Generation and use of recombinant gonadotropins in fish.

Understanding the differential roles of the pituitary gonadotropins Fsh and Lh in gonad maturation is crucial for a successful manipulation of the reproductive process in fish, and requires species-specific tools and appropriate active hormones. With the increasing availability of fish cDNAs coding for gonadotropin subunits, the production of recombinant hormones in heterologous systems has gradually substituted the approach of isolating native hormones. These recombinant hormones can be continually produced without depending on the fish as starting material and no cross-contamination with other pituitary glycoproteins is assured. Recombinant gonadotropins should be produced in eukaryotic cells, which have glycosylation capacity, but this post-translational modification varies greatly depending on the cell system, influencing hormone activity and stability. The production of recombinant gonadotropin beta-subunits to be used as antigens for antibody production has allowed the development of immunoassays for quantification of gonadotropins in some fish species. The administration in vivo of dimeric homologous recombinant gonadotropins has been used in basic studies and as a biotechnological approach to induce gametogenesis. In addition, gene-based therapies using somatic transfer of the gonadotropin genes have been tested as an alternative for hormone delivery in vivo. In summary, the use of homologous hormonal treatments can open new strategies in aquaculture to solve reproductive problems or develop out-of-season breeding programs.

Photoperiod modulates the reproductive axis of European sea bass through regulation of kiss1 and gnrh2 neuronal expression.

The onset of puberty is characterized by activation of the brain-pituitary-gonad axis. However, the molecular and endocrine mechanism involved in the process of puberty and the influence of environmental conditions, such as photoperiod signalling, are not well understood in fish. In this study, 1-year-old male European sea bass (Dicentrarchus labrax) were exposed to photoperiod manipulation in combination with size-sorting. Two treatment groups, a puberty accelerating photoperiod (AP) group and a continuous light (LL) group, were studied from August to February. Our results indicate that AP and LL are able to entrain the rhythms of both kiss1 and gnrh2 mRNA levels in the brain, while kiss2 and gnrh1 mRNA expression does not seem to be directly affected by the photoperiod, at least during testicular growth. It is likely that AP and LL photoperiod regimes affected both plasma Fsh and 11-KT profiles, which might explain, respectively, the phase shift and reduction of testes maturation seen under these conditions. We therefore hypothesize that the unbalanced production of this androgen regulated by circulating Fsh might be limiting the stimulation of germ cell proliferation in European sea bass males. In summary, our study establishes that photoperiod modulates the expression of kiss1 and gnrh2 in the forebrain-midbrain, which may be involved in the translation of the light stimulus to activate the reproductive axis.

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax).

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.

New insights into the factors mediating the onset of puberty in sea bass.

In populations of 1-year-old male European sea bass (Dicentrarchus labrax), only large males are able to acquire for the first time a functional competence of their reproductive axis; in other words, to attain puberty. To examine the causes and mechanisms involved in the onset of puberty in this species, a size sorting sampling was carried out to obtain two experimental groups of small and large male fish exhibiting different growth rates. As expected, only large fish reached full spermiogenesis (stage V of testicular development) by the end of the experiment. Our study suggests that fish size is a permissive condition to ensure full effectiveness of the hormonal (Gnrh1, gonadotropins and sexual steroids) actions. Thus, though small fish had endocrine profiles similar to those of large fish, their amplitude was much lower, and was most likely the reason why functional competence of the reproductive axis was not achieved. Moreover, this work provides evidence of the involvement of kisspeptin and Gnrh1 systems in the onset of puberty in a marine teleost fish. It also indicates that very likely kisspeptin and Gnrh1 may regulate gonadotropins and sex steroids at specific stages of testicular development.

Continuous light and melatonin: daily and seasonal variations of brain binding sites and plasma concentration during the first reproductive cycle of sea bass.

The present study reports on the daily and seasonal variations in plasma melatonin concentration, and also in optic tectum and hypothalamus melatonin binding sites, in male European sea bass maintained under natural photoperiod (NP) or continuous light (LL) from early stages of development. Samples were collected on a 24-h cycle, at four physiological phases of their first annual reproductive cycle, i.e., pre-spermatogenesis, spermatogenesis, spermiation and post-spermiation. Under NP, (1) plasma melatonin levels were higher at night than during the day regardless of the year period, and the duration of the signal matched the duration of the dark phase; (2) daily variations in Kd and Bmax were found in the optic tectum, but only during spermiation, with the acrophase being 180° out of phase with the plasma melatonin variations; and (3) significant seasonal Kd and Bmax changes were seen in the hypothalamus. Under LL, (1) plasma melatonin showed no elevation during the subjective night; and (2) Kd and Bmax exhibited seasonal variations in the hypothalamus. These results led to the conclusion that long-term exposure to LL affected both plasma melatonin and receptor oscillations; particularly, LL disrupted the receptor density circadian oscillation found in the optic tectum during spermiation under NP. This oscillation appears to be important for sea bass to pursue gametogenesis until full spermiation. The persistence of both daily and seasonal variation of receptor affinity and density in the hypothalamus under LL indicates that these variations are controlled by internal circadian and circannual clocks that do not involve melatonin.

Older triploid fish retain impaired reproductive endocrinology in the European sea bass Dicentrarchus labrax.

This paper reports on an evaluation of growth, gonadal development and reproductive endocrinology of older triploid (3n) European sea bass Dicentrarchus labrax in comparison with their diploid (2n) counterparts throughout their fifth and seventh annual cycle of life. While older triploids retained impaired reproductive endocrinology, a sexually related dimorphic growth was observed with 3n females attaining the largest sizes. Comparisons of some body indexes showed that 3n females had a significantly lower hepato-somatic index (I(H)) than 2n females but a significantly higher viscero-somatic index (I(F)). In contrast, both male and female triploids showed significantly lower gonado-somatic index (I(G)) than diploids. Accordingly, diploids produced mature gametes but triploids did not, demonstrating that despite the longer time given to triploids for gonadal development, they could not reproduce. Furthermore, older triploids had lower levels of plasma sex steroids (testosterone, T; 11-ketotestosterone, 11-KT and oestradiol-17beta, E(2)) and luteinizing hormone (LH) than their 2n counterparts with 3n females showing drastic effects of triploidization on their reproductive endocrinology. Vitellogenin (VTG) was undetectable in 3n females. Gonadal content of steroid hormones and Sparus aurata-type gonadotropin-releasing hormone (sbGnRH) in the brain and pituitary were also lower in triploids compared with diploids. These results suggest that older 3n D. labrax retain functional sterility in both sexes, and 3n females might reach larger sizes than 3n males and their 2n counterparts in this species.

Characterization of the European Sea Bass (Dicentrarchus labrax) Gonadal Transcriptome During Sexual Development.

The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17ß10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species.

Anti-Müllerian hormone (AMH/AMH) in the European sea bass: its gene structure, regulatory elements, and the expression of alternatively-spliced isoforms.

In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH/AMH), apart from inducing Müllerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5'-flanking region, and analysed its gene expression and those of alternatively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in juvenile testis, but still remarkably high in juvenile ovaries and adult testis, as well as in brain, pituitary, and heart of juvenile male sea bass. Apart from adult ovary, levels of alternatively-spliced sb-AMHexonII/-99 were marginal in comparison with sb-AMH. In contrast, the transcript variant sb-AMHexonVII/+5 was expressed to a similar extent as sb-AMH in all tissues examined. The results of this work have provided the basis for future studies concerning the regulation and function of AMH/AMH in this species.

Organochlorine compounds in liver and concentrations of vitellogenin and 17beta-estradiol in plasma of sea bass fed with a commercial or with a natural diet.

Results from previous experiments directed to determine the effect of different nutritional factors or the effect of xenobiotics on hormonal control of reproduction, lead to the hypothesis that hormonal perturbations repeatedly observed in sea bass (Dicentrarchus labrax) broodstock feeding commercial diets could have been caused by the presence of aryl hydrocarbon receptor (AhR) ligands, such as dioxins, furans and polychlorinated biphenyls (PCBs) in the diet. To evaluate this hypothesis, dioxins and related compounds were analysed in liver of female sea bass fed with a commercial or with a natural diet consisting of trash fish (bogue, Boops boops), and concentrations of vitellogenin (VTG) and 17beta-estradiol (E2) were determined in plasma obtained previously in monthly samplings of these animals. As observed in other experiments, females fed with a commercial diet exhibited lower VTG and higher E2 plasma levels than females fed with the natural diet. In liver, sea bass fed with the commercial diet exhibited a profile clearly dominated by high-chlorinated dioxins while in fish fed with the natural diet this profile was dominated by low chlorinated furans. However, typical AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin showed no differences between groups or, as is the case of planar PCBs, showed higher concentrations in the liver of fish fed with the natural diet. These results do not permit to explain the observed hormonal alterations by a possible antiestrogenic effect caused by dioxins and related compounds.

Peptide YY (PYY) and fish pancreatic peptide Y (PY) expression in the brain of the sea bass (Dicentrarchus labrax) as revealed by in situ hybridization.

Tetrapod vertebrates express three neuropeptide Y (NPY)-related peptides: NPY, peptide YY (PYY), and pancreatic polypeptide (PP). Both NPY and PYY mRNA have been localized in the brain of tetrapods whereas PP expression is restricted to the pancreas. Some teleost fish commonly produce NPY and PYY but pancreatic peptide Y (PY) instead of PP. Both NPY and PYY mRNAs are widely distributed in the brain of non-tetrapod species, but no information about PY central expression is available. In the present study, molecular riboprobes were used to study PYY and PY mRNA central distribution in the sea bass (Dicentrarchus labrax). PYY and PY gene expression was predominantly detected within the sea bass forebrain. Telencephalic PYY gene expression was restricted to the ventral part of the ventral telencephalon, and no PY expression was detected in the cerebral hemispheres. Both PYY and PY mRNAs were found within the preoptic area and lateral hypothalamus. Distinct PY or PYY mRNA cell groups were localized in the pretectal area and synencephalon or posterior tubercle, respectively. Caudally, PY gene expression was found in the medial reticular formation, whereas PYY transcripts were localized within the vagal lobe. The results demonstrate that vertebrate brain expresses three NPY-related genes and further support the hypothesis that PP and PY arose by independent gene duplications from PYY. The receptor system of the NPY family as well as gene expression within the main hypophysiotropic and feeding behavior areas suggest an involvement of both peptides in the control of food intake and pituitary secretion.

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

The expression sites of three prepro-gonadotrophin-releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser(8)-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp(7)Leu(8)-mGnRH), and chicken GnRH-II (cGnRH-II: His(5)Trp(7)Tyr(8)-mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH-associated peptide (GAP)-coding regions of the prepro-GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP-II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti-sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA-expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP-positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations.

Cloning, characterisation, and expression of three oestrogen receptors (ERalpha, ERbeta1 and ERbeta2) in the European sea bass, Dicentrarchus labrax.

Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.

Characterization of neuropeptide Y expression in the brain of a perciform fish, the sea bass (Dicentrarchus labrax).

The distribution of neuropeptide Y (NPY) gene expression was mapped in the brain of the sea bass (Dicentrarchus labrax) by in situ hybridization with 35S-UTP labeled cRNA probes. Gene expression was mainly detected within the forebrain, although NPY mRNA transcripts were also localized in the tectum and tegmentum mesencephali and posterior brain. New NPY-expressing nuclei were found in the dorsal and ventral telencephalon, preoptic area, tuberal hypothalamus, synencephalon, tegmentum mesencephali and posterior brain. The profuse NPY gene expression within the main neuroendocrine areas of the teleost fish further supports a physiological role in the control of the pituitary secretion. In addition, NPY gene was expressed within the primary visual, olfactory and gustatory circuits of teleost which, subsequently, project to hypothalamic feeding center in teleost fish. Our results extend the NPY-expressing areas known in teleost species.

Molecular evolution of the neuropeptide Y (NPY) family of peptides: cloning of three NPY-related peptides from the sea bass (Dicentrarchus labrax).

Neuropeptide Y (NPY) is a 36-amino-acid peptide that is widely and abundantly expressed in the central nervous system of all vertebrates investigated. Related peptides have been found in various vertebrate groups: peptide YY (PYY) is present in gut endocrine cells of many species and pancreatic polypeptide (PP) is made in the pancreas of all tetrapods. In addition, a fish pancreatic peptide called PY has been reported in three species of fishes. The evolutionary relationships of fish PY have been unclear and it has been proposed to be the orthologue (species homologue) of each of the three tetrapod peptides. We demonstrate here with molecular cloning techniques that the sea bass (Dicentrarchus labrax), an acanthomorph fish, has orthologues of both NPY and PYY as well as a separate PY peptide. Sequence comparisons suggest that PY arose as a copy of the PYY gene, presumably in a duplication event separate from the one that generated PP from PYY in tetrapods. PY sequences from four species of fish indicate that, similar to PP, PY evolves much more rapidly than NPY and PYY. The physiological role of PY is unknown, but we demonstrate here that sea bass PY, like NPY and PYY but in contrast to the tetrapod PP, is expressed in brain.

Time-course studies on plasma glucose, insulin, and cortisol in sea bass (Dicentrarchus labrax) held under different photoperiodic regimes.

Daily variations of insulin, cortisol, and glucose are studied in animals adapted to two different photoperiodic regimes, with the intervals between feeding times and photoperiodic events kept constant. Data support the existence of a daily rhythm of plasma glucose which seems to be photoperiodic. In contrast, the daily patterns of insulin in sea bass seem to be mainly influenced by feeding time; however, an effect of photoperiod can not be excluded. When the digestive tract is absent of food, insulin levels are generally minimal at feeding times and maximal during the inter-meals periods, suggesting the central control of insulin secretion during short-term food deprivation. Contrarily, the nadir values of plasma cortisol were reached at midday during the inter-meal period and peak plasma levels were evident at both light onset and offset. Disruption between metabolite and hormone patterns suggest that they are under different controls. Such results could be explained under the existence of a multioscillator system, including a food entrainable oscillator in addition to the master light entrainable oscillator.

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.

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.

AFLP Analysis Confirms Exclusive Maternal Genomic Contribution of Meiogynogenetic Sea Bass (Dicentrarchus labrax L.).

Meiogynogenesis was induced in the European sea bass Dicentrarchus labrax L. by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division by a cold shock. Putative gynogenetic progeny derived from three groups of breeders were analyzed for maternal inheritance using amplified fragment length polymorphism (AFLP) markers. Discrimination of fingerprints was based on male-specific bands, which were absent in females. Four of 64 MseI/EcoRI primer pairs used to analyze parental polymerase chain reaction products were selected to screen progeny for paternal AFLP markers in each group. Four to 11 diagnostic bands per fish confirmed the gynogenetic origin of the progeny. AFLP analysis determined that 89.5%, 100%, and 100% of the sea bass from groups 1, 2, and 3, respectively, were gynogenetic. Our results show that AFLP analysis is suitable for verification of gynogenesis in fish.

Cloning the neuropeptide Y exon 2 from sea bass (Dicentrarchus labrax).

A partial genomic library of sea bass DNA was constructed and screened with a goldfish NPY cDNA probe. Two identical clones were isolated and sequenced. The clones contain a segment with high identity to exon 2 of the NPY gene in tetrapods. This segment encodes a 62-amino acid peptide consisting of a signal peptide of 28 amino acids and the main portion of the mature NPY (34 amino acids). In the latter extension, sea bass NPY shows high identity with the human and deduced ancestral gnathostome sequences (88 and 91%, respectively). The open reading frame is followed by a consensus splice donor site. Northern blot hybridization to examine tissue distribution detected a 1-kb RNA transcript restricted to brain tissue. These data show that the NPY gene of this teleost fish has the same intron positions as tetrapods for at least two of the gene's three introns. In addition, the high evolutionary conservation of NPY is corroborated since sea bass NPY exhibits the same identity to both goldfish and human NPY.

Development of an in vitro system for functional studies of ovarian follicular cells in European sea bass (Dicentrarchus labrax).

The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 °C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes (cyp19a1, fshr, lhr and star). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle.