Étude fonctionnelle de l'AMP-activated protein kinase chez l'huître creuse Crassostrea gigas

L objectif de cette thèse était de caractériser les éléments appartenant à la voie de signalisation énergétique AMP-activated protein kinase chez l huître creuse Crassostrea gigas afin de comprendre son implication dans la gestion de l énergie, en particulier en réponse à des conditions physiologiques qui sollicitent de l énergie telles que la reproduction, ou à des stress environnementaux comme l hypoxie ou le jeûne. Au niveau génomique, les trois sous-unités constitutives du trimère AMPK ainsi que plusieurs éléments impliqués dans cette voie de signalisation et dans les métabolismes glucidiques et lipidiques, potentiellement cibles de l AMPK, ont été décrits. Au niveau protéique, plusieurs anticorps hétérologues ciblant les isoformes de la sous-unité a et la phosphorylation du résidu thréonine 172 de la sous-unité a, témoin indirect de l activité AMPK, ont été utilisés. Deux sous-unités a tronquées dans le domaine kinase ont été caractérisées principalement dans les tissus musculaires suggérant leurs implications dans la fonction musculaire. Au cours d un stress hypoxique, une augmentation significative des quantités de sous-unités a tronquées a été observée dans le muscle lisse. Ce résultat suggère que pendant une durée d au moins 6 h, ces protéines tronquées sont nécessaires au maintien du métabolisme aérobie dans le muscle lisse, lui permettant ainsi de remplir son rôle de fermeture statique des valves. Nous avons suggéré une hypothèse indiquant que l accumulation in vivo de ces sous-unitésa tronquées pourrait exercer un rôle de modulation ou de transdomination négative de l activité de la sous-unité a entière. Dans la gonade, nous avons observé une activation de l AMPK tout au long du processus de gamétogénèse afin de supporter les processus cataboliques de création de gamètes. Une diminution de cette activation a été observée lors du stade anabolique de mise en réserve des ovocytes. Enfin, lors d un conditionnement en milieu contrôlé, une approche physiologique par privation de nourriture et une approche pharmacologique par injection d AICAR ont été réalisées pour provoquer une modulation de l AMPK. Les analyses ont montré que ni le jeûne ni l AICAR n ont induit une augmentation de la phosphorylation de la sous-unité a. Cependant, plusieurs changements liés à l injection de l AICAR ont été observés sur la physiologie de l huître : la modification du rapport AMP:ATP chez les huîtres nourries en comparaison aux huîtres à jeun, et une mortalité dépendante de la dose injectée d AICAR chez les huîtres mises à jeun. La caractérisation de l AMPK chez C. gigas ouvre de nombreuses perspectives exigeant des études fonctionnelles poussées afin de démontrer le rôle pivot de cette kinase dans la gestion de l énergie, comme démontré chez de nombreuses espèces de vertébrés, et ainsi décrypter le métabolisme énergétique de l huître.The objective of this thesis was to characterize elements implicated in the energypathway of the AMP-activated protein kinase of the Pacific oyster Crassostrea gigas. Thecharacterization of the elements was performed in the scope of understanding their involvementin energy management, particularly in response to physiological conditions requiring energy, asreproduction or environmental stress, such as hypoxia or fasting.At genomic level, the three subunits of AMPK trimer and several elements involved inAMPK signaling pathway and in carbohydrate and lipid metabolism, supposedly under AMPKcontrol, were described. Additionally, at proteomic level, several heterologous antibodiestargeting AMPKa subunit isoforms and threonine 172 phosphorylation site of AMPKa subunit,indirect witness of AMPK activity, were assayed. Two truncated a subunits in the kinase domainwere characterized essentially in muscles, suggesting their involvement in muscle function.During a hypoxic stress, a significant increase of truncated a subunits protein amount wasobserved in smooth muscle. These results suggest that, for a period of at least 6 h, thesetruncated subunits are necessary for the maintenance of aerobic metabolism in smooth muscle ofC. gigas, allowing it to fulfill its static closing valves. We suggested that in vivo accumulation oftruncated AMPKa could serve as modulator or as transdominant negative regulator of the fulllengthAMPKa activity. In the gonad, AMPK appeared to be activated through the process ofgametogenesis, in order to support the catabolic processes of gametes creation. During theanabolic phase, when oocyte reserves were created, a signal disruption was observed. Finally,during controlled experiment, a physiological approach by food deprivation and apharmacological approach using AICAR injections were performed to modulate AMPK signal.This analysis showed that neither fasting nor AICAR induced an increase of AMPKphosphorylation, as expected. Although, several changes related to AICAR injection wereobserved in oysters physiology, such as the change of the AMP:ATP ratio in fed oysters and aAICAR dose-related mortality in fasting oysters. AMPK characterization in C. gigas opens newperspectives demanding extensive functional studies to establish the key role of AMPK in energymanagement, as demonstrated in vertebrates species, in order to understand the oyster s energymetabolism.BREST-SCD-Bib. electronique (290199901) / SudocSudocFranceF

Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns

Background: Research using the Pacific oyster Crassostrea gigas as a model organism has experienced rapid growth in recent years due to the development of high-throughput molecular technologies. As many as 56,268 EST sequences have been sequenced to date, representing a genome-wide resource that can be used for transcriptomic investigations. Results: In this paper, we developed a Pacific oyster microarray containing oligonucleotides representing 31,918 transcribed sequences selected from the publicly accessible GigasDatabase. This newly designed microarray was used to study the transcriptome of male and female gonads, mantle, gills, posterior adductor muscle, visceral ganglia, hemocytes, labial palps and digestive gland. Statistical analyses identified genes differentially expressed among tissues and clusters of tissue-enriched genes. These genes reflect major tissue-specific functions at the molecular level, such as tissue formation in the mantle, filtering in the gills and labial palps, and reproduction in the gonads. Hierarchical clustering predicted the involvement of unannotated genes in specific functional pathways such as the insulin/NPY pathway, an important pathway under study in our model species. Microarray data also accurately identified reference genes whose mRNA level appeared stable across all the analyzed tissues. Adp-ribosylation factor 1 9arf1) appeared to be the most robust reference for normalizing gene expression data across different tissues and is therefore proposed as a relevant reference gene for further gene expression analysis in the Pacific oyster. Conclusions: This study provides a new transcriptomic tool for studies of oyster biology, which will help in the annotation of its genome and which identifies candidate reference genes for gene expression analysis

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description: In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.htm l. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion: A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism

Gametogenesis in the Pacific Oyster Crassostrea gigas: A Microarrays-Based Analysis Identifies Sex and Stage Specific Genes

Background: The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. Methodology/Principal Findings: Gene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters. Conclusions/Significance: This study allowed us to identify potential markers of early sex differentiation in the oyster C. gigas, an alternative hermaphrodite mollusk. We also provided new highly valuable information on genes specifically expressed by mature spermatozoids and mature oocytes

Microarray analysis highlights immune response of pacific oysters as a determinant of resistance to summer mortality.

International audienceSummer mortality of Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. A high heritability was estimated for resistance to summer mortality, which provided an opportunity to develop lines of oysters that were resistant (R) or susceptible (S) to summer mortality. Previous genome-wide expression profiling study of R and S oyster gonads highlighted reproduction and antioxidant defense as constitutive pathways that operate differentially between these two lines. Here, we show that signaling in innate immunity also operates differentially between these lines, and we hypothesize that this is at the main determinant of their difference in survival in the field. A reanalysis of our published microarray data using separate ANOVAs at each sampling date revealed a specific "immune" profile at the date preceding the mortality. In addition, we conducted additional microarray profiling of two other tissues, gills, and muscle, and both showed an overrepresentation of immune genes (46%) among those that are differentially expressed between the two lines. Eleven genes were pinpointed to be simultaneously differentially expressed between R and S lines in the three tissues. Among them, ten are related to "Immune Response." For these genes, the kinetics of R mRNA levels between sampling dates appeared different just before the morality peak and suggests that under field conditions, R oysters had the capacity to modulate signaling in innate immunity whereas S oysters did not. This study enhances our understanding of the complex summer mortality syndrome and provides candidates of interest for further functional and genetics studies

L'huître, cette sentinelle témoin d’un littoral à préserver

Dans la catégorie des mollusques marins, l’huître est une espèce essentielle de nos côtes françaises et à forts enjeux économique et patrimonial. Espèce ingénieure, elle rend de nombreux services dans les écosystèmes côtiers. Pourtant, les huîtres subissent de fortes pressions dont les pollutions d’origine humaine. Parmi ces pollutions que nous détaillerons dans cet article : les pollutions chimiques, les pollutions biologiques comme les microalgues toxiques naturelles -dont les recrudescences peuvent avoir une origine anthropique-, et de nouveaux contaminants chimiques et particulaires, les microplastiques, complétant ainsi le précédent article montrant l’importance écologique des huîtres et les menaces liées au réchauffement climatique qui pèsent sur elles (Lire Les huîtres : ces architectes méconnus des milieux côtiers). L’huître en tant qu’espèce sentinelle indicatrice de l’état de santé (ou de dégradation) de l’écosystème côtier, se retrouve donc à la place du lanceur d’alerte exigeant des mesures fortes de protection des habitats littoraux

Characterization of a gonad-specific transforming growth factor-beta superfamily member differentially expressed during the reproductive cycle of the oyster Crassostrea gigas.

International audienceThrough differential screening between oyster families selected for high and low summer survival, we have characterized a new transforming growth factor-beta (TGF-beta) superfamily member. This novel factor, named oyster-gonadal-TGFbeta-like (og-TGFbeta-like), is synthesized as a 307 amino acid precursor and displays 6 of the 7 characteristic cysteine residues of the C-terminal, mature peptide. Sequence comparison revealed that og-TGFbeta-like has a low percentage of identity with other known TGF-beta superfamily members, suggesting that og-TGFbeta-like is a derived member of this large superfamily. Real-time PCR (RT-PCR) analysis in different oyster tissues showed that og-TGFbeta-like is specifically expressed in both male and female gonads, at distinct levels according to the reproductive stage. Og-TGFbeta-like relative expression was the lowest at the initiation of the reproductive cycle and increased as maturation proceeded to achieve a maximal level in fully mature female and male oysters. In situ hybridisation demonstrated that expression was exclusively detected in the somatic cells surrounding oocytes and spermatocytes. The role of this newly-characterized TGFbeta member in the reproduction of cupped oyster is discussed in regard to the specificity and the localization of its expression, which singularly contrasts with the pleiotropic roles in a variety of physiological processes commonly ascribed to most TGF-beta family members identified so far

Identification of a tubulin-α gene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus.

International audienceFor non-model species, as many used for aquaculture, with minimal or no genomic information, relative quantification of gene expression studies requires preliminary research including the isolation of potential reference genes and the identification of those stably expressed under the biological conditions of interest. Here we report on the isolation of five partial gene sequences from gonad tissue cDNA in the functional hermaphrodite scallop Nodipecten subnodosus to be evaluated as reference genes: 18S-rRNA, riboprotein l8 (rp-l8), actin-β (act-β), elongation factor 1α (ef-1α) and alpha-tubulin-α (tub-α). We found that 18S-rRNA was stably expressed independently of the priming method used to reverse transcribe RNA to cDNA, oligo-dT or random hexamer. Stability analysis for the five putative reference genes with geNorm and NormFinder indicated that 18S together with rp-l8 were the most stable genes for normalization of gene expression during gonad development in both, male and female sexual regions of the hermaphrodite N. subnodosus. The least stable gene was tub-α, showing a biased expression profile between sexual regions of the gonad, therefore this gene was analyzed thereafter as a target gene together with vitellogenin (vit) and a DEAD-box RNA helicase (dbx) gene. Relative expression, estimated by normalization with the combination of 18S and rp-l8 as reference genes, indicated that as gonad development advanced two of the target genes were up-regulated, tub-α in the male region and vit in the female region. Whereas an increased expression was expected during development for vit for its known role in vitellogenesis, the increased expression of tub-α in the male sexual region was unexpected, and pointed toward this gene being a testis-specific α-tubulin isotype. Further analyses of gene expression among tissues indicated that tub-α is specifically and highly expressed in the male gonad, although expression in adductor muscle was also observed at significantly lower levels. The existence of testis specific α- and β-tubulins has been previously reported in other taxa, relating their function to sperm axoneme formation. Tissue-specific tubulin genes, particularly their promoters, have recently found an application as native promoters for transgene tissue-specific expression in research and reproductive control of insect plagues. The third target gene, a putative member of the DEAD-box RNA helicase family (dbx), showed no changes in expression during gonad development or between sexual regions, therefore it was chosen to discuss the different statistical inferences resulting from the arbitrary use of 'randomly chosen' reference genes when normalizing gene expression

Co-expression and regulation of ovarian vitellogenins in the Pacific oyster Crassostrea gigas

International audienceIn addition to the reported single cDNA sequence encoding vitellogenin in the Pacific oyster Crassostrea gigas (referred as Cg-Vtg1), we isolated and characterized the 3′ end of a novel vitellogenin (Vtg) in C. gigas, named Cg-Vtg2. The transcript size is up to 6 kb long and includes a D-type von Willebrand domain, characteristic of most vitellogenins, but not occurring in Cg-Vtg1. The expression of Cg-Vtg2 was found restricted to follicular cells in maturing and ripe female gonads. Quantitative expression analysis of both vitellogenins showed a closely correlated transcription pattern during the female gametogenic cycle, with the highest expression during the ripe (mature) stage, and a declining expression, to almost undetectable levels, in post-spawning (resting) stage. Levels of vitellogenin mRNA associates with the quantity of ingested food. Mature female oysters fed either a 2% or a 12% algal biomass per female biomass, showed a 2.9-fold increase in expression of Cg-Vtg1 and a 3.7-fold increase of Cg-Vtg2 expression levels in those females fed the higher level of algae, suggesting that Vtg's could be used as quantitative indicator of vitellogenesis. Our results suggest that vitellogenins are synthesized during the late period of vitellogenesis, when most of the yolk has already been incorporated into maturing oocytes