To assess the requirement of potassium loss for the biochemical and morphological changes associated with apo-ptosis, oocytes were cultured in ion-deficient medium supplemented with 150 mM KCl to disrupt the electrochemical gradient of this ion. Data from other experimental models have shown that this medium will prevent potassium efflux during apoptosis and consequently will suppress the activation of apoptotic enzymes. Likewise, incubation of oocytes in this medium completely prevented both basal and DXR-induced oocyte fragmentation (Figs. 1C and 2).
The ability of 150 mM KCl to prevent oocyte fragmentation was mimicked by 150 mM LiCl but not by 150 mM NaCl (the latter being essentially normal culture medium; Fig. 2). Interestingly, however, some level of chromatin cleavage still occurred in DXR-treated oocytes after coculture with 150 mM KCl, as revealed by both the comet assay (Fig. 1F) and ISEL analysis (Fig. 1I), despite the complete absence of cellular fragmentation (Fig. 1C). These results differ from what has previously been found in thymocytes and suggest the existence of a novel potassium-independent pathway leading to DNA degradation in oocytes.