α-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm

Abstract

Sperm cryopreservation is associated with the production of reactive oxygen species (ROS) leading to membrane destabilization, which induces capacitation-like changes, increases protein tyrosine phosphorylation, and decreases their fertilizing ability. α-Tocopherol, a lipid peroxidation inhibitor, preserves the functionality of cryopreserved porcine sperm. Our aim was to evaluate the effect of α-tocopherol on sperm quality parameters as well as capacitation-like changes and modifications in protein tyrosine phosphorylation. Boar sperm frozen with or without 200 μg/mL of α-tocopherol were thawed and maintained at 37 °C for 10 min in BTS. Routine parameters of semen quality were evaluated by optical microscopy and membrane changes were determined by the epifluorescence chlortetracycline technique. Changes in protein tyrosine phosphorylation were examined using a specific anti-phosphotyrosine monoclonal antibody. Motility was higher (18%, P < 0.05) in semen with α-tocopherol. Viability did not differ ( P > 0.05) between treatments. However, there was less ( P < 0.05) capacitation-like changes in semen with α-tocopherol compared to control samples. A MW 32 kDa tyrosine-phosphorylated protein was detected in extracts of cryopreserved sperm; the intensity of immunostaining was lower in semen containing α-tocopherol compared to the control (0.211 ± 0.030 versus 0.441 ± 0.034 arbitrary units). Additionally, this band was not detected in fresh sperm. The addition of α-tocopherol to the extender prior to cryopreservation of boar semen protected sperm membranes against oxidative damage and reduced both tyrosine phosphorylation and the capacitation-like state.