Fig. 3. Characterisation of the dose and time dependency of WGA-stimulated sperm tyrosine phosphorylation. Purified human spermatozoa were incubated in varying concentrations of WGA (0.005-5 µg/ml) or in positive (+ve) and negative (–ve) control media for 30 minutes. Alternatively, spermatozoa were incubated in 0.5 µg/ml WGA for varying times (5-90 minutes). Following incubation, spermatozoa were either (A) solubilized in SDS extraction buffer and prepared for immunoblotting with anti-phosphotyrosine or (B) examined to determine the status of their motility and viability. This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted. Graphical data is given as the means ± s.e.m. from the three replicate experiments. *P<0.05, **P<0.01.