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A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.
Figure 2. Dose-dependent inhibition of sperm-egg binding by heat-solubilized total VE proteins. The sperm binding competition assay was carried out as described in Materials and Methods. Bars indicate the standard deviation (SD) with n = 15 eggs.
Figure 3. Effects of purified individual VE proteins on sperm-egg binding. The final concentration of each protein tested was equivalent to its concentration in either 25 or 100 μg/ml of total VE protein. The percentage of each of the proteins in the isolated X. laevis VE has been reported to be: gp120 (6.2 ± 1.5%), gp112 (6.5 ± 1.4%), gp69 (1.9 ± 0.8%), gp64 (2.6 ± 0.6%), p57 (0.9 ± 1.2%), gp41 (43 ± 2%), and gp37 (39 ± 5%) (Hedrick and Hishihara, 1991). Bars represent SD with n = 15 eggs.
Figure 4. Polyclonal anti-gp64 and anti-gp69 antibodies and their effects on sperm-egg binding. (A) The specificity of the polyclonal anti-gp69 and anti-gp64 antibodies. Total VE proteins (0.8 μg/lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the DEAE-cellulose purified anti-gp64 (lane 1) or anti-gp69 (lane 3), or with preadsorbed anti-gp64 (lane 2) or anti-gp69 (lane 4). The two bands in lane 2 indicated by arrows are gp69 and gp64, respectively. The band in lane 4 indicated by the arrow is gp69. Molecular weight standards are marked on the left. From top: 216, 97, 71, and 44 kD. (B) The effects of the pre-adsorbed polyclonal anti-gp64 and anti-gp69 antibodies on sperm-egg binding. The horizontal axis represents the antibody concentration in the MR solution in which the eggs were preincubated. Bars represent SD with n = 15 eggs.
Figure 5. Sperm binding to agarose beads containing coupled proteins. The sperm-bead binding assay was carried out as described in Materials and Methods. (A) Quantitation of the average number of sperm bound to beads containing various covalently coupled proteins. Binding was measured in the absence or presence of 70 μg/ml of polyclonal anti-gp64 antibody (which recognized both gp64 and gp69). Approximately 0.3â0.4 nmol of protein was coupled per mg of beads. Bars represent SD with n = 30 beads. The effects of the anti-gp64 antibody on gp120/112, gp41, gp37 were not determined (ND). (B) Fluorescence micrographs of sperm binding to beads coated with a 1:1 mixture of gp69 and gp64 (a) or BSA (b). Bar, 20 μm.
Figure 6. Effect of crude type I collagenase on the VE proteins and on sperm-egg binding. (A) SDS-PAGE analysis of total VE proteins isolated from a control group of eggs (lane 1) or from eggs treated with crude typeâI collagenase (lane 2). The gel was stained with Coomassie blue. Molecular weight standards are marked and shown in lane M. From top: 216, 97, 71, and 44 kD. Arrows in lane 1 indicate gp69 and gp64, respectively. Arrows in lane 2 indicate the two bands at â¼65 and 60 kD which are derived from gp69 and gp64, respectively. (B) Sperm binding levels to control eggs (lane 1) or type I collagenaseâtreated eggs (lane 2). Bars represent SD with n = 15 eggs.
Figure 8. Effect of PNGase F treatment on gp69/64. Purified gp69 and gp64 (total 1 μg , lane 1) and PNGase Fâtreated gp69/64 (total 10 μg, lane 3) were separated by 7.5% SDS-PAGE, electrotransferred to membrane, and immunoblotted with specific anti-gp69/64 antibody. The band in lane 3 was much more intense because 10 times as much protein was loaded. The molecular masses of the two bands are marked on the right. Lane 2 shows the 65-kD and 60-kD bands (total 1 μg) generated from crude collagenase digestion of gp69/64.
Figure 7. Possible role of carbohydrates on sperm binding. (A) Effect of metaperiodate treatment on sperm binding to live eggs. (B) Effects of Pronase-digested VE glycopeptides on sperm-egg binding. A total of 5 μg of gp69/64 (1:1) and proportional amounts of other VE proteins were used according to the ratio indicated in Fig. 3. The sperm binding level in the presence of an equal amount of heat-inactivated Pronase was used as a control and designated as 100%. Bars represent SD with n = 15 eggs.
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