Fig. 1. Time-dependent protein tyrosine phosphorylation of mouse spermatozoa. Cauda epididymal spermatozoa were suspended in EKRB medium supplemented with 3 mg BSA/ml (A) or 1 mM methyl-ß-cyclodextrin (B) and incubated at 37°C for 60 minutes or 90 minutes as described under Materials and Methods. At the indicated time intervals, spermatozoa were washed, extracted and subjected to SDS-PAGE. The resolved components were transferred to a nitrocellulose sheet, and the tyrosine-phosphorylated bands were revealed. A representative experiment, out of a total of at least three experiments, is shown. The sharp band at the position of 116 kDa appears in all lanes and is nonspecific. This band is also present in all lanes in Figs 2 and 3. Spermatozoa incubated in EKPB medium (without BSA or methy-ß-cyclodextrin) for 60 minutes or 90 minutes revealed little or no phosphorylation.