Determination of the intracellular dissociation constant, K-D, of the fluo-3 center dot Ca2+ complex in mouse sperm for use in estimating intracellular Ca2+ concentrations

In order to calculate the actual, rather than the relative, intracellular C a2+ concentration (Ca2+)(i) in mammalian sperm cells, using fluorescent pro bes whose fluorescence emission differs between the probe Ca2+ complex and free probe, the value of the dissociation constant for the probe Ca2+ compl ex, K-D, is required. Interaction of the probe with cellular components may change the intracellular value of K-D from that determined in buffered sol ution. We had previously shown that fluo-3, whose Ca2+ complex is highly fl uorescent whereas free fluo-3 is not, could be used to monitor changes of ( Ca2+)(i) in mouse sperm. In this report, we describe a method for determini ng K-D for the fluo-3 Ca2+ complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca2+. Th e method involved treating the sperm with ionomycin to provide a plasma mem brane Ca2+ carrier, with nigericin to eliminate pH gradient, and with grami cidin D to eliminate membrane potential, such that (Ca2+)(i) equilibrates w ith medium Ca2+ concentration (Ca2+)(e), then titrating (Ca2+)(e) with EGTA in added aliquots to near: nil concentration. At EGTA concentrations in ex cess of total medium Ca2+, an approximation algorithm was used to calculate (Ca2+)(e), based on the known K-D for the EGTA Ca2+ complex. The fluoresce nce of the intracellular fluo-3 Ca2+ complex, F, decreased with increasing additions of EGTA; (Ca2+)(i) = (Ca2+)(e) was plotted as a linear function o f F/[F-max - F]; the slope gives K-D. At 37 degrees C, intracellular K-D wa s calculated to be 0.636 +/- 0.018 mu M (+/-SEM, n = 8). At 37 degrees C an d 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 intrace llular KD value implies probe interaction with cytosol components, primaril y those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca2+)(i), monitored with fluo-3 fluorescence, tha t occur on interaction of capacitated mouse sperm with the zona pellucida a nd may now be quantified, using 0.636 mu M for K-D of the intracellular flu o-3 Ca2+ complex. (C) 1999 Wiley-Liss,Inc.