Comparative analysis of spermiogenesis and sperm ultrastructure in Callichthyidae ( Teleostei : Ostariophysi : Siluriformes )

In Corydoradinae, the presence of spermatids in the lumen of the testicular tubules together with spermatozoa suggests that spermatogenesis is of the semicystic type, whereas in Callichthyinae, sperm production occurs entirely within spermatocysts in the germinal epithelium, characterizing cystic spermatogenesis. Spermiogenesis in Callichthyinae is characterized by an initial lateral development of the flagellum, the presence of nuclear rotation to different degrees, an eccentric or medial formation of a nuclear fossa, formation of a cytoplasmic channel, and presence of centriolar migration, being more similar to type I spermiogenesis. In Corydoradinae, spermiogenesis is characterized by eccentric development of the flagellum, the absence of nuclear rotation, an eccentric nuclear fossa formation, formation of a cytoplasmic channel, and absence of centriolar migration, differing from the types previously described. The process of spermatogenesis and spermiogenesis in Corydoradinae and Callichthyinae revealed unique characters for each of these subfamilies, corroborating the hypotheses that they constitute monophyletic groups. In relation to sperm ultrastructure, the comparative analysis of the callichthyid species shows that the general characteristics found in the spermatozoa were similar, thus, reinforcing the hypothesis that the family is monophyletic.


Introduction
Siluriformes comprise an extremely large fish group that is widely distributed across the tropical regions of the world (Teugels, 1996).Among the Neotropical siluriform lineages likely to be monophyletic we find the superfamily Loricarioidea that comprises six families: Nematogenyidae, Trichomycteridae, Callichthyidae, Scoloplacidae, Astroblepidae, and Loricariidae (de Pinna, 1998;Britto, 2003a).
The current study presents the ultrastructural characterization of both spermiogenesis and spermatozoa in seven species of Callichthyidae comprised of four Corydoradinae and three Callichthyinae.Comparisons between the data ob-tained in this study and those available for other siluriform families were also conducted.
Gonad fragments were fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M Sorensen phosphate buffer, pH 7.4.The material was post-fixed for 2h in the dark in 1% osmium tetroxide in the same buffer, stained in block with an aqueous solution of 5% uranyl acetate for 2h, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% ethanol, and with lead citrate (Reynolds, 1963).Electromicrographs were obtained using a Phillips -CM 100 transmission electron microscope.

Spermiogenesis in Corydoradinae.
In the analyzed species, spermatids are found in the lumen of the testicular tubules together with spermatozoa (Figs. 1c,1d,2a).At the beginning of the process, early spermatids remain connected to the Sertoli cell surface (Figs.2b-2d).During extracystic spermiogenesis, this connection is lost and the more differentiated spermatids move toward the luminal region of the testicular tubules, mixing with spermatozoa.In early spermatids, the centriolar complex lies laterally to the nucleus and the distal centriole anchors to the plasma membrane.Development of the flagellum from the distal centriole occurs lateral to the nucleus (Fig. 2e).The centriolar complex does not move toward the nucleus, remaining anchored to the plasma membrane (Figs.2f and 2g).The centrioles are coaxial to each other, and between them a linear electron-dense structure, in transverse position to the centriolar axis is observed.Proximal and distal centrioles are fastened to the linear electron dense structure by microfibrils (Figs.2e and 2e-inset).During spermiogenesis, nuclear rotation does not occur, and the flagellum takes up an eccentric position in relation to the nucleus.A flat depression is formed in the nuclear outline, giving rise to the nuclear fossa.The nuclear fossa is eccentrically positioned, and the centriolar complex is found completely outside of it (Fig. 2e).The chromatin is highly condensed in the form of the thin fibers that confer a homogeneous aspect to the nucleus (Figs.2g, and 2i).Although the centrioles do not move toward the nucleus, the formation of a cytoplasmic channel occurs (Fig. 2g).The cyto-plasmic mass moves around the initial segment of the tail, and gives rise to the midpiece of the future spermatozoon.The midpiece has elongate mitochondria and vesicles randomly distributed along it .Within the cytoplasm of the midpiece the formation of an electron-dense circular structure is also observed (Fig. 2h).The flagellum has a classical (9+2) axoneme, with nine peripheral microtubular doublets of microtubules and a single central pair of microtubules, with neither lateral projection nor fins, and no visible membranous compartment (Fig. 2e-inset).

Spermatozoa of Corydoradinae.
The measurements of length and width of nucleus, nuclear fossa, midpiece, and cytoplasmic channel of the spermatozoa of Callichthyinae and Corydoradinae species analyzed are presented in the Table 1.
Corydoradinae spermatozoa have a round or ovoid head, asymmetric midpiece, and one flagellum eccentric to the nucleus (Figs. 3a,4a,5a,and 6a).They do not show an acrosomal vesicle.In Corydoras flaveolus and C. aeneus, the nuclei have a round shape, while in Scleromystax lacerdai and Aspidoras poecilus, they are ovoid .The chromatin has an homogeneous aspect, interspersed with electronlucent areas.In the cytoplasmic region around the nucleus, no organelles are seen 4c,4e,.The nuclear fossa is eccentric to the nucleus and forms a simple arc (Figs. 3d,4d,5d,5e,and 6d).In this species herein analyzed, there is a large number of elongate mitochondria filling all the regions of the midpiece.The mitochondria are separated from the flagellum by the cytoplasmic channel.Vesicles are observed in the basal extremity of the midpiece distributed around the flagellum .In C. flaveolus, S. lacerdai, and A. poecilus, the vesicles are small and can be isolated or interconnected to each other, whereas in C. aeneus they are interconnected to each other.In all species analyzed an electron-dense circular structure in the midpiece cytoplasm is observed, which was formed during the spermiogenesis (Figs. 3f,3g,4g,5a,.The centrioles are completely outside the nuclear fossa, coaxial to each other, and show an electron-dense structure among them (Figs. 3d,4d,5a,5d,5e,6a,6d,and 6f).The distal centriole is differentiated into the basal body and gives rise to the axoneme, which exhibits the classical 9+2 microtubular pattern.Although the flagellum of 1.9 the spermatids does not contain vesicles or tubules, the flagellum of the spermatozoon displays some regions with a membranous compartment, whereas in others this compartment is not observed 5a,5h,.

Spermiogenesis in Callichthyinae.
In the analyzed species of Callichthyinae, spermiogenesis occurs within spermatocysts in the germinal epithelium.In these cysts, groups of spermatids at the same development stage are surrounded by cytoplasmic processes of the Sertoli cells (Figs. 1a, 1b, and 7a).In the cysts, the early spermatids are interconnected by cytoplasmic bridges that result from incomplete cytokinesis during cellular divisions.In the early spermatids, the cytoplasm is symmetrically distributed around the nucleus, which contains partment in some regions of the flagellum (Figs. 8j,8k,9k,9l,10h,and 10i).Discussion Spermatogenesis and Spermiogenesis.The occurrence of spermatids in the lumen of the testicular tubules together with spermatozoa in the species of Corydoradinae suggests that spermatogenesis in this subfamily is of the semicystic type.This type of spermatogenesis was described in Cetopsidae and Aspredinidae (Spadella et al., 2006a), and also found in Nematogenyidae (our unpublished data).Although uncommon, semicystic spermatogenesis has been described in others families of Teleostei such as Opheliidae (Mattei et al., 1993), Scorpaenidae (Muñoz et al., 2002), and Bleniidae (Lahnsteiner & Patzer, 1990).
In the spermatozoa, the flagellar axis may be either perpendicular or parallel to the nucleus, depending on whether nuclear rotation during spermiogenesis occurs (type I spermiogenesis) or not (type II spermiogenesis) (Mattei, 1970).In Pimelodidae and Heptapteridae, the flagellum is medial, the nucleus does not rotate, and both the nuclear fossa and the cytoplasmic channel are absent during spermiogenesis, characterizing a third type of spermiogenesis (Quagio-Grassiotto et al., 2005;Quagio-Grassiotto & Oliveira, 2007).The process of spermiogenesis observed in Corydoradinae is characterized by an eccentric development of the flagellum, the absence of nuclear rotation, formation of an eccentric nuclear fossa, formation of a cytoplasmic channel formation, and the absence of centriolar migration.This set of characteristics is different from those previously described.This unusual process of spermiogenesis found in Corydoradinae, except for the eccentric development of the flagellum and nuclear fossa, was also observed in Cetopsidae and Aspredinidae (Spadella et al., 2006a).
It is possible that in the subfamily Corydoradinae the cytoplasmic channel results from the interconnection of the vesicles around the flagellum rather than the movement of the centrioles toward the nucleus.It is probable that a similar process occurs in Cetopsidae, Aspredinidae, and Nematogenyidae (Spadella et al., 2006a).
Spermiogenesis in Callichthyinae is characterized by an initial lateral development of the flagellum, the presence of nuclear rotation to varying degrees, formation of an eccentric or medial nuclear fossa, formation of a cytoplasmic channel and the presence of centriolar migration.This set of charac- teristics is more similar to type I spermiogenesis, which is also observed in Diplomystidae, the most basal siluriform family (Quagio-Grassiotto et al., 2001), in Scoloplacidae (Spadella et al., 2006b), and in Pseudopimelodidae (Quagio-Grassiotto et al., 2005).However, in Diplomystidae the cytoplasmic channel does not remain in the spermatozoa (Quagio-Grassiotto et al., 2001) as observed in Callichthyinae, Scoloplacidae, and Pseudopimelodidae.Furthermore, nuclear rotation in Diplomystidae, Scoloplacidae, and Pseudopimelodidae is complete, resulting in a flagellum perpendicular to the nucleus in the spermatozoon, whereas in Hoplosternum littorale and Callichthys callichthys it is partial, and in Megalechis thoracata it is complete, resulting in the eccentric position of the flagellum in H. littorale and C. callichthys and medial in M. thoracata.Mattei (1970) felt that the occurrence of intermediate processes of spermiogenesis is responsible for the formation of these intermediate spermatozoa, whose flagella are eccentric to the nucleus.
The characteristics of the process of spermatogenesis and spermiogenesis in the Corydoradinae and Callichthyinae revealed clear unique characters for each of these subfamilies, corroborating the hypotheses that they constitute monophyletic groups (Reis, 1998;Shimabukuro-Dias et al., 2004).
Spermatozoa.The comparative analyses of callichthyid sperm ultrastructure presented in Table 2 showed that species herein analyzed share the same character states in eight out of the sixteen characters observed (characters 1, 3, 6, 8, and 10 to 13).These characters are also share with other siluriform families constituting plesiomorphic characters for the family Callichthyidae.
Callichthyids sperm ultrastructure showed that character 2, arrangement of complex centriolar, is variable among the species analyzed, being co-axial in all Corydoradinae, lateral in acute angle in Hoplosternum littorale, and lateral in obtuse angle in Megalechis thoracata and Callichthys callichthys.The state "character co-axial" is only found in Corydoradinae, which represents a synapomorphic character of this subfamily.The other character state, arrangement of complex centriolar, found in Callichthyinae, is also observed in Trichomycteridae (our unpublished data).
The presence of the membranous compartment in the flagellum (character 4) is found in the majority of the species of Callichthyidae (species of Corydoradinae and Callichthys callichthys).A similar characteristic was also observed in Trichomycteridae, other members of Loricarioidea (our unpublished data), and in Characiformes of the family Curimatidae (Quagio-Grassiotto et al., 2003).
The only character not shared by all Corydoradinae is the shape of the nucleus (character 5), which is spherical in Corydoras flaveolus and C. aeneus, whereas in Scleromystax lacerdai and Aspidoras poecilus, the nucleus is ovoid (Table 2).In a phylogenetic study of the subfamily Corydoradinae conducted by Britto (2003b) with 83 morphological characters the author corroborates the monophyly of the subfamily and showed that Aspidoras is more related to Scleromystax.The data obtained in the present study corroborate this hypothesis.Some characteristics are shared by all Corydoradinae and some species of Callichthyinae (Table 2).For example, the presence of a membranous compartment in the flagellum and centrioles outside of the nuclear fossa occurs in Corydoradinae and in Callichthys callichthys (characters 4 and 9, respectively), the occurrence of eccentric nuclear fossa (Character 7), presence of asymmetric midpiece (character 14), presence of eccentric flagellum position in relation to the nucleus (character 15), and medial position of flagellum in relation to the nucleus (character 16) are found in Corydoradinae and in Hoplosternum littorale and C. callichthys.There was no characteristic exclusively found in the spermatozoa of Callichthyinae.Most of the characteristics of spermatozoon of C. callichthys (Callichthyinae) were similar with those found among the species of Corydoradinae.Character 15, presence of electron-dense circular structure in the midpiece, is also shared by all Corydoradinae species, representing an other unique character of this subfamily, not observed in any other siluriform up to the present (Poirier & Nicholson, 1982;Emel'yanova & Makeyeva, 1991a, b;Kwon et al., 1998;Lee, 1998;Lee & Kim, 1999;Kim & Lee, 2000;Quagio-Grassiotto & Carvalho, 2000;Lee & Kim, 2001;Quagio-Grassiotto et al., 2001;Santos et al., 2001;Burns et al., 2002;Mansour et al., 2002;Mansour & Lahnsteiner, 2003;Quagio-Grassiotto et al., 2005).
Considering the phylogeny of the superfamily Loricarioidea, the family Callichthyidae is a sister group of the clade formed by Scoloplacidae, Astroblepidae, and Loricariidae (de Pinna, 1998;Britto, 2003a).Table 2 shows that one or more species of Callichthyidae share eight characteristics with Nematogenyidae (another family of Loricarioidea), ten characters with Scoloplacidae, and twelve with Loricariidae.As observed above, Callichthyidae spermatozoa share more characters with Scoloplacidae and Loricariidae than with Nematogenyidae, corroborating the previous hypotheses of relationships of the superfamily Loricarioidea proposed by de Pinna (1998) and Britto (2003a).

Fig. 1 .
Fig. 1.Photomicrography showing a general view of the semi-cystic spermatogenesis in Corydoras flaveolus, Corydoradinae (a and b) and of the cystic spermatogenesis in Hoplosternum littorale, Callichthyinae (c and d).Scale bar a and c = 20 µm, b and d = 10 µm.CI: cysts of primary spermatocytes; G: spermatogonia; T: spermatids in the lumen of germinative compartment; Z spermatozoa, Arrow: spermatids cysts.
Distribution of sperm character states in the species analyzed in the present study, as well as other families of Loricarioidea.(+) present; (-) absent.*1-flagella number (a-one; b-two); 2-arrangement of centriolar complex (a-lateral and parallel; b-lateral and acute angle; c-lateral and obtuse angle; d-anterior and perpendicular; e-anterior and obtuse angle; fco-axial); 3-presence of elongated vesicles; 4-flagellar membrane specializations (a-absent; b-two lateral fins; c-membranous compartment); 5-shape of the nucleus (a-semi-ovoid; b-conic; c-ovoid; d-ovoid with its bigger axis in the horizontal direction; e-spherical); 6-pattern of chromatin condensation (a-heterogeneous; b-homogeneous); 7-nuclear fossa (aabsent; b-medial; c-eccentric); 8-nuclear fossa shape (a-simple arc; b-double arc); 9-centrioles totally inserted in the nuclear fossa; 10-midpiece size (a-short; b-long); 11-cytoplasmic channels (a-absent; b-one; c-two); 12-cytoplasmic channel size (a-short; b-long); 13-mitochondria shape (a-elongated; b-elongated and ramificated); 14-midpiece symmetry (a-symmetric; b-asymmetric); 15-Presence of electron-dense circular structure in the midpiece; 16-flagellum position in relation to the nucleus (a-medial; b-eccentric).a b c d e f -a b c a b c d e a b a b c a b -a b a b c a b a b a b -a b Nematogenyidae

Table 1 .
Spermatozoa dimensions in callichthyids, in micrometers.The "n" shows the number of structures measured.