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Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The saccus vasculosus is an organ present in gnathostome fishes, located ventral to the hypothalamus and posterior to the pituitary gland, whose structure is highly variable among species. In some fishes, this organ is well-developed; however, its physiological function is still under debate. Recently, it has been proposed that this organ is a seasonal regulator of reproduction. In the present work, we examined the histology, ultrastructure, and development of the saccus vasculosus in Cichlasoma dimerus. In addition, immunohistochemical studies of proteins related to reproductive function were performed. Finally, the potential response of this organ to different photoperiods was explored. C. dimerus presented a well-developed saccus vasculosus consisting of a highly folded epithelium, composed of coronet and supporting cells, closely associated with blood vessels, and a highly branched lumen connected to the third ventricle. Coronet cells showed all the major characteristics described in other fish species. In addition, some of the vesicles of the globules were positive for thyrotropin beta subunit, while luteinizing hormone beta subunit immunostaining was observed at the edge of the apical processes of some coronet cells. Furthermore, neuropeptide Y and gonadotropin inhibitory hormone innervation in the saccus vasculosus of C. dimerus were shown. Finally, animals exposed to the long photoperiod showed lower levels of thyrotropin beta and common alpha subunits expression in the saccus compared to those of animals exposed to short photoperiod. All these results support the hypothesis that the saccus vasculosus is involved in the regulation of reproductive function in fish., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023 Wiley Periodicals LLC.)

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., (Copyright © 2021. Published by Elsevier Inc.)

Many teleost fishes can withstand long feed deprivation periods, either due to an eventual lack of food or because of their behavior during reproduction and/or parental care. In this work, the effects of total food restriction on the oogenesis, spermatogenesis, and reproductive hormones of the neotropical cichlid fish Cichlasoma dimerus were studied. Specifically, different pairs were isolated after having a spawning event and were feed-deprived or daily fed for 3 weeks. After that period, gonadal histology, messenger levels of genes related to reproduction (gonadotropin-releasing hormone 1, gonadotropins, and insulin-like growth factor 1) and 11-ketotestosterone plasma levels were evaluated in both groups. Food restriction did not affect the reproductive axis in females since follicular maturation and gene expression showed no differences with respect to controls. However, in males, food restriction showed a stimulatory effect on the reproductive axis, reflected in a greater number of spermatozoa in their seminiferous lobes and spermatic ducts, and in an increase in follicle stimulating hormone messenger expression. Despite the negative effect reported for many fish species, C. dimerus seems to redirect their energy reserves towards gonadal development when faced with to a feed deprivation period., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Cichlasoma dimerus is a neotropical cichlid that has been used as a biological model for neuroendocrinology studies. However, its culture is problematic in terms of larval feeding to allow having enough fry quantity and quality. Larviculture requires full knowledge about the digestive system and nutrition; therefore, this study was intended to assess the digestive enzymes' changes at different ages during the early ontogeny. Acid protease activity was detectable from the first day after hatching (dah), increasing to its maximum peaks on 9 dah. In contrast, alkaline proteases had low activity in the first days of life but reached their maximum activity on 17 dah. Chymotrypsin, L-aminopeptidase, and carboxypeptidase A activities increased at 6 dah, while trypsin activity was first detected on 13 dah and reached its maximum activity on 17 dah. Lipase and α-amylase activity were detectable at low levels in the first days of life, but the activity fluctuated and reaching its maximum activity at 21 dah. Alkaline phosphatase continued to oscillate and had two maximum activity peaks, the first at 6 dah and the second at 19 dah. Zymograms of alkaline proteases on day 6 dah six revealed four activity bands with molecular weights from 16.1 to 77.7 kDa. On 13 dah, two more activity bands of 24.4 and 121.9 kDa were detected, having a total of six proteases. The enzymatic activity analyzes indicate the digestive system shows the low activity of some enzymes in the first days after hatching, registering significant increases on 6 dah and the maximum peaks of activities around at 17 dah. Therefore, we recommend replacing live food with dry feed and only providing dry feed after day 17 dah., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Morphological background adaptation is both an endocrine and a nervous response, involving changes in the amount of chromatophores and pigment concentration. However, whether this adaptation takes place at early developmental stages is largely unknown. Somatolactin (Sl) is a pituitary hormone present in fish, which has been associated to skin pigmentation. Moreover, growth hormone receptor type 1 (Ghr1) has been suggested to be the Sl receptor and was associated with background adaptation in adults. In this context, the aim of this work was to evaluate the ontogeny of morphological adaptation to background and the participation of ghr1 in this process. We found in larval stages of the cichlid Cichlasoma dimerus that the number of head melanophores and pituitary cells immunoreactive to Sl was increased in individuals reared with black backgrounds compared with that in fish grown in white tanks. In larval stages of the medaka Oryzias latipes , a similar response was observed, which was altered by ghr1 biallelic mutations using CRISPR/Cas9 . Interestingly, melanophore and leucophore numbers were highly associated. Furthermore, we found that somatic growth was reduced in ghr1 biallelic mutant O. latipes , establishing the dual function of this growth hormone receptor. Taken together, these results show that morphological background adaptation is present at early stages during development and that is dependent upon ghr1 during this period., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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.

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., (This article is protected by copyright. All rights reserved.)

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.

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6)  m and 10(-8)  m) of synthetic C. dimerus (cd-) LPQRFa-1 and LPQRFa-2 on the release of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased β-luteinising hormone (LH) and β-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of β-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits β-LH and β-FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd-LPQRF-2 could act as an β-FSH-releasing factor in this fish species., (© 2016 British Society for Neuroendocrinology.)

Neuropeptide Y (NPY) is considered the most potent orexigenic peptide, increasing before meal time and during fasting. In teleost, most studies on NPY action upon growth hormone (GH) and luteinizing hormone (LH) were conducted in females or group of animals without sex discrimination. The aim of this study was to evaluate whether NPY modulates the expression and release of GH and gonadotropins in both sexes of Cichlasoma dimerus. By double-label immunofluorescence, we first determined the association between NPY fibers and pituitary cells. In addition, we performed in vitro studies to evaluate the effect of NPY on GH and gonadotropins expression by real-time PCR, and release by Western blot, in males and females separately. Contacts between NPY fibers and GH and follicle-stimulating hormone (FSH)-producing cells were detected, indicating possible functional relationships. We observed an increase in GH release in the culture medium at 2 nM for males (p = 0.043) and 20 nM for females (p = 0.028). Pituitary FSH release was stimulated at 20 nM (p = 0.026) and 200 nM (p = 0.033) for males and females, respectively. Finally, NPY only increased β-LH mRNA expression at 20 nM in females (p = 0.028) and its release at 2 nM (p = 0.049) and 200 nM for males (p = 0.005) and 200 nM for females (p = 0.018). In conclusion, NPY acts as a GH-, LH- and FSH-releasing factor, in a dose- and sex-dependent way.

Cichlasoma dimerus is a social cichlid fish capable of growing at high rates under laboratory conditions, but knowledge on somatic growth regulation is still unclear. Growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is the key regulator of somatic growth in vertebrates. Two types of growth hormone receptors have been described in teleost fish, named GH receptor type 1 (GHR1) and type 2 (GHR2). In addition, isoforms of these receptors lacking part of the intracellular region have been described. The aim of this study was to evaluate the somatic growth, liver histology and changes in the GH/IGF-I axis after 4 weeks of food deprivation in C. dimerus. Four-week fasted fish showed reductions in specific growth rates in body weight (p < 0.001) and standard length (p < 0.001). Additionally, the hepatosomatic index (p < 0.001) and hepatocyte area (p < 0.001) decreased in fasted fish, while no changes in glucose levels were detected in plasma. The starvation protocol failed to induce changes in GH mRNA levels in the pituitary and IGF-I mRNA levels in liver. In contrast, IGF-I mRNA levels in muscle decreased in fasted fish (p = 0.002). On the other hand, GHR2 (detected with primer sets designed over the extracellular and intracellular region) was upregulated by starvation both in liver and muscle (p < 0.05), while GHR1 remained unchanged. These results show that a fasting period reduced somatic growth both in length and body weight concomitantly with alterations on liver and muscle GHR2 and muscle IGF-I mRNA expression.

Neuropeptide Y (NPY) and orexin are neuropeptides involved in the regulation of feeding in vertebrates. In this study we determined the NPY and orexin mRNA tissue expression and their immunoreactivity distribution in both preoptic area and hypothalamus, regions involved in the regulation of feeding behavior. Both peptides presented a wide expression in all tissues examined. The NPY-immunoreactive (ir) cells were localized in the ventral nucleus posterioris periventricularis (NPPv) and numerous ir-NPY fibers were found in the nucleus lateralis tuberis (NLT), the nucleus recess lateralis (NRL) and the neurohypophysis. Ir-orexin cells were observed in the NPPv, dorsal NLT, ventral NLT, lateral NLT (NLTl) and the lateral NRL. Ir-orexin fibers were widespread distributed along all the hypothalamus, especially in the NLTl. Additionally, we observed the presence of ir-orexin immunostaining in adenohypophyseal cells, especially in somatotroph cells and the presence of a few ir-orexin-A fibers in the neurohypophysis. In conclusion, both peptides have an ubiquitous mRNA tissue expression and are similarly distributed in the hypothalamus and preoptic area of Cichlasoma dimerus. The presence of ir-orexin in adenohypohyseal cells and the presence of ir-orexin and NPY fibers in the neurohypophysis suggest that both peptides may play an important neuroendocrine role in anterior pituitary., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Growth hormone (GH) is the main pituitary hormone involved in somatic growth. In fish, the neuroendocrine control of GH is multifactorial due to the interaction of multiple inhibitors and stimulators. Melanin-concentrating hormone (MCH) is a cyclic peptide involved in skin color regulation of fish. In addition, MCH has been related to the regulation of food intake in both mammals and fish. There is only one report presenting evidences on the GH release stimulation by MCH in mammals in experiments in vitro, but there are no data on non-mammals. In the present work, we report for the first time the sequence of MCH and GH cDNA in Cichlasoma dimerus, a freshwater South American cichlid fish. We detected contacts between MCH fibers and GH cells in the proximal pars distalis region of the pituitary gland by double label confocal immunofluorescence indicating a possible functional relationship. Besides, we found that MCH increased GH transcript levels and stimulated GH release in pituitary cultures. Additionally, C. dimerus exposed to a white background had a greater number of MCH neurons with a larger nuclear area and higher levels of MCH transcript than those fish exposed to a black background. Furthermore, fish reared for 3 months in a white background showed a greater body weight and total length compared to those from black background suggesting that MCH might be related to somatic growth in C. dimerus. Our results report for the first time, that MCH is involved in the regulation of the synthesis and release of GH in vitro in C. dimerus, and probably in the fish growth rate., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in the development and maintenance of vertebrate nervous systems. Although there were several studies in classical animal models, scarce information for fish was available. The main purpose of this study was to analyze the distribution of BDNF in the brain and retina of the cichlid fish Cichlasoma dimerus. By immunohistochemistry we detected BDNF-like immunoreactive cells in the cytoplasm and the nuclei of the ganglion cell layer and the inner nuclear layer of the retina. In the optic tectum, BDNF-like immunoreactivity was detected in the nucleus of neurons of the stratum periventriculare and the stratum marginale and in neurons of the intermediate layers. In the hypothalamus we found BDNF-like immunoreactivity mainly in the cytoplasm of the nucleus lateralis tuberis and the nucleus of the lateral recess. To confirm the nuclear and cytoplasm localization of BDNF we performed subcellular fractionation, followed by Western blot, detecting a 39 kDa immunoreactive-band corresponding to a possible precursor form of BDNF in both fractions. BDNF-like immunoreactivity was distributed in areas related with photoreception (retina), the integration center of retinal projections (optic tectum) and the control center of background and stress adaptation (hypothalamus). These results provide baseline anatomical information for future research about the role of neurotrophins in the adult fish central nervous system.

Somatolactin (SL) is a pituitary hormone present exclusively in fish that is involved in different physiological processes. The role of SL was evaluated in Cichlasoma dimerus (Teleostei, Perciformes) exposed for 10 days to a black and white background (BB and WB). Changes in alpha-melanophore stimulating hormone (alphaMSH) and melanin concentrating hormone (MCH) cells were also analyzed for comparison with SL. A melanin dispersing effect was observed in fish exposed to a BB, while a concentrating one was observed in those exposed to a WB. By Western blot, three SL-immunoreactive (ir) bands (32, 28 and 23.5 kD) were evidenced. Pituitary SL-ir levels were 2.66- and 2.67-fold greater in the 32 Kd and 28 kD bands, respectively, in BB fish compared with those of WB fish. The SL-ir 23.5 Kd band was not included in the analysis because of its unknown identity. In addition, SL-ir cell number and area were significantly higher in the BB condition (BB 22.73+/-1.46, WB 7.37+/-0.54 and BB 27.39+/-1.00 microm2; WB: 16.61+/-0.65 microm2). No significant differences were observed in the number of the hypothalamic MCH-ir neurons. However, a significant difference was observed in their nuclear area (BB 11.61+/-0.42 microm2, WB 17.80+/-0.84 microm2). alphaMSH-ir cells showed a marked increased in number (BB 35.96+/-1.22, WB 24.36+/-1.04), but no significant differences were observed in the cell area. In conclusion, this study presented clear evidence towards a possible involvement of SL in the adaptation to background colors in teleost together with alphaMSH and MCH.

The aim of this study was to determine the distribution of Neuropeptide-Y (NPY) immunoreactive neurons and fibres in the brain and pituitary of Odontesthes bonariensis by immunohistochemical methods. A wide distribution of immunoreactive NPY (ir-NPY) cells and fibres in the forebrain and midbrain was observed. A prominent ir-NPY nucleus was found in the ventral telencephalon and other ir-NPY cells groups were recognized at the dorso-medial telencephalon. The diencephalon showed ir-NPY cells in the Nucleus entopeduncularis, the Nucleus preopticus periventricularis and in the Nucleus lateralis tuberis. Ir-NPY fibres were conspicuous in the preoptic region and the hypothalamus. There were also numerous ir-NPY fibres at the epithalamic level running ventrally to the hypothalamus and the pituitary stalk. At the rhomboencephalic level, the ir-NPY neurons were observed in the Locus coeruleus. Double-labelled immunostaining showed a close association between ir-NPY fibres that reach the adenohypophysis and growth hormone (GH)- and gonadotropin (GtH)-expressing cells. Although our results exhibit some relevant differences when compared to other fish groups, they support the existence of a conserved NPY system in teleosts.

Distribution and development of the melanin-concentrating hormone (MCH) system were examined by immunocytochemistry of the brain, pituitary gland and skin of the South American cichlid fish Cichlasoma dimerus. In adults, the most prominent group of MCH-ir perikarya was located in the nucleus lateralis tuberis (NLT). Outside the NLT, in the posterior hypothalamic region, a group of small neurons was found between the third ventricle and the lateral ventricular recess with delicate immunoreactive fibers that did not seem to contribute to the pituitary innervation. MCH-ir perikarya were identified at day 4 after hatching (AH) in a proliferating zone of the hypothalamic floor. Pituitary innervation could be detected at this stage. Another group of small MCH-ir neurons, only detected in pre-juvenile stages, originated close to the third ventricle in the medial hypothalamic region by day 6 AH. alphaMSH-ir neurons were localized in similar regions of the NLT and in the nucleus periventricularis posterior (NPP). Free MCH-ir neuromasts were detected in the ventral and dorsal skin of larval heads. These epidermal sensory organs were in close association with blood vessels and dermal melanocytes, suggesting that MCH synthesized in larval skin might act in an endocrine way reaching different targets and/or in a paracrine mode regulating melanin concentration in dermal melanocytes.

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 cichlid fish Cichlasoma dimerus was studied using the avidin-biotin-peroxidase method with antisera raised against piscine pituitary hormones and heterologous antisera against mammalian pituitary hormones. Antiserum raised against rabbit ACTH recognized a group of cells bordering the neurohypophysis (NH) in the rostral pars distalis (RPD). Anti-chum salmon prolactin (PRL) identified a compact group of cells in the periphery of the RPD. Gonadotropin II (GTH II), thyrotropin (TSH) and growth hormone (GH)-ir cells were localized in the proximal pars distalis. Ir-GTH II cells were also observed in the dorsal area of the pars intermedia (PI). Ir-GTH I cells could not be identified using anti-chum salmon GTH I, this may be due either to a failure of the antisera to recognize the gonadotropin or to a low expression of the hormone in adults of this species. PAS positive cells from the PI bound specifically with three different antisera raised against somatolactin (SL) of four different fish species. These cells surrounded deep branches of the NH in the PI.

Previous studies in the pejerrey, Odontesthes bonariensis, have demonstrated that fibers with immunoreactivity to gonadotropin-releasing hormone (ir-GnRH) reach all areas of the pituitary gland, the rostral pars distalis (RPD), the proximal pars distalis (PPD), and the pars intemedia (PI). A close association was shown between ir-GnRH fibers and gonadotropin (GtH)-, growth hormone (GH)-, somatolactin (SL)-, and prolactin (PRL)-expressing cells. The presence of only one GnRH variant, suspected to be a novel form, has been shown in pituitary extracts of this fish. In addition, GnRH may stimulate GtHs, GH, SL, and PRL levels in different fish species. The objective of the present study was to seek GnRH receptors and therefore colocalization with GtHs, GH, SL, and PRL cells in O. bonariensis using a pituitary primary cell culture system. GnRH binding sites were revealed by autoradiography of an iodinated superactive GnRH agonist ([(125)I]GnRH-A) and pituitary cells were identified by immunocytochemistry using piscine antisera. Following autoradiography, silver grains representing specific [(125)I]GnRH-A binding were associated with anti GtH, GH, SL, and PRL positive cells. These results demonstrate the presence of GnRH binding sites on these cells. It is suggested that GnRH may play a wide role in the neuroendocrine control of different pituitary hormones in addition to the GtHs., (Copyright 1999 Academic Press.)

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.