Comparative description and discussion of spermiogenesis and spermatozoal ultrastructure in some species of Heptapteridae and Pseudopimelodidae ( Teleostei : Siluriformes )

The data obtained in the present study on spermiogenesis and spermatozoal ultrastructure of Pseudopimelodidae and Heptapteridae show that they share some characteristics, but greatly differ from each other. The main differences are the occurrence of type I spermiogenesis in Pseudopimelodidae and type III in Heptapteridae, the presence of nuclear fossa in Pseudopimelodidae and its absence in Heptapteridae, the presence of long midpiece in Pseudopimelodidae and short midpiece in Heptapteridae, the presence of cytoplasmic canal in Pseudopimelodidae and its absence in Heptapteridae, the presence of many large vesicles in the midpiece of Pseudopimelodidae and the presence of very long vesicles placed in the peripheral distal region in Heptapteridae, and mitochondria distributed all over the midpiece in Pseudopimelodidae, and very close to the nucleus in Heptapteridae. Heptapteridae and Pimelodidae share several characteristics, such as type III spermiogenesis, a similar chromatin condensation pattern, and the absence of nuclear fossa and flagellar lateral fins. The spermatozoa of Pseudopimelodidae is more similar to those of Siluridae. However, the absence of additional data on spermiogenesis and spermatozoa in siluriforms still limits a broader discussion in the order.


Introduction
Siluriformes comprise the most diverse and widely distributed ostariophysan group and represent about 32% of all freshwater fishes (Teugels, 1996).The relationships among catfish families have long been studied, but the higher-level phylogeny of siluriforms is still controversial (Diogo, 2003).It is a general consensus that the family Diplomystidae is the sister group of all other Siluriforms.According to the phylogeny proposed by Britto (2003), the families Pimelodidae, Heptapteridae and Pseudopimelodidae belong to a monophyletic clade that includes Anchariidae, Austroglanididae, Bagridae, Claroteinae, Cranoglanididae, Doradoidei, Pangasiidae, Schilbidae, and Horabagrus.In his phylogeny,

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Pseudopimelodidae and Heptapteridae appear as a monophyletic group, as well as the most basal group of this clade.Pimelodidae occupy a more derived position and is the sistergroup of the clade formed by Anchariidae, Austroglanididae, Bagridae, Claroteinae, Cranoglanididae, Pangasiidae, Schilbidae and Horabagrus (Britto, 2003).
Pseudopimelodidae may be considered the least known and one of the smallest families of neotropical freshwater catfishes (Shibatta, 2003).Lundberg et al. (1991) first demonstrated the monophyly of this group and suggested the existence of a relationship between Pseudopimelodidae and the basal Heptapteridae.This hypothesis was corroborated by Britto (2003).The family Heptapteridae comprises small to medium-sized fishes and forms one of the largest group of Neotropical catfishes (Bockmann & Guazzelli, 2003).Bockmann (1998) considered Goeldiella as the sister group of all other Heptapteridae.The remaining Heptapteridae were divided into three clades: one composed by Rhamdia quelen, another by R. laticauda and Pimelodella, and the other by the remaining species (Bockmann, 1998).
Considering their informative phylogenetic potential, the ultrastructural characters of both spermiogenesis and spermatozoa in one genus of Pseudopimelodidae (Microglanis) and two species of two genera of Heptapteridae (Rhamdia and Pimelodella) are herein described for the first time and data are compared and discussed.
Gonad fragments from newly sacrificed fish were fixed overnight in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M Sorensen phosphate buffer, pH 7.4.The material was post-fixed in the dark for 2 hours in 1% osmium tetroxide in the same buffer, stained in block with an aqueous solution of 5% uranyl acetate for two hours, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% alcohol and lead citrate.Electron micrographs were obtained using a Phillips -CM 100 transmission electron microscope.

Results
General observations.In the species analyzed, spermiogenesis occurs in cysts in the germinative epithelium.These cysts consist of a group of germ cells at the same developmental stage, which are surrounded by cytoplasmic processes of Sertoli cells.In the cysts, the early spermatids are interconnected by cytoplasmic bridges resultant from incomplete cytokinesis of both the anterior mitotic and the meiotic divisions.In all the species, spermatozoa exhibit a head, a midpiece, and one tail or flagellum.The head does not have an acrosomal vesicle.
Pseudopimelodidae spermiogenesis.In M. aff.parahybae early spermatids, the cytoplasm symmetrically encircles the nucleus, which shows diffuse homogenous chromatin, and has a circular outline.The centriolar complex lies laterally to the nucleus and anchors to the plasma membrane.The proximal centriole is anterior, oriented end-by-end and slightly oblique in relation to the distal centriole.The distal centriole differentiates into the basal body and forms the flagellum.The centriolar complex moves toward the nucleus, carrying along the plasma membrane and the initial segment of the flagellum, which invaginates.The cytoplasmic channel, a space between the flagellum and the plasma membrane, is then formed (Figs.1A-H).A depression is formed in the nuclear outline at the level of the centriolar complex.The nucleus starts to rotate, making the centriolar complex penetrate the newly formed depression.Most of the cytoplasm moves towards the region surrounding the cytoplasmic channel where a few mitochondria are located, and gives rise to the midpiece (Figs.1E-G, 1J).Many vesicles

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appear at the midpiece.These vesicles elongate and fuse to each other and to the plasma membrane (Figs.1L-O).In the final spermatids, the nucleus contains highly condensed homogeneous chromatin (Fig. 1G).The flagellum exhibits the classical (9+2) axoneme, surrounded by the flagellar membrane, and does not form lateral fins (Figs.1I-K).
Heptapteridae spermiogenesis.In Heptapteridae early sper-  , 3D).The few mitochondria initially found around the nucleus are now located around the centriolar complex, near the nucleus, in the midpiece anterior region.They are long, have many cristae and an electron-dense matrix (Figs. 2E, 2G, 2I-J, 3C, 3E, 3F).Many vesicles appear at the midpiece peripheral and distal end-regions.These vesicles elongate, fuse to each other and become very long and interspersed with a narrow strip of cytoplasm (Figs.2E, 2G-J, 3D-F).In P. gracilis, the displacement of these vesicles to the initial region of the tail may form a short pseudo-cytoplasmic channel, which does not remain in the spermatozoa (Figs.3D, 3F).
Pseudopimelodidae spermatozoa.In the Pseudopimelodidae spermatozoa, the head is formed by the nucleus that has a circular outline, is occupied by highly condensed homogeneous chromatin, and surrounded by a narrow strip of cytoplasm with no organelles (Figs.4A-B).In the nuclear outline, there is a nuclear fossa of the moderate type containing only the proximal centriole (Fig. 4D-Inset) and part of the distal centriole.The proximal centriole is anterior, oriented end-byend and slightly oblique to the basal body.The flagellum is medial and perpendicular to the nucleus (Fig. 4C).An electron-dense material are found surrounding the centriolar complex (Fig. 4C).The midpiece is long, wide, and slightly asymmetric, with a long cytoplasmic channel.Few elongate mitochondria and many vesicles that are connected to each other and also to the plasma membrane are scattered all over the midpiece (Figs.4D-F).The single tail or flagellum contains the classical axoneme (9+2), and the flagellar membrane does not have lateral fins (Figs.4A, 4C, 4E).
Heptapteridae spermatozoa.In the Heptapteridae spermatozoa, the head is formed by the nucleus that does not shows a nuclear fossa.The nucleus is surrounded by a narrow strip of cytoplasm with no organelles.The nucleus is occupied by highly condensed homogeneous chromatin and displays some electron-lucent areas (Figs.5A-B, 6A-B).R. quelen spermatozoa have an ovoid head with an ovoid nucleus (Fig. 5A), while in P. gracilis, the head and the nucleus are round (Fig. 6A).The centriolar complex remains close to the nucleus and is surrounded by a thin layer of electron-dense material (Figs. 5A, 5E, 5F, 6A, 6E).In R. quelen, the proximal centriole is anterior, lateral and perpendicular to the flagellum basal body -the differentiated distal centriole (Figs.5E-F), while in P.

Discussion
Spermiogenesis.Teleostei with external fertilization showed that the flagellum generally develops lateral to the nucleus in the early spermatids.In spermatozoa, the flagellar axis may be either perpendicular or parallel to the nucleus, depending on whether nuclear rotation occurs (type I spermiogenesis) or not (type II spermiogenesis) during spermiogenesis (Mattei, 1970).In the pimelodid (Quagio-Grassiotto and Oliveira, in press) the flagellum development is medial, the nucleus does not rotate, and both the nuclear fossa and the cytoplasmic channel are absent during spermiogenesis.These characteristics have not been found in any other fish species and represents a new type of spermiogenesis named type III.In Pseudopimelodidae, spermiogenesis is of type I.In Diplomystidae, the most basal family of Siluriformes, spermiogenesis is also of type I, though in this family the cytoplasmic channel does not remain in the spermatozoon (Quagio-Grassiotto et al., 2001).On the other hand, in Heptapteridae, spermiogenesis is of type III, as observed in Pimelodidae (Quagio-Grassiotto & Oliveira, in press).In Heptapteridae and Pimelodidae, a short pseudo-cytoplasmic channel may appear as the cytoplasmic mass moves toward the flagellum, and the midpiece vesicles fuse to each other and to the plasma membrane.No information about spermiogenesis in other Siluriform families with external fertilization is available.
In the nuclear outline, a nuclear fossa of the moderate type containing only the proximal centriole and part of the distal centriole is found in Pseudopimelodidae.A similar nuclear fossa and centriolar positioning are observed in Clariidae (Mansour et al., 2002), Siluridae (Kwon et al., 1998;Lee & Kim, 2001) 1998), and Bagridae (Lee, 1998;Kim & Lee 2000).However, in these latter families, the centriolar complex lies completely inside the nuclear fossa.In the Heptapteridae species, the nuclear fossa is absent and the centriolar complex lies close to the nucleus, at a medial position, as observed in pimelodid (Quagio-Grassiotto & Carvalho, 2000;Quagio-Grassiotto & Oliveira, in press).In Iheringichthys labrosus (Santos et al., 2001), also a Pimelodidae, the medial nuclear fossa is shallow, and the centriolar complex remains in the same position as in other Pimelodidae.In Conorhynchus conirostris (Lopes et al., 2004), the medial nuclear fossa is deep and penetrates almost to the tip of the nucleus, while the centriolar complex is completely inside of it.When the nucleus is longitudinally bisected, this kind of nuclear fossa gives rise to a horseshoeshaped image.The centriolar complex in Ictaluridae (Emel'yanova & Makeyeva, 1991a, b;Poirier & Nicholson, 1982;Mattei, 1991), Malapteruridae and Ariidae (schematic drawings- Mattei, 1991) lies outside the nuclear fossa that forms a shallow double arc at a medial position.In the inseminating Auchenipteridae (Burns et al., 2002), the nuclear fossa can be considered as of the moderate type.It lies laterally to the nuclear outline and contains only the proximal centriole and part of the distal centriole.

Position of the centrioles in relation to each other.
In Pseudopimelodidae, the proximal centriole is anterior and
The flagellum of the Pseudopimelodidae and Heptapteridae herein analyzed, as well as in Pimelodidae (Quagio-
Conclusions.The general characteristics observed in the spermatozoa of two species of Heptapteridae were very similar, which reinforces the early hypothesis developed by Baccetti et al. (1984), who suggested that species from a same family exhibit the same patterns of spermatozoon organelle distribution and agrees with the hypothesis of the monophyly of Heptapteridae (Bockmann, 1998).Britto (2003), in a phylogenetic study of morphological characters, suggested that Pseudopimelodidae and Heptapteridae were sister groups.The present data show that Pseudopimelodidae and Heptapteridae share some characteristics, but greatly differ from each other.The main differences are the occurrence of type I spermiogenesis in Pseudopimelodidae and type III in Heptapteridae, the presence of a nuclear fossa in Pseudopimelodidae and its absence in Heptapteridae, the presence of a long midpiece in Pseudopimelodidae and short midpiece in Heptapteridae, the presence of a cytoplasmic canal in Pseudopimelodidae and its absence in Heptapteridae, many large vesicles in the midpiece of Pseudopimelodidae and vesicles very long and placed in the peripheral distal region in Heptapteridae, and mitochondria distributed all over the midpiece in Pseudopimelodidae and very close to the nucleus in Heptapteridae.
On the other hand, Heptapteridae and Pimelodidae (Quagio-Grassiotto & Carvalho, 2000;Santos et al., 2001, Quagio-Grassiotto & Oliveira, in press) share several characteristics, such as the same type of spermiogenesis (Type IIIwith the formation of a medial flagellum and no nuclear rotation), the same chromatin condensation pattern, and no nuclear fossa or flagellar lateral fins.
Although the absence of additional spermiogenesis and spermatozoa data limited a broad discussion, the information currently available points to an interesting similarity between the spermatozoa of Pseudopimelodidae and Siluridae.The spermatozoa of these two families share a nuclear fossa of the moderate type, containing only the proximal centriole and part of the distal centriole, the proximal centriole anterior and slightly oblique, in an obtuse angle in relation to the distal centriole, and few elongate mitochondria distributed all over the midpiece, near the nucleus, directly around the basal body and separated from the axoneme by the cytoplasmic channel.
In addition, the ultrastructural differences observed between the spermatozoa of Conorhynchus conirostris and those of Pseudopimelodidae, Heptapteridae and Pimelodidae (previously considered to belong to the large family of Pimelodidae) support the proposal by Ferraris (2003), accord-ing to whom Conorhynchus conirostris does not appear to belong to any of those three families.A detailed analysis of C. conirostris spermatozoa does not permit a safe conclusion about the relationship of this species with other families, whose spermatozoa were previously described.On the other hand, some of the characteristics found in the spermatozoa of C. conirostris are similar to those observed in a species of Doradidae of the genus Anadoras (I.Quagio-Grassiotto, unpublished data).