Abstract
We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na+/K+ ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW. J. Exp. Zool. 311A:80-90, 2009. (c) 2008 Wiley-Liss, Inc.