes [10] as well as someCypriniformes [15, 70, 71] and also the flagellar lateral fins 1. Franzen, A., 1970. Phylogenetic aspects of theor intratubular differentiations occurred in some teleosts morphology of spermatozoa and spermatogenesis. In[11, 17, 66] are not found in A. dentex examined herein. Similar Comparative spermatology, Ed., Baccetti, B. New York,findings have been reported in many species of Characiformes Academic Press, pp: 26-46.of which data are available [10]. 2. Mattei, C., 1970. Spermiogeneses comparee des poissons.

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