PI-PLC releases a 25–40 kDa protein cluster from the hamster oolemma and affects the sperm penetration assay

The effects of phosphatidylinositol-specific phospholipase C (PI-PLC) on human sperm–hamster oocyte interaction were investigated to determine whether PI-PLC cleavable glycosylphosphatidyinositol (GPI)-anchored proteins are involved in sperm–egg binding and fusion. Two-dimensional electrophoresis was then utilized to visualize proteins released from hamster oocytes following PI-PLC treatment. For the binding and fusion assay, either spermatozoa or eggs were treated with 1 IU/ml PI-PLC for 30 min and washed prior to gamete co-incubation. Treatment of human spermatozoa with PI-PLC significantly (P ≤ 0.05) enhanced sperm–egg binding while having no effect on sperm–egg fusion. Treatment of zona-free hamster oocytes with PI-PLC blocked sperm–egg binding and fusion. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labelled with sulpho-NHS biotin and either mock treated or treated with PI-PLC. Egg protein extracts and egg supernatant proteins from each group were then analysed by two-dimensional gel electrophoresis followed by avidin blotting. Comparison of blots demonstrated that a predominant biotinylated 25–40 kDa protein cluster (pI 5–6) apparent in the mock treated egg extract blot was absent in the PI-PLC treated egg extract blot. A protein cluster of identical molecular weight and isoelectric point as the predominant 25–40 kDa protein cluster was observed in the PI-PLC supernatant blot while no proteins could be seen in the control supernatant blot. These results demonstrate that treatment of hamster oocytes with PI-PLC inhibits sperm–egg interaction and releases a 25–40 kDa protein cluster (pI 5–6) from the oolemma. It is likely that this released protein cluster represents an oolemmal GPI-linked surface protein(s) which is involved in human sperm–hamster egg interaction.