Sperm Capacitation associated increase in Tyrosine Phosphorylation: kinetic of the increase and novel members involved in the process.

Sperm capacitation is a post-ejaculatory maturational event required for successful fertilization. Specific molecular events are induced during capacitation: a cAMP-dependent activation of protein kinase A (PKA) leading downstream to the increase of tyrosine phosphorylation. Activation of PKA and tyrosine phosphorylation was shown to occur on different time scale. Here we showed that phosphorylation events during capacitation are tightly regulated over time, with fast activation of PKA and a late and continuous increase in the level of tyrosine phosphorylation to guarantee a pool of capacitated sperm at the right time/site of fertilization. Despite several studies on tyrosine phosphorylation, the identity of the tyrosine kinase(s) that mediate these increases has not been conclusively demonstrated. Recently a role for focal adhesion kinases (PYK2 and FAK) was proposed in stallion, based on the use of PF431396, a small molecule inhibitor directed against this kinase family, but critical loss of function experiment have not been reported. We used both pharmacological tools and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase of tyrosine phosphorylation in human and mouse sperm. PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation in both human and murine. On the other hand, Pyk2–/– mice showed a physiological capacitation-associated tyrosine phosphorylation and the specific inhibition of FAK by PF573228 showed no decrease in the levels of tyrosine phosphorylation, indicating that focal adhesion kinases are not responsible for this phosphorylation process. Here we show that PF431396 can also inhibit the tyrosine kinase FER and that sperm from mice targeted with a kinase inactivating mutation in FER failed to undergo capacitation-associated increases in tyrosine phosphorylation. While these mice are fertile, their sperm displayed normal levels hyperactivation but have reduced ability to fertilize metaphase-II arrested eggs in vitro