Determination of the intracellular dissociation constant, KD, of the Fluo-3 � Ca2+ complex in mouse sperm for use in estimating intracellular Ca2+ concentrations

Abstract

In order to calculate the actual, rather than the relative, intracellular Ca(2+) concentration (Ca(2+))(i) in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe. Ca(2+) complex and free probe, the value of the dissociation constant for the probe. Ca(2+) complex, K(D), is required. Interaction of the probe with cellular components may change the intracellular value of K(D) from that determined in buffered solution. We had previously shown that fluo-3, whose Ca(2+) complex is highly fluorescent whereas free fluo-3 is not, could be used to monitor changes of (Ca(2+))(i) in mouse sperm. In this report, we describe a method for determining K(D) for the fluo-3. Ca(2+) complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca(2+). The method involved treating the sperm with ionomycin to provide a plasma membrane Ca(2+) carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca(2+))(i) equilibrates with medium Ca(2+) concentration (Ca(2+))(e), then titrating (Ca(2+))(e) with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca(2+), an approximation algorithm was used to calculate (Ca(2+))(e), based on the known K(D) for the EGTA. Ca(2+) complex. The fluorescence of the intracellular fluo-3. Ca(2+) complex, F, decreased with increasing additions of EGTA; (Ca(2+))(i) = (Ca(2+))(e) was plotted as a linear function of F/[F(max) - F]; the slope gives K(D). At 37 degrees C, intracellular K(D) was calculated to be 0.636 +/- 0.018 microM (+/-SEM, n = 8). At 37 degrees C and 20 degrees C, K(D) values in MJB were calculated to be 0.502 +/- 0.022 and 0.578 +/- 0.029 (+/-SEM, n =8 and n = 6), respectively. The higher intracellular K(D) value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca(2+))(i), monitored with fluo-3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 microM for K(D) of the intracellular fluo-3. Ca(2+) complex.