Fig. 4. Tyrosine phosphorylation of live spermatozoa bound to the zona pellucida (ZP). Cauda epididymal spermatozoa were capacitated for 1 hour in BWW–Ca²⁺+Sr²⁺+ptx+cAMP followed by a 30-minute incubation with salt-stored oocytes and FITC-conjugated anti-phosphotyrosine. Sperm-ZP complexes were washed and labelling of bound spermatozoa was scored. (A) Representative fluorescent (top) and phase-contrast (bottom) images of phosphotyrosine labelling of sperm bound to ZP. Analysis revealed that 100% of the spermatozoa bound to the surface of the ZP displayed a punctate pattern of fluorescence over the head, particularly localised in the region of plasma membrane overlying the acrosome. Scale bar, 6 µm. (B) Labeled spermatozoa adhered to the ZP were compared to the free-swimming population. Phosphotyrosine was detected on the head but not the flagellum of viable spermatozoa; a highly significant increase (P<0.001) in the proportion of phosphorylated spermatozoa compared with the free-swimming sperm population was detected. The experiment was repeated three times and a minimum of 100 cells were scored for each experiment. ^**, P<0.001.