Early gametogenesis in the Pacific oyster: new insights using stem cell and mitotic markers

Abstract

While our knowledge of bivalve gametogenesis has progressed in recent times, more molecular markers are needed in order to develop tissue imaging. Here, we identified stem cell and mitotic markers to further characterize oyster early gametogenesis, mainly through immunofluorescence microscopy. Intense alkaline phosphatase activity, a non-specific marker for stem cells, was detected on the outer edge of the gonad ducts at the post-spawning stage, suggesting an abundance of undifferentiated cells very early during the sexual cycle. This observation was confirmed using an antibody against Sox2, a transcription factor specific for stem or germline cells, which labeled cells in the gonad duct inner mass and ciliated epithelium early during the initial oyster sexual cycle. Moreover, Vasa, a cytoplasmic marker for germline cells, was also detected in the gonad acini and duct cells, thus confirming that germline cells were abundant early on. In addition, the binding of the minichromosome maintenance MCM6 protein to chromatin indicated the gonad acini and duct cells were engaged in the cell cycle. DNA replication was indeed confirmed by an abundant incorporation of BrdU into the duct cell chromatin. Finally, proliferation of acini and duct cells was demonstrated by the chromatin-bound Ser10-phosphorylated histone H3, a mitotic marker. The markers for the cell cycle and mitosis used here thus indicate that acini and duct cells were already actively dividing early during the oyster sexual cycle. In addition, together with the stem cell markers, these data reveal that the epithelium delimiting the duct outer edge contains a dynamic population of undifferentiated cells.

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

“…However, the second immunodetection using fluorescence allowed us to conclude that Oyster Vasa positive labelling is exclusively observed in early germ cells, therefore, in the GSC. Recent data of Cavelier et al (2017) report similar immunolabelling of Vasa in the oyster's gonad. In contrast, unlike Milani et al 2017, we did not find any Vasa labelling in other tissues than in the gonad.…”

“…The study of the distribution of putative GSC on a Whole Slide Image (WSI) allowed us to conclude that the germinal niches in adult oyster are uniformly distributed on a gonad scale. The distribution of germinal niches in oyster from the first gametogenesis and the following ones could expand from the outermost part of the gonad (against the mantle) (Santerre et al 2013;Cavelier et al 2017) to the innermost part (against the digestive area).…”

“…Markers of stemness are usually used in histochemistry and immunochemistry approaches to identify the GSC. Recent publication of Cavelier et al (2017) reports alkaline phosphatase activity in germ cells localized in outer genital C. gigas gonad ducts. The dynamics of stem cells was also classically investigated using BrdU or phosphorylated-histone H3 labelling to detect quiescence or reduced rate of proliferation (Newmark and Alvarado 2000;Jemaà et al 2014).…”

“…Fabioux et al (2004b) showed that, in the particular case of oyster C. gigas, the vasa-like gene (Oyvlg) is specifically expressed in gonad tissue as well as the protein (Fabioux et al 2009) but the cellular location of this protein was not investigated. More recently, Cavelier et al (2017) identified Vasa-positive germ cells in gonad ducts in the earliest stages of gametogenesis. Likewise, Obata et al (2010) detected Vasa-positive cells in the Mytilus galloprovincialis mussel and Milani et al (2015Milani et al ( , 2017 confirmed by immunodetection that Vasa is a germ cell marker in several bivalve species.…”

Gene expression profiles at different stages for formation of pearl sac and pearl in the pearl oyster Pinctada fucata

“…Besides, the enrichment of these genes in epithelial cell proliferation and differentiation-related processes clarify their potentiality in the formation of pearl sac. Stem cell-specific transcription factor SOX2 was also very recently identified from proliferating gonad duct of Pacific oyster [100]. The signal transducers and activators of the transcription family gene STAT have been reported for P. fucata in a previous study, where it was up-regulated upon grafting peaking at day 3 [32].…”