Fig. 4. Comparison of the WGA-induced tyrosine phosphorylation in normal and defective spermatozoa. (A) Human spermatozoa recovered in the low density (defective) and high density (normal) fractions after Percoll density gradient centrifugation were incubated with either WGA (0.5 µg/ml) or in positive (+ve) and negative (–ve) control media for 30 minutes. An additional control, consisting of sperm incubated in complete BWW supplemented with SJA (a lectin that does not interact with the sperm surface) was also included. Following incubation, spermatozoa were solubilized in SDS extraction buffer and prepared for immunblotting with anti-phosphotyrosine; only the normal cells responded to WGA. (B) To eliminate the possibility of a contaminating leukocyte contribution to the WGA response, viable spermatozoa recovered by Percoll fractionation (pellet) and those isolated by a swim up procedure (swim up) were incubated with WGA (0.5 µg/ml) for 30 minutes at 37°C. After incubation, spermatozoa were solubilized in SDS extraction buffer and prepared for immunblotting with anti-phosphotyrosine. This comparison was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted.