Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules

Glutathione S-transferases (GSTs) are enzymes that detoxify electrophilic compounds. Earlier studies from our laboratory showed that anti-GST antibodies interfered with the fertilising ability of spermatozoa from Capra hircus (goat) in vitro, suggesting that GSTs are localised at the cell surface. In this study, we provide evidence for the presence of GSTs of 24 kDa on the sperm plasma membrane attached by non-covalent interactions. The GST activity associated with the spermatozoal plasma membrane was significantly higher than the activity present in the plasma membranes of brain cells, hepatocytes, spleenocytes and ventriculocytes. Analysis of GST isoforms demonstrates the presence of GST Pi and Mu on the sperm plasma membranes. Both isoforms were able to bind to solubilised as well as intact zona pellucida (ZP) through their N-terminal regions but failed to bind to ZP once the oocytes were fertilised. Solubilised goat ZP separates into three components, one of which, the ZP3-like component, bound to sperm GSTs. High concentrations of anti-GST antibodies or solubilised ZP led to aggregation of sperm GSTs, resulting in the release of acrosin. In contrast, inhibition of sperm GST binding to ZP, by saturation of binding sites for sperm GSTs on the solubilised ZP using peptides designed from the N-terminii of GST Pi or Mu or blocking of binding sites for ZP on sperm GSTs with antibodies raised against the N-terminal GST peptides, inhibited essential prefertilisation changes in sperm. These data therefore demonstrate the strategic location of catalytically active defensive enzymes on the sperm surface that also act as zona-binding proteins. Therefore, sperm-surface GSTs serve as bifunctional molecules in a transcriptionally inactive cell whose requirement for cellular defense and economy of molecules that it can carry is greater than that of any somatic cell type.