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.

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.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas.

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Cg-SoxE and Cg-ß-catenin, two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan, the Pacific oyster Crassostrea gigas.

Sex determination is poorly known in mollusks, lophotrochozoa and most hermaphrodites. In the oyster Crassostrea gigas, an irregular successive hermaphrodite, sex determination may occur at the end of a gametogenetic cycle to prepare the next cycle. To investigate further into these aspects we have focused on orthologs of SoxE and ß-catenin, key players of the male and female gonadic differentiation respectively in mammals. Based on phylogenetic analyses, Cg-SoxE, the oyster's SoxE ortholog, is closely related to vertebrate SoxE genes while Cg-ß-catenin, the oyster's ß-catenin ortholog, is classified amongst lophotrochozoa. The mRNA expression measured by qPCR in the gonadic area during a gametogenetic cycle is maximal for Cg-SoxE when sex is indiscernible and for Cg-ß-catenin in mature females. Both expressions are localized from early germ cells to spermatocytes and pre-vitellogenic oocytes, and potentially in somatic cells. Cg-ß-catenin is also expressed in vitellogenic oocytes. These actors may be involved in early oyster gonadic differentiation, which includes sex determination. Our results enhance the understanding of sex determination in C. gigas and in mollusks and they provide additional knowledge in compared genomics of reproduction and in molecular phylogeny.