Reactive oxygen species and their effects on boar spermatozoa function, tyrosine phosphorylation and MAPK signaling
Boar spermatozoa are very susceptible to Reactive Oxygen Species (ROS) due to their high content of polyunsaturated fatty acids (PUFAs). This study hypothesized that ROS modulate signal transduction during capacitation of fresh boar spermatozoa through the extracellular signal-regulated kinases (ERKs) of mitogen-activated protein kinase (MAPK) pathway, and that cryopreservation damage to sperm involves ROS. All experiments analysed multiple ejaculates. Flow cytometry using dual fluorescent dyes measured sperm viability, intracellular levels of O 2.-, H2O2, membrane lipid peroxidation and PLA activity. Western blotting of extracted proteins from boar spermatozoa detected and quantified MAPKs (Raf1, MEK1/2 and ERK1/2) and phosphotyrosine in boar spermatozoa after 0 and 4hr incubation in capacitating or non-capacitating media, in sperm pre-exposed for 30 minutes to buffer or an ROS generating system. The ROS generating system had multiple effects: increased fresh sperm intracellular level of H2O2; completely inhibited sperm motility; increased the percentage of sperm undergoing acrosome reactions; increased lipid peroxidation in the viable, dead and average sperm populations, and; inhibited tyrosine phosphorylation of proteins with high molecular weights. Phosphorylated forms of the ERK pathway components Raf-1, MEK1/2, and ERK1/2 are present in boar sperm. Their protein profiles, quantified using Image Quant software, differed with capacitation and ROS generating system. Boar sperm capacitated as before and preincubatied with a specific inhibitor of ERK1/2, but not with inhibitors of Raf1 or MEK1/2, significantly inhibited tyrosine phosphorylation of spermatozoa proteins of 172, 97 and 66 kDa. Combining ERK1/2 inhibitor with the ROS generating system greatly increased inhibition of tyrosine phosphorylation of these three proteins (P<0.002) and also a 111 kDa protein (P<0.028). Inhibiting MEK1/2 in the presence of the ROS generating system subsequently inhibited capacitation-induced tyrosine phosphorylation of proteins of 187(P<0.01) and 112 kDa (P<0.04). This study indicates that H2O2 is the major free radical mediating the damage of boar spermatozoa and ROS have physiological roles regulating protein tyrosine phosphorylation of capacitating boar spermatozoa. The ERK pathway regulates tyrosine phosphorylation in boar spermatozoa through its ERK1/2 component and ROS mediate cAMP-dependent PKA and ERK pathway signaling during sperm capacitation, through phosphorylation/dephosphorylation of specific proteins.