184 INHIBITION OF CALCIUM EFFLUX EXTENDS THE DURATION OF CALCIUM SIGNALS IN PIG OOCYTES

Abstract

Calcium signaling involves the transient elevation in the intracellular free-calcium concentration, which is responsible for controlling a great number of biological functions. In many cell types, a signal is generated when calcium stored in the endoplasmic reticulum is released into the cytoplasm, followed by an influx of calcium across the plasma membrane. At the same time, calcium is removed from the cytosol by ATPases, which pump it back into the intracellular store or out of the cell. The size of the calcium signal is thus determined by the amount of calcium moving into and out of the cytoplasm. In the present study, we investigated the effect of inhibiting the movement of calcium across the plasma membrane on a calcium-signal that was artificially induced in pig oocytes. In vitro-matured pig oocytes were loaded with the calcium-indicator dye, fura-2. The release of calcium from the cytoplasmic stores was stimulated by adding ethanol at a final concentration of 7% and changes in the intracellular free-calcium levels were monitored by using InCyt Im2, a dual-wavelength fluorescence imaging system. In the control group, fluorescent measurements were performed in the presence of extracellular calcium. In additional treatment groups, the ethanol treatment was performed in (1) a calcium-free medium (prevents calcium influx); (2) the presence of 1 mM gadolinium (limits calcium fluxes across the plasma membrane in both directions); and (3) the presence of gadolinium in a calcium-free medium. In each group, 15 oocytes were measured; the data were subjected to 1-way ANOVA and differences between treatment means were compared by the Tukey's test. We found that in control oocytes, 7% ethanol induced a rise of 722.1 ± 49.2 nM in the cytoplasmic calcium levels and the average duration of the calcium transient was 309.1 ± 11.3 s. In the calcium-free medium, this increase was significantly smaller (only 117.9 ± 4.8 nM; P < 0.01), probably because the calcium release was not followed by an influx of calcium across the plasma membrane. In the presence of 1 mM gadolinium in the regular calcium-containing medium, the intracellular calcium levels climbed by 278.8 ± 45.4 nM and dropped to baseline levels only after 773.3 ± 79.9 s. Finally, in the calcium-free medium and in the presence of 1 mM gadolinium, 7% ethanol induced only a small rise (64.5 ± 6.3 nM) in the cytoplasmic calcium levels. These latter increases were both significantly lower (P < 0.01) compared to that in the control group. The results indicate that (1) intracellular calcium signals are sustained by a calcium influx from the extracellular medium and (2) preventing calcium efflux extends the duration of the cytoplasmic calcium elevation in the oocyte. This may have relevance in the manipulation of intracellular calcium levels in oocytes for the development of novel parthenogenetic activation methods.