Molecular Human Reproduction, Vol. 5, No. 11, 1027-1033,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of fertilization |
PI-PLC releases a 25–40 kDa protein cluster from the hamster oolemma and affects the sperm penetration assay
Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, VA 22908, USA
Abstract
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.
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 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, 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 20x106 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, 2x106 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 oocytes (~ 12 per treatment group) at a concentration of 2x106 spermatozoa/ml in 20 µl drops of BWW with 30 mg/ml HSA and the gametes were co-incubated for 3 h.
Treatment of zona-free hamster oocytes with PI-PLC
Zona-free oocytes were treated for 30 min with either 1 IU/ml PI-PLC or 1 IU/ml of heat-inactivated PI-PLC in 20 ml drops of BWW with 30 mg/ml HSA. The eggs were then washed through five microdrops and incubated with untreated human spermatozoa at a concentration of 2x106 spermatozoa/ml for 3 h.
Quantification of sperm–egg binding and fusion
Following gamete co-incubation, loosely bound spermatozoa were removed from the oocytes by gentle pipetting. The eggs were then treated with 1 mmol/l Acridine Orange–3% in dimethyl sulphoxide (DMSO; Sigma) for 15 s to stain the chromatin and washed through three 20 µl microdrops. To quantify binding, the oocytes were placed between a microscope slide and an elevated cover slip, the oocytes were visualized at x200 using a light microscope (Zeiss Axioplan) and the number of spermatozoa bound per oocyte was recorded. The number of fused spermatozoa per egg was scored by counting the number of Acridine Orange-stained decondensed sperm heads within each oocyte using fluorescent microscopy.
Artificial activation of oocytes
In order to ensure that PI-PLC treated eggs remained viable following PI-PLC treatment, zona-free eggs were treated with either 1 IU/ml PI-PLC or 1 IU/ml heat-inactivated PI-PLC for 30 min as described in the sperm penetration assay section. Following treatment, oocytes were pre-loaded with 1 µm Hoechst dye #33342 (Sigma) for 10 min to stain chromatin and washed three times. The oocytes were then activated by placing the eggs in 0.5 µmol/l calcium ionophore A23187 (Sigma) for 5 min followed by three washes. The eggs were incubated for 3 h and oocytes were observed as described in the sperm penetration assay section. The eggs were considered activated if they had advanced from metaphase II arrest to anaphase II or telophase II (with second polar body).
Two-dimensional gel electrophoresis
Hamster oocytes were collected and the zonae were removed as described in the sperm penetration assay section. The zona-free eggs were then washed six times in BWW media containing 100 µg/ml polyvinylalcohol (PVA; Sigma), biotinylated with 2 mg/ml Sulpho-NHS biotin (Pierce, Rockford, IL, USA) in BWW/PVA for 7 min at room temperature, and washed six times in BWW/PVA. The eggs were then split into two groups of 130 and either mock treated or treated with 1 IU/ml PI-PLC in 20 µl drops for 30 min. The supernatants were removed, the eggs were washed six times, and the oocytes and the oocyte supernatants were then frozen at –70°C in BWW/PVA containing protease inhibitors (CompleteTM; Boehringer Mannheim, Mannheim, Germany). The oocytes and supernatants were extracted in Celis lysis buffer containing 2% (v/v) NP-40, 9.8 mol/l urea, 100 mmol/l dithiothreitol (DTT), 2% ampholines (pH 3.5–10), and protease inhibitors for 30 min at room temperature (Rasmussen et al., 1991). Isoelectric focusing (IEF) was performed using the Mini-PROTEAN II tube cell (Bio RAD, Richmond, CA, USA) apparatus and protocol with an ampholine mixture (Pharmacia Biotech, Uppsala, Sweden) of pH 3.5–5 (30%), 3.5–10 (40%), 5–7 (20%), and 7–9 (10%). The tube gels were placed on 12% mini slab gels and the focused proteins were separated in the second dimension at 20 mA per gel. The proteins were then electroblotted to nitrocellulose membranes at 125 mA for 45 min. The membranes were then stained with Protogold for 10 min to visualize the egg proteins and washed briefly with water. Next, the membranes were blocked in phosphate-buffered saline (PBS) with 0.1% Tween and 5% dried milk for 30 min at room temperature, washed once in PBS/0.1% Tween, and probed with 20 µg/ml strepavidin–horseradish peroxidase (HRP; Pierce) for 30 min at room temperature. The blots were washed three times in PBS/0.1% Tween (10 min per wash) and the biotinylated proteins were visualized using tetramethylbenzidene (TMB) as a substrate.
Statistical analysis
All in-vitro assays were repeated three times. Experimental and control group averages were reported as means ± SD. Student's t-test was used to identify differences in the number of bound and fused spermatozoa while the Cochran–Mantel–Haenszel test (Rosner, 1990) was used to identify differences in the proportion of activated eggs. P < 0.05 was considered to be statistically significant.
Results
Pre-treatment of spermatozoa with PI-PLC significantly enhances human sperm–hamster egg binding while having no effect on sperm–egg fusion
When capacitated human spermatozoa are treated with PI-PLC, washed and co-incubated with untreated zona-free hamster oocytes, there was a significant increase (P < 0.05) in the number of spermatozoa bound to the oolemma (21.3 spermatozoa per egg) when compared to the control group in which spermatozoa were treated with heat-inactivated PI-PLC (10.33 spermatozoa per egg) (Figure 1A). However, treatment of capacitated human spermatozoa with PI-PLC did not significantly affect sperm–egg fusion (1.7 spermatozoa fused per egg) when compared to the control group (1.4 spermatozoa per egg) (Figure 1B).
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Pre-treatment of zona-free hamster oocytes with PI-PLC blocks human sperm–hamster egg binding and fusion
When zona-free hamster oocytes are treated with PI-PLC, washed and co-incubated with untreated capacitated human spermatozoa, there was a significant decrease in the number of spermatozoa bound to the oolemma (0.3 spermatozoa per egg) when compared with the control group in which oocytes were treated with heat-inactivated PI-PLC (21.9 spermatozoa per egg; P < 0.05) (Figure 2A
). Similarly, PI-PLC treatment also significantly decreased sperm–egg fusion (0.1 spermatozoa per egg) when compared with the control group (2.1 spermatozoa per egg) (Figure 2B). This result indicates that the inhibitory effect of PI-PLC on fertilization is mediated at the oolemma and is in close agreement with results obtained using the mouse in-vitro fertilization model (Coonrod et al., 1999).
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Pre-treatment of zona-free eggs with PI-PLC has no effect on artificial egg activation
Artificial activation of oocytes was performed to ensure that the oocytes remained viable following PI-PLC treatment. When eggs were treated with PI-PLC, washed and artificially activated with 0.5 µm calcium ionophore A23187, there was no significant difference (P > 0.05) in the percentage of eggs which resumed meiotic cell division when comparing eggs (n = 41) treated with heat-inactivated PI-PLC (84.3 ± 16%; Figure 3A
) with eggs (n = 38) treated with active PIPLC (82 ± 14.1%, Figure 3B). The observation that PI-PLC treatment did not alter meiotic division supports the hypothesis that eggs remained viable following treatment and the PI-PLC effect on sperm–egg binding and fusion was authentic.
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Two-dimensional gel electrophoresis of biotinylated zona-free hamster oocyte proteins demonstrates that when oocytes are treated with PI-PLC a predominant 25–40 kDa (pI 5–6) protein cluster is released into the supernatant
Hamster oocytes were biotinylated and incubated with or without PI-PLC for 30 min. The supernatants were collected from the two groups, the eggs were washed, and the egg proteins were extracted. The egg protein extracts and the proteins from the supernatants were separated by 2-D electrophoresis and electroblotted to nitrocellulose membranes. The membranes were then stained with Protogold to visualize the egg proteins. Next the membranes were probed with strepavidin–HRP and the biotinylated egg surface proteins were visualized using TMB membrane peroxidase substrate. The repertoire of zona-free hamster egg proteins is shown in Figure 4A
with over 100 egg proteins being resolved following Protogold staining (red staining). The repertoire of surface-labelled zona-free hamster egg proteins (blue staining) can also be seen in Figure 4A. Approximately 11 biotinylated surface protein spots having molecular weights ranging from ~40 to 140 kDa can be visualized (small arrowheads). Seven of these surface-labelled protein spots were also stained with Protogold (small arrowheads labelled d). One lesser ~45–50 kDa protein cluster (c1), one predominant ~25–40 kDa protein cluster (c2), and three protein trains having masses of ~35, 20, and 15 kDa (t 1, 2, and 3 respectively) can be resolved. The predominant 25–40 kDa protein cluster (c2) can be further resolved into three smaller protein clusters, however, the clusters were not recorded as separate proteins because continuous protein staining was observed between the clusters. The two spots denoted by asterisks in Figure 3A
represent proteins that bound strepavidin–HRP non-specifically and were detected on 2-D blots of oocytes which were not biotinylated (data not shown).
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The repertoire of biotin-labelled egg proteins remaining on the egg surface following PI-PLC treatment is shown in Figure 4C
. The two arrows denote the location of the 25–40 kDa (pI 5–6) protein cluster (c2) which is prominent in the extracts of untreated eggs (Figure 4A) but is absent the extracts of eggs treated with PI-PLC. Note that the staining intensity of the remaining surface-labelled egg proteins does not appear to be affected by PI-PLC treatment. In contrast to the supernatant from untreated eggs (Figure 4B), the supernatant from eggs treated with PI-PLC (arrows, Figure 4D) reveal a 25–40 kDa (pI 5–6) protein cluster having a similar molecular weight and isoelectric point to that which was released from the egg surface following PI-PLC treatment (Figure 4C). It is likely that the PI-PLC-sensitive protein cluster seen in Figure 4D is GPI-anchored and is involved in human sperm–hamster egg binding and fusion. The train of four proteins having a mass of ~29 kDa and indicated by the asterisk in Figure 4D most probably represents isoforms of PI-PLC because identical staining patterns are observed when 1 µg of the PI-PLC preparation is separated on a 2-D gel and silver stained (data not shown). It is possible that a small amount of unbound biotin remained associated with the biotinylated oocytes following oocyte washing and became linked to PI-PLC during oocyte treatment. This could explain why, in Figure 4D, the PI-PLC isoforms appear to be double-labelled. Discussion
The findings presented here demonstrate that PI-PLC has different effects on human spermatozoa and hamster eggs during gamete interaction. When human spermatozoa were treated with PI-PLC, washed, and incubated with untreated zona-free hamster oocytes, sperm–egg binding was significantly enhanced while fusion was not affected. GPI-anchored sperm surface proteins are thought to be involved in processes such as protection of spermatozoa from the immune response (Kirchhoff and Hale, 1996), the acrosome reaction (Mendoza et al., 1993), sperm–cumulus interaction (Myles and Primakoff, 1997), and sperm–zona pellucida interaction (Mahony et al., 1991; Diekman et al., 1997). There are no reports in the literature implicating GPI-anchored sperm proteins in sperm–oolemma binding and fusion. In our previous publication, however, we found that when epididymal mouse spermatozoa were treated with PI-PLC, washed, and incubated with untreated zona-free mouse oocytes, there was a slight (but not significant) increase in sperm–egg binding compared with controls (Coonrod et al., 1999). It is possible that the enhanced increase in sperm–egg binding observed in the present study is due to the fact that ejaculated spermatozoa were treated with PI-PLC as opposed to epididymal spermatozoa which were used in the previous experiment.
One model to explain how treatment of spermatozoa with PI-PLC could cause an increase in sperm–egg binding would posit that PI-PLC treatment releases GPI-anchored proteins from the sperm surface which mask molecules required for sperm–egg binding and fusion. It is known that spermatozoa become coated with GPI-anchored proteins during passage through the epididymis (Kirchhoff, 1998). It is possible that these proteins act as capacitating factors and are released from the sperm surface during passage through the female reproductive tract. Therefore, in this study, treatment of spermatozoa with PI-PLC may have released more of these PI-PLC cleavable GPI-anchored capacitating proteins than were released from the control group, leading to enhanced sperm–egg binding. It is also possible that GPI-anchored sperm surface proteins are involved in acrosomal maintenance and loss of these proteins following PI-PLC treatment increased the percentage of acrosome-reacted spermatozoa in the PI-PLC treatment group, thus leading to enhanced binding. Studies are currently underway to establish whether PI-PLC affects capacitation or the acrosome reaction.
Perhaps the most significant finding of this study was that when zona-free hamster oocytes are treated with PI-PLC, washed, and incubated with untreated human spermatozoa, binding and fusion is blocked. Little is known about the molecular composition of the mammalian oocyte. A number of previously known adhesion molecules, e.g. cell adhesion molecule (N-CAM; Kimber et al., 1994), the C1q receptor (Fusi et al., 1991), and several integrins have been identified on the mammalian oolemma. In the mouse, the integrin 
6ß1 has been shown to function as a sperm receptor for the egg (Almedia et al. 1995). Penetration of human spermatozoa in zona-free hamster eggs was completely inhibited by RGD-containing peptides (Bronson and Fusi, 1990), indicating that integrins on the hamster oolemma may also be involved in fertilization. Integrins such as v, 5, 3 (Capmany et al, 1998) and ß1 (Ji et al, 1998) have been found on the human oocyte. Ji et al. (1998) found that human gamete fusion was only partially blocked by either RGD-containing peptide or anti-human ß1 integrin monoclonal antibodies (Ji et al., 1998), indicating that other oolemmal proteins are likely involved in fertilization.
While there are no previous reports describing GPI-anchored proteins on mammalian oocytes, there is a GPI-anchored form of N-acetylglucosaminidase which is present on the surface of Ascidian eggs (Lambert, 1989). This enzyme is PI-PLC-sensitive and is cleaved from the surface of Ascidian eggs following fertilization and occupies sperm binding sites on the vitelline coat to protect the egg against polyspermy (Lambert and Goode, 1992). Regarding the presence of GPI-anchored proteins on mammalian oocytes, two previous reports have investigated whether treatment of mammalian oocytes with PI-PLC blocks sperm–egg interaction. Clark and Koehler (1988) treated zona-free hamster oocytes with up to 1 IU/ml PI-PLC for only 3 min and found that the enzyme had a slight, but significant, inhibitory effect on hamster sperm–hamster egg fusion. However, these results are somewhat difficult to interpret due to the abbreviated treatment time. In our previous study we found that when either zona-intact or zona-free mouse oocytes were treated with PI-PLC, fertilization was blocked. To demonstrate that the effect of PI-PLC was specific to the release of GPI-anchored proteins, we also performed several control experiments. As with the hamster oocytes in this study, PI-PLC treated mouse oocytes are fully capable of being artificially activated, thus indicating that the oocytes are viable following treatment. Also, the decrease in mouse sperm–egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 1 IU per ml. Finally, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, 6ß1 (Coonrod et al., 1999). Therefore, in-vitro data from our previous study as well as this study indicate that there is a PI-PLC-cleavable GPI-anchored protein(s) on the mammalian oocyte which is required for sperm–egg binding and fusion.
In this study, the repertoire of biotinylated hamster oolemmal proteins has been resolved using 2-D gel electrophoresis followed by avidin blotting. Results show that ~ 11 isolated protein spots, two protein clusters, and three protein trains are surface-labelled (Figure 4A). The two protein clusters (c1 and c2) probably represent separate proteins each with multiple isoforms containing varying degrees of glycosylation while the protein trains (t1, t2, and t3) likely represent non-glycosylated proteins each consisting of multiple isoforms (Shackelford et al., 1980; Negm et al., 1991). The number of surface-labelled proteins which can be visualized on the 2-D avidin blots of hamster oocytes is consistent with results obtained from one dimensional blots of surface-labelled mouse (Boldt et al., 1989; Flaherty and Swan, 1993; Ya Zhong et al., 1997) and hamster (Ya Zhong et al., 1997) oocytes. Also, the 2-D repertoire of hamster oolemmal proteins is quite similar to that which was observed in the mouse (Coonrod et al., 1999), however, there were notable differences in the masses of the protein clusters. In the mouse, the predominant oolemmal protein cluster is ~70 kDa with a less prominent protein cluster seen at 35–45 kDa. The hamster oolemma, on the other hand, contains a predominant protein cluster at ~25–40 kDa with a less prominent protein cluster at 45–50 kDa.
When evaluating the 2-D avidin blots in Figure 4, a most striking observation is that the predominant 25–40 kDa (pI 5–6) protein cluster (c2) which is evident in the blot of untreated oocytes (Figure 4A) is absent from the blot in which hamster oocytes were treated with PI-PLC (Figure 4B). Further, the supernatant from eggs treated with PI-PLC (arrows, Figure 4D) revealed a protein cluster of similar molecular weight and isoelectric point (25–40 kDa, pI 5–6) to that which was released from the surface of the egg following PI-PLC treatment (Figure 4D). The enzyme appears to be specifically affecting the 25–40 kDa protein cluster (c2) because the staining intensity of the remaining surface-labelled proteins in Figure 4C is similar to that which is seen in the untreated eggs in Figure 4A. Of interest is the observation that, in the hamster, the predominant protein cluster (c2) is PI-PLC sensitive while the less prominent protein cluster (c1) is not affected by PI-PLC treatment (see Figure 4B). However, in the mouse, both the 70 and 35–40 kDa protein clusters are PI-PLC sensitive (Coonrod et al., 1999).
In conclusion, when human spermatozoa are treated with PI-PLC, sperm–egg binding is enhanced while sperm–egg fusion is not effected. When zona-free hamster oocytes are treated with PI-PLC, sperm–egg binding and fusion is blocked. Results from the 2-D avidin blots show that a predominant protein cluster is released from the hamster oolemma following PI-PLC treatment. It seems likely that this PI-PLC-sensitive protein cluster (~25–40 kDa, pI 5–6) mediates human sperm–hamster egg binding and fusion. However, it is also possible that the observed ~25–40 kDa protein cluster is not involved in sperm–egg interaction and the true fertilization-related PI-PLC cleavable protein(s) cannot be visualized using our 2-D avidin blotting technique.
One further possibility is that PI-PLC affects fertilization by altering properties of the egg by some mechanism other than the release of GPI-anchored proteins. For example exogenous PI-PLC may directly or indirectly affect calcium release mechanisms within the oocyte leading to a block to polyspermy (Wilding and Dale, 1997). It will be necessary to identify the PI-PLC-sensitive ~25–40 kDa protein if we are to ascertain the role of the protein (if any) in fertilization. Therefore, we are currently attempting to obtain microsequence information from the protein using mass spectrometry.
Acknowledgments
This work supported by NIH U54 HD 29099, P30-28934, P32-DK07642, T32-HD07382, F32-HD08002, and the Andrew W.Mellon Foundation.
Notes
1 To whom correspondence should be addressed 
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Submitted on May 4, 1999; accepted on August 4, 1999.
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