- Email: [email protected]
Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae)
Micron 62 (2014) 1–6
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae) Mônica Cassel a,∗ , Adelina Ferreira a , Mahmoud Mehanna b Instituto de Biociências, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, n◦ 2367, Bairro Boa Esperanc¸a, Cuiabá, MT 78060-900, Brazil Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Campus de Botucatu, Distrito de Rubião Junior, S/N, Botucatu, SP 18618-970, Brazil a
b
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 17 February 2014 Accepted 17 February 2014 Available online 25 February 2014 Keywords: Germ cells Spermatozoa Aquasperm Morphology Phylogeny
a b s t r a c t Fish belonging to the family Rivulidae possess one of the most complex reproductive systems. Rivulus, a genus of freshwater fish in the Rivulidae family, was recently reclassified into five genera, including Melanorivulus. Its type species, M. punctatus, is widely distributed and probably represents a species complex. The ultrastructure of sperm has been broadly used in systematics, and we hereby describe the ultrastructural features of spermatogenesis in M. punctatus. Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira, municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and prepared for analysis by light microscopy and transmission electron microscopy. M. punctatus undergoes cystic spermatogenesis. Its cysts consist of groups of germ cells that are in synchronous development and are surrounded by cytoplasmic projections of Sertoli cells. With the breakdown of the cysts, the spermatozoa are released and their maturation is completed in the duct, where part of the cytoplasmic material is discarded through the vesicles. The mature spermatozoon is characterized by a spherical head with homogeneously condensed chromatin, a symmetric midpiece consisting of a pair of perpendicular centrioles, a ring of mitochondria, several vesicles, and one flagellum medial to the nucleus. Early stages of spermatogenesis show no peculiarities; however, in spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges and have pockets of residual cytoplasm. The sperm is of the aquasperm type and is similar to that observed in the members of the family Rivulidae. The spermatozoa have a single flagellum that consists of a classic axoneme (9 + 2), as found in most groups of fish, despite the lateral extensions. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The order Cyprinodontiformes consists of families with peculiarities in their reproductive strategy (i.e., Casatti et al., 2006). Of these, family Rivulidae is known for its complexities, which led to the definition of annualism (Costa, 2002). This family is comprised of the subfamilies Kryptolebiatinae, Cynolebiatinae, and Rivulinae (Costa, 2003, 2004a, 2004b, 2011) and 36 genera (Costa, 2011; Eschmeyer, 2013). The Neotropical genus Rivulus Poey 1860 was recognized as a paraphyletic group based on molecular data (Hrbek and Larson, 1999; Hrbek et al., 2004). However, groupings within the genus were further classified into monophyletic subunits or subgenera (Costa, 2006). Due to observed discrepancies, Costa (2011) proposed the reclassification of genus Rivulus into
∗ Corresponding author. Tel.: +55 6536158878. E-mail addresses: [email protected], monica [email protected] (M. Cassel), [email protected] (A. Ferreira), [email protected] (M. Mehanna). http://dx.doi.org/10.1016/j.micron.2014.02.012 0968-4328/© 2014 Elsevier Ltd. All rights reserved.
five genera, which includes the new genus Melanorivulus, which was restricted to continental basins. The type species, M. punctatus, is widely distributed and probably represents a species complex (Costa, 2011). According to Schindler and Etzel (2008), Rivulus punctatus (=Melanorivulus punctatus) presents a morphological variability that raises the question about the limits of this species within the genus, which added to the absence of physiogeographical barriers that eventually prevented the possible isolation of populations, thereby making the presence of low specific differentiation more likely. Thus, species identification can be satisfactorily achieved only when a direct complement of their evolutionary and biogeographic history becomes available. The ultrastructural features of spermatozoa have been widely used in systematics because these are cells that possess morphological characteristics that are more likely conserved than the macroscopic characters traditionally used (Jamieson et al., 1996). Likewise, the use of spermatozoan ultrastructural features have been proven effective in resolving issues relating to
2
M. Cassel et al. / Micron 62 (2014) 1–6
Fig. 1. Spermatogenesis of Melanorivulus punctatus. (a) General arrangement of the cells in the lobules, the spermatogonia (Sg) are confined to the bottom of the lobes, followed by the other germ cells, primary (Pc) and secondary (Sc) spermatocytes, early (Et) and late (Lt) spermatids and, finally, spermatozoa (Sz), all of which are lined by Sertoli cells (S); (b/c) Sertoli cells (S) forming the cysts are polymorphic, with evident nucleoli (Nc), mitochondria (M) with lamellar cristae, and numerous vesicles (V) with residual bodies in degradation; (d/e) Spermatogonia: characterized by a round nucleus (N) with decondensed chromatin, well-developed nucleolus (Nc) and numerous mitochondria (M) and nüages (Nu) in the cytoplasm; (f/g/h) Primary (Pc) and secondary (Sc) spermatocytes are much smaller than spermatogonia and are linked together by means of cytoplasmic bridges (circles); (g) Primary spermatocytes: have a round and centralized nucleus with minimally condensed chromatin consisting of small granules (Csg), and numerous mitochondria (M) distributed across the cytoplasm; (h) Secondary spermatocytes: the cytoplasm is electron-dense manly because of the presence of numerous nüages (Nu) and mitochondria (M), and the nucleus shows chromatin undergoing a heterogenous condensation process that results in the development of gross granules (Cgg).
taxonomic positioning and relationships among different groups of fish (Jamieson, 1991; Mattei, 1991). Considering the great diversity of fish fauna, data on the ultrastructural features of spermatogenesis in teleosts are scarce and restricted to few species (Magalhães et al., 2011). For the family Rivulidae, there are only three studies that have used the ultrastructural features of spermatozoa as a tool for reproductive and phylogenetic analysis of species (Kweon et al., 1998; Arezo et al., 2007; Garcia et al., 2009). Thus, we describe the ultrastructural characteristics of spermatogenesis in M. punctatus.
2. Material and methods Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira (15◦ 27 58 S, 55◦ 44 46 W), municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and fixed in a 2% glutaraldehyde and 4% paraformaldehyde solution in Sorensen’s buffer (0.1 M, pH 7.2) for 24 h. The gonads were extracted and prepared according to the protocol for inclusion in plastic resin, cut with glass knives into 3 m thick sections and stained with the combination of dyes iron hematoxylin/eosin, toluidine
blue/borax, and Periodic Acid–Schiff (PAS) + hematoxylin + metanil yellow (Quintero-Hunter et al., 1991). Some fragments of the same gonads were prepared according to the routine procedure for transmission electron microscopy, as described by Cassel et al. (2013).
3. Results M. punctatus shows cystic spermatogenesis and these cysts consist of groups of germ cells at the same stage of development. The cysts are enclosed by cytoplasmic projections of Sertoli cells (Fig. 1). Sertoli cells are polymorphic, with the nuclear envelope well defined and cytoplasmic projections more electron-dense than those of the germ cells (Figs. 1a–c). These cells possess evident nucleoli, mitochondria with lamellar cristae, and numerous vesicles with residual bodies in degradation (Fig. 1c). The spermatogonia are the largest cells that are characterized by a high nucleus to cytoplasm ratio; their nuclei are round, eccentrically located and show decondensed chromatin and well-developed nucleoli (Fig. 1a, d and e). Their cytoplasm contains a large amount of mitochondria and nüages (Fig. 1e). The nüages are patches of dense granular-fibrillar material close to the
M. Cassel et al. / Micron 62 (2014) 1–6
3
Fig. 2. Spermiogenesis of Melanorivulus punctatus. (a/b) Early spermatids: are interconnected by cytoplasmic bridges (circles), have a cytoplasm with spherical or slightly elongated mitochondria (M) that are symmetrically distributed around the nucleus (N), which has a circular outline and contains chromatin with dispersed granules that gradually increase in number and size; (c) Movement of the cytoplasmic mass (arrows) that gives rise to the midpiece of the future spermatozoon; (d/e) Late spermatids: the nucleus (N) remains spherical and will contain filamentous clusters of highly condensed chromatin (Cc), cells also have a pocket or vesicles of residual cytoplasm (Pr) to the side of the nucleus that will be eventually eliminated at the end of the duct; at the same time, a depression is formed in the nuclear outline, giving rise to the nuclear fossa (Nf), which is medially positioned.
nuclear membrane, either free in the cytoplasm or associated with mitochondria. Primary and secondary spermatocytes (Fig. 1a and f–h) are much smaller than spermatogonia and are linked together by means of cytoplasmic bridges (Fig. 1g and h), and marked by local thickening on the inner side of the plasma membrane. The nuclear morphology is variable as meiosis proceeds. The primary spermatocytes show an electron-dense cytoplasm and are characterized by round, centralized nuclei with minimally condensed chromatin appearing as small granules, and many mitochondria distributed in the cytoplasm (Fig. 1g). The secondary spermatocytes, which are produced after the first meiotic division, show smaller cytoplasmic volumes,
yet remain electron-density due to the presence of numerous nüages and mitochondria (Fig. 1h). The nuclei of secondary spermatocytes show heterogeneously condensed chromatin consisting of large granules (Fig. 1h). Spermatids are found in cysts, separated from the spermatozoa (Fig. 1a). The early spermatids are interconnected by cytoplasmic bridges that result from incomplete cytokinesis (Fig. 2a and b). These cells have cytoplasm with spherical or slightly elongated mitochondria that are symmetrically distributed around the nucleus, which has a circular outline and contains chromatin that consists of dispersed granules that gradually increase in number and size (Fig. 2a and b). A displacement of the cytoplasmic mass
4
M. Cassel et al. / Micron 62 (2014) 1–6
Fig. 3. Ultrastructure of the sperm of Melanorivulus punctatus. (a/b) Immature spermatozoon: part of the cytoplasmic material (C), mainly mitochondria (M), is discarded through vesicles (V) reducing its amount in the midpiece of the mature spermatozoa; (c) cross-section of the midpiece: characterized by a flap formed by the cytoplasmic membrane (Cm), a ring of mitochondria (Mr) and axoneme (Ax); (d) cross-section of middle part of the flagellum: presents the axoneme (Ax) added to the bilateral fins (Bf); (e) cross-section of the end of the flagellum: presents the axoneme (Ax) without bilateral fins; (f) Spermatozoon: composed of a (1) spherical head consisting of a rounded nucleus (N) with a homogeneously condensed chromatin, interpersed with electron-lucent areas (circles), encased in a cytoplasmic membrane (Cm), an arrangement of microtubules (Mt) spread near to the insertion area of the flagellum and lateral to the nuclear fossa (Nf), which in turn is medial to the nucleus and has an simple arc shape, (2) the midpiece that is comprised of an annulus (An), a ring of mitochondria (M), together with some vesicles (V) that are separated from the flagellum by a cytoplasmic channel (Cch), a set of typical centrioles with the proximal centriole (Pct) inserted into the nuclear fossa and the distal centriole (Dct) immersed in a fibrous sheath that serves as basis for (3) the characteristic axoneme (Ax) (9 + 2), plus bilateral fins (Bf) and covered by the cytoplasmic membrane.
occurs that gives rise to the midpiece of the future spermatozoon (Fig. 2c). In spermiogenesis, nuclear rotation occurs and simultaneously, a depression is formed at the nuclear outline, giving rise to the nuclear fossa, which is medially positioned (Fig. 2d). In late spermatids, the nucleus remains spherical and will contain filamentous clusters of highly condensed chromatin, which appear as electron-dense structures under an electron microscope (Fig. 2d and e). These cells also have a pocket or vesicle of residual cytoplasm to the side of the nucleus that will be eventually eliminated (Fig. 2d and e). The spermatozoa of M. punctatus are found individualized (Fig. 1a) and complete their maturation in the duct. Part of the cytoplasmic material, mainly the mitochondria, is discarded through vesicles, thus resulting in a reduction in its number in the midpiece of mature spermatozoa (Fig. 3a and b). The mature spermatozoon (Fig. 3f) is characterized by a spherical head, a symmetric midpiece, and one flagella located medial to the nucleus. There is no acrosome vesicle. The nucleus is round shaped, contains condensed chromatin, and is encased in a cytoplasmic membrane (Fig. 3f). The chromatin is homogeneously condensed in the form of chromatin clusters that are interspersed with electron-lucent areas. The nuclear fossa is located medial to the nucleus and shows a simple arc shape. Large amounts of microtubules are distributed near the site of insertion of the flagellum and lateral to the nuclear fossa (Fig. 3f). In the midpiece (Fig. 3c and f), we observed the annulus, which is located in front of the ring of mitochondria. This ring consists of a row with five spherical or slightly elongated mitochondria that are located in the apical region of the midpiece. Several vesicles are present in the basal region of the midpiece, either isolated or interconnected with each other, thus forming a membranous compartment. The mitochondria and vesicles are separated from the
flagella by the cytoplasmic channel, which is a space between the cytoplasmic sheath and the initial region of the flagellum (Fig. 3f). The midpiece also shows pair of perpendicular centrioles that are positioned lateral and perpendicular to each other. The proximal centriole is inserted into the initial portion of the nuclear fossa, it is oblique to the flagellum, and the distal centriole is immersed in a fibrous sheath, which appears as an electron-dense ring (Fig. 3f). The fibrous sheath is the basis for the typical axoneme (9 + 2) conformation and is the main piece of the flagellum, to which bilateral fins are added and covered by the cytoplasmic membrane (Fig. 3d and f). The final piece of the flagellum does not present these fins anymore (Fig. 3e). 4. Discussion Early stages of spermatogenesis in M. punctatus showed no peculiarities, wherein the spermatogonia and spermatocytes show features that are similar to those described in other teleosts (Grier, 1981; Schulz et al., 2010). During the initial stages of spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges, as previously described for other fish species, and have pockets of residual cytoplasm, which was also observed by Thiaw et al. (1988) in Aphyosemion riggenbachi and A. splendopleure of the family Cyprinodontidae (Cyprinodontiformes). These pockets are also observed in species of Pimelodidae (Quagio-Grassiotto and Carvalho, 1999; Santos et al., 2001; Quagio-Grassiotto and Oliveira, 2008). Based on the general characteristics of the spermatozoa of M. punctatus, we have classified this species as an aquasperm (i.e., a spermatozoa that is involved with external fertilization, according to Mattei, 1991) that is devoid of an acrosome vesicle, with the
M. Cassel et al. / Micron 62 (2014) 1–6
head containing a spherical nucleus, a short middle piece with the presence of cytoplasmic channel, and a long flagellum (Jamieson, 1991). The nuclear rotation for the adjustment of organelles can be seen because the centriolar arrangement that gives rise to the flagellum is perpendicular to the nucleus during the final stages of sperm maturation (Mattei, 1970). For subfamily Rivulinae, the ultrastructural description of Kweon et al. (1998) for the species Rivulus marmoratus (=Kryptolebias marmoratus) includes characteristics of spermatozoa that function for external fertilization and are very similar to that of M. punctatus in this study. We observed in the family Rivulidae as well that the characteristic spermatozoa found in this study are similar to that of Austrolebias charrua, A. reicherti, and A. viarius (Arezo et al., 2007; Garcia et al., 2009). In Teleostei, the position of the centriolar complex is related to the shape of the nuclear fossa (Quagio-Grassiotto and Oliveira, 2008). In M. punctatus, the nuclear fossa is moderately deep, contains a part of the centriolar complex, and the centrioles show a perpendicular arrangement. Next to these structures, we also observed that the microtubules lined the basal part of the nucleus. The features of the nuclear fossa have also been previously described by Kweon et al. (1998) for the Rivulinae subfamily; however, these are not shared with the other subfamilies of Rivulidae or families of Cyprinodontiformes. However, the perpendicular position of the centrioles, as noted, is the most common (QuagioGrassiotto et al., 2003; Gusmão-Pompiani et al., 2005; Greven and Schmahl, 2006; Fishelson et al., 2007; Chung, 2008; Vázquez et al., 2012). According to descriptions of Mattei (1970), Jamieson (1991), and Mattei (1991), the midpiece of the sperm of teleosts can either be a short, symmetrical, with a reduced cytoplasmic channel or long and without a cytoplasmic channel. For Cyprinodontiformes, the second type of midpiece is more commonly observed (Billard, 1973; Grier, 1973; Jamieson, 1989; Vicentini et al., 2010), although in M. puntatus, the midpiece can be characterized as short with a cytoplasmic channel. Mitochondria and vesicles are other structures that are present in the midpiece of teleost spermatozoa (Jamieson, 1991; Mattei, 1991). M. punctatus presents a ring with five mitochondria, followed by several vesicles on the cytoplasmic sheath. The number of mitochondria present in spermatozoa varies among species, but their arrangement in a single ring seems to occur among rivulids (Kweon et al., 1998; Arezo et al., 2007). In terms of vesicles in the midpiece, these structures have not been described in other Cyprinodontiformes. M. punctatus possesses spermatozoa with a single flagellum that is comprised of a classic axoneme with two central microtubules and nine pairs of peripheral microtubule surrounded by the plasma membrane, as found in most groups of fish (Mattei, 1988, 1991; Jamieson, 1991). We also observed that the flagellum may have lateral extensions, known as lateral fins, and that widely vary in number among species. However, according to Thiaw et al. (1986), these variations can occur even within a species and could not be associated with any other structural changes in the spermatozoa, thus characterizing these sperm as similar to the other species.
5. Conclusion In summary, the stages of spermatogenesis in M. punctatus did not differ from what has already been previously described in other fish species. Its spermatozoa is of the aquasperm type and is similar to what has been previously described by Kweon et al. (1998), Arezo et al. (2007), and Garcia et al. (2009) for members of family Rivulidae, despite the variation in the number of side fins.
5
Acknowledgments We thank Irani Quagio-Grassiotto (UNESP-Botucatu) for the space provided for the histological preparations for light and electron microscopy analysis.
References Arezo, M.J., D’Alessandro, S., Papa, N., Sá, R., Berois, N., 2007. Sex differentiation pattern in the annual fish Austrolebias charrua (Cyprinodontiformes: Rivulidae). Tissue Cell 39, 89–98. Billard, R., 1973. La spermatogenese des poissons Poeciliidae: qualques comparaisons avec celle d’autres especes de poissons. Bull. Fr. Piscic. Ichthyol. Hydrobiol. Appl. 250, 17–34. Casatti, l., Carvalho, F.R., Veronezi Jr., J.L., Lacerda, D.R., 2006. Reproductive biology of the neotropical superfetaceous Pamphorichthys hollandi (Cyprinodontiformes: Poeciliidae). Ichthyol. Explor. Freshw. 17, 59–64. Cassel, M., Silva, D.F.N., Ferreira, A., 2013. Cytoarchitectonical dynamic of Sertoli cells in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae). Micron. 45, 115–118. Chung, E.Y., 2008. Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae). Tissue Cell 40, 195–205. Costa, W.J.E.M., 2002. Peixes anuais brasileiros: Diversidade e conservac¸ão. editora da UFPR, Curitiba. Costa, W.J.E.M., 2003. Family Rivulidae (South American annual fishes). In: Reis, R.E., Kullander, S.O., Ferraris Jr., C.J. (Eds.), Check list of the Freshwater Fishes of South and Central America. Edipucrs, Porto Alegre, pp. 526–548. Costa, W.J.E.M., 2004a. A new killifish genus and species from the coastal plains of north-eastern (Teleostei: Cyprinodontiformes: Rivulidae). Zootaxa 642, 1–10. Costa, W.J.E.M., 2004b. Relationships and redescription of Fundulus brasiliensis Valenciennes (Cyprinodontiformes: Rivulidae), with description of a new genus and notes on the classification of the Aplocheiloidei. Ichthyol. Explor. Freshw. 15, 105–120. Costa, W.J.E.M., 2006. Relationships and taxonomy of the killifish genus Rivulus (Cyprinodontiformes: Aplocheiloidei: Rivulidae) from the Brazilian Amazonas river basin, with notes on historical ecology. Aqua 11, 133–175. Costa, W.J.E.M., 2011. Phylogenetic position and taxonomic status of Anablepsoides, Atlantirivulus, Cynodonichthys, Laimosemion and Melanorivulus (Cyprinodontiformes: Rivulidae). Ichthyol. Explor. Freshw. 22, 233–249. Eschmeyer, W.N., 2013. Catalog of Fishes. California Academy of Sciences (http://research.calacademy.org/research/ichthyology/catalog/fishcatmain.asp) Electronic version dated 09.09.13, accessed 03.10.13. Fishelson, L., Gon, O., Holdengreber, V., Delarea, Y., 2007. Comparative spermatogenesis, spermatocytogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei). Anat. Rec. 290, 311–323. Garcia, G., Loureiro, M., Berois, N., Arezo, M.J., Casanova, G., Clivio, G., Oliveira, A., 2009. Pattern of differentiation in the annual killifish genus Austrolebias (Cyprinodontiformes: Rivulidae) from a biosphere reserve site in South America: a multidisciplinary approach. Biol. J. Linn. Soc. 98, 620–635. Greven, H., Schmahl, G., 2006. A note on the spermatozoon ultrastructure of the foureyed fish Anableps anableps (Atherinomorpha, Cyprinodontiformes). Z. Fischk. 8, 83–88. Grier, H.J., 1973. Ultrastructure of the testis in the teleost Poecilia latipinna: spermiogenesis with reference to the intercentriolar lamellated body. J. Ultrastruct. Res. 45, 82–92. Grier, H.J., 1981. Cellular organization of the testis and spermatogenesis in fishes. Am. Zool. 21, 345–357. Gusmão-Pompiani, P., Oliveira, C., Quagio-Grassiotto, I., 2005. Spermatozoa ultrastructure in Sciaenidae and Polynemidae (Teleostei:Perciformes) with some consideration on Percoidei spermatozoa ultrastructure. Tissue Cell 37, 177–191. Hrbek, T., Deus, C.P., Farias, I.P., 2004. Rivulus duckensis (Teleostei; Cyprinodontiformes): new species from the Tarumã basin of Manaus, Amazonas, Brazil, and its relationships to other neotropical Rivulidae. Copeia 2004, 569–576. Hrbek, T., Larson, V., 1999. The diapause in the killifish family Rivulidae (Atherinomorpha, Cyprinodontiformes): a molecular phylogenetic and biogeographic perspective. Evolution 53, 1200–1216. Jamieson, B.G.M., 1989. Complex spermatozoon of the live-bearing kalfbeak, Hemirhamphodon pogonognathus (Bleeker): ultrastructural description (Euteleostei, Atherinomorpha, Beloniformes). Gamete Res. 24, 247–259. Jamieson, B.G.M., 1991. Fish Evolution and Systematics: Evidence from Spermatozoa, 1st ed. Cambridge University Press, Cambridge. Jamieson, B.G.M., Oliver, S.C., Scheltinga, D.M., 1996. The ultrastructure of the spermatozoa of Squamata – I. Scincidae, Gekkonidae and Pygopodidae (Reptilia). Acta Zool. 77, 85–100. Kweon, H.S., Park, E.H., Peters, N., 1998. Spermatozoon ultrastructure in the internally self-fertilizing hermaphroditic teleost, Rivulus marmoratus (Cyprinodontiformes, Rivulidae). Copeia 1998, 1101–1106. Magalhães, A.L.B., Andrade, R.F., Gomes, B.V.C., Perini, V.R., Rizzo, E., Bazzoli, N., 2011. Ultrastructure of the semicystic spermatogenesis in the South American freshwater characid Hemigrammus marginatus (Teleostei, Characiformes). J. Appl. Ichthyol. 27, 1041–1046.
6
M. Cassel et al. / Micron 62 (2014) 1–6
Mattei, X., 1970. Spermiogenese comparee des poisons. In: Baccetti, B. (Ed.), Comparative Spermatology. Academic Press, New York, pp. 57–69. Mattei, X., 1988. The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution. Biol. Cell. 63, 151–158. Mattei, X., 1991. Spermatozoon ultrastructure and its systematic implications in fishes. Can. J. Zool. 69, 3038–3055. Quagio-Grassiotto, I., Antoneli, F.N., Oliveira, C., 2003. Spermiogenesis and sperm ultrastructure in Cichla intermedia with some considerations about Labroidei spermatozoa (Teleostei, Perciformes, Cichlidae). Tissue Cell 35, 441–446. Quagio-Grassiotto, I., Carvalho, E.D., 1999. The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: Premeiotic and meiotic period. Tissue Cell 31, 561–567. Quagio-Grassiotto, I., Oliveira, C., 2008. Sperm ultrastructure and a new type of spermiogenesis in two species of Pimelodidae, with a comparative review of sperm ultrastructure in Siluriformes (Teleostei: Ostariophysi). Zool. Anz. 247, 55–66. Quintero-Hunter, I., Grier, H., Muscato, M., 1991. Enhancement of histological detail using metanil yellow as a counterstain in periodic acid/Schiff’s hematoxylin staining of glycol methacrylate tissue sections. Biotech. Histochem. 66, 169–172.
Santos, J.E., Bazzoli, N., Rizzo, E., Santos, G.B., 2001. Morphofunctional organization of the male reproductive system of the catfish Iheringichthys labrosus (Lütken, 1874) (Siluriformes: Pimelodidae). Tissue Cell 33, 533–540. Schindler, I., Etzel, V., 2008. Re-description and distribution of Rivulus punctatus Boulenger, 1895 (Teleostei: Rivulidae) and its habitats in Paraguay. Vert. Zool. 58, 33–43. Schulz, R.W., Franc¸a, L.R., Lareyre, J.J., LeGac, F., Chiarini-Garcia, H., Nóbrega, R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165, 390–411. Thiaw, O.T., Mattei, X., Romand, R., 1988. Process of cytoplasmic elimination during spermiogenesis in two Cyprinodontidae (teleostean fishes). J. Ultrastruct. Mol. Struct. Res. 101, 192–198. Thiaw, O.T., Mattei, X., Romand, R., Marchand, B., 1986. Reinvestigation of spermatic flagella structure: the teleostean Cyprinodontidae. J. Ultrastruct. Mol. Struct. Res. 97, 109–118. ˜ R.H., Meijide, F.J., Guerrero, G.A., 2012. Spermatogenesis Vázquez, G.R., Da Cuna, and changes in testicular structure during the reproductive cycle in Cichlasoma dimerus (Teleostei, Perciformes). Acta Zool. 93, 338–350. ˜ Vicentini, C.A., Munoz, M.E., Vicentini, I.B.F., 2010. Ultrastructural aspects of spermatogenesis in Phalloceros caudimaculatus (Teleostei, Poeciliidae). Int. J. Morphol. 28, 951–956.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae) Mônica Cassel a,∗ , Adelina Ferreira a , Mahmoud Mehanna b Instituto de Biociências, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, n◦ 2367, Bairro Boa Esperanc¸a, Cuiabá, MT 78060-900, Brazil Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Campus de Botucatu, Distrito de Rubião Junior, S/N, Botucatu, SP 18618-970, Brazil a
b
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 17 February 2014 Accepted 17 February 2014 Available online 25 February 2014 Keywords: Germ cells Spermatozoa Aquasperm Morphology Phylogeny
a b s t r a c t Fish belonging to the family Rivulidae possess one of the most complex reproductive systems. Rivulus, a genus of freshwater fish in the Rivulidae family, was recently reclassified into five genera, including Melanorivulus. Its type species, M. punctatus, is widely distributed and probably represents a species complex. The ultrastructure of sperm has been broadly used in systematics, and we hereby describe the ultrastructural features of spermatogenesis in M. punctatus. Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira, municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and prepared for analysis by light microscopy and transmission electron microscopy. M. punctatus undergoes cystic spermatogenesis. Its cysts consist of groups of germ cells that are in synchronous development and are surrounded by cytoplasmic projections of Sertoli cells. With the breakdown of the cysts, the spermatozoa are released and their maturation is completed in the duct, where part of the cytoplasmic material is discarded through the vesicles. The mature spermatozoon is characterized by a spherical head with homogeneously condensed chromatin, a symmetric midpiece consisting of a pair of perpendicular centrioles, a ring of mitochondria, several vesicles, and one flagellum medial to the nucleus. Early stages of spermatogenesis show no peculiarities; however, in spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges and have pockets of residual cytoplasm. The sperm is of the aquasperm type and is similar to that observed in the members of the family Rivulidae. The spermatozoa have a single flagellum that consists of a classic axoneme (9 + 2), as found in most groups of fish, despite the lateral extensions. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The order Cyprinodontiformes consists of families with peculiarities in their reproductive strategy (i.e., Casatti et al., 2006). Of these, family Rivulidae is known for its complexities, which led to the definition of annualism (Costa, 2002). This family is comprised of the subfamilies Kryptolebiatinae, Cynolebiatinae, and Rivulinae (Costa, 2003, 2004a, 2004b, 2011) and 36 genera (Costa, 2011; Eschmeyer, 2013). The Neotropical genus Rivulus Poey 1860 was recognized as a paraphyletic group based on molecular data (Hrbek and Larson, 1999; Hrbek et al., 2004). However, groupings within the genus were further classified into monophyletic subunits or subgenera (Costa, 2006). Due to observed discrepancies, Costa (2011) proposed the reclassification of genus Rivulus into
∗ Corresponding author. Tel.: +55 6536158878. E-mail addresses: [email protected], monica [email protected] (M. Cassel), [email protected] (A. Ferreira), [email protected] (M. Mehanna). http://dx.doi.org/10.1016/j.micron.2014.02.012 0968-4328/© 2014 Elsevier Ltd. All rights reserved.
five genera, which includes the new genus Melanorivulus, which was restricted to continental basins. The type species, M. punctatus, is widely distributed and probably represents a species complex (Costa, 2011). According to Schindler and Etzel (2008), Rivulus punctatus (=Melanorivulus punctatus) presents a morphological variability that raises the question about the limits of this species within the genus, which added to the absence of physiogeographical barriers that eventually prevented the possible isolation of populations, thereby making the presence of low specific differentiation more likely. Thus, species identification can be satisfactorily achieved only when a direct complement of their evolutionary and biogeographic history becomes available. The ultrastructural features of spermatozoa have been widely used in systematics because these are cells that possess morphological characteristics that are more likely conserved than the macroscopic characters traditionally used (Jamieson et al., 1996). Likewise, the use of spermatozoan ultrastructural features have been proven effective in resolving issues relating to
2
M. Cassel et al. / Micron 62 (2014) 1–6
Fig. 1. Spermatogenesis of Melanorivulus punctatus. (a) General arrangement of the cells in the lobules, the spermatogonia (Sg) are confined to the bottom of the lobes, followed by the other germ cells, primary (Pc) and secondary (Sc) spermatocytes, early (Et) and late (Lt) spermatids and, finally, spermatozoa (Sz), all of which are lined by Sertoli cells (S); (b/c) Sertoli cells (S) forming the cysts are polymorphic, with evident nucleoli (Nc), mitochondria (M) with lamellar cristae, and numerous vesicles (V) with residual bodies in degradation; (d/e) Spermatogonia: characterized by a round nucleus (N) with decondensed chromatin, well-developed nucleolus (Nc) and numerous mitochondria (M) and nüages (Nu) in the cytoplasm; (f/g/h) Primary (Pc) and secondary (Sc) spermatocytes are much smaller than spermatogonia and are linked together by means of cytoplasmic bridges (circles); (g) Primary spermatocytes: have a round and centralized nucleus with minimally condensed chromatin consisting of small granules (Csg), and numerous mitochondria (M) distributed across the cytoplasm; (h) Secondary spermatocytes: the cytoplasm is electron-dense manly because of the presence of numerous nüages (Nu) and mitochondria (M), and the nucleus shows chromatin undergoing a heterogenous condensation process that results in the development of gross granules (Cgg).
taxonomic positioning and relationships among different groups of fish (Jamieson, 1991; Mattei, 1991). Considering the great diversity of fish fauna, data on the ultrastructural features of spermatogenesis in teleosts are scarce and restricted to few species (Magalhães et al., 2011). For the family Rivulidae, there are only three studies that have used the ultrastructural features of spermatozoa as a tool for reproductive and phylogenetic analysis of species (Kweon et al., 1998; Arezo et al., 2007; Garcia et al., 2009). Thus, we describe the ultrastructural characteristics of spermatogenesis in M. punctatus.
2. Material and methods Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira (15◦ 27 58 S, 55◦ 44 46 W), municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and fixed in a 2% glutaraldehyde and 4% paraformaldehyde solution in Sorensen’s buffer (0.1 M, pH 7.2) for 24 h. The gonads were extracted and prepared according to the protocol for inclusion in plastic resin, cut with glass knives into 3 m thick sections and stained with the combination of dyes iron hematoxylin/eosin, toluidine
blue/borax, and Periodic Acid–Schiff (PAS) + hematoxylin + metanil yellow (Quintero-Hunter et al., 1991). Some fragments of the same gonads were prepared according to the routine procedure for transmission electron microscopy, as described by Cassel et al. (2013).
3. Results M. punctatus shows cystic spermatogenesis and these cysts consist of groups of germ cells at the same stage of development. The cysts are enclosed by cytoplasmic projections of Sertoli cells (Fig. 1). Sertoli cells are polymorphic, with the nuclear envelope well defined and cytoplasmic projections more electron-dense than those of the germ cells (Figs. 1a–c). These cells possess evident nucleoli, mitochondria with lamellar cristae, and numerous vesicles with residual bodies in degradation (Fig. 1c). The spermatogonia are the largest cells that are characterized by a high nucleus to cytoplasm ratio; their nuclei are round, eccentrically located and show decondensed chromatin and well-developed nucleoli (Fig. 1a, d and e). Their cytoplasm contains a large amount of mitochondria and nüages (Fig. 1e). The nüages are patches of dense granular-fibrillar material close to the
M. Cassel et al. / Micron 62 (2014) 1–6
3
Fig. 2. Spermiogenesis of Melanorivulus punctatus. (a/b) Early spermatids: are interconnected by cytoplasmic bridges (circles), have a cytoplasm with spherical or slightly elongated mitochondria (M) that are symmetrically distributed around the nucleus (N), which has a circular outline and contains chromatin with dispersed granules that gradually increase in number and size; (c) Movement of the cytoplasmic mass (arrows) that gives rise to the midpiece of the future spermatozoon; (d/e) Late spermatids: the nucleus (N) remains spherical and will contain filamentous clusters of highly condensed chromatin (Cc), cells also have a pocket or vesicles of residual cytoplasm (Pr) to the side of the nucleus that will be eventually eliminated at the end of the duct; at the same time, a depression is formed in the nuclear outline, giving rise to the nuclear fossa (Nf), which is medially positioned.
nuclear membrane, either free in the cytoplasm or associated with mitochondria. Primary and secondary spermatocytes (Fig. 1a and f–h) are much smaller than spermatogonia and are linked together by means of cytoplasmic bridges (Fig. 1g and h), and marked by local thickening on the inner side of the plasma membrane. The nuclear morphology is variable as meiosis proceeds. The primary spermatocytes show an electron-dense cytoplasm and are characterized by round, centralized nuclei with minimally condensed chromatin appearing as small granules, and many mitochondria distributed in the cytoplasm (Fig. 1g). The secondary spermatocytes, which are produced after the first meiotic division, show smaller cytoplasmic volumes,
yet remain electron-density due to the presence of numerous nüages and mitochondria (Fig. 1h). The nuclei of secondary spermatocytes show heterogeneously condensed chromatin consisting of large granules (Fig. 1h). Spermatids are found in cysts, separated from the spermatozoa (Fig. 1a). The early spermatids are interconnected by cytoplasmic bridges that result from incomplete cytokinesis (Fig. 2a and b). These cells have cytoplasm with spherical or slightly elongated mitochondria that are symmetrically distributed around the nucleus, which has a circular outline and contains chromatin that consists of dispersed granules that gradually increase in number and size (Fig. 2a and b). A displacement of the cytoplasmic mass
4
M. Cassel et al. / Micron 62 (2014) 1–6
Fig. 3. Ultrastructure of the sperm of Melanorivulus punctatus. (a/b) Immature spermatozoon: part of the cytoplasmic material (C), mainly mitochondria (M), is discarded through vesicles (V) reducing its amount in the midpiece of the mature spermatozoa; (c) cross-section of the midpiece: characterized by a flap formed by the cytoplasmic membrane (Cm), a ring of mitochondria (Mr) and axoneme (Ax); (d) cross-section of middle part of the flagellum: presents the axoneme (Ax) added to the bilateral fins (Bf); (e) cross-section of the end of the flagellum: presents the axoneme (Ax) without bilateral fins; (f) Spermatozoon: composed of a (1) spherical head consisting of a rounded nucleus (N) with a homogeneously condensed chromatin, interpersed with electron-lucent areas (circles), encased in a cytoplasmic membrane (Cm), an arrangement of microtubules (Mt) spread near to the insertion area of the flagellum and lateral to the nuclear fossa (Nf), which in turn is medial to the nucleus and has an simple arc shape, (2) the midpiece that is comprised of an annulus (An), a ring of mitochondria (M), together with some vesicles (V) that are separated from the flagellum by a cytoplasmic channel (Cch), a set of typical centrioles with the proximal centriole (Pct) inserted into the nuclear fossa and the distal centriole (Dct) immersed in a fibrous sheath that serves as basis for (3) the characteristic axoneme (Ax) (9 + 2), plus bilateral fins (Bf) and covered by the cytoplasmic membrane.
occurs that gives rise to the midpiece of the future spermatozoon (Fig. 2c). In spermiogenesis, nuclear rotation occurs and simultaneously, a depression is formed at the nuclear outline, giving rise to the nuclear fossa, which is medially positioned (Fig. 2d). In late spermatids, the nucleus remains spherical and will contain filamentous clusters of highly condensed chromatin, which appear as electron-dense structures under an electron microscope (Fig. 2d and e). These cells also have a pocket or vesicle of residual cytoplasm to the side of the nucleus that will be eventually eliminated (Fig. 2d and e). The spermatozoa of M. punctatus are found individualized (Fig. 1a) and complete their maturation in the duct. Part of the cytoplasmic material, mainly the mitochondria, is discarded through vesicles, thus resulting in a reduction in its number in the midpiece of mature spermatozoa (Fig. 3a and b). The mature spermatozoon (Fig. 3f) is characterized by a spherical head, a symmetric midpiece, and one flagella located medial to the nucleus. There is no acrosome vesicle. The nucleus is round shaped, contains condensed chromatin, and is encased in a cytoplasmic membrane (Fig. 3f). The chromatin is homogeneously condensed in the form of chromatin clusters that are interspersed with electron-lucent areas. The nuclear fossa is located medial to the nucleus and shows a simple arc shape. Large amounts of microtubules are distributed near the site of insertion of the flagellum and lateral to the nuclear fossa (Fig. 3f). In the midpiece (Fig. 3c and f), we observed the annulus, which is located in front of the ring of mitochondria. This ring consists of a row with five spherical or slightly elongated mitochondria that are located in the apical region of the midpiece. Several vesicles are present in the basal region of the midpiece, either isolated or interconnected with each other, thus forming a membranous compartment. The mitochondria and vesicles are separated from the
flagella by the cytoplasmic channel, which is a space between the cytoplasmic sheath and the initial region of the flagellum (Fig. 3f). The midpiece also shows pair of perpendicular centrioles that are positioned lateral and perpendicular to each other. The proximal centriole is inserted into the initial portion of the nuclear fossa, it is oblique to the flagellum, and the distal centriole is immersed in a fibrous sheath, which appears as an electron-dense ring (Fig. 3f). The fibrous sheath is the basis for the typical axoneme (9 + 2) conformation and is the main piece of the flagellum, to which bilateral fins are added and covered by the cytoplasmic membrane (Fig. 3d and f). The final piece of the flagellum does not present these fins anymore (Fig. 3e). 4. Discussion Early stages of spermatogenesis in M. punctatus showed no peculiarities, wherein the spermatogonia and spermatocytes show features that are similar to those described in other teleosts (Grier, 1981; Schulz et al., 2010). During the initial stages of spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges, as previously described for other fish species, and have pockets of residual cytoplasm, which was also observed by Thiaw et al. (1988) in Aphyosemion riggenbachi and A. splendopleure of the family Cyprinodontidae (Cyprinodontiformes). These pockets are also observed in species of Pimelodidae (Quagio-Grassiotto and Carvalho, 1999; Santos et al., 2001; Quagio-Grassiotto and Oliveira, 2008). Based on the general characteristics of the spermatozoa of M. punctatus, we have classified this species as an aquasperm (i.e., a spermatozoa that is involved with external fertilization, according to Mattei, 1991) that is devoid of an acrosome vesicle, with the
M. Cassel et al. / Micron 62 (2014) 1–6
head containing a spherical nucleus, a short middle piece with the presence of cytoplasmic channel, and a long flagellum (Jamieson, 1991). The nuclear rotation for the adjustment of organelles can be seen because the centriolar arrangement that gives rise to the flagellum is perpendicular to the nucleus during the final stages of sperm maturation (Mattei, 1970). For subfamily Rivulinae, the ultrastructural description of Kweon et al. (1998) for the species Rivulus marmoratus (=Kryptolebias marmoratus) includes characteristics of spermatozoa that function for external fertilization and are very similar to that of M. punctatus in this study. We observed in the family Rivulidae as well that the characteristic spermatozoa found in this study are similar to that of Austrolebias charrua, A. reicherti, and A. viarius (Arezo et al., 2007; Garcia et al., 2009). In Teleostei, the position of the centriolar complex is related to the shape of the nuclear fossa (Quagio-Grassiotto and Oliveira, 2008). In M. punctatus, the nuclear fossa is moderately deep, contains a part of the centriolar complex, and the centrioles show a perpendicular arrangement. Next to these structures, we also observed that the microtubules lined the basal part of the nucleus. The features of the nuclear fossa have also been previously described by Kweon et al. (1998) for the Rivulinae subfamily; however, these are not shared with the other subfamilies of Rivulidae or families of Cyprinodontiformes. However, the perpendicular position of the centrioles, as noted, is the most common (QuagioGrassiotto et al., 2003; Gusmão-Pompiani et al., 2005; Greven and Schmahl, 2006; Fishelson et al., 2007; Chung, 2008; Vázquez et al., 2012). According to descriptions of Mattei (1970), Jamieson (1991), and Mattei (1991), the midpiece of the sperm of teleosts can either be a short, symmetrical, with a reduced cytoplasmic channel or long and without a cytoplasmic channel. For Cyprinodontiformes, the second type of midpiece is more commonly observed (Billard, 1973; Grier, 1973; Jamieson, 1989; Vicentini et al., 2010), although in M. puntatus, the midpiece can be characterized as short with a cytoplasmic channel. Mitochondria and vesicles are other structures that are present in the midpiece of teleost spermatozoa (Jamieson, 1991; Mattei, 1991). M. punctatus presents a ring with five mitochondria, followed by several vesicles on the cytoplasmic sheath. The number of mitochondria present in spermatozoa varies among species, but their arrangement in a single ring seems to occur among rivulids (Kweon et al., 1998; Arezo et al., 2007). In terms of vesicles in the midpiece, these structures have not been described in other Cyprinodontiformes. M. punctatus possesses spermatozoa with a single flagellum that is comprised of a classic axoneme with two central microtubules and nine pairs of peripheral microtubule surrounded by the plasma membrane, as found in most groups of fish (Mattei, 1988, 1991; Jamieson, 1991). We also observed that the flagellum may have lateral extensions, known as lateral fins, and that widely vary in number among species. However, according to Thiaw et al. (1986), these variations can occur even within a species and could not be associated with any other structural changes in the spermatozoa, thus characterizing these sperm as similar to the other species.
5. Conclusion In summary, the stages of spermatogenesis in M. punctatus did not differ from what has already been previously described in other fish species. Its spermatozoa is of the aquasperm type and is similar to what has been previously described by Kweon et al. (1998), Arezo et al. (2007), and Garcia et al. (2009) for members of family Rivulidae, despite the variation in the number of side fins.
5
Acknowledgments We thank Irani Quagio-Grassiotto (UNESP-Botucatu) for the space provided for the histological preparations for light and electron microscopy analysis.
References Arezo, M.J., D’Alessandro, S., Papa, N., Sá, R., Berois, N., 2007. Sex differentiation pattern in the annual fish Austrolebias charrua (Cyprinodontiformes: Rivulidae). Tissue Cell 39, 89–98. Billard, R., 1973. La spermatogenese des poissons Poeciliidae: qualques comparaisons avec celle d’autres especes de poissons. Bull. Fr. Piscic. Ichthyol. Hydrobiol. Appl. 250, 17–34. Casatti, l., Carvalho, F.R., Veronezi Jr., J.L., Lacerda, D.R., 2006. Reproductive biology of the neotropical superfetaceous Pamphorichthys hollandi (Cyprinodontiformes: Poeciliidae). Ichthyol. Explor. Freshw. 17, 59–64. Cassel, M., Silva, D.F.N., Ferreira, A., 2013. Cytoarchitectonical dynamic of Sertoli cells in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae). Micron. 45, 115–118. Chung, E.Y., 2008. Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae). Tissue Cell 40, 195–205. Costa, W.J.E.M., 2002. Peixes anuais brasileiros: Diversidade e conservac¸ão. editora da UFPR, Curitiba. Costa, W.J.E.M., 2003. Family Rivulidae (South American annual fishes). In: Reis, R.E., Kullander, S.O., Ferraris Jr., C.J. (Eds.), Check list of the Freshwater Fishes of South and Central America. Edipucrs, Porto Alegre, pp. 526–548. Costa, W.J.E.M., 2004a. A new killifish genus and species from the coastal plains of north-eastern (Teleostei: Cyprinodontiformes: Rivulidae). Zootaxa 642, 1–10. Costa, W.J.E.M., 2004b. Relationships and redescription of Fundulus brasiliensis Valenciennes (Cyprinodontiformes: Rivulidae), with description of a new genus and notes on the classification of the Aplocheiloidei. Ichthyol. Explor. Freshw. 15, 105–120. Costa, W.J.E.M., 2006. Relationships and taxonomy of the killifish genus Rivulus (Cyprinodontiformes: Aplocheiloidei: Rivulidae) from the Brazilian Amazonas river basin, with notes on historical ecology. Aqua 11, 133–175. Costa, W.J.E.M., 2011. Phylogenetic position and taxonomic status of Anablepsoides, Atlantirivulus, Cynodonichthys, Laimosemion and Melanorivulus (Cyprinodontiformes: Rivulidae). Ichthyol. Explor. Freshw. 22, 233–249. Eschmeyer, W.N., 2013. Catalog of Fishes. California Academy of Sciences (http://research.calacademy.org/research/ichthyology/catalog/fishcatmain.asp) Electronic version dated 09.09.13, accessed 03.10.13. Fishelson, L., Gon, O., Holdengreber, V., Delarea, Y., 2007. Comparative spermatogenesis, spermatocytogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei). Anat. Rec. 290, 311–323. Garcia, G., Loureiro, M., Berois, N., Arezo, M.J., Casanova, G., Clivio, G., Oliveira, A., 2009. Pattern of differentiation in the annual killifish genus Austrolebias (Cyprinodontiformes: Rivulidae) from a biosphere reserve site in South America: a multidisciplinary approach. Biol. J. Linn. Soc. 98, 620–635. Greven, H., Schmahl, G., 2006. A note on the spermatozoon ultrastructure of the foureyed fish Anableps anableps (Atherinomorpha, Cyprinodontiformes). Z. Fischk. 8, 83–88. Grier, H.J., 1973. Ultrastructure of the testis in the teleost Poecilia latipinna: spermiogenesis with reference to the intercentriolar lamellated body. J. Ultrastruct. Res. 45, 82–92. Grier, H.J., 1981. Cellular organization of the testis and spermatogenesis in fishes. Am. Zool. 21, 345–357. Gusmão-Pompiani, P., Oliveira, C., Quagio-Grassiotto, I., 2005. Spermatozoa ultrastructure in Sciaenidae and Polynemidae (Teleostei:Perciformes) with some consideration on Percoidei spermatozoa ultrastructure. Tissue Cell 37, 177–191. Hrbek, T., Deus, C.P., Farias, I.P., 2004. Rivulus duckensis (Teleostei; Cyprinodontiformes): new species from the Tarumã basin of Manaus, Amazonas, Brazil, and its relationships to other neotropical Rivulidae. Copeia 2004, 569–576. Hrbek, T., Larson, V., 1999. The diapause in the killifish family Rivulidae (Atherinomorpha, Cyprinodontiformes): a molecular phylogenetic and biogeographic perspective. Evolution 53, 1200–1216. Jamieson, B.G.M., 1989. Complex spermatozoon of the live-bearing kalfbeak, Hemirhamphodon pogonognathus (Bleeker): ultrastructural description (Euteleostei, Atherinomorpha, Beloniformes). Gamete Res. 24, 247–259. Jamieson, B.G.M., 1991. Fish Evolution and Systematics: Evidence from Spermatozoa, 1st ed. Cambridge University Press, Cambridge. Jamieson, B.G.M., Oliver, S.C., Scheltinga, D.M., 1996. The ultrastructure of the spermatozoa of Squamata – I. Scincidae, Gekkonidae and Pygopodidae (Reptilia). Acta Zool. 77, 85–100. Kweon, H.S., Park, E.H., Peters, N., 1998. Spermatozoon ultrastructure in the internally self-fertilizing hermaphroditic teleost, Rivulus marmoratus (Cyprinodontiformes, Rivulidae). Copeia 1998, 1101–1106. Magalhães, A.L.B., Andrade, R.F., Gomes, B.V.C., Perini, V.R., Rizzo, E., Bazzoli, N., 2011. Ultrastructure of the semicystic spermatogenesis in the South American freshwater characid Hemigrammus marginatus (Teleostei, Characiformes). J. Appl. Ichthyol. 27, 1041–1046.
6
M. Cassel et al. / Micron 62 (2014) 1–6
Mattei, X., 1970. Spermiogenese comparee des poisons. In: Baccetti, B. (Ed.), Comparative Spermatology. Academic Press, New York, pp. 57–69. Mattei, X., 1988. The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution. Biol. Cell. 63, 151–158. Mattei, X., 1991. Spermatozoon ultrastructure and its systematic implications in fishes. Can. J. Zool. 69, 3038–3055. Quagio-Grassiotto, I., Antoneli, F.N., Oliveira, C., 2003. Spermiogenesis and sperm ultrastructure in Cichla intermedia with some considerations about Labroidei spermatozoa (Teleostei, Perciformes, Cichlidae). Tissue Cell 35, 441–446. Quagio-Grassiotto, I., Carvalho, E.D., 1999. The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: Premeiotic and meiotic period. Tissue Cell 31, 561–567. Quagio-Grassiotto, I., Oliveira, C., 2008. Sperm ultrastructure and a new type of spermiogenesis in two species of Pimelodidae, with a comparative review of sperm ultrastructure in Siluriformes (Teleostei: Ostariophysi). Zool. Anz. 247, 55–66. Quintero-Hunter, I., Grier, H., Muscato, M., 1991. Enhancement of histological detail using metanil yellow as a counterstain in periodic acid/Schiff’s hematoxylin staining of glycol methacrylate tissue sections. Biotech. Histochem. 66, 169–172.
Santos, J.E., Bazzoli, N., Rizzo, E., Santos, G.B., 2001. Morphofunctional organization of the male reproductive system of the catfish Iheringichthys labrosus (Lütken, 1874) (Siluriformes: Pimelodidae). Tissue Cell 33, 533–540. Schindler, I., Etzel, V., 2008. Re-description and distribution of Rivulus punctatus Boulenger, 1895 (Teleostei: Rivulidae) and its habitats in Paraguay. Vert. Zool. 58, 33–43. Schulz, R.W., Franc¸a, L.R., Lareyre, J.J., LeGac, F., Chiarini-Garcia, H., Nóbrega, R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165, 390–411. Thiaw, O.T., Mattei, X., Romand, R., 1988. Process of cytoplasmic elimination during spermiogenesis in two Cyprinodontidae (teleostean fishes). J. Ultrastruct. Mol. Struct. Res. 101, 192–198. Thiaw, O.T., Mattei, X., Romand, R., Marchand, B., 1986. Reinvestigation of spermatic flagella structure: the teleostean Cyprinodontidae. J. Ultrastruct. Mol. Struct. Res. 97, 109–118. ˜ R.H., Meijide, F.J., Guerrero, G.A., 2012. Spermatogenesis Vázquez, G.R., Da Cuna, and changes in testicular structure during the reproductive cycle in Cichlasoma dimerus (Teleostei, Perciformes). Acta Zool. 93, 338–350. ˜ Vicentini, C.A., Munoz, M.E., Vicentini, I.B.F., 2010. Ultrastructural aspects of spermatogenesis in Phalloceros caudimaculatus (Teleostei, Poeciliidae). Int. J. Morphol. 28, 951–956.