Ultrastructural aspects of oogenesis and oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The ultrastructural characteristics of the organelles present in Serrasalmus spilopleura oogonia and oocytes undergoing primary growth were described in detail, considering its role in the nuclear and cytoplasmic metabolic processes that occur in these cell types. Even though these cells do not significantly differ from those similar to them that are found in other teleost groups, the analysis of their ultrastructure makes available new data on the reproductive biology of Characiformes.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Ultrastructure of the epithelium from the ovary wall of Dendrochirus brachypterus (Pteroidae,Teleostei)

Summary The inner epithelium of the ovary wall in Dendrochirus brachypterus does not take part in egg production, as it does in other teleosts. The epithelium consists of columnar cells rich in mitochondria and secretory organelles. The distal ends of these cells hang free in the ovary lumen, separated from each other and densely covered by large microvilli. During reproduction the epithelium secretes large amounts of mucus that forms an envelope around the eggs produced from a spongy stroma of the ovary, and keeps the spawn afloat for 24 h.Thanks are due to Mrs. H. Segal and Mr. N. Sharon for technical assistance, and Mr. D. Fridman, for help during the collection of the fish. Also appreciation is given to the technicians of the Electron Microscopy Laboratory of Tel-Aviv University for help in preparation of electron and scanning microscope figures     

Morphology,molecules and the phylogeny of Characidae (Teleostei,Characiformes)

This is the most comprehensive phylogenetic analysis of the Characidae to date and the first large-scale hypothesis of the family, combining myriad morphological data with molecular information. A total of 520 morphological characters were analysed herein, of which 98 are newly defined. Among the analysed taxa, 259 species were coded by examining specimens, three fossil species were coded from the literature, one species was coded almost completely from published figures, 122 were partially coded from the literature, and 88 were analysed exclusively from molecular data. The total number of species in the analysed dataset is 473. Analyses were made by parsimony under equal and extended implied weighting with a broad range of parameters. The final hypothesis was selected using a stability criterion that chooses among the most parsimonious trees of all searches. It was found by weighting molecular characters with the average homoplasy of entire partitions (markers). The resulting hypothesis is congruent with previous molecular-based phylogenies of the family. The Characidae are monophyletic, with four main clades: the Spintherobolinae new subfamily; an expanded Stethaprioninae including the Grundulini, Gymnocharacini, Rhoadsiini and Stethaprionini; the Stevardiinae; and a clade composed of the Aphyocharacinae, Characinae, Cheirodontinae, Exodontinae and Tetragonopterinae. Also, a stem Characidae was found, as formed by the Eocene–Oligocene genera †Bryconetes and †Paleotetra as successive sister groups of extant members of the family. A subfamilial classification is proposed, but deep changes in the systematics that are beyond the scope of this study are still needed to classify the Characidae into monophyletic genera.     

Morphofunctional changes in Leydig cells throughout the continuous spermatogenesis of the freshwater teleost fish, Serrasalmus spilopleura (Characiformes, Characidae): an ultrastructural and enzyme study

The freshwater fish Serrasalmus spilopleura (piranha) has a continuous type of reproduction; gametes are constantly produced and released during the reproductive cycle. The testes do not undergo seasonal morphological changes but exhibit two constant regions throughout the year: the medullar region (involved with spermatogenesis) and the cortical region (involved with spermiation and sperm storage). We have evaluated the ultrastructure of the Leydig cells and the activity of 3beta-HSD (an essential enzyme related to steroid hormone biosynthesis) and acid phosphatase (AcPase; lysosomal marker enzyme) in these two regions. The activity of 3beta-HSD is stronger in the medullar region, and the Leydig cells in this region have a variety of cytological features that reflect differences in hormone synthesis and/or that could be linked to steroidogenic cells under various degrees of hormonal activity. In the cortical region, 3beta-HSD activity is weak and the Leydig cells exhibit signs of degeneration, as confirmed by their ultrastructure and intense AcPase activity. These degenerative signs are indicative of cytoplasmic remodelling to degrade steroidogenic enzymes, such as 3beta-HSD, that could lead to senescence or even to autophagic cell degeneration. S. spilopleura thus constitutes an interesting model for increasing our understanding of steroidogenesis control in freshwater teleost fish.     

Identification and characterization of a unique Xenopus laevis egg envelope component,ZPD

We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.     

Phylogenetic study of the Characinae (Teleostei: Characiformes: Characidae)

The Characinae is a subunit of the Characidae of special significance in including Charax, the type genus of the family and the order Characiformes. Twelve genera and 79 species have been traditionally assigned to the Characinae, but the subfamily still lacks a phylogenetic diagnosis. Herein, a data matrix including 150 morphological characters and 64 taxa (35 species representing all genera of the Characinae and 29 included in other lineages within the Characiformes) was submitted to two cladistic analyses that differ in the inclusion/exclusion of Priocharax due to the difficulty of coding most of the character states in the miniature species of this genus. Both analyses resulted in a non‐monophyletic Characinae and this subfamily is herein restricted to only seven of the original 12 genera forming the clade (Phenacogaster((Charax Roeboides)(Acanthocharax(Cynopotamus(Acestrocephalus Galeocharax))))), which is supported by ten non‐ambiguous synapomorphies and is more closely related to other genera of the Characidae than those traditionally placed in the subfamily. A second clade includes the members of the tribe Heterocharacini (Lonchogenys(Heterocharax Hoplocharax)) as the sister‐group of Gnathocharax, supported by seven non‐ambiguous synapomorphies. This clade is more closely related to a taxon formed by Roestes and Gilbertolus based on seven non‐ambiguous synapomorphies. Results do not corroborate a close relationship between Roestes–Gilbertolus and the Cynodontinae. Inclusion of the genus Priocharax suggests that it is related more closely to the Heterocharacini, but the profound modifications in its anatomy possibly related to ontogenetic truncations obscure a better understanding of its relationships. A new classification of the Characinae and the Heterocharacinae is proposed. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 809–915.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

Ecological aspects between Contracaecum sp. (Nematoda, Anisakidae) and the host Serrasalmus spilopleura Kner, 1860 (Pisces, Characidae) in natural populations of northeastern Argentina

From February 1987 to February 1989, the populational biology of Contraceacum sp. (larvae) in its paratenic host, the fish Serrasalmus spilopleura Kner, 1860, was studied in two ponds in a subtropical permanent habitat northeastern of Argentina. Fishes from Ramada Paso pond presented 80% of prevalence and 1 to 132 larvae per fish while fishes from Aeroclub pond presented 63% of prevalence and 1 to 184 larvae per fish. Fishes collected from Aeroclub pond have shown a high prevalence of infection during the first period of study (1987), diminishing the following year. In fishes from Ramada Paso pond the prevalence varied not significatively during the two years. Prevalence and mean intensity of infection increase with body length and weight of the hosts. Sex of hosts is not an influential factor in parasitic level. The lenitic "closed" environmental (Ramada Paso pond) evidenced the greatest larvae mean intensity and prevalence. Although, the lenitic "open" environmental (Aeroclub pond) showed the greatest parasitic number of individuals in an infrapopulation. The spatial dispersion in both ponds were aggregated and fit well a negative binomial model. Nevertheless, the Aeroclub pond presented the greatest overdispersion.     

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, Zl-1,2 and Zl-3. On SDS-PAGE, the Zl-1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). Zl-3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of Zl-1,2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT-PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full-length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro-X-Y repeat sequences in two-fifths of the whole length from its N-terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.     

Spermiogenesis and spermatozoa ultrastructure in Salminus and Brycon, two primitive genera in Characidae (Teleostei: Ostariophysi: Characiformes)

In Salminus, spermiogenesis is cystic and gives origin to a type I aquasperm. Spermatid differentiation is characterized by chromatin condensed into thick fibres, nuclear rotation, nuclear fossa formation, cytoplasmic channel formation, mitochondrial fusion producing long and ramified mitochondria, and the presence of several membranous concentric rings around the plasma membrane that encircles the cytoplasmic channel. In Salminus and Brycon, spermatozoa are very similar. They exhibit a spherical nucleus and chromatin condensed into fibre clusters, and a deep nuclear fossa. They show a long midpiece with few elongate mitochondria at the initial region and a cytoplasmic channel completely encircled by one or two membranous concentric rings. The flagellar axis is perpendicular to the nucleus and exhibits the classic axoneme (9 + 2). The very strong similarity observed between Salminus and Brycon spermatozoa supports the hypothesis that these subfamilies are likely to have a monophyletic origin.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Osteology of Priocharax and remarkable developmental truncation in a miniature Amazonian fish (Teleostei: Characiformes: Characidae)

Establishing phylogenetic relationships of miniature fishes is challenging in taxa with developmental truncation. Within the Characiformes, developmental truncation appears to be relatively rare, with the Neotropical genus Priocharax being an example. Priocharax includes three miniature species among the smallest of the order and has been hypothesized to belong to the Heterocharacinae. The pronounced reduction in its skeleton, however, prevented a clearer evaluation of its relationships. The present detailed osteological study was designed to address this question and revealed that 21 bones are absent and nine other skeletal structures are simplified in Priocharax when compared to other characids. Comparison of the skeleton of adult Priocharax with early developmental stages of other characids demonstrated that most of the absences and simplifications can be interpreted as developmental truncations. The most striking developmental truncations are in the pectoral girdle, in which the endoskeleton remains entirely cartilaginous. Other interesting truncations are in the ethmoid region of the skull, infraorbital series, and Weberian apparatus, in which the claustrum is absent. Our study also revealed some unusual sexual dimorphisms in the pelvic girdle. Two cladistic analyses were performed to assess the relationships of Priocharax within the Heterocharacinae. The first consisted of a traditional analysis in which all absences and reductions of Priocharax were coded in the same way as in the remaining taxa. This resulted in three equally most parsimonious topologies, all of which have Priocharax as the most basal taxon of the Heterocharacinae. The second analysis incorporated ontogenetic information, and most absences and reductions of Priocharax were reinterpreted as apomorphic conditions and thus, coded differently from similar conditions in outgroups. This resulted in a single phylogenetic hypothesis with Priocharax and Gnathocharax as sister groups based on seven synapomorphies. Our approach demonstrates the importance of developmental studies to better understand morphological evolution of miniaturized, truncated taxa, and to generate hypotheses of their relationships. J. Morphol. 277:65–85, 2016. © 2015 Wiley Periodicals, Inc.     

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.     

Concentration of Ran on chromatin induces decondensation,nuclear envelope formation and nuclear pore complex assembly

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.     

On the lectotype designation of Bryconamericus rubropictus (Berg) (Characiformes,Characidae, Stevardiinae)

The lectotype specimen of Bryconamericus rubropictus (Berg) and its designation have remained imprecisely documented since its publication. The lack of a photograph or an unambiguous illustration, correct size, inaccurate labelling, and proper specimen separation has led to an uncertainty about the identity and nomenclatural status of the lectotype. We recovered and provided detailed morphological data on the specimen that must be recognised as the lectotype. This contribution brings stability and clarity on the nomenclatural status of the species and its type material.     

Preliminary survey of egg envelope morphology in the Macrouridae and the possible implications of its ornamentation

The eggs of only 13 macrourid species have been reported hitherto, eight identified from free buoyant eggs and five from ripening oocytes. Eggs of 11 species had raised, hexagonally patterned ornamentation on the envelope, while two species bore only partial ornamentation. Ripening oocytes of a further eight species showed that, overall, the eggs of 13 species had characteristic hexagonal ornamentation and three partial ornamentation, but four were smooth. Fertilized eggs of Coryphaenoides (Coryphaenoides) rupestris were found with smooth envelopes, while ornamented eggs have been reported for this species also. Little taxonomic significance could be attached to these results. Some correlation was found between the three categories of egg envelope ornamentation and species' bathymetric distributions. A recent model of egg development in a slope dwelling trachichthyid species suggested analagous potential ecological advantage for ornamentational variation in macrourids.     

Occurrence of macro B chromosomes in Astyanax scabripinnis paranae (Pisces,Characiformes, Characidae)

B chromosomes occur in several Neotropical fish species. Cytogenetic analysis of 27 specimens (15 females and 12 males) of Astyanax scabripinnis paranae from the Araquá river (a small headwater tributary of the Tietê river) shows that this population has 2n=50 chromosomes (4M+30 SM+4ST+12A), two chromosome pairs with NORs and conspicuous C-band positive blocks in the terminal position of the long arm of four chromosome pairs. In this population, eight females presented 2n=51 chromosomes and the extra chromosome was a large metacentric similar in size and morphology to the first chromosome pair in the karotype. This accessory chromosome is entirely heterochromatic in C-banded metaphases and shows a late replication pattern evidenced by BrdU incorporation. There was no significant correlation between the presence of B chromosomes and increased NOR activity at the P>0.05 level. Some aspects related to these B chromosomes are discussed.