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

Abstract

While our knowledge of bivalve gametogenesis recently progressed, more molecular markers are needed in order to develop tissue imaging. Here, we identified stem cell and mitotic markers to further characterize the oyster early gametogenesis, mainly through immunofluorescence microscopy. Intense alkaline phosphatase activity, a nonspecific marker for stem cells, was detected on the outer edge of the gonad ducts at the post-spawning stage, suggesting the abundance of undifferentiated cells very early during the sexual cycle. This observation was confirmed using Sox2, a transcription factor specific for stem or germline cells, which decorated 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 already 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 cell cycle. DNA replication was indeed confirmed by an abundant in vivo incorporation of BrdU in 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 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, altogether with the stem cell markers, these data revealed that the epithelium delimiting the duct outer edge contains a dynamic population of undifferentiated cells.