| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Biology, University of California, Riverside, California 92521
b Department of Biology, University of Central Florida, Orlando, Florida 32816
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
acrosome reaction, fertilization, gamete biology, signal transduction, sperm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chlortetracycline fluorescence of the mouse sperm acrosome has shown that there are several intermediate stages during acrosomal exocytosis [10, 11]. Biochemical and microscopic studies have indicated differential release of acrosomal contents during and after membrane fusion [12, 13]. More recently, studies have shown that acrosomal contents are accessible in sperm that would be considered AI by Coomassie blue staining; beads coated with antibodies to acrosomal proteins were able to bind to these apparently intact sperm [14]. These data suggest that there are transitional stages in this exocytotic process during which newly exposed or modified sperm proteins could mediate the transition between ZP3 and ZP2 binding while maintaining sperm adhesion to the ZP.
As a first step in identifying mouse sperm proteins involved in both initial and subsequent adhesion events at the ZP, we have generated membrane vesicles from AI mouse sperm by sonication in hypotonic medium [15]. In the present study, we investigated the presence of ZP-binding sperm proteins in these preparations. Previous work has demonstrated that sperm membranes retain the ability to interact with ZP glycoproteins, as indicated by their ability to inhibit binding of sperm to the intact ZP [16], to activate heterotrimeric G-proteins in response to ZP3 [17], or to bind ZP glycoproteins directly [18–20]. In the studies presented here, we characterized ZP-binding properties of the 3 membrane-containing fractions generated by sonication of AI mouse sperm. Our data indicate that intact mouse sperm possess 2 different ZP-binding activities, which suggests that complex binding interactions mediate changes in ZP-binding affinity during and after acrosomal exocytosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sodium 125I was purchased from New England Nuclear (Dupont, Boston, MA). Electrophoresis chemicals were purchased from Bio-Rad (Hercules, CA). Protease inhibitors, DNase I, hyaluronidase, BSA, Percoll, desoxycholate, and prestained markers for SDS-PAGE were purchased from Sigma (St. Louis, MO). NHS-Sepharose columns were purchased from Pharmacia (Uppsala, Sweden). Chloramine-T was purchased from Aldrich (Milwaukee, WI). Quantigold protein assay reagent was purchased from Diversified Biotech (Piscataway, NJ). Whatman GF/C filters for binding assays and all other chemicals were purchased from Fisher (Pittsburgh, PA).
ZP Collection and Labeling
ZPs were collected as previously described [21]. Ovaries from 3-wk-old ICR mice were homogenized in the presence of leupeptin and aprotinin (20 µg/ml), DNaseI (0.1 mg/ml), and hyaluronidase (0.1 mg/ml) and incubated at 20°C for 15 min. Desoxycholate was added to a final concentration of 1%, and the homogenate was layered onto a 3-step Percoll gradient (2 ml 3% Percoll, 4 ml 10% Percoll, 2 ml 25% Percoll) and centrifuged at 200 x g for 2 h. ZPs were collected from the 10% step by dilution with buffer and centrifugation at 16 000 x g for 20 min to pellet ZPs. ZPs were 125I labeled, quantified, and stored in liquid N2.
Isolation of 125I-ZP3
A ZP3-enriched fraction (125I-ZP3) was isolated from whole solubilized 125I-labeled ZPs by affinity chromatography using the rat monoclonal antibody IE-10 [22] (gift from Dr. J. Dean, National Institutes of Health) covalently coupled to Sepharose 4B according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden). 125I-Labeled solubilized ZP glycoproteins were loaded onto the column in 20 mM Na2HPO4, pH 7.5. After washing (10 column volumes), bound 125I-ZP3 was eluted with 0.1 M glycine, pH 2.6, neutralized, and concentrated (Ultrafree Biomax; Millipore, Bedford, MA). Samples from the bound and flow-through fractions were analyzed by gel autoradiography and immunoblotting. The distribution of radioactive material in the fractions was determined by quantifying gamma emissions of each fraction. The purity of the eluted fractions was determined by densitometry of gel autoradiographs of the pooled ZP3 peak material and by immunoblotting. The purity of the ZP3 fraction was determined by densitometry of the autoradiogram (Labworks software; UVP, Upland, CA).
Collection of Sperm and Membrane Preparations
Mice (ICR strain) were purchased from Harlan Sprague-Dawley (San Diego, CA). Cauda epididymides were collected from 12- to 15-wk-old mice as previously described [21, 23]. Sonicated sperm membranes were prepared, fractionated, and stored as previously described [15]. Mouse liver membranes were used as control membrane fractions and were prepared by the same protocol as used for sperm membrane preparations, with the exception that liver samples were first minced and homogenized (10 strokes, Wheaton 2-ml ground glass homogenizer) before sonication.
Binding Assays
Competitive binding assays were performed as previously described [23]. Cauda epididymal mouse sperm were incubated in capacitating conditions (Hepes-buffered Whittingham buffer, 37°C, 5%CO2 for 1 h) and were referred to as intact capacitated sperm after this incubation. The fraction of AR sperm (%AR sperm) was quantified following this incubation. Sperm were 
20–25% AR (n = 4) at the initiation of the binding assays, and the change in acrosomal status over the time course of the binding assays was less than 5% (n = 4) as indicated by Coomassie staining [24]. All membrane fractions were added, based on protein mass, at a final concentration of 10 µg/ml in a total assay volume of 100 µl. In initial studies, only minor changes in binding were detected at lower concentrations of membrane (data not shown), and 10-µg/ml membranes have been successfully used to detect ZP binding in the pig [19]. Further, this concentration of membranes did not disrupt acrosomal structure over the time course of the assays. We have not assessed whether this concentration of membranes is saturating, but because they promoted significant changes in sperm-ZP binding and did not appear to disrupt the sperm, we conducted these studies with the 10 µg/ml concentration of membrane competitors. The intact capacitated sperm were incubated with 125I-ZPs, 125I-ZP3, or a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (125I-ZP1/2) in the presence or absence of membranes from bands 1–4 at 37°C for 5 min. The sperm and bound ZPs were captured on Whatman GF/C filters, unbound ZPs were washed through the filter, and the amount of bound and unbound ZP material was quantified using a gamma counter. The binding was expressed as fractional binding (cpm bound/cpm unbound). Fractional binding values ranged from 0.01 to 0.16. All samples contained approximately 5 ng/µl ZP glycoprotein, which yielded an initial radioactivity of 4 x 106 cpm for 125I-ZP assays, 5.5 x 105 cpm for 125I-ZP3 assays, and 8 x 106 cpm for 125I-ZP1/2. Control fractional binding in the absence of any membrane competitor was normalized to 100%. ZP binding in the presence of membrane fraction competitors is presented as the percentage of binding relative to controls.
Statistical Analysis
Differences were analyzed using the Mann-Whitney rank sum test because not all treatment groups showed a normal distribution. Significant differences are reported at P < 0.05. Analyses were based on the following numbers of replicates: 125I-ZP assays: n = 12 for control (no membranes); sperm membranes, n = 6 for B1, B2, and B3; n = 4 for B4; liver membranes, n = 5 for B1, B2, B3; n = 4 for B4; 125I-ZP1/2 assays: n = 7 for control; sperm membranes, n = 5 for B1, B2, B3; liver membranes, n = 4 for B1, B2, B3; 125I-ZP3 assays: n = 10 for control; sperm membranes, n = 5 for B1, B2, B3; liver membranes, n = 4 for B1, B2, B3.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
125I-ZP binding to AI sperm in the presence of sperm membrane competitors was quantified relative to 125I-ZP binding in the absence of a competitor (Fig. 1). Mouse liver membranes were used as a control for nonspecific binding. None of the mouse liver membrane fractions significantly altered mouse sperm binding to 125I-ZPs. Similarly, the sperm band 4 fraction, which contains primarily membrane-free cell fragments, axonemes, and nuclei [15], had no affect on sperm-ZP interactions. In contrast, sperm membranes from band 1, band 2, or band 3 were each able to significantly inhibit 125I-ZP binding to capacitated intact sperm (P < 0.05). 125I-ZP binding to capacitated sperm was reduced by 51% in the presence of band 1 membranes, 58% in the presence of band 2 membranes, and 70% in the presence of band 3 membranes. These data indicate that the isolated sperm membrane vesicles contain ZP-binding proteins that compete for 125I-ZP binding to intact sperm.
|
125I-ZP3 and 125I-ZP1/2 Binding Assays
Current models of the molecular basis of sperm-ZP binding would suggest that the results obtained in the 125I-ZP binding assays above are a consequence of ZP3 binding to the membrane competitors because only ZP3 binds to intact sperm [3, 5, 25, 26]. To test this hypothesis, we purified 125I-ZP3 using antibody affinity chromatography (Fig. 2). Fractions collected from the antibody column were quantified in a gamma counter (Fig. 2A). The first peak, material that did not bind to the column, contained ZP1 and ZP2, as detected by gel autoradiography (Fig. 2B). ZP3 was not detected in the flow-through fraction either by autoradiography (Fig. 2B, lane 1) or by immunoblotting (Fig. 2B, lane 1'). The second peak, material that bound to the antibody column, contained 84% ZP3, as determined by densitometry of the autoradiogram (Labworks software, UVP). The remainder of the material in the eluted fraction consisted of ZP2.
|
These 2 fractions were used in competitive binding assays with the membrane-containing fractions (bands 1–3). Using 125I-ZP1/2 as the ligand, none of the membrane fractions were able to compete for binding to live capacitated intact mouse sperm (Fig. 3). Sperm binding to the 125I-ZP1/2 fraction in the presence of membrane fractions was not significantly different from control binding in the absence of membranes. Sperm binding to 125I-ZP1/2 was 108% in the presence of band 1, 82% in the presence of band 2, and 83% in the presence of band 3. The presence of liver membranes tended to increase binding of 125I-ZP1/2 to intact sperm, but these trends were not significant.
|
To demonstrate ZP3 binding directly, isolated 125I-ZP3 was used in binding assays with intact sperm and competitor sperm membranes. 125I-ZP3 binding to live capacitated intact sperm was quantified using the immunopurified 125I-ZP3 fraction as the ligand. In the presence of band 2 or band 3 membranes, there was a significant decrease (P < 0.05) in 125I-ZP3 binding to intact sperm (Fig. 4). There was no significant change in 125I-ZP3 binding to sperm in the presence of band 1 membranes. Further, there were no significant differences in sperm-125I-ZP3 interactions in the presence of mouse liver membrane fractions.
|
These data indicate that components of the band 2 and band 3 membrane fractions are able to bind to ZP3 alone and suggest that band 2 and band 3 membranes contain sperm proteins required for the initial sperm-ZP interactions. In contrast, band 1 membranes did not interact with ZP3 or ZP2 alone but were able to interact with whole solubilized ZP glycoproteins, suggesting that sperm proteins in band 1 may recognize a complex of ZP2 and ZP3.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To understand the specific interactions between sperm proteins and ZP glycoproteins during initial ZP adhesion and subsequent signaling events, we used a competitive binding assay incorporating intact capacitated sperm, solubilized ZP glycoproteins, and biochemically characterized membrane fractions [15] of mouse sperm to compete for ZP glycoprotein binding. Although the membrane fractions are biochemically complex, they have enabled us to identify subsets of sperm membrane proteins that bind to ZP glycoproteins and have provided starting material for further isolation and characterization of the ZP binding components of the sperm plasma membrane.
The fractionated sperm membrane vesicles contain proteins that display high-affinity interactions with ZP glycoproteins as demonstrated by their ability to competitively inhibit binding of 125I-ZPs to capacitated sperm. In these assays, sperm membrane fraction band 1, band 2, and band 3 each were able to inhibit binding of 125I-ZPs to live capacitated sperm. When 125I-ZP1/2 was used as the ZP ligand, none of the membrane fractions were able to compete for binding sperm. These data are consistent with those from previous studies showing that isolated ZP1 or ZP2 did not interact with AI sperm [2, 4]. ZP2 has previously been implicated in binding of AR but not AI sperm to the ZP. The 125I-ZP1/2 fraction did appear to bind to sperm possibly because of the presence of a minor population of AR sperm (20–25%) and the small amount of ZP2 material that has been shown to bind to AI sperm [4]. The data are also consistent with the assumption that only ZP3 binds to AI sperm and that the bioactivity of ZP3 is solely responsible for the behavior of the sperm membrane vesicles when used as competitors in the 125I-ZPs binding assays.
However, when immunopurified 125I-ZP3 was used as the ZP ligand in the competitive binding assays, only the band 2 and band 3 membranes were capable of inhibiting sperm-ZP binding. Band 1 was not able to compete for binding to isolated ZP3. These data suggest that ZP3 has a high-affinity binding partner among the band 2 and band 3 proteins, but only when ZP3 and ZP1/2 are present together are they recognized by a high-affinity binding partner in band 1. The matrix of the ZP has been proposed to consist of ZP3/ZP2 heterodimers that assemble into long filaments and are infrequently crosslinked by ZP1 [27]. The surface of the ZP presents a highly ordered, immobilized array of ZP3 and ZP2, and ZP recognition by the sperm components in band 1 may depend on interactions with a spatial distribution of ZP glycoproteins resembling the intact ZP surface. Additionally, because the assay captures sperm and bound ZPs while unbound ZPs or ZPs bound to membrane vesicles are washed away, the assay only measures competition with components available on the target cell population, the capacitated intact sperm. Thus, although these data support the long-standing model that ZP3 binds to AI sperm [3, 5], the data also indicate that there is an additional ZP-binding activity present on intact sperm, activity that requires ZP3 and the ZP1/2 fraction.
Previous evidence has indicated that ZP3 alone is sufficient to block adhesion of intact sperm to the ZP matrix and to induce acrosomal exocytosis [1, 2]. More recent studies demonstrated that ZP1 knockout mice are fertile [28], suggesting that ZP1 is not required for sperm-egg interactions during fertilization. These data and those presented in this study lead us to propose that band 2/3 sperm proteins are responsible for the primary adhesion events between sperm and ZP3 and the induction of the acrosome reaction, whereas band 1 sperm proteins interact with a ZP3/ZP2 complex in subsequent binding events. Given that ZP1 does not appear to be required for sperm-egg interactions [28] and given that band 1 proteins clearly do not bind to ZP1, ZP2, or ZP3 alone (these studies), it seems reasonable to assume that band 1 proteins bind to a ZP3/ZP2 complex. Although the affinity-purified ZP3 fraction contains some residual ZP2, the sperm-binding behavior of the ZP3 fraction is clearly distinct from the behavior of the whole ZPs. Thus, either the residual ZP2 does not form ZP3/ZP2 complexes or there are so few complexes they cannot block enough binding to be detectable in our assay. The current results do not reveal any significant differences in the competitive behavior of band 2 and band 3, which may indicate that they contain the same ZP3-binding component. However, we cannot exclude the possibility that band 2 and band 3 contain different ZP3-binding proteins.
The band 1 and band 2 membrane fractions contain the majority of the membrane material recovered in our preparations and the majority of plasma membrane material [15]. Using lectins, we have found that band 1 and band 2 both contained glycoproteins that were found only on the head and that the identity of these glycoproteins was different [15]. The observation that band 1 and band 2 comprise overlapping but distinct subsets of sperm head plasma membrane proteins is not only consistent with but would be required for the differential ZP-binding behavior of the membranes observed in these studies.
Based on these data, we cannot exclude the possibility that these band 1 components are involved with the initial sperm-ZP adhesion. However, the previous data indicating that ZP3 is sufficient to block adhesion to the ZP [3] suggest that the band 1 proteins are involved in subsequent interactions. Such interactions might include maintaining sperm adhesion during acrosomal exocytosis and subsequent penetration of the zona matrix. Recent studies have demonstrated that acrosomal contents may be accessible early during the AR [14]. For example, beads coated with sp56 antibodies bind to the sperm anterior head at times when histologic stains or in vivo staining of the acrosome with GFP fusion proteins indicate that the sperm acrosomal vesicle is intact, suggesting that sp56 in the acrosome is accessible. Several studies have shown biochemically the differential release of acrosomal contents during AR [12, 13]. Components in band 1 may bind to a complex of ZP glycoproteins during acrosomal exocytosis. Previous studies have indicated that ZP2 may be involved in binding to AR sperm, but no data yet have addressed the molecular events that allow sperm to undergo acrosomal exocytosis, change their adhesion ligand from ZP3 to ZP2, and remain in contact with the ZP matrix during these events. Because band 1 proteins cannot bind to either ZP3 or ZP2 alone but can bind to a complex of ZP2 and ZP3, these sperm proteins may be involved in these transitional adhesive events during the AR. Because of the ability of isolated ZP3 to block sperm-ZP binding, interactions of a sperm protein component with a ZP2-ZP3 heterodimer would not have been detected by previous assays. Only by using fractionated sperm components as competitors could such additional interactions be detected.
In a recent study using putative ligand oligosaccharides as competitive inhibitors of sperm-ZP binding, 2 different oligosaccharides each partially inhibited sperm-ZP binding but had additive effects when used together. Thus, there must be 2 different sites on intact sperm that are able to interact with the putative ZP ligands [29]. The results presented here are consistent with multiple ZP3-binding proteins on the sperm plasma membrane [30, 31]. These studies have demonstrated that there are 2 different ZP-binding activities present on AI mouse sperm. Our working model of sperm-ZP interactions is that initial adhesion is mediated by 1 or more ZP3-binding proteins (bands 2 and 3) and that as sperm begin to undergo acrosomal exocytosis, additional proteins are recruited to bind ZP2-ZP3 (band 1) before finally establishing interactions with ZP2-binding proteins.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was funded by National Institutes of Health Grant HD 27244 and by grants from the University of California and the University of Central Florida. 
2 Correspondence: Catherine D. Thaler, Department of Biology, University of Central Florida, Orlando, FL 32816. FAX: 407 823 5769; cthaler{at}pegasus.cc.ucf.edu
Accepted: August 13, 2001.
Received: March 8, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles. Mol Reprod Dev 1993; 36:382-389[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or liver membranes (
) from different fractions. Inhibition of binding was quantified relative to samples that contained no membrane fractions (control). Bands 1, 2, and 3 significantly inhibited 125I-ZP binding to live, acrosome intact, capacitated sperm. Band 4 did not significantly inhibit binding. None of the liver membrane fractions inhibited binding. Data are presented as mean ± SEM. *P < 0.05.
