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

Molecular Human Reproduction vol.5 no.11 pp. 1027–1033, 1999 PI-PLC releases a 25–40 kDa protein cluster from the hamster oolemma and affects the sperm penetration assay Scott Coonrod, Soren Naaby-Hansen, Jagatpala Shetty and John Herr1 Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, VA 22908, USA 1To whom correspondence should be addressed 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. Key words: GPI-anchored/sperm–egg interaction/two-dimensional electrophoresis Introduction The hamster oocyte is unique in that zona-free eggs from other species such as the mouse, rat, and guinea pig do not incorporate heterologous spermatozoa as readily (Yanagimachi, 1972; Hanada and Chang, 1976; Quinn, 1979). Because of this promiscuity, the zona-free hamster egg has been used extensively in the sperm penetration assay (SPA) to assess the fertilizing capacity of human spermatozoa (Yanagimachi et al., 1976; Rodgers et al., 1979; Liu and Baker, 1992). In spite of the widespread use of this assay, the molecular interactions which occur between the human spermatozoa and hamster oocyte during gamete interaction remain largely unknown. Presumably, however, there are molecules on the hamster egg plasma membrane (oolemma) which specifically interact with molecules on the human sperm plasma membrane during sperm–egg binding and fusion. In a previous publication we investigated the effects of phosphatidylinositol-specific phospholipase C (PI-PLC) on mouse sperm–egg interaction to investigate whether glycosylphosphatidyinositol (GPI)-anchored proteins were required for fertilization (Coonrod et al., 1999). Results showed that treatment of mouse spermatozoa with PI-PLC had no significant effect on either sperm–zona pellucida binding or sperm–egg binding and fusion. However when zona-intact or zona-free © European Society of Human Reproduction and Embryology oocytes were treated with PI-PLC, fertilization was blocked. We then began characterization of the PI-PLC cleavable oolemmal proteins by two-dimensional (2-D) avidin blotting and found that biotinylated mouse oocytes released protein clusters of ~70 kDa (pI 5) and 35–45 kDa (pI 5.5) following PI-PLC treatment. In the present study we investigated the effects of PI-PLC on human sperm–hamster egg interaction. Upon finding that treatment of zona-free hamster oocytes with PI-PLC blocked sperm–egg binding and fusion we began to characterize the PI-PLC-sensitive hamster oolemmal proteins using 2-D avidin blotting. We found that when biotinlyated hamster oocytes are treated with PI-PLC, a predominant protein cluster (~25–40 kDa, pI 5–6) is released from the oocyte into the supernatant. It is likely that this protein(s) is required for human sperm–hamster egg binding and fusion. The resolution of this ~25–40 kDa (pI 5–6) protein cluster by 2-D gel electrophoresis provides a basis for proceeding with the microsequencing, identification, and cloning of this protein(s) from hamster oocytes. Materials and methods PI-PLC preparation The phosphatidylinositol-specific phospholipase C preparation used for this study was purchased from Boehringer Mannheim (Indianapolis, 1027 S.Coonrod et al. IN, USA). The PI-PLC was isolated from the cultured filtrate of Bacillus cerus and migrates as a single band on a sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS–PAGE) gel at 29 kDa. Sperm penetration assay Gamete preparation Gamete incubations were carried out in microdrops under paraffin oil at 37°C and 5% CO2. Ejaculated human semen was allowed to liquefy for at least 30 min. The ejaculate (500 µl) was placed under 2 ml of Biggers–Whitten–Whittingham (BWW) medium (Irvine Scientific, Santa Ana, CA, USA) with 5 mg/ml human serum albumin (HSA; Sigma, St Louis, MO, USA) for 30 min and the spermatozoa were allowed to swim up. The swim-up spermatozoa were then washed twice by centrifugation (8 min at 600 g) in 10 ml volumes of BWW in 15 ml centrifuge tubes. The spermatozoa were capacitated overnight in 250 µl microdrops of BWW with 30 mg/ml HSA at a concentration of 203106 spermatozoa/ml. Cumulus–oocyte complexes were collected from at least three superovulated Golden Syrian hamsters per repetition and placed in BWW with 5 mg/ml HSA. Cumulus cells were removed by treating eggs with 1 mg/ml hyaluronidase (Sigma) for 3 min. The oocytes were then pooled and washed (for this treatment and all subsequent treatments) by passing the eggs through 20 µl drops of media covered with mineral oil using a pulled, heat-polished, Pasteur pipette. Zonae pellucida were removed by treating eggs with 1 mg/ml trypsin (Sigma) for 30 s followed by five washes. The eggs were then randomly allotted into two groups. Treatment of human spermatozoa with PI-PLC Following overnight capacitation, 23106 spermatozoa were treated for 30 min with either 1 IU/ml PI-PLC or 1 IU/ml of heat-inactivated (95°C for 5 min) PI-PLC in 100 µl of BWW with 30 mg/ml HSA. The spermatozoa were then washed twice by centrifugation in 5 ml volumes of BWW in 15 ml centrifuge tubes to remove the PI-PLC. Treated spermatozoa were then added to untreated zona-free hamster oo (...truncated)