Stimulation of plasminogen activator activity by free radicals in boar spermatozoa

Plasminogen activators (PAs), commonly found on the membrane of spermatozoa, convert plasminogen into plasmin and may participate in mammalian fertilization. Correlations have been reported between reactive oxygen species (ROS) and spermatozoa function, although the relationship between PA activity and ROS is unknown. We investigated the effects of ROS on PA activity. We used an in vitro model of free radical generation whereby boar spermatozoa were preincubated in xanthine and xanthine oxidase (X-XO) and PA activity was then measured. The acrosome reaction of boar spermatozoa was significantly promoted by 100 mU/mL plasmin (P < 0.01), similar to levels achieved when stimulated with the positive calcium (2 mM) control. The addition of plasminogen to the fertilization medium significantly promoted both spermatozoa binding (157.5 ± 14.0 spermatozoa/oocyte) and the percentage of oocytes with a male pronucleus (74.5 ± 6.4%) compared with control (98.4 ± 21.8 spermatozoa/oocyte and 51.4 ± 5.3%, respectively; P < 0.05). The acrosome reactions of spermatozoa were significantly higher when incubated with calcium (2 mM; 60.2 ± 2.7%), calcium (2 mM) + EDTA (6 mM; 29.4 ± 4.2%), sodium nitroprusside (0.1 μM; 38.0 ± 4.2%), H₂O₂ (100 μM; 56.0 ± 3.0%), and X-XO (0.5 mM and 0.05 U/mL, respectively; 31.8 ± 3.7%) compared with non-capacitation medium as control (19.0 ± 2.7%; P < 0.05). However, when spermatozoa were incubated with only X-XO, PA activity was significantly higher than with other treatments (P < 0.05). Moreover, the addition of the antioxidant superoxide dismutase to the X-XO system significantly blocked the PA activity of spermatozoa (P < 0.05). The PA activity of spermatozoa treated with X-XO was significantly reduced by the addition of MEK inhibitor (55.2 ± 5.6 ng/mL) and p38 inhibitor (57.4 ± 2.7 ng/mL), but not PI3K inhibitor, compared to the control (X-XO; 68.0 ± 5.8 ng/mL; P < 0.05). The induction of PA activity in boar spermatozoa by free radical generation suggests the PA/plasmin system plays a role in mammalian fertilization.