[Show abstract][Hide abstract] ABSTRACT: Kisspeptin regulates reproductive events, including puberty and ovulation, primarily via GnRH neurons. Prolonged treatment of pre-pubertal striped bass females with Kiss1 or Kiss2 peptides failed to enhance puberty but suggested a gnrh-independent pituitary control pathway. Kiss2 inhibited, but Kiss1 stimulated FShβ expression and gonadal development, although hypophysiotropic gnrh1 and gnrh receptor expression remained unchanged. In situ hybridization and immunohistochemistry on brains and pituitaries revealed a differential plasticity between the two kisspeptin neurons. The differences were most pronounced at the pre-spawning phase in two regions along the path of gnrh1 axons: the nucleus lateralis tuberis (NLT) and the neurohypophysis. Kiss1 neurons appeared in the NLT and innervated the neurohypophysis of pre-spawning males and females, reaching Lh gonadotropes in the proximal pars distalis. Males, at all reproductive stages, had Kiss2 innervations in the NLT and the neurohypophysis, forming large axonal bundles in the former and intermingling with gnrh1 axons. Unlike in males, only pre-ovulatory females had massive NLT-neurohypophysis staining of kiss2. Kiss2 neurons showed a distinct appearance in the NLTv-equivalent region only in spawning zebrafish, indicating that this phenomenon is wide-spread. These results underscore the NLT as important nuclei for kisspeptin action in two facets: 1) kisspeptin - gnrh interaction: both kisspeptins are involved in the regulation of gnrh release, in a stage- and sex-dependent manner, especially at the pre-spawning phase and 2) gnrh-independent effect of Kiss peptides on the pituitary, which together with the plastic nature of their neuronal projections to the pituitary, implies that a direct gonadotropic regulation is plausible.
[Show abstract][Hide abstract] ABSTRACT: Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day-night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day-night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.
International Journal of Molecular Sciences 01/2013; 14(4):7603-16. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.
PLoS ONE 01/2013; 8(7):e70177. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3million quality sequences with an average size of 328bp, representing more than 1Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).
[Show abstract][Hide abstract] ABSTRACT: The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass.
General and Comparative Endocrinology 11/2011; 175(3):398-406. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pineal organ of fish is a photosensitive structure that receives light information from the environment and transduces it into hormonal (rhythmic melatonin secretion) and neural (efferent projections/neurotransmitters) signals. In this study, we focused on this neural output. Thus, we performed a tract-tracing study using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a fluorescent carbocyanine dye, in order to elucidate the efferent and afferent connections of the pineal organ in the European sea bass. The axonal transport of DiI revealed extensive bilateral projections in the sea bass brain. The efferent projections of the sea bass pineal organ reach the habenula, ventral thalamus, periventricular pretectum, central pretectal area, posterior tubercle and medial and dorsal tegmental areas. In addition, in this study we also examined the pinealopetal system in sea bass. This analysis demonstrated that the sea bass pineal organ receives central projections from neurons located, to a large extent, in brain areas innervated by pineal efferent projections, i.e. the thalamic eminence, habenula, ventral thalamus, dorsal thalamus, periventricular pretectum, posterior commissure, posterior tubercle and medial tegmental area. This study is the first description of pinealofugal projections in a representative of Perciformes, which constitutes a derived order within teleosts. Moreover, it represents the first evidence for the presence of pinealopetal neurons in the brain of a teleost species. Our findings, together with the analysis of retinal connections, represent a step forward in the understanding of the integration of photoperiodic signals into the central nervous system of sea bass.
Brain Behavior and Evolution 09/2011; 78(4):272-85. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The existence of two arylalkylamine N-acetyltransferase 1 (Aanat1) genes in the genome of some teleosts has been reported recently by in silico analysis. However, there are no data concerning the similarities and/or differences between them and many questions remain to be answered, such as their expression sites, development, or kinetics. Here, we report the cloning of Aanat1a and Aanat1b cDNAs from the sole retina and show for the first time that at least three Aanat genes are expressed in a vertebrate species. Because melatonin is involved in fish ontogeny, we analyzed the developmental transcript levels of Aanat1a and Aanat1b by quantitative real-time PCR, showing their inverse and stage-specific expression patterns. Aanat1a was more abundant during early than late larval stages. Before metamorphosis, nocturnal expression was higher. At metamorphosis, Aanat1a expression decreased and lost these day-night variations. In contrast, the abundance of Aanat1b transcripts, low during early developing stages, rose significantly throughout metamorphosis. This situation seemed to apply to the adult because Aanat1a expression was lower than Aanat1b expression in the retina of adults, where the former did not exhibit day-night variations, while the latter did so with much higher nocturnal transcript levels. In situ hybridization analysis detected Aanat1a and Aanat1b messengers in the outer and inner nuclear layers of retina. The differences in abundance and distinct day-night expression patterns between Aanat1a and Aanat1b during sole development suggest different functions for these two enzymes as well as the existence of interactions between the melatoninergic and thyroid hormone systems during flatfish metamorphosis.
Journal of Pineal Research 05/2011; 51(4):434-44. · 7.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.
[Show abstract][Hide abstract] ABSTRACT: The pineal organ of fish is a photosensory and neuroendocrine epithalamic structure that plays a key role in the temporal organisation of physiological and behavioural processes. In this study performed in the European sea bass, Dicentrarchus labrax, we provided an in-depth description of the macroscopic and microscopic anatomy of the pineal organ and identified the presence of photoreceptor and presumed melatonin-producing cells using histological and immunohistochemical techniques. In addition, we analysed in the pineal the day-night expression (using quantitative real-time PCR) of two key enzymes in the melatonin-synthesising pathway; arylalkylamine-N-acetyltransferase 2 (AANAT2) and hydroxyindole-O-methyltransferase (HIOMT). The pineal complex of sea bass consisted of a narrow and short pineal stalk that adopts a vertical disposition, a small-sized pineal end vesicle firmly attached to the skull by connective tissue, a parapineal organ and a convoluted dorsal sac. Immunohistochemical study showed the presence of abundant serotonin-positive cells. Cone opsin-like and rod opsin-like photoreceptor cells were also evidenced in the pineal stalk and vesicle. Both Aanat2 and Hiomt were expressed in sea bass pineal organ. Aanat2 exhibited higher nocturnal transcript levels, while no significant day-night differences were found for Hiomt. These results, together with ongoing studies analysing neural and neurohormonal outputs from the pineal organ of sea bass, provide the basic framework to understand the transduction integration of light stimulus in this relevant species for marine aquaculture.
Journal of chemical neuroanatomy 02/2011; 41(3):170-80. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because a large proportion of potential endocrine disruptors (EDC) end up in surface waters, aquatic species are particularly vulnerable to their potential adverse effects. Recent studies identified a number of brain targets for EDC commonly present in environmentally relevant concentrations in surface waters. Among those neuronal systems disrupted by EDC are the gonadotropin-releasing hormone (GnRH) neurons, the dopaminergic and serotoninergic circuits, and more recently the Kiss/GPR54 system, which regulates gonadotropin release. However, one of the most striking effects of EDC, notably estrogen mimics, is their impact on the cyp19a1b gene that encodes the brain aromatase isoform in fish. Moreover, this is the only example in which the molecular basis of endocrine disruption is fully understood. The aims of this review were to (1) synthesize the most recent discoveries concerning the EDC effects upon neuroendocrine systems of fish and (2) provide, when possible, the underlying molecular basis of disruption for each system concerned. The potential adverse effects of EDC on neurogenesis, puberty, and brain sexualization are also described. It is important to point out the future environmental, social, and economical issues arising from endocrine disruption studies in the context of risk assessment.
Journal of Toxicology and Environmental Health Part B 01/2011; 14(5-7):370-86. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation.
PLoS ONE 01/2011; 6(11):e28375. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Before kisspeptins became new players in the field of reproductive biology, GnRH was acknowledged in all
vertebrates as the major initiator of the hormonal cascade modulating the reproductive axis. Originally identified as a metastasis suppressor in mammals , the KISS1 gene produces several peptides named kisspeptins (kisspeptin -54, 14, 13, 10), which activate the KISS1 receptor (GPR54 or KISSR) previously known as an orphan receptor . Recent phylogenetical analysesprovided evidence that the number of kiss genes andkiss receptors varies from one class of vertebrate to the other.
According to these studies  modern mammals have only one KISS gene, monotrems have two, birds would have none, reptiles have one, amphibians have three and fishes have two KISS genes. Similarly, the number of genes encoding GPR54 receptors (or Kissr) also varies from one class to the other. With the aim to enlarge our knowledge on organization and potential functions of Kiss systems in relation to GPR54 receptors in non-mammalian species, so far very poorly investigated,we have focused our interest on the elucidation of these systems in zebrafish, taken as model of study. Zebrafish have two kiss genes, kiss1 and kiss2 and two kiss receptors (GPR54), kiss1r and kiss2r.
Indian Journal of Science and Technology. 01/2011; 4:52-53.
[Show abstract][Hide abstract] ABSTRACT: Unlike that of mammals, the brain of adult teleost fish exhibits an intense and widespread neurogenic activity as a result of the persistence of radial glial cells acting as neural progenitors throughout life. Because chemokines, notably CXCL12, and their receptors, such as CXCR4, play key roles in mammalian embryonic neurogenesis, we investigated Cxcr4 and Cxcl12 expressions in the brain of adult zebrafish and their potential relationships with cell proliferation. Cxcr4 expression was found to be restricted to radial glial cells in the adult zebrafish, where it is co-expressed with established radial glial cell markers, such as brain lipid-binding protein (Blbp) or the estrogen-synthesizing enzyme aromatase B (Cyp19a1b). Double stainings combining proliferating cell nuclear antigen (PCNA) and Cxcr4 immunolabelling indicated that there is no obvious association between Cxcr4 expression and radial glial cell proliferation. Interestingly, cxcl12a messengers were detected in ventricular regions, in cells corresponding to aromatase B-immunoreactive radial glial cells. Altogether, our data demonstrate Cxcl12 and Cxcr4 expression in radial glial cells of the brain of adult zebrafish, supporting important roles for the Cxcl12/Cxcr4 pair in brain development and functioning.
The Journal of Comparative Neurology 12/2010; 518(24):4855-76. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the exception of modern mammals, most vertebrate species possess two GnRH genes, GnRH-1 and GnRH-2. In addition, in many teleost fish, there is a third gene called GnRH-3. If the main function of GnRH-1 is unambiguously to stimulate gonadotropin release, the other two GnRH forms still lack clear functions. This is particularly true for the highly conserved GnRH-2 that encodes chicken GnRH-II. This GnRH variant is consistently expressed in neurons of the dorsal synencephalon in most vertebrate groups but still has no clear functions supported by anatomical, pharmacological, and physiological data. In this study performed on a perciform fish, the European sea bass, we show for the first time that the pineal organ receives GnRH-2-immunoreactive fibers originating from the synencephalic GnRH-2 neurons. This was shown through a combination of retrograde tracing and immunohistochemistry, using highly specific antibodies. Supporting the presence of GnRH-2 functional targets, RT-PCR data together with the in situ hybridization studies showed that the sea bass pineal gland strongly expressed a GnRH receptor (dlGnRHR-II-2b) with clear selectivity for GnRH-2 and, to a lesser extent, the dlGnRHR-II-1a subtype. Finally, in vitro and in vivo experiments demonstrate stimulatory effects of GnRH-2 on nocturnal melatonin secretion by the sea bass pineal organ. Altogether, these data provide, for the first time in a vertebrate species, converging evidence supporting a role of GnRH-2 in the modulation of fish pineal functions.
[Show abstract][Hide abstract] ABSTRACT: Long term cell cultures could be obtained from brains of adult sea bass (Dicentrarchus labrax) up to 5 days post mortem. On three different occasions, sea bass brain tissues were dissected, dispersed and cultured in Leibovitz's L-15 media supplemented with 10% fetal bovine serum. The resulting cellular preparations could be passaged within 2 or 3 weeks of growth. The neural cells derived from the first trial (SBB-W1) have now been passaged over 24 times within two years. These cells have been cryopreserved and thawed successfully. SBB-W1 cells are slow growing with doubling times requiring at least 7 days at 22 degrees C. These long term cell cultures could be grown in suspension as neurospheres that were immunopositive for nestin, a marker for neural stem cells, or grown as adherent monolayers displaying both glial and neural morphologies. Immunostaining with anti-glial fibrillary acidic protein (a glial marker) and anti-neurofilament (a neuronal marker), yielded positive staining in most cells, suggesting their possible identity as neural stem cells. Furthermore, Sox 2, a marker for neural stem cells, could be detected from these cell extracts as well as proliferating cell nuclear antigen, a marker for proliferating cells. SBB-W1 could be transfected using pEGFP-N1 indicating their viability and suitability as convenient models for neurophysiological or neurotoxicological studies.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 12/2008; 152(2):245-54. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the
five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day–night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day–night fluctuations, suggesting that exogenous melatonin reinforces physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.
36ème Colloque de la Société de Neuroendocrinologie.