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.
Coonrod
S
S
Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, VA 22908, USA.
Naaby-Hansen
S
S
Shetty
J
J
Herr
J
J
D000818:Animals; D019921:Biotinylation; D006224:Cricetinae; D004527:Egg Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008297:Male; D008970:Molecular Weight; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D010063:Ovum / Q000187:drug effects; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D017421:Sequence Analysis; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology; D010738:Type C Phospholipases / Q000494:pharmacology*
D004527:Egg Proteins; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase