Sperm-binding regions on bovine egg zona pellucida glycoprotein ZP4 studied in a solid supported form on plastic plate

Abstract

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. The bovine ZP consists of the glycoproteins ZP2, ZP3, and ZP4. Sperm-binding mechanisms of the bovine ZP are not yet fully elucidated. In a previous report, we established the expression system of bovine ZP glycoproteins using Sf9 insect cells and found that the ZP3/ZP4 heterocomplex inhibits the binding of sperm to the ZP in a competitive inhibition assay, while ZP2, ZP3, ZP4, the ZP2/ZP3 complex, and the ZP2/ZP4 complex do not exhibit this activity. Here, we show that bovine sperm binds to plastic plates coated with ZP4 in the absence of ZP3. We made a series of ZP4 deletion mutants to study the sperm-binding sites. The N-terminal region, Lys-25 to Asp-136, and the middle region, Ser-290 to Lys-340, of ZP4 exhibit sperm-binding activity. These results suggest that among the three components of bovine ZP glycoproteins, ZP4 contains the major potential sperm-binding sites, and the formation of a multivalent complex is necessary for the sperm-binding activity of ZP4.

Fig 1. Schematic representation of the bovine zona pellucida (ZP) 4 polypeptide precursor and the recombinant ZP4 mutants examined in this study.

The bovine ZP4 precursor is synthesized as a transmembrane protein consisting of a signal peptide, the N-terminal ZP-N-like domain (ZP-N1), the trefoil domain, the N-terminal half domain of the ZP module (ZP-N2), the flexible hinge region, an internal hydrophobic patch (IHP) in the C-terminal half domain of the ZP module (ZP-C), a consensus furin cleavage site (CFCS), an external hydrophobic patch (EHP), and a transmembrane domain. The mature ZP4 polypeptide includes Lys-25 to Arg-464. The ZP4 fragments examined in this study are shown by open bars. Translation initiation Met is numbered as 1.

Fig 2. Sperm-binding activity of bovine ZP proteins in a solid supported form.

(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant bovine ZP proteins. Each single component of bovine ZP, ZP2, ZP3, and ZP4; combinations of two components, ZP2/ZP3, ZP2/ZP4, and ZP3/ZP4; and combinations of all three components, ZP2/ZP3/ZP4; were expressed in Sf9 cells by infecting the corresponding baculoviruses and partially purified using TALON resin specific to the His-tag. SDS-PAGE of elution fractions are shown. Gels were silver-stained. Molecular mass standards (kDa) are indicated on the left side of each panel. In the gels loaded with two or three component mixtures, each band is shown by an arrow with a number (ZP2:2, ZP3:3, and ZP4:4). (B) Adsorption of recombinant bovine ZP proteins to plastic wells. The amount of each recombinant ZP protein or mixture added to one plastic well is indicated under each group of bars. The amount of proteins necessary for adsorption saturation was examined by detecting the adsorbed proteins with antibodies specific to N-terminal His-tag and N-terminal FLAG-tag. The experiment was performed three times and average±standard deviation (SD) of absorbance at 405 nm is shown. The amounts of ZP2, ZP3, ZP4, ZP2/ZP3, ZP2/ZP4, ZP3/ZP4, and ZP2/ZP3/ZP4 adsorbed to plastic wells are shown by bars with the gradation from dark gray (left) to light gray (right), respectively, in each group of seven bars. (C) Sperm-binding activity of bovine ZP proteins. Plastic wells were coated with the ZP proteins indicated in the graph. The number of sperm bound to wells coated with solubilized native ZP varied from 28 to 72 among experiments but was designated as 100% at each experiment. Assays were repeated at least three times. Data are presented as the mean ± SD, with statistical significance relative to “solubilized ZP” indicated as P < 0.01 (**) on the right side of the SD bars. The statistical significance between ZP4 and ZP2/ZP4, and between ZP3/ZP4 and ZP2/ZP3/ZP4, is indicated as P < 0.01 (**) and P < 0.05 (*) above the respective lines.

Fig 3. Sperm-binding activity of bovine ZP proteins multimerized in a solution using streptavidin-biotin binding.

(A) Biotinylation of bovine ZP proteins. Bovine ZP2, ZP3, and ZP4 were biotinylated as described in Materials and methods section. These proteins were subjected to westernblot and detected with anti-His tag antibody (anti-His) used as a primary antibody (left panel). The same membrane was stripped and reprobed with horseradish peroxidase-conjugated streptavidin (Streptavidin, right panel). Molecular mass markers are indicated on the left of the membrane in kDa. (B) Effect of streptavidin on the competitive inhibition of biotinylated ZP proteins on sperm–ZP binding. Plastic wells were coated with recombinant bovine ZP2/ZP3/ZP4 mixture. Bovine sperm were preincubated with each inhibitor indicated below each bar and then added to the coated wells. bZP2, biotinylated ZP2; bZP3, biotinylated ZP3; bZP4, biotinylated ZP4; SA, streptavidin. The count of sperm bound to the bovine ZP2/ZP3/ZP4-coated wells in the absence of inhibitors (Without inhibitors) varied from 39 to 57 among experiments but was designated as 100% at each experiment. Assays were repeated four times. Data are presented as the mean ± SD. Neither bZP2 nor bZP3 showed significant increase in inhibitory activity by preincubation with SA, while bZP4 showed significant increase in inhibitory activity by preincubation with SA, as indicated by P < 0.01 (**) above the line. (C) Indirect immunofluorescent detection of ZP4 bound to bovine sperm. Biotinylated bovine ZP4 was preincubated with streptavidin (+SA, upper panels) or without streptavidin (–SA, lower panels) and then mixed with bovine sperm. After washings, ZP4 bound to sperm was detected with anti-porcine ZP4 antiserum as a primary antibody and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody as a secondary antibody. Arrows show that acrosomal region is fluorescently stained. Phase, phase contrast image; Fluorescence, fluorescence image; SA, streptavidin. Bar in the upper left panel indicates 10 μm.

Fig 4. Sperm-binding activity of N-terminal fragments of bovine ZP4.

(A) SDS-PAGE of N-terminal fragments of bovine ZP4. Four N-terminal fragments of bovine ZP4, ZP4 (25–340), ZP4 (25–289), ZP4 (25–184), and ZP4 (25–136) were expressed in Sf9 cells and partially purified by TALON resin specific to His-tag. Gels were silver-stained. The objective bands are indicated by arrows. Molecular mass standards (kDa) are indicated on the left side of each panel. (B) Sperm-binding activity of the four N-terminal fragments of bovine ZP4. Plastic wells were coated with the ZP proteins (0.8 μg for each protein) indicated in the graph. The number of sperm bound to wells coated with ZP4 varied from 33 to 65 but was designated as 100% at each experiment. Assays were repeated at least three times. Data are presented as the mean ± SD, with statistical significance relative to ZP4 indicated as P < 0.05 (*) and P < 0.01 (**) on the right side of the SD bars. The statistical significance between ZP4 (25–340) and ZP4 (25–289) is indicated as P < 0.05 (*) above the line. There was no statistical significance among ZP4 (25–289), ZP4 (25–184), and ZP4 (25–136).

Fig 5. Sperm-binding activity of C-terminal fragments of bovine ZP4.

(A) SDS-PAGE of C-terminal fragments of bovine ZP4. Two C-terminal fragments of bovine ZP4, ZP4 (136–464) and ZP4 (286–464) were expressed in Sf9 cells and partially purified by using TALON resin specific to His-tag. Gels were silver-stained. The objective bands are indicated by arrows. Molecular mass standards (kDa) are indicated on the left side of each panel. (B) Sperm-binding activity of the two C-terminal fragments of bovine ZP4. Plastic wells were coated with the ZP4 fragments (0.8 μg for each fragment) indicated in the graph. The number of sperm bound to wells coated with ZP4 varied from 33 to 65 but was designated as 100% at each experiment. Assays were repeated at least three times. Data are presented as the mean ± SD, with statistical significance relative to ZP4 indicated as P < 0.01 (**) on the right side of the SD bars. There was no statistical significance between ZP4 (136–464) and ZP4 (286–464).

Fig 6. Sperm-binding activity of the region from Ser-290 to Lys-340 of bovine ZP4.

(A) SDS-PAGE of enhanced green fluorescent protein (EGFP) and the fragment from Ser-290 to Lys-340 of bovine ZP4 fused to EGFP (EGFPZP4 (290–340)). These proteins were expressed in Sf9 cells and partially purified by using TALON resin specific to His-tag. Gels were silver-stained. The objective bands are indicated by arrows. Molecular mass standards (kDa) are indicated on the left side of the gel. df, dye front. (B) Sperm-binding activity of EGFP and EGFPZP4 (290–340). Plastic wells were coated with the recombinant proteins (1.0 μg for each protein) indicated in the graph. The number of sperm bound to wells coated with ZP4 varied from 32 to 64 among experiments but was designated as 100% at each experiment. Assays were repeated five times. Data are presented as the mean ± SD, with statistical significance between EGFP and EGFPZP4 (290–340) indicated as P < 0.01 (**) above the line.