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 3α (GSK3α), 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 μg/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3α(Ser21) and increased p-GSK3α(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3α(Ser21) but not p-GSK3α(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3α and p-GSK3α(Ser21) but increased immunoreactivity of p-GSK3α(Tyr279) in spermatozoa. DBP at a concentration of 100 μg/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3α(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3α(Ser21) and sperm motility. On western blot, 1–100 μg/mL DBP decreased GSK3α in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 μg/mL increased polyubiquitinated sperm proteins including GSK3α. The MG115, proteasome inhibitor attenuated degradation of GSK3α in DBP-treated spermatozoa. Hydrogen peroxide at 10 μM increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3α via ROS induction. Together, DBP may decrease the cellular amount of GSK3α through the ubiquitin-proteasome pathway and p-GSK3α(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility.