A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation

Abstract

Capacitation is a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus-oocyte complex. The attainment of a capacitated state is dependent upon an increase in tyrosine phosphorylation and results in the acquisition of responsiveness to physiological agonists such as progesterone and ZP3. In this study we have shown that this capacitation-dependent increase in tyrosine phosphorylation is controlled by a unique redox-regulated, cAMP-mediated, signal transduction cascade. Either stimulation of reactive oxygen species generation or elevation of intracellular cAMP induced increases in phosphotyrosine expression by human spermatozoa and enhanced their responsiveness to progesterone. Ultimate convergence of the redox- and cAMP-regulated pathways was indicated by the ability of the protein kinase A inhibitor, H89, to block both modes of signal transduction. Furthermore, the fact that the redox-regulated pathway could be silenced by catalase, while this enzyme had no effect on the cAMP-mediated response, indicated that oxidant generation must lie upstream from cAMP in the reaction sequence. In keeping with this conclusion, a functional association was demonstrated between the redox status of human spermatozoa and their cAMP content. The continuous production of reactive oxygen species was also shown to be necessary for the protein kinase A-tyrosine phosphorylation axis to remain functional. If the generation of oxidising conditions during capacitation was prevented with 2-mercaptoethanol, 2-deoxyglucose or the flavoprotein inhibitor, diphenylene iodonium, then cAMP could no longer trigger tyrosine phosphorylation. These data support a model for human sperm capacitation as a redox-regulated process, involving a unique sequence of interactive events including reactive oxygen species production, elevation of intracellular cAMP, stimulation of protein kinase A and the induction of tyrosine phosphorylation. This is the first report of such a signal transduction cascade and may have implications for the functional significance of reactive oxygen metabolites in other cell types.