436. Investigation of the role of SRC in capacitation associated tyrosine phosphorylation of human spermatozoa

Abstract

Capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilise an oocyte. A fundamental part of this mechanism is a dramatic increase in tyrosine phosphorylation. Implicated in this process in the mouse is a unique cAMP/PKA-mediated pathway involving a PKA-activated tyrosine kinase suggested to be pp60^c-src (SRC). The Src kinases examined were predominantly expressed in the human sperm tail, a site compatible with a role in mediating the capacitation-associated tyrosine phosphorylation cascade. Co-immunoprecipitation revealed that PKA-c could be isolated from sperm and this interaction was restricted to capacitated cells, suggesting PKA-mediated activation of SRC forms an integral part of the signalling cascade assembled during capacitation. Upon activation, SRC undergoes autophosphorylation of Y416 and thus phosphorylation of this residue indicates the presence of active SRC kinase. The phosphorylation status of SRC was compared using both 2D-immunoblotting and immunocytochemical studies, both revealing a significant increase in SRC activation during capacitation. Furthermore, suppression of PKA and SRC through application of SU6656, or H89, a PKA inhibitor, led to a dramatic decrease in tyrosine phosphorylation and SRC activity. In conclusion, this study has provided evidence for the involvement of non-receptor tyrosine kinase, SRC, in regulating tyrosine phosphorylation associated with capacitation. Inhibition of SRC did not completely suppress tyrosine phosphorylation suggesting this complex signal transduction pathway exhibits a degree of functional redundancy.