Fig. 10. Sperm function and calcium-dependent ATP binding proteins. (A) Approximately 10x10⁶ human spermatozoa were harvested, washed and lysed in either 5% CHAPS, with 0.6 mM CaCl₂ (lane 1) or without CaCl₂ (lane 2). Approximately 2 µCi -³²P-labeled 8-azidoadenosine-5'-triphosphate [8-N₃ATP; 20 Ci/mmol, 2 mCi/ml] was added. Following 10 minutes incubation, the cells lysates were UV irradiated (2 minutes at 1200 MW/m²). An equal amount of loading dye was then added to 1 µg of protein, which was subject to 10% SDS-PAGE. Autoradiography was performed using X-ray film according to the manufacturer's instructions. (B) Cells were incubated in BWW -Ca²⁺, (1) or BWW medium (vehicle control, 2), or BWW medium containing 1 (3), 5 (4) or 10 (5) µM thapsigargin. After 3 hours, 50 µl of cell suspension was taken and ATP measured as described in Materials and Methods. Inset shows relative increase in [Ca²⁺]_i following the addition of thapsigargin (Tps, 10 µM). The graph represents data from three experiments performed in duplicate; ^**P<0.01. (C) Cells were incubated in BWW medium (vehicle control, lane 1,), or BWW medium containing 1 (lane 3), 5 (lane 4) or 10 (lane 5) µM thapsigargin. As a positive control, cells were incubated in BWW -Ca²⁺ (lane 2). After 3-hours incubation, cells were harvested, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected by western blot analysis using the anti-phosphotyrosine antibody, 4G10. The positions of the molecular mass markers (kDa) are shown on the left. (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.