Fig. 2. The rapid, dose-dependent stimulatory action of bicarbonate increases with duration of exposure and with the age of the sperm preparation. (A) Depolarization-evoked responses were monitored in sperm stored in medium Na7.4 at ambient conditions for the indicated times (t=0 marks the start of indo-1 loading). Stimulus with medium K8.6 was applied (to a total of 175 cells from 11 animals) after 2-minute perfusions with medium Na7.4 containing 15 mM HCO₃^-. Mean rates of rise were determined for age bins of 10 minutes duration. Regression analysis indicates that the rate of rise increases linearly with age (r=0.9917). (B) Anti-phosphotyrosine immunoblot of extracts from sperm prepared as follows: lane 1, after swim-out (15 minutes at 37°C with 5% CO, 95% air) and washing in medium Na7.4; lane 2, after 15 minutes further incubation at ambient conditions in medium Na7.4 containing 15 mM HCO₃^-; lane 3, after 90 minutes incubation of the washed cells in capacitation medium at 37°C with 5% CO₂/95% air; lane 4, after 180 minutes incubation in storage medium Na7.4 at ambient conditions. The arrow indicates migration of a 120 kDa marker protein. Results are representative of three independent experiments. (C) Ca²⁺ channel activity was determined as in Fig. 1B, except that the third stimulus followed perfusions of varied durations using medium Na7.4 with 15 mM NaHCO₃. The depolarization-evoked rate of rise was normalized to the mean rate for the two preceding stimuli applied before exposure to HCO₃^- (each time point is averaged from 12-15 trials examined in three independent experiments). (D) In a similar protocol, the third stimulus followed a 2 minute perfusion using medium Na7.4 with 0, 1 or 15 mM NaHCO₃. Values shown are mean±s.e.m. (n=11, in three experiments): 1.30±0.17; 2.45±0.20; 6.41±0.87.