Climate change impacts on fish reproduction are mediated at multiple levels of the brain-pituitary-gonad axis.

Anthropogenic emissions of carbon dioxide in the atmosphere have generated rapid variations in atmospheric composition which drives major climate changes. Climate change related effects include changes in physico-chemical proprieties of sea and freshwater, such as variations in water temperature, salinity, pH/pCO2 and oxygen content, which can impact fish critical physiological functions including reproduction. In this context, the main aim of the present review is to discuss how climate change related effects (variation in water temperature and salinity, increases in duration and frequency of hypoxia events, water acidification) would impact reproduction by affecting the neuroendocrine axis (brain-pituitary-gonad axis). Variations in temperature and photoperiod regimes are known to strongly affect sex differentiation and the timing and phenology of spawning period in several fish species. Temperature mainly acts at the level of gonad by interfering with steroidogenesis, (notably on gonadal aromatase activity) and gametogenesis. Temperature is also directly involved in the quality of released gametes and embryos development. Changes in salinity or water acidification are especially associated with reduction of sperm quality and reproductive output. Hypoxia events are able to interact with gonad steroidogenesis by acting on the steroids precursor cholesterol availability or directly on aromatase action, with an impact on the quality of gametes and reproductive success. Climate change related effects on water parameters likely influence also the reproductive behavior of fish. Although the precise mechanisms underlying the regulation of these effects are not always understood, in this review we discuss different hypothesis and propose future research perspectives.

The gonadotropin-inhibitory hormone system of fish: The case of sea bass (Dicentrarchus labrax).

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide belonging to the RFamide peptide family that was first discovered in quail by Tsutsui and co-workers in the year 2000. Since then, different GnIH orthologues have been identified in all vertebrate groups, from agnathans to mammals. These GnIH genes synthesize peptide precursors that encompass two to four C-terminal LPXRFamide peptides. Functional and behavioral studies carried out in birds and mammals have demonstrated a clear inhibitory role of GnIH on GnRH and gonadotropin synthesis and secretion as well as on aggressive and sexual behavior. However, the effects of Gnih orthologues in reproduction remain controversial in fish with both stimulatory and inhibitory actions being reported. In this paper, we will review the main findings obtained in our laboratory on the Gnih system of the European sea bass, Dicentrarchus labrax. The sea bass gnih gene encodes two putative Gnih peptides (sbGnih1 and sbGnih2), and is expressed in the olfactory bulbs/telencephalon, diencephalon, midbrain tegmentum, rostral rhombencephalon, retina and testis. The immunohistochemical study performed using specific antibodies developed in our laboratory revealed Gnih-immunoreactive (ir) perikarya in the same central areas and Gnih-ir fibers that profusely innervated the brain and pituitary of sea bass. Moreover, in vivo studies revealed the inhibitory role of centrally- and peripherally-administered Gnih in the reproductive axis of male sea bass, by acting at the brain (on gnrh and kisspeptin expression), pituitary (on gnrh receptors and gonadotropin synthesis and release) and gonadal (on androgen secretion and gametogenesis) levels. Our results have revealed the existence of a functional Gnih system in sea bass, and have provided evidence of the differential actions of the two Gnih peptides on the reproductive axis of this species, the main inhibitory role in the brain and pituitary being exerted by the sbGnih2 peptide. Recent studies developed in our laboratory also suggest that Gnih might be involved in the transduction of photoperiod and temperature information to the reproductive axis, as well as in the modulation of daily and seasonal rhythmic processes in sea bass.

Daily rhythms of expression in reproductive genes along the brain-pituitary-gonad axis and liver of zebrafish.

The brain-pituitary-gonadal (BPG) axis regulates the activation of the endocrine machinery that triggers reproduction, which is a typical rhythmic process. In this research we focused on investigating the daily expression rhythms of the key reproductive genes involved in the BPG axis and the liver of zebrafish. To this end, male and female zebrafish were subjected to a stimulating photoperiod with a 14 h light:10 h dark cycle. Brain, pituitary and gonads, as well as female liver samples, were taken every 4 h during a 24 h cycle. The results revealed that most genes exhibited statistically significant daily rhythms. Most of the brain reproductive genes (gnrh2, gnrh3, kiss1, kiss2 and gnrhr3) displayed a daily rhythm of expression with a nocturnal acrophase (between Zeitgeber Time [ZT] 14:34 h and ZT18:34 h, lights off at ZT = 14 h). The male kiss2 gene presented neither significant rhythms nor daily variations, while the male gnrh3 and female kiss2 genes exhibited diurnal peaks of expression at ZT06:34 h and ZT04:34 h, respectively. In contrast, the pituitary genes (fshß, lhß, gnrhr2) showed daily rhythms of expression with an acrophase during the light phase (between ZT02:10 h and ZT10:35 h). The female gnrhr3 gene exhibited neither significant rhythms nor daily variations. The male gnrhr3 gene presented a nocturnal acrophase (ZT14:32 h). The gonad genes (star, cyp17a1, 20ßhsd, lhr, fshr, cyp19a1a, foxl2, amh, dmrt1 and 11ßhsd) revealed statistically significant daily rhythms with nocturnal acrophases, except for female cyp17a1a (ZT06:21 h) and 20ßhsd (ZT05:19 h). Lastly, the female liver genes presented daily rhythms with a maximum peak of expression around the transition phase from darkness to light (ZT01:00 h for erα and at ZT23:09 h for vtg2). These findings are consistent with the daily reproduction rhythms displayed by zebrafish, which are timed by the reproductive axis. Considering that reproductive success is critical for survival of the species, the knowledge of the rhythms of the endocrine BPG machinery provides useful information to understand the reproduction process and to establish optimal protocols and conditions for reproductive treatments.

Subfunctionalization of arylalkylamine N-acetyltransferases in the sea bass Dicentrarchus labrax: two-ones for one two.

Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N-acetyltransferase (Aanat: serotonin→N-acetylserotonin) and acetylserotonin-O-methyltransferase (Asmt: N-acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost-specific whole-genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co-expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N-acetylserotonin and N-acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.

Melatonin inhibits GnRH-1, GnRH-3 and GnRH receptor expression in the brain of the European Sea Bass, Dicentrarchus labrax.

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.

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone species.

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.

The retina is a target for GnRH-3 system in the European sea bass, Dicentrarchus labrax.

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.

Afferent and efferent connections of the pineal organ in the European sea bass Dicentrarchus labrax: a carbocyanine dye tract-tracing study.

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.

The Highly conserved gonadotropin-releasing hormone-2 form acts as a melatonin-releasing factor in the pineal of a teleost fish, the european sea bass Dicentrarchus labrax.

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.

Neuroendocrinology of reproduction in teleost fish.

This review aims at synthesizing the most relevant information regarding the neuroendocrine circuits controlling reproduction, mainly gonadotropin release, in teleost fish. In teleosts, the pituitary receives a more or less direct innervation by neurons sending projections to the vicinity of the pituitary gonadotrophs. Among the neurotransmitters and neuropeptides released by these nerve endings are gonadotrophin-releasing hormones (GnRH) and dopamine, acting as stimulatory and inhibitory factors (in many but not all fish) on the liberation of LH and to a lesser extent that of FSH. The activity of the corresponding neurons depends on a complex interplay between external and internal factors that will ultimately influence the triggering of puberty and sexual maturation. Among these factors are sex steroids and other peripheral hormones and growth factors, but little is known regarding their targets. However, very recently a new actor has entered the field of reproductive physiology. KiSS1, first known as a tumor suppressor called metastin, and its receptor GPR54, are now central to the regulation of GnRH, and consequently LH and FSH secretion in mammals. The KiSS system is notably viewed as instrumental in integrating both environmental cues and metabolic signals and passing this information onto the reproductive axis. In fish, there are two KiSS genes, KiSS1 and KiSS2, expressed in neurons of the preoptic area and mediobasal hypothalamus. Pionneer studies indicate that KiSS and GPR54 expression seem to be activated at puberty. Although precise information as to the physiological effects of KiSS1 in fish, notably on GnRH neurons and gonadotropin release, is still limited, KiSS neurons may emerge as the "gatekeeper" of puberty and reproduction in fish as in mammals.

Iodothyronine deiodinases and thyroid hormone receptors regulation during flatfish (Solea senegalensis) metamorphosis.

Thyroid hormone-induced metamorphosis seems to represent an ancestral feature of chrordates (urochordates, cephalochordates and vertebrates), but also of nonchordate animals. Although thyroid hormones and thyroid hormone receptor profiles during metamorphosis have been analyzed in different vertebrate taxa, including fish, developmental expression and activity of type 2 (dio2, D2) and type 3 (dio3, D3) iodothyronine deiodinases, two key enzymes in anuran metamorphosis, remain unknown in any fish species. The aim of this work was to investigate the development of thyroid hormone system during the metamorphosis of a flatfish species, the Senegalese sole, focusing on the deiodinases developmental profile. We have cloned sole D2 and D3 and analyzed several parameters of thyroid hormones system in pre-, early-, middle-, and late-metamorphic larvae. Both deiodinases contain in their catalytic centers an UGA triplet encoding for a selenocystein (Sec) residue as expected. Left eye migration and rotation in body position were associated with a significant increase in both thyroid hormones and thyroid hormone receptors at the middle-late metamorphic stages. Although dio2 expression slightly increased during metamorphosis, D2 activity augmentation was much more significant. Sole dio3 expression declined only slightly, whereas the D3 activity clearly decreased at mid-late metamorphic period. This developmental profile of deiodinases sustained the rise of thyroid hormones levels observed during sole metamorphosis. No clear cut daily rhythms were observed in the parameters analyzed although it seemed that thyroid hormone system was more active during daytime, in particular at late metamorphic stages. These developmental changes point out the importance not only of thyroid hormones and their receptors but also of dio2 and dio3 in mediating flatfish metamorphosis, as it has been described in amphibians.

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax.

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.

A cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax): the midbrain and hindbrain.

This study is the third part of a comprehensive series of publications on the cytoarchitectonic organization of the brain of the European sea bass, Dicentrarchus labrax. This study provides an atlas of the brain stem based on Nissl-stained transverse sections as well as a description of cell masses and a discussion on comparative aspects of brain stem nuclei, including methodological studies in other species. By external examination, the sea bass exhibits a prominent Optic tectum and Corpus cerebelli as expected in a predator species with a highly developed visual system. However, no hypertrophy of the facial and vagal lobes was observed as reported in other non-perciform teleosts. The general organization pattern of the mesencephalon and rhombencephalon of the sea bass brain resembles that reported for other perciform teleosts. However, the Valvula cerebelli has been subdivided into anterior, central and posterior parts. In addition, the ventricular surface of the granular layer of the Valvula cerebelli appears to be in contact with those of the Torus longitudinalis. This cell apposition could be interpreted as a direct connection, but more studies demonstrating the absence of ependyma between both structures are needed. Furthermore, we have tentatively described the electro/mechano receptive pre-eminential nucleus in the rhombencephalon of the sea bass. This study completes one of the few descriptions, as well as the most complete and detailed available, of the brain of any marine perciform species.

GnRH systems of Cichlasoma dimerus (Perciformes, Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides.

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.

New insights in developmental origins of different GnRH (gonadotrophin-releasing hormone) systems in perciform fish: an immunohistochemical study in the European sea bass (Dicentrarchus labrax).

The knowledge of the roles and origins of different gonadotrophin-releasing hormone (GnRH) systems could greatly contribute to improve the understanding of mechanisms involved in the physiological control of early development, puberty and spawning. Thus, in this study, we have analyzed the distribution of the cells expressing salmon GnRH, seabream GnRH and chicken GnRH-II forms in the brain and pituitary of developing sea bass using specific antibodies to their corresponding GnRH-associated peptides. The first prepro-chicken GnRH-II-immunoreactive cells arose in the germinal zone of the third ventricle at 4 days after hatching, increasing their number from days 10 to 30, in which they adopted their adult position. The prepro-chicken GnRH-II-immunoreactive fibers became conspicuous in the first week and from day 26 they reached almost all brain areas, especially the hindbrain, being never detected in the pituitary. First prepro-salmon GnRH-immunoreactive cells were detected in the olfactory placode at day 7 after hatching and reached the olfactory bulbs at day 10. Migrating prepro-salmon GnRH cells arrived at the ventral telencephalon at day 15, and became apparent in the preoptic area from day 45. The prepro-salmon GnRH innervation was more evident in the forebrain and increased notably between 10 and 30 days, at which fibers already extended from the olfactory bulbs to the medulla. A few prepro-salmon GnRH-immunoreactive fibers were observed in the pituitary from day 30. The prepro-seabream GnRH-immunoreactive cells were first detected at day 26 in the rostral olfactory bulbs. On day 30, prepro-seabream GnRH-immunoreactive cells were also present in the ventral telencephalon, reaching the preoptic area and the hypothalamus at 45 and 60 days, respectively. The prepro-seabream GnRH innervation appeared restricted to the ventral forebrain, increasing notably during the sixth week, when fibers also reached the pituitary. A significant prepro-seabream GnRH innervation was not detected in the pituitary until day 60.

Melatonin binding sites in the brain of European sea bass (Dicentrarchus labrax).

Characteristics, day-night changes, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[(125)I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radioligand binds to a single class of receptor site with the affinity (Kd) of 9.3 +/-0.6 pM and total binding capacity (Bmax) of 39.08 +/-0.86 fmol/mg protein (mean+/-SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPgammaS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.

Cloning and expression of gonadotropin-releasing hormone receptor in the brain and pituitary of the European sea bass: an in situ hybridization study.

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the pituitary of the European sea bass, Dicentrarchus labrax. The complete cDNA is 1814 base pairs (bp) in length and encodes a protein of 416 amino acids. The 5' UTR and 3' UTR are 239 bp and 324 bp in size, respectively. The expression sites of this GnRH-R were studied in the brain and pituitary of sea bass by means of in situ hybridization. A quantitative analysis of the expression of the GnRH-R gene along the reproductive cycle was also performed. The GnRH-R brain expression was especially relevant in the ventral telencephalon and rostral preoptic area. Some GnRH-R messenger-expressing cells were also evident in the dorsal telencephalon, caudal preoptic area, ventral thalamus, and periventricular hypothalamus. A conspicuous and specific GnRH-R expression was detected in the pineal gland. The highest expression of the GnRH-R gene was observed in the proximal pars distalis of the pituitary. This expression was evident in all LH cells and some FSH cells but not in somatotrophs. In the pituitary, the quantitative analysis revealed a higher expression of GnRH-R gene during late vitellogenesis in comparison with maturation, spawning, and postspawning/resting periods. However, in the brain, the highest GnRH-R expression was evident at spawning or postspawning/ resting periods. These results suggest that the expression of this GnRH-R is regulated in a different manner in the brain and the pituitary of sea bass.

Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish.

Gonadotrophin-releasing hormone (GnRH) was originally believed to be released by a unique set of hypophysiotrophic neurons to stimulate the release of gonadotrophins from the pituitary, therefore acting as a major initiator of the hormonal cascade controlling the reproductive axis. However, it now appears that each vertebrate species expresses two or three GnRH forms in multiple tissues and that GnRHs exert pleiotropic actions via several classes of receptors. This new vision of the GnRH systems arose progressively from numerous comparative studies in all vertebrate classes, but fish in general, and teleosts in particular, have often plaid a leading part in changing established concepts. To date fish still appear as attractive models to decipher the evolutionary mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest variety of GnRH variants, but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptors. This paper intends to summarize the current situation with special emphasis on interspecies comparisons which provide insights into the possible evolutionary mechanisms leading to the diversification of GnRH functions.

Developmental expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the European sea bass (Dicentrarchus labrax).

In this study, we have analyzed the ontogenic expression of three gonadotrophin-releasing hormones (GnRH) systems expressed in the brain of a perciform fish, the European sea bass, using in situ hybridization. The riboprobes used correspond to the GnRH-associated peptide (GAP) coding regions of the three prepro-GnRH cDNAs cloned from the same species: prepro-salmon GnRH, prepro-seabream GnRH and prepro-chicken GnRH II. On day 4 after hatching, the first prepro-chicken GnRH-II mRNA-expressing cells appeared in the germinal zone of the third ventricle. They increased in number and size from 10 to 21 days, reaching at day 30 their adult final position, within the synencephalic area, at the transitional zone between the diencephalon and the mesencephalon. First prepro-salmon GnRH mRNA-expressing cells became evident on day 7 arising from the olfactory placode and migrating towards the olfactory nerve. On day 10, this cell group reached the olfactory bulb, being evident in the ventral telencephalon and preoptic area from days 15 and 45, respectively. Weakly labeled prepro-seabream GnRH mRNA-expressing cells were first detected at 30 days in the olfactory area and ventral telencephalon. On day 45, prepro-seabream GnRH mRNA-expressing cells were also present in the preoptic region reaching the ventrolateral hypothalamus on day 60. The results obtained in sea bass indicate that sGnRH and sbGnRH cells have a common origin in an olfactory primordium suggesting that both forms might arise from a duplication of a single ancestral gene, while cGnRH-II cells develop from a synencephalic primordium.

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides.

The distribution of the cells expressing three prepro-gonadotrophin-releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH-II (cGnRH-II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross-reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH-associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken-II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP-immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP-ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP-ir fibers. The seabream GAP-ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP-ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP-ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken-II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP-ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species.