Cell Culture and Gene Expression Studies in Relation to Biomineralization in the Black-lip Pearl Oyster, Pinctada margaritifera

Abstract

Nacre is composed of aragonite platelets and organic material formed by molluscs as inner shell layer through biomineralization process, and mantle epithelial cells control nacre formation. A primary culture of granulated epithelial cells was established from mantle tissue of Pinctada margaritifera, through a process of continuous sub-culturing at 28°C in yeast supplemented sea water culture medium. Nuclear beads were placed in culture wells containing semi-solid agar medium and incubated in vitro with cultured granulated epithelial cells in order to evaluate the nacre secretion. On visual observation, a brown coloration was observed on the surface of the bead after 7-10 days. Evaluation of the surface of the nuclear beads by scanning electron microscopy (SEM) after 60 days of incubation revealed a good brick and mortar pattern, characteristic of nacreous layer formation. A lustrous hue was also seen to develop on bead surfaces after this stage. SEM images of a cross section of the nacre-coated bead showed a pattern of arrangement of aragonite tablets similar to that seen in cross sections of the nacre layer of shell of molluscs. The functional ability of cultured granulated epithelial cells was further confirmed by detecting gene expression of two matrix proteins, nacrein and amorphous calcium carbonate binding proteins (ACCBP), which play an important role in formation of the nacreous layer, in both cultured cells and in native mantle tissue. Amplification products for nacrein (480 bp) and ACCBP (500 bp) genes were obtained in both native mantle tissue and in vitro cultured mantle epithelial cells. There was good correlation between the expression patterns of the two genes in in vitro cultured cells and in native mantle tissue, signifying that cultured mantle epithelial cells retain their functional characteristics of biomineralization.