Fig. 3. Localisation of phosphotyrosine residues on live spermatozoa. Cauda epididymal spermatozoa were capacitated for 90 minutes in either complete BWW (1), BWW–HCO₃^– (2) or BWW–Ca²⁺+Sr²⁺+ptx+cAMP (4). Tyrosine phosphorylation was assessed by immunofluorescence or by a 30-minute capacitation with anti-phosphotyrosine-coated magnetic beads. Controls were included where beads were preabsorbed with 20 mM O-phospho-L-tyrosine (pY). (A) Representative images of live spermatozoa displaying phosphotyrosine residues on the surface of the head visualised by immunofluorescence and immunobead assay. Scale bar, 10 µm. (B) Percentage of viable spermatozoa expressing phosphotyrosine residues on the head as assessed by immunobead assay. The experiment was repeated three times, with a minimum of 200 viable cells scored for each experiment. Surface tyrosine phosphorylation was observed on the head of approximately 9% of spermatozoa capacitated in complete media. This was significantly (P<0.05) decreased in uncapacitated cells (solution 2). By contrast, capacitation in solution 4 induced significantly (P<0.01) increased surface labelling compared with uncapacitated cells. Pre-incubation of phosphotyrosine beads with O-phospho-L-tyrosine significantly (P<0.01) reduced the labelling of cells in solution 4 to approximately 2.5%. ^*, P<0.05; ^**, P<0.01.