Fig. 2. Impact of extracellular calcium on intracellular pH. (A) Spermatozoa (10x10⁶/ml) were incubated in either complete BWW (circles) or BWW -Ca²⁺ (squares). Approximately 40 minutes before the time shown, 1 ml of sperm was harvested, washed and incubated with 9 µM BCECF. Following 40 minutes incubation, the cells were washed to remove free dye. A ratiometric analysis of 440/530 nm was performed and plotted against known standards to determine the [pH]_i. The results are presented as the average of three experiments performed in triplicate; ^*P<0.05. (B) Spermatozoa (10x10⁶/ml) were incubated in either BWW -Ca²⁺ (lane 1), complete BWW (lane 2) or BWW with a pH of 8.2 (lane 3). After 2 hours, 1 ml of sperm from each treatment was harvested, washed and incubated with 9 µM BCECF. Following 40 minutes incubation, the cells were washed to remove free dye. A ratiometric analysis of 440/530 nm was performed and plotted against known standards to determine the [pH]_i. The results are presented as the average of three experiments performed in triplicate; ^*P<0.05. (C) Spermatozoa (10x10⁶/ml) were incubated in either BWW -Ca²⁺ (lane 1), complete BWW (lane 2) or BWW with a pH of 8.2 (lane 3). Following a 3-hour incubation the cells were lysed (2% SDS) and subject to anti-phosphotyrosine western blot analysis as described in Materials and Methods. (D) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three independent experiments.