Prior to fertilisation, mammalian spermatozoa undergo a series of molecular and biochemical events that render them capable of interacting with the oocyte. This process, termed capacitation, has been correlated with the tyrosine phosphorylation of multiple sperm proteins. The current study aimed to dissect this association and determine specifically how tyrosine phosphorylation of sperm proteins impacts on their ability to recognise and bind the zona pellucida, a question that has remained largely unexplored. When spermatozoa bound to the zona pellucida in vitro were recovered, fixed and immunostained using monoclonal anti-phosphotyrosine antibody, all cells were labelled along the entire length of the flagellum. Preparation of sperm populations in which tyrosine phosphorylation was significantly reduced (bicarbonate-free medium) or increased (calcium-deficient medium with pentoxifylline and dibutyryl cAMP) resulted in analogous changes in the zona binding affinity of the population. This suggests that tyrosine phosphorylation of sperm proteins is a pre-requisite for gamete binding. To further examine this phemonenon, the localisation of tyrosine phosphoproteins on the surface of live spermatozoa was investigated. We report for the first time a capacitation-dependent appearance of phosphotyrosine residues on the head of live mouse spermatozoa, as assessed by binding to anti-phosphotyrosine coated beads. Importantly, 100% of live sperm bound to the zona showed this head pattern of phosphotyrosine expression. These data suggest a role for tyrosine phosphoproteins in the assemblage of a functional zona pellucida receptor complex on the surface of spermatozoa. We have identified two chaperone proteins that become tyrosine phosphorylated during capacitation: heat shock protein 60 and endoplasmin. Both proteins are localised on the sperm head and are exposed on the cell surface following capacitation. Identification of binding partners for these chaperones represents an important step towards the elucidation of the nature of the zona pellucida receptor.
37th Annual Meeting of the Society for the Study of Reproduction. Biology of Reproduction 2004 Special Issue: Program for the Thirty-Seventh Annual Meeting of the Society for the Study of Reproduction (Vancouver, Canada 1-4 August, 2004) p. 144-144