Potassium and Apoptosis in Germ Cells (Oocytes)
We have recently used in vitro culture of oocytes as a model to dissect the intracellular pathways responsible for chemotherapy-induced female germ cell apoptosis. Apoptosis is clearly apparent in these cells by observing the retraction of the oolemma away from the zona pellucida (cytoplasmic condensation), membrane budding, chromatin cleavage, and eventual fragmentation of the oocyte into apo-ptotic bodies of unequal sizes (Fig. 1B). Oocytes cultured in medium alone exhibited negligible levels (<3%) of apoptosis after 24 h (Figs. 1A and 2), whereas exposure of oocytes to 200 nM DXR for 24 h induced cellular fragmentation in over 60% of the oocytes (Fig. 2B). To visualize changes in intracellular potassium, the cells were loaded with a potassium-binding fluorescence indicator dye (PBFI) 1 h before microscopic analysis.
Control (non-drug treated) oocytes stained brightly and uniformly with PBFI, whereas those stimulated to die by DXR treatment consistently displayed reduced fluorescence indicative of a net loss of intracellular potassium (Fig. 3). The decrease in intracellular potassium occurred coincident with a decrease in cell size and prior to cell budding (and remained low in oocytes that had undergone fragmentation into apoptotic bodies), consistent with the proposed role for the loss of this ion as an early death event mediating cell shrinkage.
FIG. 1. Cytochemical analysis of mouse oocytes treated with DXR in the absence or presence of potassium. A-C) Representative DIC photomicrographs of oocytes after a 24-h incubation in vehicle (control, A), 200 nM DXR (B), or 200 nM DXR in the presence of 150 mM KCl (C). Note that DXR-induced oocyte fragmentation into apoptotic bodies is completely prevented by potassium. D-F) Chromatin cleavage, as assessed by the comet assay in oocytes incubated in control medium (D) or in medium containing 200 nM DXR in the absence (E) or presence of 150 mM KCl (F). Note that although the plume of low-molecular weight DNA fragments visible in the DXR-treated oocyte (E) is absent when oocytes are coincubated with 150 mM KCl (F), some level of DNA fragmentation still occurs in oocytes treated with DXR in the presence of potassium (evidenced by the electrophoretic shift of chromatin to the oocyte plasma membrane). G-I) DNA cleavage, as assessed by ISEL, in control oocytes (G), oocytes incubated with DXR (H), or DXR with 150 mM KCl (I). Note that ISEL detects DNA cleavage in DXR-treated oocytes in the absence or presence of KCl. These data are representative of results obtained in three independent experiments.
FIG. 2. Quantitative analysis of the effects of potassium on the incidence of in vitro murine oocyte fragmentation induced by DXR. Pools of mature (metaphase II) oocytes harvested by superovulation from adult female mice were denuded of cumulus cells and then cultured without or with DXR (DXR, 200 nM) in the absence or presence of 150 mM KCl, NaCl, or LiCl for 24 h. After culture, the number of oocytes that underwent fragmentation out of the total number of oocytes cultured in each treatment group was determined by light-microscopic analysis. The data represent the combined results from at least four independent experiments (mean ± SEM) with the total number of oocytes analyzed per group provided over the respective bar (*P < 0.05 versus DXR alone).
FIG. 3. Representative fluorescence microscopic analysis of intracellular potassium levels in control (A) and DXR-treated (B) oocytes. Oocytes were cultured without (A) or with (B) DXR (200 nM) for 20 h, after which PBFI was added to the culture at a final concentration of 5 ^M. Cells were examined 1 h later using live-mount microscopy with an excitation of 340 nm and an emission of 505 nm. Arrows indicate drug-treated oocytes that have undergone fragmentation.