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.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

Summary The development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.     

Embryonic development of the sea bass Dicentrarchus labrax

The embryonic development of the sea bass Dicentrarchus labrax during the endotrophic period is discussed. An 8 cells stage, not reported for other studied species, results from two rapid successive cleavages. Blastula occurs at the eighth division when the embryo is made of 128 cells. During gastrulation, the infolded blastoderm creates the endomesoblastic layer. The Kupffer??s vesicle is reported to drive the left/right patterning of brain, heart and digestive tract. Heart formation starts at 8 pairs of somites, differentiation of myotomes and sclerotomes starts at the stage 18 pairs of somites; main parts of the digestive tract are entirely formed at 25 pairs of somites. At 28 pairs of somites, a rectal region is detected, however, the digestive tube is closed at both ends, the jaw appears the fourth day after hatching, but the mouth is not opened before the fifth day. Although cardiac beating and blood circulation are observed, gills are not reported in newly hatched individuals; eye melanization appears concomitant with exotrophic behavior.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na⁺/K⁺-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na⁺/K⁺-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na⁺/K⁺‐ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na⁺/K⁺/2Cl^? cotransporters (NKCCs) and the chloride channel “cystic fibrosis transmembrane regulator” (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.     

Double staining protocol for developing European sea bass (Dicentrarchus labrax) larvae

The alcian blue‐alizarin red technique was successfully adjusted to stain developing European sea bass (Dicentrarchus labrax) larvae. For an optimal staining protocol design both larval size and their morphological characteristics at each developmental stage were considered, since such parameters notably influence the staining of tissues. The incubation times of the different solutions were adjusted to allow the stain penetration for revealing the integrity of cartilaginous and bony tissues without significant tissue degradation. Three developmental windows were determined for an optimal staining procedure: (i) 4.5–6.4 mm, (ii) 6.7–8.7 mm, and (iii) 12.8–15.5 mm total length (TL). In order to validate the continuity of staining along the larval development, quantification of bone mineralization and osteocalcin gene expression were also monitored. Quantitative analysis revealed that ossification followed an exponential kinetic that was positively correlated with the osteocalcin gene expression pattern (Rs = 0.9762, P < 0.05). The mineralized tissue increased from 6.4 mm TL onwards, corresponding with the detection of the first ossified structures. The quantity of bony tissue increased gradually until 7.6 mm TL, since mineralization remained limited to the skull. From 8.3 to 15.5 mm TL, the mineralized bone was notable and nearly concerned the whole larval skeleton (skull, vertebral column and caudal complex). Since it was possible to detect the first cartilaginous and mineralized structures in specimens as small as 4.5 and 6.4 mm TL, respectively, this procedure is a useful tool to study the European sea bass skeletal ontogenesis, to precociously diagnose skeletal malformations in small larvae and eventually to better characterize the effect of different environmental and/or nutritional factors on the ossification status of specific skeletal components.     

Cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax). II. The diencephalon

The cytoarchitecture of nuclei in the preoptic area, ventral thalamus, dorsal thalamus, epithalamus, hypothalamus, posterior tuberculum, synencephalon, and pretectum and the accessory optic nuclei was analyzed in a perciform teleost, the sea bass Dicentrarchus labrax, by using serial sections stained with cresyl-violet. In general, the cytoarchitecture of the preoptic area, ventral and dorsal thalamus, epithalamus, and synencephalon resembles the histological pattern of other teleosts. However, the parvocellular preoptic nucleus of sea bass has been subdivided into parvocellular and anteroventral parts for morphological and functional reasons. The hypothalamus of the sea bass seems to differ slightly from that of other teleosts. An elaborated lateral tuberal nucleus, with five subdivisions, and three different nuclei around the lateral recesses were recognized. A medial nucleus of the inferior lobe, which has been reported previously in the perciform Sparus aurata, is also present in the hypothalamus of sea bass but has not been described before in another advanced teleost. The organization of the pretectum and the accessory optic system is essentially similar in sea bass to that described in other perciforms with highly developed vision. The migrated portion of the posterior tuberculum of sea bass appears to differ from this region of the diencephalon in other teleosts. In sea bass, three cell masses that have been described previously only in the perciform Sparus aurata have been assigned to the migrated area of the posterior tuberculum. This study will provide the neuroanatomical basis for future morpho-functional studies to be done in the sea bass brain.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax). I. The telencephalon

A cytoarchitectonic analysis of the telencephalon of the sea bass Dicentrarchus labrax, based on cresyl violet-stained serial transverse sections, is presented. Rostrally, the brain of the sea bass is occupied by sessile olfactory bulbs coupled to telencephalic hemispheres. The olfactory bulbs comprise an olfactory nerve fiber layer, a glomerular layer, an external cellular layer, a secondary olfactory fiber layer, and an internal cellular layer. Large terminal nerve ganglion cells are evident in the caudomedial olfactory bulbs. We recognized 22 distinct telencephalic nuclei which were classified in two main areas, the ventral telencephalon and the dorsal telencephalon. The ventral telencephalon displays four periventricular cell masses: the dorsal, ventral, supracommissural, and postcommissural nuclei; and four migrated populations: the lateral, central, intermediate, and entopeduncular nuclei. In addition, a periventricular cell population resembling the lateral septal organ reported in birds is observed in the ventral telencephalon of the sea bass. The dorsal telencephalon contains 13 nuclei, which can be organized into five major zones: the medial part, dorsal part, lateral part and its ventral, dorsal, and posterior divisions, the central part, and posterior part. Based on histological criteria, two cell masses are recognized in the ventral division of the lateral part of the dorsal telencephalon. The nucleus taenia is found in the caudal area of the dorsal telencephalon, close to the ventral area. This study represents a useful tool for the precise localization of the neuroendocrine territories and for the tracing of the neuronal systems participating in the regulation of reproduction and metabolism in this species.     

Eleven new microsatellites of the sea bass (Dicentrarchus labrax L.)

Eleven polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with an (AC)₁₂ probe. The loci showed different variation patterns in 21 unrelated sea bass individuals, with a mean number of alleles of 8.6 and a mean observed heterozygosity of 0.68. These microsatellite markers should be useful for population genetic analysis and biodiversity studies of sea bass.     

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.     

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.     

Action of leptin on in vitro luteinizing hormone release in the European sea bass (Dicentrarchus labrax).

The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptin-related peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10(-8)-10(-6) M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10(-6) M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.