Fig. 2. Tyrosine phosphorylation and zona pellucida binding. Cauda epididymal spermatozoa were capacitated for 90 minutes in complete BWW (1), BWW–HCO₃^– (2), BWW–Ca²⁺ + pentoxifylline (ptx) + dibutyryl cyclic-AMP (cAMP) (3) or BWW–Ca²⁺+Sr²⁺+ptx+cAMP (4). (A) Representative western blot of sperm proteins probed with anti-phosphotyrosine. The experiment was repeated three times. (B) Percentage of spermatozoa displaying partial (white bars) and complete (black bars) flagellum labelling following immunofluorescence with anti-phosphotyrosine. The experiment was repeated three times, with a minimum of 200 cells scored for each one. Treatment 2 led to a significant (P<0.05) reduction in the percentage of cells exhibiting phosphotyrosine expression over the entire tail when compared to control treated cells (1). By contrast, treatment 3 and 4 resulted in significant (P<0.005) increases in the proportion of spermatozoa exhibiting partial and complete labelling. ^*, P<0.05; ^**, P<0.005. (C) Correlation between tyrosine phosphorylation and zona pellucida binding. Cauda spermatozoa were prepared as described followed by a 30-minute capacitation with salt-stored oocytes. The mean number of sperm bound to each zona is expressed as a percentage of the control (treatment 1) for four repeats. Cells capacitated in solution 2 express low levels of flagellar tyrosine phosphorylation exhibited a significant (P<0.05) decrease in zona binding compared with the controls. Conversely, the enhanced tyrosine phosphorylation status of spermatozoa capacitated in solution 4 was associated with a significant (P<0.005) elevation in the zona binding capacity (Fig. 2C). ^*, P<0.05; ^**, P<0.005.