Cell Proliferation Detected with Flow Cytometric Cell Cycle Analysis and Immunohistochemical Detection of Proliferating Cell Nuclear Antigen (PCNA) from Somatic Tissues of Eastern Oysters,Crassostrea virginica∗

Abstract

Two novel biomarker of response assays were developed and compared for use with bivalves. Bivalve mollusks are often used as bioindicators to monitor contaminant body burdens and are employed globally in pollution monitoring and as sentinels of environmental quality. The prevalence of proliferating cells in tissues of the eastern oyster, Crassostrea virginica, (n = 10) was investigated by using immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and by flow cytometric analysis of DNA in cell cycle phases. The PCNA protein is highly conserved and central to DNA synthesis in dividing cells. This protein was detected by using a commercial antibody in the histological staining of gills, mantle, digestive gland, stomach, and labial palps. Cells obtained from these same organs and from the hearts of the oysters were used for flow cytometry (FCM). The proportions of nuclei in the S plus G2/M and G2/M fractions were compared with percentages of PCNA-positive cells scored through microscopy. Proliferation responses generated by microscopy and FCM (S plus G2/M) were equal for digestive gland, gills, stomach, and mantle. The proliferation response in labial palps was significantly higher by PCNA microscopy than by DNA FCM (S plus G2/M). For PCNA microscopy results, a significantly higher proliferation response was noted both for labial palps as compared to gills and mantle and for stomach and digestive gland as compared to gills (P < 0.005). For DNA FCM results, the percentages of proliferating cells in G2M fractions were significantly higher for labial palps and heart when compared to gills, mantle, and digestive gland. By FCM (S plus G2M), the percentages of proliferating cells followed a similar trend, although no significant differences were found between organs. The choice of a relatively highly proliferative tissue, such as labial palps as determined by this study, has utility for developing a cell line, for examining proliferation as a biomarker due to stressor impacts in feral oysters, and for detecting impacts of compounds on proliferative mechanisms.