A Quantitative Histologic Analysis of Oogenesis in the Flatfish Species Pleuronectes platessa as a Tool for Fisheries Management.

The following paper gives a detailed description of the oogenesis cycle for the European Plaice (Pleuronectes platessa), from oogonia to post-ovulatory follicle, including ovarian follicle and zona pellucida sizes. Noteworthy particularities were the difficulty in identifying cortical alveoli due to their very small size. Quantitative histology (stereology) on histological slides was used to determine a first size at maturity for females from the English Channel, which was found to be smaller compared to the literature (19 cm). Stereology also determined a first spawning event starting in January, with a peak in February and ongoing until March. Moreover, the use of stereology showed misclassifications for individuals categorized into a maturity phase using a macroscopic visual method. Misclassifications were found with individuals that had spawned (D) but were put under the immature (A) phase, and individuals in development (B) classified under D.

A macroscopic and stereological imaging dataset of Pleuronectes platessa ovaries.

The North Sea plaice, Pleuronectes platessa (Linnaeus, 1758), is a commonly studied commercial flatfish with poorly known ovarian histology. The following dataset is a collection of female plaice gonad images and their corresponding histological slides, collected during a complete season of the plaice's reproduction cycle. Stereology was used to determine the percentage of different structures found throughout the ovaries. Inter-agent calibrations were accomplished in order to harmonize the stereological readings, and were based on a comprehensive reading protocol and histological lexicon that were specifically written for the plaice's ovaries. The distribution and homogeneity of the different cell types found throughout the ovaries were also evaluated. This dataset can be used to automate the stereological reading process (through statistical learning methods for example) or to objectively determine the plaice's maturity phase, and link that information to either macroscopic measurements or through image analysis of the full ovaries.

Data for evolutive analysis of insulin related peptides in bilaterian species.

In bilaterian species, the amino acid sequence conservation between Insulin related peptides is relatively low except for the cysteine residues involved in the disulphide bonds. In the A chain, the conserved cystein residues are included in a signature motif. Investigating the variations in this motif would give insight into the phylogenetic history of the family. The table presented in this paper contains a large set of insulin-related peptides in bilateral phylogenetic groups (deuterostomian, ecdysozoan, lophotrochozoan). NCBI databases in silico wide screening combined with bibliographic researches provided a framework for identifying and categorising the structural characteristics of these insulin related peptides. The dataset includes NCBI IDs of each sequence with hyperlinks to FASTA format. Moreover, the structural type (α, ß or γ), the A chain motif, the total number of cysteins, the C peptide cleavage mode and the potential additional domains (D or E) are specified for each sequence. The data are associated with the research article "Molecular evolution and functional characterisation of insulin-related peptides in molluscs: contributions of Crassostrea gigas genomic and transcriptomic-wide screening" [1]. The table presented here can be found at http://dx.doi.org/10.17632/w4gr8zcpk5.4#file-21c0f6a5-a3e3-4a15-86e0-e5a696458866.

Molecular evolution and functional characterisation of insulin related peptides in molluscs: Contributions of Crassostrea gigas genomic and transcriptomic-wide screening.

Insulin Related Peptides (IRPs) belong to the insulin superfamily and possess a typical structure with two chains, B and A, linked by disulphide bonds. As the sequence conservation is usually low between members, IRPs are classified according to the number and position of their disulphide bonds. In molluscan species, the first IRPs identified, named Molluscan Insulin-related Peptides (MIPs), exhibit four disulphide bonds. The genomic and transcriptomic data screening in the Pacific oyster Crassostrea gigas (Mollusc, Bivalvia) allowed us to identify six IRP sequences belonging to three structural groups. Cg-MIP1 to 4 have the typical structure of MIPs with four disulphide bonds. Cg-ILP has three disulphide bonds like vertebrate Insulin-Like Peptides (ILPs). The last one, Cg-MILP7 has a significant homology with Drosophila ILP7 (DILP7) associated with two additional cysteines allowing the formation of a fourth disulphide bond. The phylogenetic analysis points out that ILPs may be the most ancestral form. Moreover, it appears that ILP7 orthologs are probably anterior to lophotrochozoa and ecdysozoa segregation. In order to investigate the diversity of physiological functions of the oyster IRPs, we combine in silico expression data, qPCR measurements and in situ hybridization. The Cg-ilp transcript, mainly detected in the digestive gland and in the gonadal area, is potentially involved in the control of digestion and gametogenesis. The expression of Cg-mip4 is mainly associated with the larval development. The Cg-mip transcript shared by the Cg-MIP1, 2 and 3, is mainly expressed in visceral ganglia but its expression was also observed in the gonads of mature males. This pattern suggested the key roles of IRPs in the control of sexual reproduction in molluscan species.

Morphological and molecular criteria allow the identification of putative germ stem cells in a lophotrochozoan, the Pacific oyster Crassostrea gigas.

While our knowledge of bivalve gametogenesis recently progressed, data on early stages of gametogenesis remain to be developed, especially when dealing with germinal stem cells (GSC) and their niche in these organisms. Here, we wish to develop a strategy to identify putative GSC in Pacific oyster Crassostrea gigas based on morphological criteria combined with vasa marker expression. A histological quantitative approach, based on stereology, allowed us to identify two types of early germ cells in the germinal epithelium, one presenting round nuclei and the other irregular ones. Both early germ cell types present slightly condensed chromatin in nucleus, are vasa-positive and the Oyvlg (oyster vasa-like gene) expression in these cells is recorded throughout the whole gametogenesis process. The microenvironment of an early germ cell in oyster includes an associated somatic cell presenting an immunolabeling for BMP2/4 and a close myoid cell. In agreement with the GSC characteristics in other species, we postulate that putative germ stem cells in C. gigas correspond to the early germ cell type with irregular nucleus shape; those early germ cells with a round nucleus may consist in progenitors.

The endocrine-disrupting effect and other physiological responses of municipal effluent on the clam Ruditapes decussatus.

In order to document the potential endocrine disrupting and toxic effect of the municipal wastewater effluents discharged into the Sfax coastal area (South of Tunisia), specimens of clam R. decussatus were collected from a reference site and were in vivo exposed to treated sewage effluent for 30 days. To this end, estrogenic and androgenic activities were measured in the gills to assess potential accumulation and regulation of active compounds. After effluent exposure androgenic activity in organic extracts increased up to fivefold compared to controls and remained elevated, while estrogenic activity was not significantly affected by exposure. As a consequence, remarkable disruptions in the gametogenesis activity, glycogen content, and Vitellogenin-like protein levels in male clams were observed. A parallel analysis of heavy metals in clam tissues was determined. A significant uptake of Ni, Zn, and Pb in soft tissues of exposed clams was observed. The significant increase of malondialdehyde (MDA) concentrations as a function of exposure time implies that clams have been exposed to an oxidative stress probably due to the presence of high metal concentrations in sewage effluent. Correlation analysis has revealed a statistically significant and positive relationship between MDA levels and metal concentrations in clams' tissues. The acetylcholinesterase activity was not significantly affected by exposure. Altogether, these results showed that a short-term exposure to a mixture of chemical compounds released by the Sfax wastewater treatment plant induce adverse physiological and reproductive effects in R. decussatus. Further studies are underway in order to evaluate its long-term impacts on aquatic wildlife in the gulf of Gabes area.

Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. BIOLOGICAL SIGNIFICANCE: This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa in the Pacific oyster. This work improves the current knowledge in marine bivalve hatchery.

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.

Intragonadal somatic cells (ISCs) in the male oyster Crassostrea gigas: morphology and contribution in germinal epithelium structure.

The ultrastructure of somatic cells present in gonadal tubules in male oyster Crassostrea gigas was investigated. These cells, named Intragonadal Somatic Cells (ISCs) have a great role in the organization of the germinal epithelium in the gonad. Immunological detection of α-tubulin tyrosine illustrates their association in columns from the basis to the lumen of the tubule, stabilized by numerous adhesive junctions. This somatic intragonadal organization delimited some different groups of germ cells along the tubule walls. In early stages of gonad development, numerous phagolysosomes were observed in the cytoplasm of ISCs indicating that these cells have in this species an essential role in the removal of waste sperm in the tubules. Variations of lipids droplets content in the cytoplasm of ISCs were also noticed along the spermatogenesis course. ISCs also present some mitochondria with tubullo-lamellar cristae.

Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues.

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.