Fig. 4. Tyrosine phosphorylation and sperm hyperactivation. (A,B) Treatment of murine spermatozoa with inhibitors of SRC such as (A) lavendustin A and (B) SU6656 inhibited phosphotyrosine expression. For this analysis, spermatozoa were pre-incubated with the inhibitors for 5 minutes before the addition of 1 mM dbcAMP and 1 mM PTX. After a 40-minute incubation the spermatozoa were analyzed for hyperactivated motility to confirm induction of capacitation in the vehicle-only controls. Approximately 2 µg of lystate was run in a 10% SDS-PAGE and western-blot analysis was performed with anti-phosphotyrosine antibodies as described. Arrowheads indicate the position of protein bands that were reduced in the presence of inhibitor; Hx indicates the location of hexokinase, a constitutively phosphorylated protein that served as a loading control (Nixon et al., 2006). When caudal epididymal murine spermatozoa were treated with PTX (1 mM) and dbcAMP (1 mM) tyrosine phosphorylation was observed along the length of the sperm tail in >95% of cells examined. (C,D) Phase-contrast and phosphotyrosine images of mouse spermatozoa, respectively. (E) Suppression of tyrosine phosphorylation had a dramatic effect on hyperactivated movement. Spermatozoa were pre-incubated for 5 minutes with the inhibitors indicated before the addition of 1 mM dbcAMP and 1 mM PTX to capacitate the cells. Following a 40-minute incubation, a 20 µl aliquot was taken and the spermatozoa were assessed for hyperactivated motility (presented as a percentage of the motile sperm population) as described in Materials and Methods.