Dibutyl phthalate disrupts glycogen synthase kinase 3α essential for sperm motility

To unravel the toxic mechanism of phthalate ester plasticizer endocrine disruptor in spermatozoa, we examined the effect of dibutyl phthalate (DBP) on the stability and inhibitory phosphorylation of glycogen synthase kinase 3 alpha (GSK3 alpha), a protein kinase crucial for sperm motility in mice. In DBP-treated spermatozoa, reactive oxygen species (ROS) and lipid peroxide were significantly increased. In computer-assisted sperm analysis, DBP at concentrations of 10 - 100 mu g/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3 alpha(Ser21) and increased p-GSK3 alpha(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3 alpha(Ser21) but not p-GSK3 alpha(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3 alpha and p-GSK3 alpha(Ser21) but increased immunoreactivity of p-GSK3 alpha(Tyr279) in spermatozoa. DBP at a concentration of 100 mu g/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3 alpha(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3 alpha (Ser21) and sperm motility. On western blot, 1-100 mu g/mL DBP decreased GSK3 alpha in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 mu g/mL increased polyubiquitinated sperm proteins including GSK3 alpha. The MG115, proteasome inhibitor attenuated degradation of GSK3 alpha in DBP-treated spermatozoa. Hydrogen peroxide at 10 mu M increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3 alpha via ROS induction. Together, DBP may decrease the cellular amount of GSK3 alpha through the ubiquitin-proteasome pathway and p-GSK3 alpha(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility.