BackgroundThe 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 FindingsGene 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/SignificanceThis 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.

The aim of the present study was to investigate the effect of size of from which goat oocytes originate on their subsequent ability to be fertilized and to undergo early embryonic development in vitro. Nonatretic follicles larger than 2 mm in diameter were dissected and distributed into three groups according to size (small: 2-3 mm; medium: 3.1-5 mm; large: > 5 mm). Cumulus-oocyte complexes were isolated from the follicles and only those with a compact multilayered cumulus were selected for in vitro maturation. After maturation, 70%, 83% and 97% of oocytes from small, medium and large follicles, respectively, were at metaphase II. After in vitro fertilization, no significant difference was observed in the cleavage rate 40 h after insemination between oocytes from small (46%) and medium (55%) follicles, and between oocytes from large follicles (69%) and ovulated oocytes (75%). After in vitro culture, significantly more embryos from small follicles arrested before or at the 8-16 cell stage (84% compared with 53%, 45% and 39% of embryos from medium and large follicles and ovulated oocytes, respectively). The proportion of morulae and blastocysts obtained was 10% and 6% from small follicles, 35% and 12% from medium follicles, 29% and 26% from large follicles and 20% and 41% from ovulated oocytes. Oocytes from small and medium follicles yielded a significantly lower proportion of hatched blastocysts (0% and 3%, respectively) than did those from large follicles and from ovulated oocytes (15% and 34%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40).To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.Growth factors orchestrate growth and development in metazoan organisms. Despite the huge variety of growth factors characterized in vertebrates, only a few have been identified in Protostome lineages and are mainly restricted to the Ecdysozoan model organisms Caenorhabditis elegans and Drosophila melanogaster. Thus, the third branch of bilaterian, named Lophotrochozoa, is an obviously understudied group of animals, since none of the major model organisms presently belong to this clade (1). Recent reports show that Lophotrochozoan animals exhibit biological characteristics that are considered ancestral, or at least less derived from the ancestral state than in other established systems that are known (2). Since bivalve mollusks belong to the Lophotrochozoa, they are good candidates to characterize growth factors and then contribute to our understanding of the evolution of growth and developmental regulations in bilaterian animals. Furthermore, a better knowledge of the molecular control of mollusk physiology may help improve the hatchery production of these economically important animals. Unfortunately, the number of growth factor genes identified so far at the molecular level in bivalve mollusks is extremely limited. In the pacific oyster, Crassostrea gigas, only four members of the transforming growth factor-␤ superfamily and their corresponding receptors have already been described (3-6). This lack of knowledge is above all the consequence of both the paucity of genomic sequence information and the difficu...

Members of the short neuropeptide F (sNPF) family of peptides and their cognate receptors play key roles in a variety of physiological processes in arthropods. In silico screening of GigasDatabase, a specific expressed sequence tag database from the Pacific oyster Crassostrea gigas, resulted in the identification of a receptor (CgsNPFR-like) phylogenetically closely related to sNPF receptors (sNPFRs) of insects. A reverse endocrinology approach was undertaken to identify the peptide ligand(s) of this orphan receptor. Though structurally distinct from insect sNPFs, three RFamide peptides derived from the same precursor, i.e. GSLFRFamide, SSLFRFamide and GALFRFamide, specifically activate the receptor in a dose-dependent manner, with respective EC 50 values (halfmaximal effective concentrations) of 1.1, 2.1 and 4.1 μmol l −1 . We found that both Cg-sNPFR-like receptor and LFRFamide encoding transcripts are expressed in the oyster central nervous system and in other tissues as well, albeit at lower levels. Mass spectrometry analysis confirmed the wide distribution of LFRFamide mature peptides in several central and peripheral tissues. The Cg-sNPFRlike receptor was more abundantly expressed in ganglia of females than of males, and upregulated in starved oysters. In the gonad area, highest receptor gene expression occurred at the start of gametogenesis, when storage activity is maximal. Our results suggest that signaling of LFRFamide peptides through the CgsNPFR-like receptor might play a role in the coordination of nutrition, energy storage and metabolism in C. gigas, possibly by promoting storage at the expense of reproduction.