Fig. 5

Effects of BIO, a GSK3 inhibitor on p-GSK3α(Ser21), CypD, mPTP opening, MMP, and ATP production. A Representative western blot images of the effect of GSK3 inhibitor BIO on inhibitory phosphorylation of GSK3α. GSK3 inhibitor BIO increased p-GSK3α(Ser21) in spermatozoa. At least three independent experiments were subjected to western blot analysis. B Representative western blot images of the effect of GSK3 inhibitor BIO on amount of CypD. GSK3 inhibitor BIO decreased the protein amount of CypD in spermatozoa. At least three independent experiments were subjected to western blot analysis. (C) Effect of GSK3 inhibitor BIO on sperm motility. BIO significantly increased progressive motility but not total motility (n = 4). D Representative images of calcein fluorescence in BIO-treated spermatozoa. BIO markedly increased calcein fluorescence intensity in the sperm midpiece. Bar, 5 μm. E Effect of BIO on mPTP opening. BIO significantly increased calcein fluorescence, indicative of decreased mPTP opening in spermatozoa under capacitation condition (n = 4). F Representative images of TMRE fluorescence in BIO-treated spermatozoa. BIO markedly increased TMRE fluorescence intensity in the sperm midpiece. Bar, 5 μm. G Effect of GSK3 inhibitor BIO on sperm mitochondrial membrane potential (MMP). BIO significantly increased sperm MMP (n = 4). H Effect of GSK3 inhibitor BIO on sperm ATP production. BIO significantly increased ATP production in spermatozoa (n = 4). Data are presented by mean ± SD. *p < 0.05 and **p < 0.05 indicate significant difference from control by Student’s t-test. AU, arbitrary units