MONOLAYER FORMATION AND DNA-SYNTHESIS OF THE OUTER EPITHELIAL-CELLS FROM PEARL OYSTER MANTLE IN COCULTURE WITH AMEBOCYTES

In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalve s. The outer epithelia of the pallial mantle of the pearl oyster, Pinc tada ficata martensii, were separated in cell sheets from the inner co nnective tissue layers by digestion with Dispase. Clumps of the separa ted mantle epithelia were inoculated onto the amebocyte layers prepare d on the bottom of culture dishes and maintained at 20 degrees C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) gluc ose was used as a culture medium, The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squ amous, anti migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to ce ll junction in the sheets, 6 d after the inoculation, 5-bromo-2'-deoxy uridine was added to the culture at 30 mu M. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2'-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in th e epithelial cell sheets, indicating active DNA synthesis in the epith elial sheets. Thus, cocultured with amebocytes; the outer epithelial c ells from pallial mantle tissue formed a monolayer sheet and started D NA synthesis. The morphological features of the mantle outer epithelia l cells are analogous to those described for the in vivo cutaneous wou nd healing process, suggesting that the epithelium-amebocyte interacti on is important in the regeneration of epithelium in bivalves.