Fig. 4. Change in Ser21 phosphorylation and priming phosphorylated substrate-specific kinase activity in human spermatozoa with different motility. (A) Fluorescent gel kinase assay of glycogen synthase kinase 3 (GSK3) from human spermatozoa with high and low-motility. After incubation in Tyrode’s complete medium (TCM), the kinase activity of GSK3 decreased in the high-motility spermatozoa but increased in the low-motility spermatozoa. T-Pep(pp) band intensities were measured by densitometric analysis and calculated as ratio compared to those of T-Pep(p). T-Pep, T-Pep(p), and T-Pep(pp) were loaded as marker. (B) Phosphorylation of GSK3 in low- and high-motility spermatozoa after incubation in TCM. p-GSK3α(Ser21), but not p-GSK3α(Tyr279), increased in the high-motility spermatozoa. In the low-motility spermatozoa, no visible change in p-GSK3 was observed. (C) The ratio of p-GSK3α(Ser21)/GSK3α was increased significantly after incubation in TCM, whereas the ratio of p-GSK3α(Tyr279)/GSK3α ratio was not. (D) Western blots of p-GSK3α(Ser21) and phosphotyrosine (p-Tyr) proteins after incubation with the 6-bromo-indirubin-3′-oxime (BIO), an inhibitor of GSK3 kinase activity in TCM. BIO (5–500 nM) increased p-GSK3α(Ser21) and p-Tyr proteins in a dose-dependent manner. AU: arbitrary units. ^**Significantly different from fresh spermatozoa by Mann–Whitney U-test (p<0.01).