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First published online 24 December 2003
doi: 10.1242/dev.00937

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Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat

Department of Cell Biology, Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA

View larger version (16K): [in a new window]   Fig. 1. Ovulated, but not ovarian, zona pellucida (ZP) glycoproteins contain a ligand that competitively inhibits GalT I-null sperm binding to ovulated eggs. (A) Inhibition of wild-type and GalT I-null sperm binding to ovulated eggs with 0-75 ng/µl solubilized ovarian ZP. (B) Inhibition of wild-type and GalT I-null binding with 0-5 zonae equivalents solubilized ovulated ZP. Each point represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value for each data point represents the average of three determinations (for a total of nine assays per data point).

View larger version (15K): [in a new window]   Fig. 2. Wild-type and GalT I-null sperm binding to ovulated eggs is competitively inhibited by a ligand peripherally associated with the ovulated zona pellucida (ZP). (A) Inhibition of wild-type sperm binding by glycoproteins solubilized from either the matrix fraction or peripheral fraction. (B) Inhibition of GalT I-null sperm binding by the matrix and peripheral fractions. Each point represents the mean±s.e.m.; n=3 experiments. In a single experiment, the value for each data point represents the average of three determinations (for a total of nine assays per data point).

View larger version (12K): [in a new window]   Fig. 3. The ligand in the peripheral fraction of the ovulated zona pellucida (ZP) is not a contaminant of the cortical granules. (A) N-acetylglucosaminidase activity of fractions of the ovulated ZP (three ZP equivalents/µl) prepared from eggs treated with 10 µM A23187 to induce the cortical reaction or DMSO, as control. (B) Biological activity of the ovulated ZP fractions (three ZP equivalents/µl) prior to and after treatment of the eggs with 10 µM A23187. Each bar represents the mean±s.d.; n=2 experiments. In a single experiment, the value for each data point represents the average of three determinations (for a total of six assays per data point). The peripheral fraction contains variable amounts of GlcNAc'ase, but this contribution does not account for its biological activity.

View larger version (32K): [in a new window]   Fig. 4. The biological activity in the peripheral fraction is not due to ZP3 contamination. (A) Wild-type sperm, but not GalT I-null sperm, are inhibited from binding ovulated eggs by a monoclonal antibody against mouse ZP3 (East et al., 1985). Controls were performed with a non-specific rat IgG. Each bar represents the mean±s.d.; n=1 experiment, which was carried out in triplicate. (B) Immunoblot of matrix and peripheral fraction proteins from ovulated zonae pellucidae with a rat monoclonal antibody against mouse ZP3. Molecular weight markers are shown.

View larger version (14K): [in a new window]   Fig. 5. Oviduct-secreted glycoprotein (OGP) is not the peripherally associated, ZP3-independent ligand. Inhibition of wild-type and GalT I-null sperm binding to eggs by ovulated zona glycoproteins (three ZP/µl) prepared from wild-type and OGP-null females (Araki et al., 2003). Each bar represents the mean±s.d.; n=1 experiment, which was carried out in triplicate. Wild-type and GalT I-null sperm are equally inhibited from binding to ovulated eggs by zona glycoproteins isolated from wild-type and OGP-null females. The broken line indicates the number of sperm bound in the control (buffer).

View larger version (15K): [in a new window]   Fig. 6. The peripheral fraction contains a WGA-reactive high molecular weight glycoprotein that correlates with the biological activity of the ligand. (A) Biological activity of matrix and peripheral fractions before and after depletion with WGA-agarose or BS-I agarose. WGA depletes ligand activity, assayed against wild-type sperm, from both the matrix and peripheral fractions, whereas BS-I fails to deplete activity from either fraction. Each bar represents the mean±s.e.m. of triplicate assays from one experiment. The broken line indicates the number of sperm bound in the control (buffer). (B) WGA and BSI lectin blots of peripheral fraction glycoproteins. WGA reacts with high molecular weight glycoproteins in the peripheral fraction, whereas BSI does not react with any glycoprotein species. Each blot is representative of at least two experiments. Molecular weight markers are shown.

View larger version (39K): [in a new window]   Fig. 7. Ovulated, but not ovarian, zona pellucida (ZP) glycoproteins contain a basic, WGA-reactive, 250 kDa glycoprotein. Ovarian (A) and 500 zonae equivalents of ovulated (B) zona proteins (2.5 µg) were separated by two-dimensional polyacrylamide gel electrophoresis, transferred to PVDF and visualized by staining with biotinylated-WGA. This blot is representative of five experiments. The arrow indicates a 250 kDa, basic, WGA-reactive protein that is present in ovulated ZP, but not ovarian ZP. The pH gradient, the theoretical location of the matrix proteins [according to Bleil and Wassarman (Bleil and Wassarman, 1980b)], and molecular weight markers are shown.

View larger version (16K): [in a new window]   Fig. 8. Proteins isolated from the basic region of an IEF gel containing ovulated zona pellucida (ZP) glycoproteins inhibit wild-type and GalT I-null sperm binding to ovulated eggs. Proteins were isolated from acidic (fraction 1), neutral (fractions 2 and 3) and basic (fraction 4) regions of an IEF gel containing 5000 ovulated ZP and tested for biological activity in the sperm-egg binding assay. The acidic fraction (containing ZP3) inhibits wild-type, but not GalT I-null, sperm from binding to ovulated eggs. By contrast, the basic fraction (containing the basic ligand) prevents both wild-type and GalT I-null sperm from binding to ovulated eggs. Doubling the amount of starting material lead to 69% inhibition of sperm-egg binding by fraction 4. Each bar represents the mean±s.e.m.; n=4 experiments. In a single experiment, the value for each bar represents the average of three determinations (for a total of 12 assays per bar). The broken line indicates the number of sperm bound in the control, i.e. material obtained from a blank IEF gel treated identically to gels containing zona glycoproteins.

View larger version (15K): [in a new window]   Fig. 9. Binding of GalT I-null sperm to the egg is independent of other members of the GalT family. UDP-galactose inhibits wild-type sperm, but not GalT I-null sperm, from binding to ovulated eggs. Each data point represents the mean±s.d.; n=2 experiments. In a single experiment, the value for each data point represents the average of three determinations (for a total of six assays per data point). UDP-glucose had no effect on the binding of either sperm genotype to eggs (data not shown).

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