Genomic resources for a commercial flatfish, the Senegalese sole (Solea senegalensis): EST sequencing, oligo microarray design, and development of the Soleamold bioinformatic platform

Background: The Senegalese sole, Solea senegalensis, is a highly prized flatfish of growing commercial interest for aquaculture in Southern Europe. However, despite the industrial production of Senegalese sole being hampered primarily by lack of information on the physiological mechanisms involved in reproduction, growth and immunity, very limited genomic information is available on this species. Results: Sequencing of a S. senegalensis multi-tissue normalized cDNA library, from adult tissues (brain, stomach, intestine, liver, ovary, and testis), larval stages (pre-metamorphosis, metamorphosis), juvenile stages (post-metamorphosis, abnormal fish), and undifferentiated gonads, generated 10,185 expressed sequence tags (ESTs). Clones were sequenced from the 3'-end to identify isoform specific sequences. Assembly of the entire EST collection into contigs gave 5,208 unique sequences of which 1,769 (34%) had matches in GenBank, thus showing a low level of redundancy. The sequence of the 5,208 unigenes was used to design and validate an oligonucleotide microarray representing 5,087 unique Senegalese sole transcripts. Finally, a novel interactive bioinformatic platform, Soleamold, was developed for the Senegalese sole EST collection as well as microarray and ISH data. Conclusion: New genomic resources have been developed for S. senegalensis, an economically important fish in aquaculture, which include a collection of expressed genes, an oligonucleotide microarray, and a publicly available bioinformatic platform that can be used to study gene expression in this species. These resources will help elucidate transcriptional regulation in wild and captive Senegalese sole for optimization of its production under intensive culture conditions

Growth differentiation factor 9 and bone morphogenetic protein 15 mRNA and protein: cellular localization and developmental expression in the ovary of European sea bass

Trabajo presentado como póster en la "Session V: Gametogenesis-II (Folliculogenesis)" del "9th International Symposium on Reproductive Physiology of Fish" celebrado en Thrissur (Cochin-India) del 9 al 14 de agosto de 2011.Funded by the former Spanish Ministry of Education and Science. Grant AGL2007-61192/ACU to F.P. and CSIC-JAE program contract to Á.G.-L.Peer Reviewe

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): Cellular localization, developmental profiles, and response to unilateral ovariectomy

9 páginas, 5 figuras. La versión del editor incluye apendice suplementario.Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.Funding for the present work was provided by the Spanish Ministry of Education and Science to F.P through the grant AGL2007-61192/ACU. Á.G.-L. was supported by a contract from the CSIC-JAE program.Peer reviewe

Transcriptomic Characterization of the Larval Stage in Gilthead Seabream (Sparus aurata) by 454 Pyrosequencing

Gilthead seabream (Sparus aurata) is a teleost belonging to the family Sparidae with a high economical relevance in the Mediterranean countries. Although genomic tools have been developed in this species in order to investigate its physiology at the molecular level and consequently its culture, genomic information on post-embryonic development is still scarce. In this study, we have investigated the transcriptome of a marine teleost during the larval stage (from hatching to 60 days after hatching) by the use of 454 pyrosequencing technology. We obtained a total of 68,289 assembled contigs, representing putative transcripts, belonging to 54,606 different clusters. Comparison against all S. aurata expressed sequenced tags (ESTs) from the NCBI database revealed that up to 34,722 contigs, belonging to about 61% of gene clusters, are sequences previously not described. Contigs were annotated through an iterative Blast pipeline by comparison against databases such as NCBI RefSeq from Danio rerio, SwissProt or NCBI teleost ESTs. Our results indicate that we have enriched the number of annotated sequences for this species by more than 50% compared with previously existing databases for the gilthead seabream. Gene Ontology analysis of these novel sequences revealed that there is a statistically significant number of transcripts with key roles in larval development, differentiation, morphology, and growth. Finally, all information has been made available online through user-friendly interfaces such as GBrowse and a Blast server with a graphical frontend.This research has been funded by the Spanish Ministry of Science and Innovation MICINN + FEDER/ERDF, Consolider Ingenio 2010 Program (Project Aquagenomics, CSD2007-0002). This study also benefited from participation in LARVANET-COST action FA0801 (EU RTD framework Programme).Peer reviewe

Gene expression of estrogen receptors α and β during early sexual differentiation in the European sea bass (Dicentrarchus labrax)

A dimorphic expression pattern of ERα was found during sexual development in the European sea bass. It is therefore suggested that ERα plays an important role in sexual differentiation in this species.This work was supported by an EU grant (Q5RS-2000-31365)

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.We would like to thank Dr. Adelino Canario, University of Algarve, Portugal, for providing the sea bass testicular cDNA library. This work has been carried out with the financial support of the European Union, Q5RS-2000-31365, QLK5-CT-2001-51019 and AGL2002-10024E

Molecular characterisation of growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass

Members of the transforming growth factor-β superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in primary follicle growth in mammals. To initiate investigations into their significance in teleost oogenesis, we set out to clone and characterise the cDNAs of gdf9 and bmp15 and analysed their patterns of gene expression during the ovarian reproductive cycle in the European sea bass (Dicentrachus labrax). Sea bass gdf9 and bmp15 cDNAs were 2200 and 2049 bp long, coding for 438 and 459 amino acids (aas), respectively, and were most similar to zebrafish gdf9 and bmp15 (64.4 and 56.1%, respectively). By Northern analysis, sea bass gdf9 and bmp15 mRNA transcripts were detected in the ovary only of the tissues analysed and their sizes were 2.2 and 2.1 kb, respectively. Dot-blot analysis revealed high levels of gdf9 and bmp15 expression in the ovary during primary oocyte growth and previtellogenesis (July to October), with a significant decline at the onset of vitellogenesis (November) and remaining low until the beginning of new oocyte growth (April/May). There was a highly significant positive correlation (r = 0.939) between gdf9 and bmp15 gene expression in individual samples. The high levels of gdf9 and bmp15 mRNA transcripts in the ovary, especially during the previtellogenic growth period suggest an important role for these factors in early primary oocyte growth in the European sea bass. © 2008 Elsevier Ireland Ltd. All rights reserved.This work was funded by a grant of the Spanish Ministry of Science and Education to F.P. (AGL2005-03747/ACU).Peer Reviewe

Cloning of oestrogen receptors beta in the European sea bass (Dicentrarchus labrax) for the study of sex differentiation in fish

Resumen del trabajo presentado en la 21st Conference of European Comparative Endocrinologists, celebrada en Bonn (Alemania), del 26 al 30 de agosto de 2002The mechanism of sex differentiation in fish is only poorly understood, mainly due to a high plasticity and sensitivity of the gonads compared with the more stable gonadal development found in marnmals. However, oestrogen receptors [ERs] have been found to be expressed very early during embryonic development, suggesting a role for oestrogens in sexual differentiation in fish. Moreover, the recent discovery of oestrogen receptors different from the "c1assical" ERa, as well as different ER isoforms, imply that oestrogen action may be rather complex encompassing diverse functions at distinct phases during sexual development. In this study we set out to clone ERs beta [ERb] from the European sea bass [sb] with the aim of studying ER gene expression during early sexual differentiation in this species. By RT-PCR and 3'-RACE-PCR, we obtained two 1934 bp and 1926 bp partial-Iength cDNAs, called ER beta-1 [ERb1] and -2 [ERb2], from the ovary of sb. These two ERs beta encode proteins of 517 and 608 amino acids [aa], respectively, representing approximately 90 % of the complete protein. They display highest aa-identity with ERb in fish, i.e. 86.4 % with sea bream ERb (for sb-ERb1), and 90 % with Atlantic croaker ERb (for sb-ERb2). In addition, we isolated a sb-ERb2 isoform from liver, lacking the complete exon 5, but otherwise identical to the sb-ERb2 cDNA fragment isolated from ovary.Supported by the EU (FAIR-CT961410 & Q5RS-2000-31365

Bone morphogenetic protein 15 and growth differentiation factor 9 in European sea bass oocyte development

Trabajo presentado en el 9th International Congress on the Biology of Fish, celebrado en Barcelona del 5 al 9 de julio de 2010.It is well known that vertebrate oocytes contribute actively to follicle development and maturation by secreting a variety of growth factors, among which growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic factor 15 (BMP15/bmp15) have been paid particular attention both in mammals and in zebrafish. Recently, we have cloned the cDNA sequences encoding for gdf9 and bmp15 in the European sea bass Dicentrarchus labrax, the most important commercial marine fish species in the Mediterranean area, and reported their high mRNA levels in early stages of ovarian development. Here, we provide further evidences supporting the potential roles of gdf9 and bmp15 in sea bass folliculogenesis. Specific antibodies were raised and used to quantify protein levels throughout oocyte development as well as for cellular localization studies together with in situ hybridization approaches. In addition, the roles of both factors on the compensatory ovarian growth induced by unilateral ovariectomy were evaluated.Peer Reviewe

Cloning, characterisation, and expression of three oestrogen receptors (ERα, ERβ1 and ERβ2) in the European sea bass, Dicentrarchus labrax

Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERα, and two ERβ subtypes, called ERβ1 and ERβ2 (or ERβ and ERγ 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-ERα cDNA) and ovary (partial sb-ERβ1 and sb-ERβ2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERα, ERβ1 and ERβ2. Deduced amino acid numbers for sb-ERα, sb-ERβ1 and sb-ERβ2 were 639, 517 and 608, respectively, representing in the case of sb-ERβ1 and sb-ERβ2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERα with eelpout ERα (88.7%), for sb-ERβ1 with Atlantic croaker ERγ (85.8%), and for sb-ERβ2 with Atlantic croaker ERβ (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, ERα was predominantly expressed in liver and pituitary, while sb-ERβ1 and sb-ERβ2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERα expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERβ1 and sb-ERβ2 highest expression levels were observed in gonads at 250 dph. For sb-ERβ2, 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. © 2004 Elsevier Ireland Ltd. All rights reserved.Peer Reviewe