From zero to ossified: Larval skeletal ontogeny of the Neotropical Cichlid fish Cichlasoma dimerus.

The identification of skeletal elements, the analysis of their developmental sequence, and the time of their appearance during larval development are essential to broaden the knowledge of each fish species and to recognize skeletal abnormalities that may affect further fish performance. Therefore, this study aimed to provide a general description of the development of the entire skeleton highlighting its variability in Cichlasoma dimerus. Larvae of C. dimersus were stained with alcian blue and alizarin red from hatching to 25 days posthatching. Skeletogenesis began with the endoskeletal disk and some cartilage structures from the caudal fin and the splachnocranium, while the first bony structure observed was the cleithrum. When larvae reached the free-swimming and exogenous feeding stage, mostly bones from the jaws, the branchial arches, and the opercle series evidenced some degree of ossification, suggesting that the ossification sequence of C. dimerus adjusts to physiological demands such as feeding and ventilation. The caudal region was the most variable regarding meristic counts and evidenced higher incidence of bone deformities. In conclusion, this work provides an overview of C. dimerus skeletogenesis and lays the groundwork for further studies on diverse topics, like developmental plasticity, rearing conditions, or phylogenetic relationships.

Developmental aspects of the hypothalamic-pituitary network related to reproduction in teleost fish.

The hypothalamic-pituitary-gonadal axis is the main system that regulates reproduction in vertebrates through a complex network that involves different neuropeptides, neurotransmitters, and pituitary hormones. Considering that this axis is established early on life, the main goal of the present work is to gather information on its development and the actions of its components during early life stages. This review focuses on fish because their neuroanatomical characteristics make them excellent models to study neuroendocrine systems. The following points are discussed: i) developmental functions of the neuroendocrine components of this network, and ii) developmental disruptions that may impact adult reproduction. The importance of the components of this network and their susceptibility to external/internal signals that can alter their specific early functions and/or even the establishment of the reproductive axis, indicate that more studies are necessary to understand this complex and dynamic network.

Fish skin pigmentation in aquaculture: The influence of rearing conditions and its neuroendocrine regulation.

Skin pigmentation pattern is a species-specific characteristic that depends on the number and the spatial combination of several types of chromatophores. This feature can change during life, for example in the metamorphosis or reproductive cycle, or as a response to biotic and/or abiotic environmental cues (nutrition, UV incidence, surrounding luminosity, and social interactions). Fish skin pigmentation is one of the most important quality criteria dictating the market value of both aquaculture and ornamental species because it serves as an external signal to infer its welfare and the culture conditions used. For that reason, several studies have been conducted aiming to understand the mechanisms underlying fish pigmentation as well as the influence exerted by rearing conditions. In this context, the present review focuses on the current knowledge on endocrine regulation of fish pigmentation as well as on the aquaculture conditions affecting skin coloration. Available information on Iberoamerican fish species cultured is presented.

Gonadotropin-inhibitory hormone (GnIH) distribution in the brain of the ancient fish Atractosteus tropicus (Holostei, Lepisosteiformes).

The Holostei group occupies a critical phylogenetic position as the sister group of the Teleostei. However, little is known about holostean pituitary anatomy or brain distribution of important reproductive neuropeptides, such as the gonadotropin-inhibitory hormone (GnIH). Thus, the present study set out to characterize the structure of the pituitary and to localize GnIH-immunoreactive cells in the brain of Atractosteus tropicus from the viewpoint of comparative neuroanatomy. Juveniles of both sexes were processed for general histology and immunohistochemistry. Based on the differences in cell organization, morphology, and staining properties, the neurohypophysis and three regions in the adenohypophysis were identified: the rostral and proximal pars distalis (PPD) and the pars intermedia. This last region was found to be innervated by the neurohypophysis. This organization, together with the presence of a saccus vasculosus, resembles the general teleost pituitary organization. A vast number of blood vessels were also recognized between the infundibulum floor of the hypothalamus and the PPD, evidencing the characteristic presence of a median eminence and a portal system. However, this well-developed pituitary portal system resembles that of tetrapods. As regards the immunohistochemical localization of GnIH, we found four GnIH-immunoreactive (GnIH-ir) populations in three hypothalamic nuclei (suprachiasmatic, retrotuberal, and tuberal nuclei) and one in the diencephalon (prethalamic nucleus), as well as a few scattered neurons throughout the olfactory bulbs, the telencephalon, and the intersection between them. GnIH-ir fibers showed a widespread distribution over almost all brain regions, suggesting that GnIH function is not restricted to reproduction only. In conclusion, the present study describes, for the first time, the pituitary of A. tropicus and the neuroanatomical localization of GnIH in a holostean fish that exhibits a similar distribution pattern to that of teleosts and other vertebrates, suggesting a high degree of phylogenetic conservation of this system.

The Gonadotropin-Inhibitory Hormone: What We Know and What We Still Have to Learn From Fish.

Gonadotropin-inhibitory hormone, GnIH, is named because of its function in birds and mammals; however, in other vertebrates this function is not yet clearly established. More than half of the vertebrate species are teleosts. This group is characterized by the 3R whole genome duplication, a fact that could have been responsible for the great phenotypic complexity and great variability in reproductive strategies and sexual behavior. In this context, we revise GnIH cell bodies and fibers distribution in adult brains of teleosts, discuss its relationship with GnRH variants and summarize the few reports available about the ontogeny of the GnIH system. Considering all the information presented in this review, we propose that in teleosts, GnIH could have other functions beyond reproduction or act as an integrative signal in the reproductive process. However, further studies are required in order to clarify the role of GnIH in this group including its involvement in development, a key stage that strongly impacts on adult life.

Morphological relationship between GnIH and GnRH neurons in the brain of the neotropical cichlid fish Cichlasoma dimerus.

Reproduction is regulated by the hypothalamic-pituitary-gonadal axis. The first neuropeptide identified that regulates this function was the decapeptide gonadotropin-releasing hormone (GnRH). Nowadays, in gnatostomates, a number of GnRH variants have been identified and classified into three different types: GnRH1, GnRH2, and GnRH3. Almost 30 years later, a new peptide that inhibits gonadotropin synthesis and secretion was discovered and thus named as gonadotropin-inhibitory hormone (GnIH). In avians and mammals, the interaction and regulation between GnRH and GnIH neurons has been widely studied; however, in other vertebrate groups there is little information about the relationship between these neurons. In previous works, three GnRH variants and a GnIH propeptide were characterized in Cichlasoma dimerus, and it was demonstrated that GnIH inhibited gonadotropins release in this species. Because no innervation was detected at the pituitary level, we speculate that GnIH would inhibit gonadotropins via GnRH. Thus, the aim of the present study was to evaluate the anatomical relationship between neurons expressing GnIH and the three GnRH variants by double labelling confocal immunofluorescence in adults of C. dimerus. Our results showed no apparent contacts between GnIH and GnRH1, fiber to fiber interactions between GnIH and GnRH2, and co-localization of GnIH and GnRH3 variant in neurons of the nucleus olfacto-retinalis. In conclusion, whether GnIH regulates the expression or secretion of GnRH1 in this species, an indirect modulation seems more plausible. Moreover, the present results suggest an interaction between GnIH and GnRH2 systems. Finally, new clues were provided to investigate the role of nucleus olfacto-retinalis cells and putative GnIH and GnRH3 interactions in the modulation of the reproductive network in teleost fish.

Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives.

The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH forms that are classified into three main groups, namely GnRH1, GnRH2, and GnRH3. In order to gain more insights into the GnRH gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we searched the genomes and/or transcriptomes of three representative species of this group, the small-spotted catshark, Scyliorhinus canicula, the whale shark, Rhincodon typus and the elephant shark Callorhinchus milii. In each species, we report the identification of three GnRH genes. In catshark and whale shark, phylogenetic and synteny analysis showed that these three genes correspond to GnRH1, GnRH2, and GnRH3. In both species, GnRH1 was found to encode a novel form of GnRH whose primary structure was determined as follows: QHWSFDLRPG. In elephant shark, the three genes correspond to GnRH1a and GnRH1b, two copies of the GnRH1 gene, plus GnRH2. 3D structure prediction of the chondrichthyan GnRH-associated peptides (GAPs) revealed that catshark GAP1, GAP2, and elephant shark GAP2 peptides exhibit a helix-loop-helix (HLH) structure. This structure observed for many osteichthyan GAP1 and GAP2, may convey GAP biological activity. This HLH structure could not be observed for elephant shark GAP1a and GAP1b. As for all other GAP3 described so far, no typical 3D HLH structure was observed for catshark nor whale shark GAP3. RT-PCR analysis revealed that GnRH1, GnRH2, and GnRH3 genes are differentially expressed in the catshark brain. GnRH1 mRNA appeared predominant in the diencephalon while GnRH2 and GnRH3 mRNAs seemed to be most abundant in the mesencephalon and telencephalon, respectively. Taken together, our results show that the GnRH gene repertoire of the vertebrate ancestor was entirely conserved in the chondrichthyan lineage but that the GnRH3 gene was probably lost in holocephali. They also suggest that the three GnRH neuronal systems previously described in the brain of bony vertebrates are also present in cartilaginous fish.

Ontogeny of Gonadotropin-Inhibitory Hormone (GnIH) in the cichlid fish Cichlasoma dimerus.

RFamide peptides are expressed in the early stages of development in most vertebrates. Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family, and its role in reproduction has been widely studied in adult vertebrates, ranging from fish to mammals. As only three reports evaluated GnIH during development, the aim of this study was to characterise the ontogeny of GnIH in a fish model, Cichlasoma dimerus. We detected the presence of two GnIH-immunoreactive (GnIH-ir) cell clusters with spatial and temporal differences. One cluster was observed by 3 days post-hatching (dph) in the nucleus olfacto-retinalis (NOR) and the other in the nucleus posterioris periventricularis by 14 dph. The number of GnIH-ir neurons increased in both nuclei, whereas their size increased only in the NOR from hatchling to juvenile stages. These changes occurred from the moment larvae started feeding exogenously and during development and differentiation of gonadal primordia. We showed by double-label immunofluorescence that only GnIH-ir neurons in the NOR co-expressed GnRH3 associated peptide. In addition, GnIH-ir fibre density increased in all brain regions from 5 dph. GnIH-ir fibres were also detected in the retina, optic tract and optic tectum, suggesting that GnIH acts as a neuromodulator of photoreception and the integration of different sensory modalities. Also, there were GnIH-ir fibres in the pituitary from 14 dph, which were in close association with somatotropes. Moreover, GnIH-ir fibres were observed in the saccus vasculosus from 30 dph, suggesting a potential role of GnIH in the modulation of its function. Finally, we found that gnih was expressed from 1 dph, and that the pattern of variation of its transcript levels was in accordance with that of cell number. Present results are the starting point for the study of new GnIH roles during development. This article is protected by copyright. All rights reserved.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Cloning, phylogenetic analysis and expression of somatolactin and its receptor in Cichlasoma dimerus: their role in long-term background color acclimation.

Somatolactin (SL) and SL receptor (SLR) belong to the growth hormone and cytokine type I receptor superfamilies, respectively. However, further research is required to define the duplications and functions of SL and its receptors in basal vertebrates including environmental background color adaptation in fish. In the present study, we cloned and sequenced SL and its putative receptor (SLR), classified and compared the sequences phylogenetically, and determined SL and SLR mRNA expression levels during long-term background color exposure in Cichlasoma dimerus, a freshwater South American cichlid. Our results show that C. dimerus SL and SLR share high sequence similarity with homologous from other perciform fish. Phylogenetic analysis indicates that C. dimerus SL belongs to the SLα clade sub-group. C. dimerus SLR is clearly a member of the GHR1 receptor subgroup, which includes the experimentally validated SLR from salmonids. Higher transcript levels of SLα in the pituitary and SLR in the epidermis and dermis cells of fish scales were observed in fish following long-term black background color exposure compared to those exposed to a white background. A higher number of melanophores was also observed in fish exposed for 10days to a black background compared to those exposed to a white background. These changes were concomitant to differences in SL or SLR transcript levels found in fish exposed to these two different background colors. Our results suggest, for the first time, that SLR is expressed in fish scales, and that there is an increase in SL in the pituitary and the putative SLR in likely target cells, i.e., melanophores, in long-term black background exposure in C. dimerus.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae).

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.(AU)

Presence of beta-follicle-stimulating hormone and beta-luteinizing hormone transcripts in the brain of Cichlasoma dimerus (Perciformes: Cichlidae): effect of brain-derived gonadotropins on pituitary hormone release.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for beta-FSH and beta-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary beta-LH, beta-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in beta-LH release, dose-dependent increases in beta-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

Relation between the reproductive status and somatolactin cell activity in the pituitary of pejerrey, Odontesthes bonariensis (Atheriniformes).

The aim of this study was to analyze the relationship between somatolactin (SL) expressing cells and the reproductive status in a multiple spawning fish, the pejerrey Odontesthes bonariensis. Somatolactin cells were identified in adults of both sexes by immunocytochemistry using a heterologous piscine antiserum. The area of the cells that showed immunoreactivity to SL (ir-SL) was compared in specimens with different degrees of reproductive activity as inferred from histological examination of the gonads and calculation of the gonadosomatic index (GSI %). The results showed a significant difference between the area of ir-SL cells of resting/regressing (62.9 +/- 2.1 micron 2) and sexually active/vitellogenic (76.8 +/- 2.3 micron 2) females and a significant positive correlation between the ir-SL cellular area and the GSI % (P < 0.01 in both cases). In males, the correlation between the area of ir-SL cells and the GSI % was not statistically significant. However, in those animals with the highest GSI % values, the ir-SL cells appeared more numerous and showed an increase in the immunostained area when compared to individuals with lower GSI % values. The present in morphological observations are in accordance with biochemical data obtained from other species and support the assumption that SL might be involved in the regulation of reproduction in fish.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.(AU)