Fig. 3. Change in Ser21 phosphorylation and priming phosphorylated substrate-specific kinase activity of glycogen synthase kinase 3 (GSK3) after capacitation in human spermatozoa. (A) Efficacy of the priming phosphorylated peptide substrate (T-Pep(p)) in determining GSK3 kinase activity in sperm extracts. T-Pep showed no remarkable change in phosphorylation by recombinant GSK3 regardless of ATP. In contrast, T-Pep(p) was phosphorylated by recombinant GSK3 and ATP. T-Pep and T-Pep(p) were loaded in control (Con) lane for comparison. (B) Fluorescent gel kinase assay for GSK3 substrate specific kinase activity to T-Pep(p) in fresh human spermatozoa lysates. LiCl (0–10 mM) decreased GSK3 kinase activity in dose dependent manner. T-Pep(p) was loaded in Con lane without sperm extracts and LiCl. (C) Western blots of p-GSK3α(Ser21) and phosphotyrosine (p-Tyr) proteins in human spermatozoa after incubation in Tyrode’s basal medium (TBM) or Tyrode’s complete medium (TCM). p-GSK3α(Ser21) and p-Tyr proteins levels in spermatozoa incubated in TBM were lower than those incubated in TCM. (D) Gel kinase assay of GSK3 in high-motility human spermatozoa after incubation in TBM or TCM. Reduction of GSK3 kinase activity of spermatozoa following incubation was apparent in TCM compared to TBM. ATP: adenosine 5′-triphosphate, TAMRA: tetramethyl-6-carboxyrhodamine.