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Biology of Reproduction 66, 1585-1595 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Characterization of Zona Pellucida Glycoprotein 3 (ZP3) and ZP2 Binding Sites on Acrosome-Intact Mouse Sperm1

Candace L. Kerra, William F. Hannaa, Joel H. Shaperb, and William W. Wright2,,a

a Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205-2179 b The Cell Structure and Function Laboratory, The Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-1000


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (Bmax ~185 000 sites per sperm; Kd ~67 nM) and ZP2 (Bmax ~500 000 sites per sperm; Kd ~200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.

developmental biology, fertilization, ovum, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An essential event in mouse fertilization is the tight binding of capacitated, acrosome-intact sperm to the zona pellucida (ZP) [1–3]. This extracellular matrix is composed of three glycoproteins, ZP1, ZP2, and ZP3 [1]. Considerable evidence supports the hypothesis that acrosome-intact sperm initially bind to ZP3 and in response to this binding undergo the acrosome reaction, a calcium-dependent, exocytotic event that causes vesiculation and subsequent loss of the outer acrosomal membrane and the overlying plasma membrane [4]. As a result, ZP3 binding sites on the overlying plasma membrane are lost, and ZP2 binding sites on the inner acrosomal membrane are exposed [5]. It has been proposed that these secondary ZP2 binding sites allow acrosome-reacted sperm to remain bound to the ZP [6, 7]. Thus, the initial events in the mouse fertilization cascade involve, at minimum, two different types of ZP binding sites on sperm.

A complete and systematic characterization of ZP3 and ZP2 binding sites is a necessary step in the elucidation of the molecules and mechanisms that mediate the interactions of sperm with the ZP. Thus, a goal of the experiments described herein was to define fundamental characteristics of ZP3 and ZP2 binding sites on acrosome-intact mouse sperm. These characteristics include the distribution of these binding sites over the sperm surface, the numbers of sites per sperm, and the affinities of these sites for their respective ligands. Additionally, our experiments tested whether ZP3 and ZP2 bind these sperm surface sites in a ZP3- and ZP2-specific manner, respectively. We recognized in beginning these studies that other investigators had examined the binding of zona glycoproteins to sperm (see for example [5, 8–10]). However, to date, none have demonstrated that ZP3 and ZP2 bind to mouse sperm in both a saturable and ZP glycoprotein-specific manner. Saturability and ligand specificity are hallmarks of all functional cell surface binding sites or receptors [11]. Documentation of these hallmarks is a prerequisite for accurately estimating the numbers of binding sites on any cell and to conclude that a particular ligand binds a single type of site.

The experiments described in this paper use fluorescence microcopy and image analysis to quantify the binding of fluorochrome-labeled mouse ZP3 and ZP2 to living mouse sperm. These methods allow us to describe the fundamental characteristics of the aforementioned ZP3 and ZP2 binding sites and to test whether extracellular calcium or maturation or capacitation of sperm affects the functions of these two types of binding sites. These final experiments are prompted by the fact that although calcium and maturation and capacitation of sperm are required for sperm to fertilize an egg, there is no consensus as to whether they are also required for mouse sperm to bind to ZP3 or to ZP2 [9, 12, 13]. Results presented in this paper establish that acrosome-intact sperm express distinct and ZP glycoprotein-specific binding sites for ZP3 and ZP2 and describe the distribution of both types of sites on the surface of sperm. Furthermore, our experiments demonstrate that extracellular calcium and the maturation and capacitation of sperm are prerequisites for these two types of sites to bind their respective ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Heat-Solubilized ZP Glycoproteins

ZP were isolated from frozen ovaries obtained from 6- to 7-wk-old ICR female mice (Harlan BioProducts for Science, Indianapolis, IN) using Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) gradient centrifugation [8]. Isolated ZP were then heat-solubilized at 60°C for 15 min in 0.1% SDS, 0.2 M NaH2PO4, pH 6.5, and ZP1, ZP2, and ZP3 were separated by HPLC using a 250 Bio-Sil Select size exclusion column (Bio-Rad Laboratories, Hercules, CA) as previously described [6]. Duplicate samples of each fraction were characterized using 7.5% SDS-PAGE run under nonreducing conditions [14], and proteins were detected by silver staining or by Western blot analysis using rat monoclonal immunoglobulin G (IgG) antibodies against mouse ZP1, ZP2, and ZP3 [15–17]. Antibody-antigen complexes were detected using biotinylated rabbit anti-rat IgG and the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Fractions from several HPLC runs containing only ZP1, ZP2, or ZP3 were then pooled, dialyzed for 48 h against four 4-L changes of water, and then concentrated using Centricon-10 (Millipore, Bedford, MA).

Fluorescence Labeling of ZP

The Alexa Fluor 568 fluorochrome (Alexa) was conjugated to a mixture of total ZP proteins, to individual ZP glycoproteins, and to BSA according to the manufacturer's instructions (Molecular Probes, Eugene, OR). To compare binding of different ZP glycoproteins, only fluorescence-labeled ZP glycoproteins with approximately 1 mole of fluorochrome attached per mole of protein were used. Protein concentrations and levels of substitution of Alexa-ZP glycoproteins and Alexa-BSA was determined using the following formulas provided by the manufacturer:

91 300 cm-1 M-1: molar extinction coefficient of the Alexa Fluor 568 at 577 nm

A280 and A577: absorbance of the Alexa-labeled protein solution at 280 and 577 nm, respectively

E(280,ptn): molar extinction coefficient of the protein at 280 nm (cm-1 M-1), where ptn = BSA, ZP1, ZP2, or ZP3

Df: dilution factor

Estimated molar extinction coefficients for BSA or individual ZP glycoproteins were calculated based on the numbers of tyrosine and tryptophan residues per mole of protein and the molar extinction of these two amino acids [18]. The number of these residues for BSA was determined from its amino acid sequence [19]. The number for each ZP glycoprotein was derived from reported coding sequences for ZP1 [20], ZP2 [21], and ZP3 [22, 23]. Only sequences included in the mature proteins were used in the calculations. Therefore, the extinction coefficients calculated for BSA, ZP1, ZP2, and ZP3 were 48 150, 60 980, 74 230, and 35 010 cm-1 M-1, respectively. The molar extinction coefficient for estimating the concentration of fluorescence-labeled total ZP (Alexa-ZPtotal) preparations were based on a weighted average of ZP1, ZP2, and ZP3 extinction coefficients using a 1:5:5 molar ratio of each ZP glycoprotein in ZP, respectively [24]. Unlabeled ZP1, ZP2, and ZP3 concentrations were estimated using the Quantigold Protein Assay (Diversified Biotech, Boston, MA) and BSA as the standard. Similar results were obtained when protein concentration of a heat-solubilized ZP preparation was measured by the Quantigold assay and by the measurement of A280.

Microscopic Analysis

Fluorescence images were captured at 400x magnification using a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY) equipped with a 40x Plan Apo lens and a Princeton 5-MHz cooled interlined CCD camera (Princeton Instruments, Trenton, NJ). Alexa fluorescence was detected using a G2E-RHOD excitation filter, a 565-nm dichroic mirror, and a barrier filter with a band width of 600–660 nm (Nikon). The binding to sperm of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) (Vector) or SYTOX-Green (Molecular Probes) was detected using an FITC excitation filter, a 505-nm dichroic mirror, and a barrier filter with a band width of 515–555 nm. Both barrier filters were manufactured by Chroma, Inc. (Burlington, VT).

Quantitative Analysis of Fluorescent Proteins Boundto Sperm

To quantify the binding of fluorescent ZP glycoproteins to individual sperm heads, images of sperm heads were captured and imported into IPLab Spectrum analysis software (Scanalytics, Fairfax, VA). Three 0.0385-mm2 fields were captured from each slide, and every sperm in the field (3–30 sperm per field) was analyzed. To quantify fluorescence, a rectangular area of approximately 48 µm2 (~2000 pixels) was drawn around each sperm head. Total fluorescence units (FUs) in this box were divided by the number of pixels in the rectangle, giving mean fluorescence intensity. An adjacent area of the field that did not contain sperm was also measured to establish background fluorescence. In a given field, a box of the same size was used to measure fluorescence from sperm and from the background. Because we used a range of concentrations of ZP glycoproteins in our experiments, a range of exposure times of the digital camera were required. To correct data for time of exposure, mean fluorescence intensities were divided by seconds of exposure. These data were further corrected for autofluorescence of sperm to give units of fluorescence emission (FE) per sperm. Thus,

FE: fluorescence emission (FU area-1 sec-1)

Auto: autofluorescence

C: mean fluorescence intensity, which equals total FU divided by area encompassing the sperm head (FU/area)

Back: background fluorescence

Exp1 and Exp2: exposure times in seconds for Alexa-ZP proteins on sperm and autofluorescence of sperm, respectively.

We noted no photobleaching of Alexa-ZP-labeled sperm during the time that images were recorded.

To determine the linear range of the detection system, a standard curve was produced using Alexa-BSA. For this purpose, known amounts of Alexa-BSA in water were dried on slides inside a manufactured circle with a 15-mm diameter (FisherBrand ProbeOn Plus; Fisher, Pittsburgh, PA), mounted with Vectashield mounting medium (Vector) and immediately analyzed under the microscope. The fluorescence emission of six 0.0385-mm2 fields per slide was then measured. During the analysis, fluorescence emission outside of the 15-mm-diameter circle remained at background, indicating that Alexa-BSA was not dissolved during the 2–3 min required for imaging. Results of the dose-response analysis demonstrated a linear response from ~10–2500 FU/area x sec, which includes the range of measurements (~20–1500 FU/area x sec) described in this paper (Fig. 1).



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  		FIG. 1. Demonstration of the linearity of the fluorescence microscopic detection system. Alexa-BSA (~1 mole fluorochrome per mole of BSA) in water was dried to 15-mm circles etched on glass slides, and the slides were mounted in Vectashield mounting medium just before analysis. The FE from six 0.0385-mm2 fields per slide was then measured. Shown is FE (mean ± SEM) from a mass of Alexa-BSA in the 0.0385-mm2 fields. A linear response from ~10–4000 relative FE units was observed (R2 = 0.995)

Conversion of the FE to Molecules of Protein Bound

Conversion of FE into moles of ZP glycoprotein bound per sperm head was accomplished by using Alexa-BSA, labeled with approximately 1 mole of fluorochrome per mole of BSA, as a fluorescent standard. An FE of 100 ng Alexa-BSA (1.5 x 10-12 moles) was established by drying 100 ng of BSA within a 15-mm-diameter circle (176 mm2) on glass slides; the slides were mounted in Vectashield, and total fluorescence emitted from six 0.0385-mm2 fields per slide was determined as described previously. These fields omitted the edge and immediate middle of the circle but otherwise were selected randomly. There was some variation in the distribution of dried Alexa-BSA within the circle; a 0.2-µm rim of BSA surrounded the dried spot, and fluorescence from this rim was approximately 50% greater than that in an adjacent area that contained Alexa-BSA. Additionally, there were detectable differences in the fluorescence emitted from Alexa-BSA from different parts of this circle. These differences resulted in a coefficient of variation (SD divided by mean) for this method of 8%.

The average total fluorescence per exposure time across all fields was ~7.02 x 108 FU/sec. Similar results were obtained when Alexa-ZP3 was used as a standard. This average was corrected by the number of fields by 4588 (area covered by the entire sample divided by the area of one microscopic field) to calculate the total fluorescence emitted by the 1.5 x 10-12 moles of Alexa-BSA. One FU per second was therefore equal to 4.67 x 10-25 moles of Alexa-BSA. This conversion factor was used to convert fluorescence, measured as FU sec-1 area-1, emitted by Alexa-ZP proteins bound to a sperm to moles of bound ZP protein. Thus,

To convert data to molecules bound per sperm, moles bound per sperm were multiplied by the Avagadro number, 6.023 x 1023.

Estimation of the Percentage of ZP3 Binding Sites over the Acrosomal Cap and the Postacrosomal Domain of the Sperm Head

To calculate the percentage of ZP3 binding sites over the postacrosomal domain, the mean fluorescence intensities for the acrosomal cap and for the postacrosomal domain were first estimated. FUs for a series of randomly selected pixels over these domains were measured, and the FE for both domains was calculated as the means of these pixel intensities. The area of each domain was measured by use of the image analysis software, and each area was multiplied by the FE for its domain. The estimate of the percentage of the total ZP3 binding sites that were on the postacrosomal domain were then calculated.

Culture Media

Two culture media were used for the isolation and incubation of mouse sperm with fluorescent ZP proteins. All but two experiments required capacitated sperm and, thus, used Medium 199 (Life Technologies, Gaithersburg, MD) supplemented with 4 mg/ml crystalline BSA (Sigma, St. Louis, MO) and 30 µg/ml sodium pyruvate (M199-M). Incubations of sperm in this medium were conducted at 37°C in 95% air, 5% CO2. Except where noted in the results, 1 µg/ml Bordetella pertussis toxin (Calbiochem, San Diego, CA) was added with the ZP proteins to sperm to prevent the ZP-induced acrosome reaction [25]. One experiment tested whether binding of sperm to ZP3 or ZP2 required extracellular calcium, and in this experiment, 0.1 M Na2EGTA (pH 7.5) (Sigma) was added simultaneously with ZP proteins to obtain a final EGTA concentration of 3.75 mM. (The concentration of Ca2+ in M199-M is 1.8 mM.)

Two experiments tested whether noncapacitated sperm bound ZP proteins. For this purpose, sperm were isolated and incubated in medium M199-NC, which lacked NaHCO3 but contained 1 g/L polyvinyl alcohol, 5.95 g/L Hepes, 1.53 g/L NaCl, 100 µg/ml BSA, and 30 µg/ml sodium pyruvate. All incubations of sperm in M199-NC were carried out at 37°C in 100% air.

Isolation of Spermatozoa

Use of mice as a source of sperm for these studies was approved by the Institutional Animal Care Use Committee. Sperm were isolated from 8- to 12-wk-old proven-breeder CD-1 mice (Charles River Breeding Labs, Wilmington, MA). Except where noted in results, sperm were collected from the cauda epididymides using a swim-up technique [26]. Briefly, cauda epididymides of two mice were minced, and sperm allowed to swim out into 0.5 ml medium for 15 min. Two hundred fifty microliters of the pooled sperm suspension was submerged under 1.5 ml fresh media in a polyethylene tube (12 x 75 mm) and incubated undisturbed at 37°C for 45 min. Viable sperm that swam up only into the 0.5 ml of medium were collected, counted, and used in the experiments described herein.

Sperm from caput epididymides were expressed from tubules into either M199-M or M199-NC and incubated for 45 min before addition of ZP proteins.

Incubation of Sperm with ZP Glycoproteins

To establish the conditions for all of the experiments described in this paper, preliminary studies were conducted using Alexa-ZPtotal as a ligand for sperm. If binding was detected under those conditions, experiments were repeated with Alexa-ZP3, Alexa-ZP2, or Alexa-ZP1. The general protocol for all but two experiments follows: ~75 000 sperm were pipetted into a 500-µl microcentrifuge tube and incubated at 37°C for 1.5 h in 25 µl of medium containing Alexa-ZP proteins. Preliminary experiments (data not shown) indicated that 1.5 h was required to detect binding of Alexa-ZP proteins to >95% of sperm, although some sperm bound ZP proteins in as little as 5 min. After 1.5 h, sperm were pelleted by centrifugation at 500 x g for 5 min. Fifteen microliters of supernatant was removed, and the sperm were resuspended by addition of an additional 50 µl of M199-M and repelleted. After the second centrifugation step, 50 µl of medium was removed, and the sperm were suspended and fixed for 30 min by addition of 50 µl of 4% formaldehyde in Ca2+-Mg2+-free Dulbecco PBS, pH 7.5. Sperm were then transferred to FisherBrand SuperFrost/Plus slides (Fisher), air dried overnight, rinsed for 5 min in Dulbecco PBS, and mounted with Vectashield mounting medium for microscopic analysis. In some experiments, sperm in one tube were resuspended in 60 µl of Dulbecco PBS after the second centrifugation step and placed as a drop on a glass slide. The motility of this sample of sperm was immediately confirmed by phase-contrast microscopy.

In experiments to localize ZP3 and ZP2 binding sites on sperm, one half of the sperm samples were incubated in M199-M supplemented with pertussis toxin, and the other half were incubated in M199-M supplemented with the calcium ionophore A23187 (5 µM) (Sigma) to induce the acrosome reaction. To analyze dose-dependency of ZP glycoprotein binding, sperm were incubated with 3.0–360 nM of Alexa-ZP1, Alexa-ZP2, Alexa-ZP3, or Alexa-ZPtotal. The concentration of Alexa-ZPtotal was the calculated sum of the molar concentrations of all three ZP glycoproteins. To determine whether ZP3 and ZP2 binding sites preferentially bound to their respective ligands, competitive binding assays were performed in which sperm were incubated with either 36 nM Alexa-ZP3 or Alexa-ZP2 and with or without a 5-fold (180 nM) or 10-fold (360 nM) excess of unlabeled ZPtotal, ZP1, ZP2, or ZP3.

The general protocol described previously was modified in two experiments. The objective of the first experiment was to visualize the distribution of Alexa-ZP3 and Alexa-ZP2 binding sites on live, unfixed sperm. In this experiment, after incubation and washing of the sperm with M199-M, sperm were pelleted and resuspended in M199-NC supplemented with 50 nM SYTOX Green, a fluorescent, membrane-impermeable nucleic acid stain. Spermatozoa were then allowed to adhere to CellTak-coated glass coverslips (BD Biosciences, Bedford, MA) and immediately examined by fluorescence microscopy. The objective of the second experiment was to examine the distribution of Alexa-ZP3 and Alexa-ZP2 binding sites on sperm when diffusion of membrane proteins was inhibited by cold temperature. In this experiment, the general protocol described previously was followed with the exception that after capacitation, the sperm were cooled on ice, and the incubation, washing, and fixation of sperm were conducted at 4°C.

Determination of Whether Individual Sperm Have Undergone the Acrosome Reaction

To correlate the location ZP3 and ZP2 binding sites with the presence or absence of the acrosome, sperm were incubated for 1.5 h in M199-M with 360 nM Alexa-ZP3 or Alexa-ZP2, pelleted by centrifugation at 500 x g for 5 min, washed twice in M199-NC, fixed in 70% ethanol, and dried overnight onto slides. Sperm were then incubated for 10 min in 100-µl drops of 100 µg/ml of FITC-PNA, rinsed for 5 min in Ca2+-Mg2+-free Dulbecco PBS, and mounted [27]. Sperm were located on the slides by their binding of Alexa-ZP3 or Alexa-ZP2 and scored as acrosome intact if FITC-PNA stained the entire acrosomal cap.

Statistical Analysis

For each experiment, all treatments were analyzed in duplicate, and every experiment was replicated three times. Data from all binding experiments were obtained from ~200–900 sperm per treatment and were presented as average fluorescence emission (FU/area x sec) ± SEM. The affinities of Alexa-ZP3 and Alexa-ZP2 for sperm and the number of binding sites on sperm were estimated by Scatchard plot analysis. Differences among treatments were compared using ANOVA, and effects of individual treatments were compared using the Fisher least significant difference test. For all analyses, statistically significant differences were defined as P < 0.05 using Statistical Analysis Systems, Statistical Package version 6.1 (SAS Institute, Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preparation and Biologic Characterization of Alexa-ZP1, Alexa-ZP2, and Alexa-ZP3

ZP1, ZP2, and ZP3 were isolated from heat-solubilized ZP by size-exclusion HPLC as detailed in Materials and Methods. Individual column fractions were analyzed by SDS-PAGE under nonreducing conditions, and resolved proteins were detected by silver staining and by Western blot analysis using monoclonal antibodies specific for each ZP glycoprotein. Appropriate fractions containing a single ZP glycoprotein were pooled and reanalyzed by SDS-PAGE, and the proteins were detected by silver staining or by Western blot analysis to demonstrate homogeneity (Fig. 2A).



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  		FIG. 2. A) Characterization of isolated ZP glycoproteins. Isolated ZP from frozen mouse ovaries were solubilized and subjected to HPLC. ZP1, ZP2, and ZP3 from several HPLC runs were pooled, and approximately 10 ng of protein was analyzed by nonreducing SDS-PAGE. Purity of the protein in each pool was assessed by silver staining (A, C, E), and the identity of the proteins was confirmed by Western blot analysis using monoclonal antibodies to ZP1, ZP2, and ZP3 (B, D, F). Additional Western blot analyses confirmed that a pool of one ZP glycoprotein did not contain detectable levels of the other two ZP glycoproteins (data not shown). Numbers represent the position of the relative molecular mass standards (Mr x 103). B) Biologic activities of Alexa-ZP2 and Alexa-ZP3 as measured by the induction of the acrosome reaction. Control mouse sperm (open bars) were incubated in M199-M alone, in M199-M supplemented with 5 µM A23187 (Ca-I) to induce the acrosome reactions, or in M199-M supplemented with 1 µg/ml pertussis toxin (PT) to inhibit the acrosome reaction. Test sperm were incubated in either 360 nM Alexa-ZP2 (ZP2) (hatched bars) or Alexa-ZP3 (ZP3) (black bars) in the presence or absence of pertussis toxin. The presence of the acrosome was assessed by staining with FITC-PNA. Percentages were calculated from 100 sperm per sample, two samples per experiment, and then averaged across three experiments. Data are presented as means ± SEMs. Letters represent statistically significant differences between treatments

The fluorochrome Alexa was conjugated to ZP1, ZP2, and ZP3 (~1 mole fluorochrome per mole of protein) to enable us to visualize and quantify the binding of individual ZP glycoproteins to living sperm. Before using Alexa-ZP proteins for this purpose, we posed two questions. First, did the purification process introduce factors (e.g., detergent, salt) that increase the frequency of a spontaneous acrosome reaction? Second, did the presence of the fluorochrome on ZP3 block its ability to trigger the acrosome reaction in an inhibitory guanine nucleotide binding regulatory (Gi) protein-dependent manner? The results (Fig. 2B) showed that 10% of control sperm incubated without a ZP glycoprotein and with or without pertussis toxin underwent the acrosome reaction. In contrast, 67% of control sperm incubated in the absence of pertussis toxin but in the presence of A23187 (5 µM) underwent the acrosome reaction. This effect of calcium ionophore is consistent with published results [28–30].

In testing the effect of Alexa-ZP2 or Alexa-ZP3, it was observed that 25% of sperm incubated only with 360 nM Alexa-ZP2 underwent the acrosome reaction. This percentage was not reduced by the addition of pertussis toxin (Fig. 2B). In contrast, 56% of sperm incubated with Alexa-ZP3 alone underwent the acrosome reaction, and this effect was reduced to 25% when pertussis toxin was also added. Additional experiments (data not shown) demonstrated that 67% of sperm incubated with A23187 and either Alexa-ZP2 or Alexa-ZP3 underwent the acrosome reaction. Thus, neither Alexa-ZP protein diminished the maximal response of sperm to the ionophore. We therefore concluded that Alexa-ZP3 retained its capacity to stimulate the acrosome reaction in a Gi protein-dependent manner, whereas, as anticipated, Alexa-ZP2 lacked this capacity. Additionally, the data showed that incubation of sperm with either 360 nM Alexa-ZP3 or 360 nM Alexa-ZP2 significantly increased (from 10% to 25%) the percentage of sperm that undergo the acrosome reaction in a Gi protein-independent manner. This increase may reflect the presence of residual components of the chromatography buffer that remain with the ZP protein after dialysis or an effect of the fluorochrome conjugated to each ZP protein.

Analysis of the Binding of Alexa-ZP3, Alexa-ZP2,and Alexa-ZP1 to Capacitated Sperm

To evaluate the topographic distribution of ZP3 binding sites, live sperm were incubated with 360 nM Alexa-labeled ZP3, washed with M199-M, fixed, and dried onto glass slides. Figure 3A shows a typical fluorescence micrograph obtained from sperm incubated with both Alexa-ZP3 and pertussis toxin. Alexa-ZP3 binding is clearly evident over the acrosomal cap and over the postacrosomal region of acrosome-intact sperm. Quantitative image analysis indicated that approximately 20% of Alexa-ZP3 bound over the acrosomal cap, and 80% bound over the postacrosomal region.



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  		FIG. 3. Distribution of Alexa-ZP3 and Alexa-ZP2 binding sites on mouse sperm that were fixed after the incubation with these ligands. A) Distribution of Alexa-ZP3 binding sites on sperm incubated in both 360 nM Alexa-ZP3 and pertussis toxin. The boxed area around one sperm illustrates the dimensions of the area that was sampled to quantify binding of Alexa-ZP3 or Alexa-ZP2 to a single sperm. Binding of Alexa-ZP3 (B) and FITC-PNA (C) to an individual sperm head incubated in both 360 nM Alexa-ZP3 and pertussis toxin. Binding of Alexa-ZP3 (D) and lack of binding of FITC-PNA (E) to an individual sperm head that was incubated in both Alexa-ZP3 and the calcium ionophore A23187. Note that Alexa-ZP3 bound both over the acrosomal cap and to the postacrosomal region of acrosome-intact sperm. In contrast, Alexa-ZP3 bound only to the postacrosomal region of acrosome-reacted sperm. F) Distribution of Alexa-ZP2 binding sites on sperm incubated in 360 nM Alexa-ZP2 and pertussis toxin. Binding of Alexa-ZP2 (G) and FITC-PNA (H) to an individual sperm head that was incubated in Alexa-ZP3 and pertussis toxin. Binding of Alexa-ZP2 (I) and lack of binding of FITC-PNA (J) to an individual sperm head that was incubated in Alexa-ZP2 and A23187. Note that Alexa-ZP2 bound only to the postacrosomal region of acrosome-intact sperm. In contrast, in acrosome-reacted sperm, Alexa-ZP2 bound to the remainder of the acrosome cap and to the postacrosomal region of acrosome-reacted sperm

To determine whether the binding of ZP3 to these two domains was specific for acrosome-intact sperm, sperm were incubated with Alexa-ZP3 and with either pertussis toxin or A23187 to prevent or trigger the acrosome reaction, respectively. The presence of an acrosome was monitored by staining with FITC-PNA. Figure 3, B and C, show typical fluorescence micrographs obtained from acrosome-intact sperm, which were identified by the intense staining of the acrosome by FITC-PNA. Alexa-ZP3 binds over the acrosomal cap and to the postacrosomal region of the same sperm. Figure 3, D and E, show typical fluorescence micrographs obtained for acrosome-reacted sperm. Neither Alexa-ZP3 nor FITC-PNA bound to the acrosomal region of these sperm. However, binding sites for Alexa-ZP3 were detected over the postacrosomal region of the sperm head.

Similar experiments were conducted with sperm incubated in 360 nM Alexa-ZP2. Figure 3F presents a typical fluorescence micrograph obtained when living sperm were incubated with both pertussis toxin and Alexa-ZP2. For most sperm, Alexa-ZP2 bound only to the postacrosomal region of sperm. To ascertain whether this represented the distribution of ZP2 binding sites on acrosome-intact sperm, incubations with Alexa-ZP2 were supplemented either with pertussis toxin or with A23187. Figure 3, G and H, show the binding of Alexa-ZP2 and FITC-PNA, respectively, to a typical acrosome-intact sperm. Alexa-ZP2 binding sites were detected only on the postacrosomal region. In contrast, Alexa-ZP2 bound both to the acrosomal ridge and to the postacrosomal region of acrosome-reacted sperm (Fig. 3, I and J).

Living, capacitated sperm were also incubated with 360 nM Alexa-ZP1. In contrast to what was observed with Alexa-ZP3 and Alexa-ZP2, there was no detectable binding of Alexa-ZP1 to living mouse sperm (data not shown).

Analysis of the Distribution of ZP3 and ZP2 Binding Sites on Live Sperm and on Sperm That Were Incubated at 4°C with Alexa-ZP3 or Alexa-ZP2

Our observation that 80% of ZP3 binding sites are present on the postacrosomal membrane is in disagreement with previous conclusions that greater than 50% of ZP3 binding sites are located over the acrosomal cap [5]. We therefore asked whether the results in Figure 3 accurately reflected the distribution of ZP3 binding sites on living sperm or were an artifact that resulted from the fixation and drying of the sperm. To answer this question, spermatozoa were incubated with either Alexa-ZP3 or Alexa-ZP2, washed with M199-M, and then allowed to stick to CellTak-coated coverslips. Sperm were immediately analyzed in medium that contained SYTOX Green, a membrane-impermeable fluorescent dye for nucleic acids. Figure 4A shows the typical distribution of ZP3 binding sites on living sperm. Whereas Alexa-ZP3 bound over the acrosomal cap, most ZP3 binding sites were detected in the postacrosomal region of the sperm head. The sperm was defined as alive because its heads did not stain with SYTOX Green (Fig. 4B). In contrast, heads of dead or dying sperm were brightly fluorescent (Fig. 4C). Figure 4E shows the distribution of ZP2 binding sites on a typical live sperm. Almost all ZP2 binding sites were detected over the postacrosomal region of the sperm head. In contrast to binding of ZP3, there was no binding of ZP2 to the acrosomal cap. This sperm was defined as alive because it did not stain with SYTOX Green (Fig. 4F).



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  		FIG. 4. Distribution of Alexa-ZP3 and Alexa-ZP binding sites on living mouse sperm and on sperm that have incubated at 4°C with these ligands. To assay the distribution of ZP3 and ZP2 binding sites on living sperm, sperm were incubated for 1 h with 180 nM Alexa-ZP3 (A) or 180 nM Alexa-ZP2 (E), washed once in Dulbecco PBS, and mounted in M199-NC with 50 nM SYTOX Green nucleic acid stain. Viability of sperm from A and E was determined by the absence of SYTOX Green staining of the sperm heads (B and F, respectively). C) In contrast, the heads of dead or dying sperm stained intensely with SYTOX Green. To examine the distribution of ZP3 and ZP2 binding sites under conditions that inhibit the diffusion of plasma membrane proteins, sperm were incubated at 4°C for 2 h with 180 nM Alexa-ZP3 (D) or 180 nM Alexa-ZP2 (G), washed with ice-cold Dulbecco PBS, and fixed in 4% formaldehyde before microscopic analysis

The previous analysis of live sperm confirmed that after a 60-min incubation, most ZP3 and ZP2 binding sites were localized on the postacrosomal region. The question remained, however, whether these sites were resident in that domain before binding their ligands. It was possible that ZP3 and ZP2 binding sites were initially concentrated over the acrosomal cap but once bound by ligand, diffused to the postacrosomal region. To address this possibility, we initially tried to examine the binding of Alexa-ZP3 and Alexa-ZP2 to sperm that had been fixed with 4% formaldehyde or with 4% glutaraldehyde and then washed exhaustively before incubation with Alexa-ZP3 or Alexa-ZP2. However, neither ligand bound to fixed sperm. As an alternative approach, we incubated sperm with Alexa-ZP3 and Alexa-ZP2 at 4°C and washed and fixed sperm at that temperature. Since diffusion of proteins within a plasma membrane is substantially reduced at 4°C, potential movement of ZP3 and ZP2 binding sites from the acrosomal cap to the postacrosomal region would be inhibited at this temperature. Results from this experiment showed once again that most ZP3 and ZP2 binding sites were concentrated over the postacrosomal region of the sperm head (Fig. 4, D and G, respectively). Since these two experiments confirmed that most ZP3 and ZP2 binding sites reside on the postacrosomal region, the remaining studies were conducted by incubating sperm at 37°C with Alexa-ZP3 or Alexa-ZP2 and analyzing sperm after they had been fixed with 4% formaldehyde and dried onto slides.

Dose-Response Analyses of Individual Alexa-Labeled ZP Glycoproteins to Sperm

The differential binding of Alexa-ZP3 and Alexa-ZP2 on sperm, in combination with the lack of binding of Alexa-ZP1, suggested the presence of distinct binding sites for ZP3 and ZP2. Thus, the binding of Alexa-ZP3 and Alexa-ZP2 to sperm should be saturable. To test this prediction, capacitated sperm were incubated with both pertussis toxin and a range of concentrations (3–360 nM) of Alexa-ZP3, Alexa-ZP2, or Alexa-ZP1, and binding of these ligands to individual sperm was measured. (Figure 3A gives a pictorial definition of the area of the microscopic field that was sampled to quantify binding of Alexa-ZP3 to an individual sperm.) The concentrations of ZP proteins used in these studies were consistent with those used in previous dose-response analyses of sperm-ZP interactions [7, 10, 30, 31]. Binding of Alexa-ZP3 to sperm was dose-dependent and saturable at 100–200 nM (Fig. 5A). Binding of Alexa-ZP3 over the acrosomal cap and to the postacrosomal region exhibited similar dose-response curves (data not shown). Titration of Alexa-ZP2 was also dose-dependent, but saturation appeared to be approached at a concentration equal to or greater than 360 nM (Fig. 5A). Saturable binding of Alexa-ZP2 was confirmed by the observation that unlabeled ZP2 competes with Alexa-ZP2 for binding sites on sperm (Fig. 6B). As anticipated (Fig. 3 legend), the results confirmed that incubation of sperm with 3–360 nM Alexa-ZP1 did not result in detectable binding of ZP1 to sperm (Fig. 5A).



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  		FIG. 5. A) Dose-response analysis of binding of Alexa-ZP1 ({blacksquare}), Alexa-ZP2 ({blacktriangledown}), and Alexa-ZP3 (•) to capacitated, living mouse sperm. Sperm were allowed to capacitate in M199-M containing 1 µg/ml pertussis toxin for 45 min and then incubated with 3–360 nM Alexa-ZP1, Alexa-ZP2, or Alexa-ZP3 for 1.5 h. Binding was converted from FE units (left axis) to molecules bound per sperm head (right axis) as described in Materials and Methods. Each concentration of Alexa-ZP glycoprotein was tested in duplicate in each experiment, and three separate experiments were performed. Data are expressed as mean ± SEM. B) Scatchard plot analysis of the binding of Alexa-ZP3 to the 75 000 sperm included in each incubation. The estimated Bmax and Kd were ~185 000 sites per sperm and 67 nM, respectively (R2 = 0.83). C) Scatchard plot analysis of the binding of Alexa-ZP2. The estimated Bmax and Kd were ~500 000 sites per sperm and 200 nM, respectively (R2 = 0.90)



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  		FIG. 6. Comparison of the abilities of ZP1, ZP2, ZP3, and ZPtotal to inhibit the binding of Alexa-ZP3 (A) and Alexa-ZP2 (B) to sperm. Sperm were incubated in 36 nM Alexa-ZP3 or Alexa-ZP2 and in the presence or absence of a 5- or 10-fold molar excess of unlabeled ZP1, ZP2, ZP3, or ZPtotal. Data (mean ± SEM) are from three replicate experiments. Bars labeled with different letters identify means that are statistically different

To estimate the affinities (Kd) and numbers of binding sites (Bmax) for Alexa-ZP3 and Alexa-ZP2 on sperm, the dose-response curves were transformed by Scatchard plot analysis (Fig. 5, B and C). The transformation of the data for the binding of Alexa-ZP3 was best described by a straight line (R2 = 0.83). Based on the negative slope and intercept of this line, we estimate that there are ~185 000 sites that bind Alexa-ZP3 with a Kd of 63 nM. Transformation of the data for the binding of Alexa-ZP2 to sperm also produced a straight line (R2 = 0.9). This analysis indicated that there were ~500 000 sites on the sperm that had a Kd of 200 nM. These differences in the numbers and affinities of Alexa-ZP3 and Alexa-ZP2 binding sites provide further evidence that these two ZP glycoproteins bind different receptors on the surface of acrosome-intact sperm.

Demonstration That Alexa-ZP3 and Alexa-ZP2 Bindto Different Sites on Acrosome-Intact Sperm

Data presented in Figures 3–5 predicted but did not prove that ZP3 and ZP2 bound separate sites on sperm in a ZP glycoprotein-specific manner. If the sites that bound ZP3 were specific for that ZP glycoprotein, ZP3 but not ZP2 or ZP1 would be an effective competitive inhibitor of Alexa-ZP3 binding. Likewise, if sites that bound ZP2 were specific for that ZP glycoprotein, only ZP2 would be an effective competitive inhibitor of Alexa-ZP2 binding. Therefore, to directly examine the specificity of Alexa-ZP3 and Alexa-ZP2 binding to sperm, capacitated sperm were incubated with 36 nM Alexa-ZP3 or 36 nM Alexa-ZP2 in the presence or absence of a 5- or 10-fold molar excess of unlabeled ZP3, ZP2, or ZP1. Figure 6A shows the inhibition of Alexa-ZP3 binding to sperm by these unlabeled ZP glycoproteins. Substantial inhibition of Alexa-ZP3 binding was observed when a 5- and 10-fold molar excess of unlabeled ZP3 was added as a competitor (80% and 90% inhibition, respectively). This inhibition was similar to what was observed when ZPtotal was used as a competitor. In contrast, addition of a 5- and 10-fold molar excess of ZP1 or ZP2 reduced binding of Alexa-ZP3 by 10%–45%.

Figure 6B illustrates the inhibition of Alexa-ZP2 binding to sperm by unlabeled ZP glycoproteins. Seventy percent and 80% inhibition of Alexa-ZP2 to sperm was observed with 5- and 10-fold molar excesses of ZP2, respectively. This inhibition was similar to what was observed when ZPtotal was used as competitor. The inhibition of binding of Alexa-ZP2 by both competitors confirms the conclusion from the Scatchard plot analysis that binding of Alexa-ZP2 to sperm is saturable. In contrast to the results obtained when ZP2 or ZPtotal was used as a competitor, addition of a 5- or 10-fold molar excess of ZP1 or ZP3 reduced Alexa-ZP2 binding by only approximately 25%. Therefore, data in Figure 6, A and B, when combined with data in Figures 3–5 demonstrate that Alexa-ZP3 and Alexa-ZP2 bind in a saturable and ZP-glycoprotein specific manner to distinct receptors on mouse sperm.

Binding of ZP Proteins to Sperm Is a Calcium-Dependent Process That Requires Both Epididymal Maturation and Capacitation of Sperm

As noted in the Introduction, three requirements of fertilization are extracellular calcium and the maturation and capacitation of sperm. To date, however, it has not been established whether ZP3 and ZP2 binding sites on mouse sperm share these same requirements. To determine whether the binding of Alexa-ZP3 or Alexa-ZP2 is calcium-dependent, sperm were allowed to capacitate for 1.0 h in M199-M (which contains 1.8 mM Ca2+ and 1.2 mM Mg2+). To chelate the calcium in the medium, we then added excess EGTA (3.75 mM) simultaneously with either Alexa-ZP3 or Alexa-ZP2. Comparison of the dose-response curves for Alexa-ZP3 and Alexa-ZP2 in the presence and absence of EGTA demonstrated that chelation of calcium in the medium reduced the binding of both ZP proteins to the sperm surface by approximately 70%–80% (Fig. 7, A and B). To assess whether sperm were still capacitated when incubated with EGTA, sperm were analyzed for the capacitation-dependent increase in proteins containing phosphotyrosine residues [32, 33]. Results from noncapacitated (negative control) and capacitated sperm were similar to those previously described. Sperm that had been incubated in capacitating conditions and then with EGTA were not different from sperm incubated only in capacitating conditions (data not shown).



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  		FIG. 7. The requirement of extracellular calcium for the maximal binding of Alexa-ZP3 and Alexa-ZP2 to sperm. A) Dose-response analysis of the binding of Alexa-ZP3 to sperm incubated in the presence (dotted line) or absence (solid line) of 3.75 mM EGTA. B) Dose-response analyses of the binding of Alexa-ZP2 to sperm incubated in the presence (dotted line) or absence (solid line) of 3.75 mM EGTA. Data are presented as mean ± SEM

In the experiments summarized in Figure 8, A and B, we tested the hypothesis that only mature, capacitated sperm bind ZP proteins. In the first experiment (Fig. 8A), cauda epididymal sperm were incubated in either capacitating medium or noncapacitating medium and in the presence of 3–180 nM of Alexa-ZPtotal. Whereas a dose-response of total ZP binding was observed with capacitated sperm, noncapacitated sperm did not bind Alexa-ZPtotal. Based on these data, we conclude that capacitation is a prerequisite for sperm to bind both ZP3 and ZP2. To test the effect of sperm maturation on the binding of ZP3, sperm were collected from both the caput (immature sperm) and cauda (mature sperm) epididymides into both capacitating and noncapacitating medium. The results (Fig. 8B) showed that caput sperm collected in either medium did not bind Alexa-ZP3. In contrast, cauda sperm incubated in capacitating medium, but not noncapacitating medium, bound Alexa-ZP3. We therefore conclude that important characteristics of ZP3 and ZP2 binding sites are requirements for extracellular calcium and the capacitation of sperm. Additionally, only mature sperm bind to Alexa-ZP3.



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  		FIG. 8. Capacitation and sperm maturation affects the binding of ZP glycoproteins to sperm. A) Dose-response analysis of Alexa-ZPtotal binding to mature sperm collected from the cauda epididymis and incubated under capacitating (solid line) and noncapacitating (dotted line) conditions. B) Alexa-ZP3 binding to caput (immature) and cauda epididymal (mature) sperm incubated under capacitating and noncapacitating conditions. Data (mean ± SEM) are results from three independent experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ZP3 Binding Sites Are Concentrated on the Postacrosomal Region of the Head of Mouse Sperm

The experiments described in this paper were initiated because of convincing evidence that interactions of mouse sperm with ZP3 and ZP2 are essential for fertilization. However, despite this evidence, the sperm surface binding sites for these ZP glycoproteins had not been completely and systematically characterized. This report is the first to estimate numbers of ZP3 and ZP2 binding sites on acrosome-intact mouse sperm under conditions in which the ligand is saturating. Additionally, our studies are the first to prove that ZP3 and ZP2 bind to mouse sperm in a ZP glycoprotein-specific manner. Thus, these studies document two hallmarks of all functional cell surface ligand binding molecules. Finally, we identified three important characteristics of these binding sites. As with fertilization, the functions of both types of sites requires the presence of extracellular calcium and the maturation and capacitation of sperm.

Examination of the binding of Alexa-ZP3 and Alexa-ZP2 allowed us to describe the distribution of ZP3 and ZP2 binding sites on the entire surface of the sperm head. Our data indicate that 80% of ZP3 binding sites are present on the postacrosomal region. This distribution was observed on living sperm, on sperm that had been fixed after incubation with Alexa-ZP3, and on sperm that had been incubated with ligand at 4°C to inhibit the movement of ZP3 binding sites within the plasma membrane. Our conclusion that 80% of ZP3 binding sites are over the postacrosomal domain differs from that of previous investigators [5] who estimated that 60% of ZP3 binding sites were on the plasma membrane overlying the acrosome, and 40% were on the postacrosomal domain. However, it must be recognized that the previous estimate was based on an analysis of sagittal sections of sperm and not on the analysis of the entire sperm surface. As a result, those previous experiments may have underestimated the percentage of the sites that reside on the postacrosomal region. Additionally, the previous study did not examine the binding of ZP3 at saturating concentrations of ligand. Nonetheless, the results of both our study and the previous study agree that significant numbers of ZP3 binding sites are located on the postacrosomal region of the sperm head.

Our conclusion that most ZP3 binding sites are present on the postacrosomal region raises the question of the function of these sites. A function for the ZP3 binding sites on the plasma membrane overlying the acrosome is evident from the fact that free-swimming, acrosome-reacted mouse sperm cannot bind to ZP-encased eggs [7]. Thus, the initial attachment of sperm to the ZP requires ZP3 binding sites on the plasma membrane that overlies the acrosome. However, the ZP3 binding sites on the postacrosomal region of the sperm head are retained during the acrosome reaction. One potential function of these sites is to couple bound ZP3 to second messenger pathways that trigger the acrosome reaction. Levels of important second messengers required for this process are regulated by Gi proteins and low voltage-activated T-type calcium channels [34]. Although current data indicate that Gi proteins are localized only to the acrosomal cap [35], there is some evidence for regulated calcium entry in the postacrosomal domain. The influx of calcium into hamster sperm that is induced by soluble hamster ZP begins in the equatorial segment but then encompasses the postacrosomal domain [36]. Additionally, both N-type and Trp2 calcium channels have been detected in the postacrosomal domain [37, 38]. However, the distribution of the T-type calcium channels in mouse sperm has yet to be reported.

The second potential function of the ZP3 binding sites on the postacrosomal domain is to ensure that a spermatozoon remains bound to the ZP during the acrosome reaction.

Electron microscopic studies have provided two different views of the orientation of acrosome-intact mouse sperm on an intact ZP. Some studies have observed that sperm bind to the ZP via the plasma membrane overlying the apical portion of the acrosome [39, 40]. As a result, these sperm face perpendicular to the ZP. Other studies have observed mouse sperm bound to the ZP by the convex surface over the acrosomal cap or by the plasma membrane over the postacrosomal domain of the sperm head [41–44]. These latter observations are consistent with reports that mouse sperm traverse the matrix of the ZP at an oblique angle. Therefore, these data lead us to suggest the following scenario. Initially, sperm bind to the intact ZP via ZP3 binding sites over the apical portion of the acrosome. Once initial tight binding occurs, sites on the postacrosomal domain then form a second area of tight binding between the acrosome-intact sperm and the ZP. The question remains, however, as to why this second area of tight binding would be needed. Baltz and colleagues [45] have calculated that fewer than 200 noncovalent bonds are sufficient to bind sperm to the ZP. It is likely that before the acrosome reaction, these bonds could be formed by ZP3 binding sites on the apical portion of the acrosomal membrane. However, during the acrosome reaction, the plasma membrane overlying the acrosome with its associated ZP3 binding sites become separated from the sperm. During this separation, those ZP3 binding sites will likely lose their ability to mediate sperm-ZP adhesion. Nonetheless, sperm-ZP adhesion is maintained. We propose that ZP3 binding sites on the postacrosomal region of the head maintain sperm-ZP adhesion during the acrosome reaction until ZP2 binding sites on the inner acrosomal membrane are exposed and able to bind to the ZP.

Characteristics of the Binding of ZP3 to Sperm Are Consistent with the Biology of Sperm-ZP3 Interactions

Thaler and Cardullo [9] concluded that fixed sperm have both high- and low-affinity binding sites for ZP3. In contrast, our results suggest that, based on affinity, the mouse sperm expresses a single class of ZP3 binding sites. The affinity of ZP3 for live sperm, Kd = 63 nM, is similar to the ED50 of murine ZP3 estimated in other biochemical and biologic assays. In a competitive sperm-ZP binding assay, 1.5 solubilized ZP per microliter (~37 nM ZP3) reduces the binding of sperm to ZP-encased eggs by approximately 50% ([46] and unpublished data). Similarly, one to two solubilized ZP per microliter (~25–50 nM ZP3) have been reported to trigger a half-maximal response of sperm as measured by induction of the acrosome reaction and activation of Gi proteins [47, 48]. Thus, the similarity of the affinity of Alexa-ZP3 for sperm with the biologic activities of ZP3 argues that Alexa-ZP3 binds the sites that mediate both the initial binding of sperm to the ZP and the acrosome reaction. Our experiments provide the first direct demonstration that these binding sites bind their respective ligands in a ZP glycoprotein-specific manner. Whereas a 5-fold molar excess of ZP3 inhibited 80% of Alexa-ZP3 binding, a similar concentration of ZP1 and ZP2 inhibited only 30% and 10% of binding, respectively. The weak cross-reactivity of these other two ZP glycoproteins for ZP3 binding sites may be explained by the nature of the sperm-binding domains on ZP3, which are one or more O-linked glycans covalently attached to the polypeptide backbone [49]. It is possible that glycans on ZP1 and ZP2 are weak agonists for the high-affinity sperm-binding glycans on ZP3 and, thus, allow some cross-reactivity of the other two ZP glycoproteins for the ZP3 binding site. Despite this cross-reactivity, however, our data indicate that ZP3 binding sites have a substantial preference for ZP3 over ZP2 and ZP1.

Functional Sperm Surface Binding Sites for ZP3 Require Extracellular Calcium and Maturation and Capacitation of Sperm

This report provides the first direct proof that extracellular calcium and the maturation and capacitation of sperm are required for sperm to bind isolated ZP3. Our observation that calcium is required is consistent with the report of Saling et al. [13] that binding of sperm to ZP-encased eggs requires extracellular calcium. However, our results are in contrast to those of Thaler and Cardullo [9], who reported that binding of total solubilized ZP to glutaraldehyde-fixed sperm was calcium-independent. It should be noted, however, that those authors posited that this lack of requirement for calcium may have been an artifact resulting from fixation of sperm. The new insight from our studies is that ZP3 binding sites on sperm do not function when calcium, but not magnesium, in the medium is chelated by EGTA. As O-linked carbohydrates constitute an essential part of the binding determinant of ZP3, this calcium dependence raises the possibility that the ZP3 binding sites on mouse sperm may be C-type lectins [50].

It is well established that mouse fertilization requires maturation and capacitation of sperm [44, 51–58]. This observation led to the hypothesis that maturation and capacitation of sperm are prerequisites for sperm to express functional ZP3 binding sites. However, this hypothesis was inconsistent with the report by Saling et al. [13] that uncapacitated sperm adhere to ZP-encased eggs. Our experiments were the first to directly examine the binding of ZP3 to noncapacitated mouse sperm, and the results support the hypothesis previously articulated. Additionally, since immature sperm cannot undergo capacitation, we interpret the inability of caput sperm to bind ZP3 as resulting from the inability of these cells to undergo capacitation. What do our results say about the report from Saling et al.? It should be noted that in studying in vitro sperm-ZP binding, those authors did not include two-cell embryos as negative controls for adhesion of sperm to zona-encased eggs. Thus, it is possible that the assay used by those authors did not measure sperm-ZP adhesion but rather looser attachment of sperm to egg, an event that is prerequisite for tight binding [59].

ZP2 Binding Sites Are Localized on the Postacrosomal Region of Acrosome-Intact Sperm

Although sperm surface receptors for ZP3 have been the subject of extensive study, only a few investigators have examined the binding of ZP2 to sperm [13]. Consistent with the findings of Mortillo and Wassarman [5], we demonstrated that Alexa-ZP2 bind to the acrosomal ridge of acrosome-reacted sperm. The new insight provided by our study is that there are also considerable numbers of ZP2 binding sites expressed on the postacrosomal region of live, acrosome-intact sperm. Although these sites reside in a similar location as the ZP3 binding sites, they differ in their affinity and ligand specificity. Our data indicates that the affinity of ZP2 for sperm (Kd = 200 nM) is 3-fold lower than the affinity of ZP3. This lower affinity is consistent with the proposal that ZP2 is a secondary ligand for acrosome-reacted sperm as they move through the mass of the ZP [6]. Although the affinities of the binding sites for ZP2 and ZP3 differ, the two binding sites share a similar specificity for their respective ligands. ZP2, but not ZP1 or ZP3, was a potent competitive inhibitor of the binding of Alexa-ZP2 to sperm. Additionally, ZP2 and ZP3 binding sites share similar requirements for both capacitation and extracellular calcium. Thus, the binding activities of both primary and secondary ZP receptors are subject to physiologic regulation.

In summary, the experiments described in this paper are the first to show both saturable and ligand structure-specific binding of isolated ZP3 and ZP2 to mouse sperm. We have documented that these two glycoproteins bind separate sites on sperm, and we have estimated the affinities of the binding reactions and the numbers of binding sites on sperm. Additionally, we have demonstrated that extracellular calcium and maturation and capacitation of sperm are prerequisites for the normal function of these binding sites. These physiologically important characteristics may be keys to the eventual identification of the individual sperm surface proteins that constitute these ZP3 and ZP2 binding sites on mouse sperm.


   ACKNOWLEDGMENTS
 
We thank Dr. Jurrien Dean for the hybridoma cell lines that secrete antibodies to the ZP glycoproteins. We also thank Ms. Janet Folmer for excellent technical assistance with fluorescence microscopy and Dr. Barry Zirkin and Dr. Richard Cone for useful discussions.


   FOOTNOTES
 
First decision: 28 August 2001.

1 This work was supported by grant 1 R01 HD-35699 from NICHD; by the Hopkins Population Center (P30-HD-06268); and by an institutional training grant from the National Institutes of Health (5T32 AG 00250) to C.L.K. Back

2 Correspondence: William W. Wright, The Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 N. Wolfe St., Baltimore, MD 21205-2179. FAX: 410 614 2356; bwright{at}jhmi.edu Back

Accepted: December 19, 2001.

Received: July 25, 2001.


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 ABSTRACT
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 RESULTS
 DISCUSSION
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