Quantitation of bovine sperm cytoplasmic calcium with Quin-2 and Fura-2: evidence that external calcium does not have direct access to the sperm cytoplasm.

Internal calcium levels of sperm loaded with Quin-2 in the absence or presence of exogenous calcium were 63 +/- 5 and 189 +/- 19 nM, respectively. These values were similar to those determined by Fura-2. Surprisingly, however, dye loaded sperm depleted of internal calcium did not take up calcium from the medium into the cytoplasm upon re-addition of this ion. Uptake was rapid and maximal, however, if these cells were exposed to the calcium ionophores A23187 or ionomycin. Increasing [Quin-2]i progressively lowered [Ca2+]i in spite of the presence of exogenous calcium during dye loading. This anomaly was not due to interference of the fluorophores with calcium uptake, since exogenous 45Ca2+ was taken up at the same rate and to the same extent by control and fluorophore loaded sperm. This 45Ca2+ uptake was mitochondrial and energy dependent. Also, inhibition of mitochondrial calcium accumulation during dye loading lowered [Ca2+]i to values similar to those observed for calcium depleted sperm. These results suggest an extreme impermeability of the sperm plasma membrane to direct calcium entry into the cytoplasm while substantial amounts of calcium entry occurs into the sperm mitochondria.