Evidence for a role of the major glycoprotein in the structural maintenance of the pig zona pellucida B. S. Dunbar and D. S. Bundman Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A.

Summary. The functional domains of the glycoproteins of the pig zona pellucida have been analysed using lectin binding, peptide mapping, and immunoblotting in conjunction with analysis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and protein detection with the silver-based colour stain. Two of the pig zona pellucida glycoproteins identified in 2D-PAGE were differentially proteolysed within the intact matrix by a variety of enzymes. This proteolysis of specific proteins, however, did not affect the suprastructure of the matrix, or inhibit spermatozoa from adhering to the surface of the zona pellucida. The major glycoprotein appears to be involved in the structural maintenance of the zona pellucida because dissolution of the matrix correlated with proteolysis of this glycoprotein by proteinase K. These glyco- proteins were further evaluated by lectin blotting with Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) before and after proteolysis of zona pel- lucida with trypsin. The lectins bound to all charge species of the three major zona pellucida glycoproteins. Only the most acidic components of the major glycoprotein family, which are not extensively digested, were recognized by these lectins after proteolysis. These studies provide evidence that the major glycoprotein family I of the pig zona pellucida is primarily responsible for maintaining the integrity of the matrix.

Introduction

The zona pellucida is the complex extracellular glycoprotein matrix which surrounds the mammalian oocyte. This matrix is formed during the early stages of oocyte growth and follicular cell differen¬ tiation and serves to protect the oocyte and embryo until implantation (Odor, 1965; Kang, 1974; Austin, 1982; Wolgemuth et al, 1984). In addition, the zona pellucida plays an important role in the fertilization process since the spermatozoa must first adhere to and penetrate the zona pellucida. The penetration of the zona pellucida has been proposed as being due to the limited hydrolysis of zona pellucida components by sperm enzymes (Srivastava et al, 1965; Schleuning et al, 1973; McRorie & Williams, 1974; Stambaugh, 1978), aided by the mechanical force of the spermatozoa (Green & Purves, 1984; Green, 1987). The role of proteolytic enzymes in fertilization has been questioned because early studies showed that treatment of zona pellucida of some species did not result in the dissolution of the zona pellucida matrix as observed by light microscopy (see review by Moore & Bedford, 1983). It is now apparent that there are major biological, morphological, physicochemical and immuno- chemical variations in properties amongst the zona pellucida of rodents as compared with the zona pellucida of other species including non-human primates and humans (Sacco et al, 1981; Drell et al, 1984; see also reviews by Dunbar (1983a, b); Dunbar & Wolgemuth (1984)). Although it is now generally accepted that mammalian zona pellucida is composed of a limited number of major glycoproteins (Bleil & Wassarman, 1980a, b,c; Dunbar et al, 1981; Sacco étal, 1981), it is apparent that the structural and functional relationships of zona pellucida proteins of different species vary. Methods have now been developed to isolate large numbers of native zona pellucida matrices

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access from pig oocytes (Dunbar et al, 1980; Wood et al, 1981 ). Although the isolated zonae pellucidae are obtained from ovarian oocytes, the method is selective for zona pellucida-coated oocytes of a particular size (150 µ ) which are from antral follicles. The zonae pellucidae isolated from these preparations have been shown to be homogeneous in their ability to bind spermatozoa, lectins, and antibodies (Dunbar, 1980; Dunbar & Raynor, 1980). These preparations can also be used to develop specific polyclonal and monoclonal antibodies to purified zona pellucida proteins and to study the structure and function of these molecules (Dunbar, 1983a, b; Drell & Dunbar, 1984; Skinner et al, 1984; Wolgemuth et al, 1984). Because of the availability of large amounts of material, the present studies have been designed to use intact, non-solubilized zonae pellucidae to evaluate the suprastructure of this unique extra¬ cellular matrix. The present experiments have been designed to: (a) characterize and determine which molecules of the native pig zona pellucida matrix are involved in the maintenance of the zona pellucida suprastructure; (b) determine which glycoproteins of the native matrix are susceptible to proteolysis by enzymes of different substrate specificities; and (c) determine what effect the proteolysis of intact zona pellucida matrices has on homologous and heterologous sperm binding. Such information should provide an insight into the biochemical nature of the zona pellucida glycoproteins which may be involved in sperm-zona pellucida interaction and sperm penetration of the zona pellucida as well as which molecules play roles in maintenance of the structure of this unique extracellular matrix.

Materials and Methods

Rabbit ovaries were obtained from PelFreez Biologicals (Rogers, AK). Pig ovaries for zona pellucida isolation were obtained from slaughter houses. The large scale isolation of zonae pellucidae was carried out as previously described by Wood et a! (1981). Acrylamide was from Serva Chemicals (Garden City Park, Long Island, NY) and wide range ampholytes (pH 3-5-10) from LKB (Bromma, Sweden). Other electrophoresis reagents were from BioRad (Richmond, CA). Staphylococcus aureus 125I-labelled protein A was obtained from Amersham (Arlington Heights, IL). Trypsin (270 U/mg) which cleaves at lysine and arginine side chains and chymotrypsin which catalyses hydrolysis of peptide bonds of L-isomers of tyrosine, phenylalanine, and tryptophahe were obtained from Worthington (Freehold, NJ). Proteinase (Boehringer-Mannheim, Indianapolis, IN) which cleaves primarily at asparagine-glycine bonds and protease Staphylococcus aureus V8 was used as a non-specific protease (Miles Laboratories, Naperville, IL) (see discussion by Fey et a!, 1984). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-resolution two-dimensional polyacrylamide gel electrophoresis. The exact conditions used are those outlined by Dunbar et a! (1981) and Dunbar (1987) as modified from Anderson & Anderson (1977). Isoeiectric focussing (lst-dimension electrophoresis) was carried out at 25°C for 16 h at 750 V for a total of 12 000 volt-hours after solubilization at pH 9-5 (95°C, 10 min) with isoeiectric focussing (IEF) solubilization buffer containing 2% SDS, 2% ß-mercaptoethanol, 1% cyclohexaminoethylsulphonic acid, and 10% glycerol. Second-dimension slab gels of 10-20% polyacrylamide (including bis-crosslinker) were prepared using the Pace Linear Gradient Maker (Isolabs, Akron, OH) and the Anderson DALT gel casting system (Electronucleonics, Inc., Oak Ridge, TN), and electrophoresis was carried out using the Anderson DALT electrophoresis tank (Electro- nucleonics, Inc.). The silver-based colour stain as described by Sammons et a! (1981, 1984) or the Gelcode® stain kit (Health Products, Pierce Chemical Co., Rockford, IL) were used to detect zona pellucida proteins and peptides. Enzymic digestion ofzona pellucida matrices. Intact zona pellucida matrices (approximately 10 000 zonae pellucidae or 330 µg protein) were pelleted in a microfuge tube and incubated for 1 h at 37°C in 50 µ Dulbecco's phosphate-buffered saline (DPBS), pH 7-5, containing 14 µ of a 1 % solution of trypsin to give a final concentration of 37 U/330 µg zona pellucida protein. (Protein concentrations were estimated based on content of intact protein in pig zona pellucida which has been previously measured (Dunbar et a!, 1980). The consistency ofprotein patterns ofhundreds of2D-PAGE gels run in these experiments suggests that this is a reliable method ofestimating amounts ofprotein.) After 1 h, the zonae pellucidae were observed to be microscopically intact. The zonae pellucidae were prepared for isoeiectric focussing by adding 50 µ IEF solubilization buffer, pH 9-5 and heating for 10 min in a boiling water bath. A final volume of 30 µ was loaded on each isoeiectric focussing gel. Enzymic digestions were also carried out by adding 50 µ -chymotrypsin (0-26 U) or protease, Staphylococcus aureus, V8 (2-5 U) in DPBS, pH 7-4 to a pellet of 10 000 intact native pig zona pellucidae matrices and incubating them for 1 h at 37°C. Zonae pellucidae which were microscopically intact were prepared for isoeiectric focussing as described above. Alternatively, pig zonae pellucidae were incubated at 37°C for 10 min with 20 µ proteinase (0-5 U/330 pg zona pellucida protein) in DPBS, pH 7-4. After 10 min, an additional 20 µ (0-5 U) enzyme were added. After a second 10-min incubation at 37°C, zona pellucida matrices were no longer

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access morphologically intact. The sample was prepared for isoeiectric focussing by adding 60 µ IEF solubilization buffer and boiling for 10 min, then 30 µ were added to each isoeiectric focussing gel. The enzymic digestion with proteinase was carried out using two different concentrations of enzyme: 004 U per 200 µg zona pellucida protein and 2U per 330 µg zona pellucida protein. In the first experiment, the intact zonae pellucidae were incubated at 37°C for 15 min and the sample was centrifuged at 10 000 g to separate the supernate and pellet and each was prepared for IEF. In the second experiment, the zonae pellucidae were no longer discernible as intact matrices after enzyme treatment and were prepared for isoeiectric focussing by adding 80 µ IEF solubilization buffer. A volume of 30 µ! was loaded on each gel. Identification ofglycoproteins on 2D-PAGEby lectin binding. Pigzonae pellucidae were separated by high-resolution two-dimensional PAGE and electrophoretically transferred to nitrocellulose as previously described (Dunbar, 1987). The biotin-avidin system used to enhance lectin binding to zona pellucida glycoproteins, followed by visualization with horseradish peroxidase and 3,3'-diaminobenzidine, has been described in complete detail elsewhere (Dunbar, 1987). Briefly, the nitrocellulose transfer was blocked with periodic acid-treated bovine serum albumin (20-30 ml of a 3% solution in Tris-saline) for 2 h at 25°C. The BSA must be treated with periodic acid before use or the lectins will bind to the carbohydrate on the BSA, resulting in unusually high background. The BSA was treated by adding 10 mM- periodic acid to a solution of 4% BSA in 0T M-sodium acetate, pH 4-5, and stirring at room temperature. After 3 h, another lOmM-periodic acid was added and the mixture stirred for an additional 3 h. The treated BSA is dialysed extensively against Tris-saline. Transfers were then washed two times with 150 ml Tris-saline for 20 min each. Biotinylated lectins (Vector Labs, Burlingame, CA) were diluted 1:200 in Tris-saline with 3% periodic acid-treated BSA and incubated with each transfer for 3 h while shaking at 25°C. Excess lectin was removed using 20-min washes of Tris-saline (150 ml each). Horseradish peroxidase conjugated to avidin D (Vector Labs) was diluted 1:200 in Tris- saline containing 3% periodic acid-treated BSA and 20-30 ml were incubated with each transfer and allowed to shake for 3 h at 25°C. The colour reaction was developed by adding 100 ml of 001% substrate, 3,3'-diaminobenzidine (DAB) first dissolved in a small amount of dimethylsulphoxide and containing 10 µ 30% hydrogen peroxide. Evaluation of sperm-zona pellucida interaction. Sperm-zona pellucida interaction was evaluated using methods previously described by Swenson & Dunbar ( 1982) and Drell & Dunbar ( 1984). Ejaculated, motile rabbit spermatozoa were obtained with an artificial vagina and/or boar spermatozoa were obtained by hand from a trained boar. Seminal fluid was removed by washing the spermatozoa twice with minimal essential medium (Gibco Labs, NY). A 50 µ suspension containing 1 IO5 spermatozoa was added to a 50 µ suspension of isolated zonae pellucidae which had been previously treated with enzymes as described above and washed on Nytex screens to remove excess enzyme. After a 2-h incubation in a 3% C02 incubator, supernumerary spermatozoa were removed by micropipetting as previously described (Swenson & Dunbar, 1982; Drell & Dunbar, 1984). Sperm adherence to the zonae pellucidae was monitored microscopically.

Results

Identification of zona pellucida glycoproteins by lectin blotting of2D-PAGEgels The extensive post-translational modification of the zona pellucida proteins may be critical for the biological properties and structural maintenance of this matrix. The glycoproteins of the zona pellucida were therefore analysed using lectin blotting of zona pellucida proteins separated by 2D-PAGE. Figure 1 illustrates that both Ricinus communis agglutinin (RCA, sugar competition specificity, ß-D-galactose > -D-galactose) and wheat germ agglutinin (WGA, sugar competition specificity, ß-[l-+4]-D-Af-acetylglucosamine) bind to all charge species of the three major glyco¬ protein families of the pig zona pellucida. As with silver-stained protein patterns previously shown (Dunbar et al, 1981, 1985), three major protein families of the zona pellucida are identified which exhibit extreme charge and molecular weight heterogeneity. Using internal molecular weight markers, these proteins have been shown in 3 previous studies to have ranges of molecular weights: Protein Family I, 49 000-120 000; Protein Family II, 70 000-101000; and Protein Family III, 95 000-118 000.

Role of zona pellucida glycoproteins in the structural maintenance of the extracellular matrix Initially, native zonae pellucidae were treated with enzymes since this would be the state of the matrix with which the spermatozoa would interact in vivo and visual as well as biochemical parameters of matrix dissolution could be monitored. Trypsin digestion of the native zona pellucida

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 1. Pattern of lectin binding to the major glycoproteins of pig zonae pellucidae separated by high-resolution 2D-PAGE: isoeiectric focussing in the first dimension, second dimension SDS- PAGE in 10-20% gradient gels, (a) Ricinus communis lectin (biotinylated) and (b) wheat germ agglutinin (biotinylated). Lectins were detected with horseradish peroxidase conjugated to avidin using 3,3'-diaminobenzidine substrate. ( + ) = relative pH of 3-8, ( ) = relative pH of 9. —

Table 1. Identification of specific peptides analysed by 2D-PAGE and silver staining following treatment of native or heat-solubilized zonae pellucidae (ZP) with trypsin*

Proteins or peptides

Trypsin cone. Time at 25°C 20 000- ( /µ8 ZP) (min) la lb III 40 000f 30 000Í

Intact ZP 00 0 (control) 00001 U 60 + + 0001 U 60 + + + § + + 001 U 60 + + + § + + 0-1 U 0-25 + § + 15 + + + § + + 60 + + + + 240 + + + 480 + + 1440 + + 10 U 4h +

Heat-solubilized* ZP 0001 60 + + 001 240 + + 01 0-25 + 15 + + 60 + + 120 + + 480 + 1440 +

Summary of analyses of > 100 2D-PAGE gels: + = presence of glycoproteins at original charge and molecular weight; = glycoprotein no longer apparent on 2D-PAGE gel at original molecular weight. tPeptide of—intermediate molecular weight as shown in Fig. 2. ÎPeptides of molecular weight as shown in Fig. 2. §Peptides of M, ~ 50 000 as shown in Fig. 2. cZona pellucida matrices treated in 0-01 M-Na2C03, pH 9-5 for 60 min at 65°C to cause macroscopic dissolution of zona pellucida before treatment with trypsin. Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 2. High-resolution 2D-PAGE of pig zona pellucida proteins detected with the colour-based silver stain, (a) Intact zona pellucida (150µg) treated with trypsin (0-1 U^g zona pellucida, 37°C, 1 h). Circle = intermediate 40 000 peptide; arrow = intermediate 50 000 peptide; Bracket = peptides of 20 000-30 000; asterisk = position of Protein III before proteolysis. (b) Heat-solubilized zonae pellucidae treated with trypsin (0-1 U^g zona pellucida protein, 37°C, 1 h). = trypsin. matrices did not result in gross morphological changes of this structure unless a high concentration of enzyme and long incubation was used (Table 1). If trypsin-treated, intact zonae pellucidae were analysed by 2D-PAGE, however, the constituent glycoproteins were altered as compared to untreated zonae pellucidae (Fig. 2a). At all enzyme concentrations tested, the glycoprotein family labelled III was proteolysed in that it was no longer detected at 95 000-118 000 (see Table 1).

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 3. Pattern of lectin binding to glycoproteins of trypsin-treated pig zona pellucida. Intact zonae pellucidae heated with trypsin (0-1 U^g zona pellucida for 60 min): (a) Ricinus communis lectin (biotinylated); (b) wheat germ agglutinin (biotinylated). Lectins were detected with horseradish peroxidase conjugated to avidin utilizing diaminobenzidine as substrate. ( + ) = pH3-8, (-) = pH5.

Protein family II ( 70 000-85 000) was partly proteolysed to an M, of 60 000 and 55 000 at low concentrations of enzyme or at short incubation times (Fig. 2a; Table 1), while at higher enzyme concentrations, this protein was more completely proteolysed. The major glycoprotein family I was proteolysed into two distinct peptides (la and lb) at most enzyme concentrations. One of these (Peptide la) appeared to be more resistant to enzymic digestion since its charge and molecular weight patterns were not changed, except at high concentrations of trypsin and for long incubation times. Peptide lb had an apparent reduction in molecular weight of approximately 10 000-30 000 after enzymic digestion. An intermediate peptide having an apparent molecular weight of 40 000 (circled region in Fig. 2a) was seen in protein patterns ofzonae pellucidae in which a low concentration of trypsin or short times of incubations were used. Colour silver staining (Gelcode®) patterns showed that Peptide la stained a characteristic orange-rust while lb stained a darker brown as previously described (Dunbar et al, 1985). If zonae pellucidae were heat-solubilized using conditions known to affect dissolution detectable by light microscopy (pH 9-5 for 60 min at 65°C) (Dunbar et al, 1980) before trypsin digestion, all glycoproteins including the major glycoprotein family were more susceptible to enzymic digestion (Fig. 2b; Table 1). The patterns obtained by lectin probing of trypsin-treated, intact zona pellucida matrices separ¬ ated by 2D-PAGE and transferred to nitrocellulose are shown in Fig. 3. These studies demonstrate that the Peptides la and lb which were resolved by 2D-PAGE still contained carbohydrate moieties which are recognized by lectins. None of the low molecular weight peptides generated by proteolysis of proteins of the native, intact matrix were detected by this method and therefore these areas of these gels are not shown. Similar proteolysis experiments were carried out using a-chymotrypsin and V8 protease digestion of intact zonae pellucidae (Figs 4a and b). While these also did not cause dissolution of the intact matrix as visualized by light microscopy, the peptide patterns generated by treatment with these enzymes and analysed by 2D-PAGE were distinct from those of the trypsin-treated intact zonae pellucidae. These studies are summarized in Table 2. Treatment with a-chymotrypsin resulted in the proteolysis of Glycoproteins II and III as shown in Fig. 4. Glycoproteins II and III were no

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 4. High-resolution 2D-PAGE analysis of proteins and peptides of pig zonae pellucidae detected by silver staining after treatment of intact matrices with a-chymotrypsin (a) and V8 protease (b). Zona pellucida matrices in both samples were morphologically intact before elec¬ trophoresis (+ = pH 3-8; = pH 9). Asterisk = silver stain artefact; open arrows = distinct zona pellucida peptides resolved.— longer apparent at their native molecular weights ( > 68 000) as they had appeared in untreated samples of zona pellucida proteins. The treatment of intact matrices with V8 protease under the conditions described resulted in the proteolysis of Glycoprotein III but not Glycoprotein II. However, V8-protease proteolysed Glycoprotein I in the region where the enzyme migrates during

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 5. Intact pig oocytes surrounded by zona pellucida treated with proteinase illustrating the microscopic visualization of swelling and dissolution of the zona pellucida matrix (37°C) at 0 time (a); 15 min (b); and 30 min (c). At 1 h, zonae pellucidae were no longer morphologically intact. OC = oocyte cytoplasma; ZP = zona pellucida.

isoeiectric focussing. Because V8 protease is enzymically active in the presence of sodium dodecyl sulphate (Cleveland et al, 1977), it is apparent that proteolysis of a portion ofzona pellucida Protein I occurred during the second-dimension electrophoresis. This proteolysis therefore appeared to occur after detergent solubilization and denaturation ofproteins of the native matrix which occurred during first-dimension isoeiectric focussing. To confirm this, an isoeiectric focussing gel containing zona pellucida protein was incubated in V8 protease along with the SDS solubilization buffer. Under this condition, all proteins were proteolysed to multiple small peptides (data not shown). To determine whether Peptide la is involved in the structural integrity of the matrix, zonae pellucidae were incubated with enzymes which caused dissolution of this structure. As shown in Fig. 5, proteinase caused progressive 'swelling' and macroscopic dissolution of the pig zona pellucida matrix until the zona pellucida was no longer visible by light microscopy (Dunbar, 1983a; Drell & Dunbar, 1984). The protein patterns of isolated zonae pellucidae obtained after digestion with this enzyme are shown in Fig. 6. Figure 6(a) illustrates the pellet (x 10 000g) taken after a 20-min incubation of intact zonae pellucidae in proteinase K. Figure 6(b) demonstrates an aliquant taken from a sample treated with a higher concentration of this protease in which zona pellucida matrices were no longer morphologically intact. These studies demonstrate that the sequential dissolution of the intact matrix corresponds with the proteolysis of the major glycoprotein family I. These data are summarized in Table 2.

Role of zona pellucida glycoproteins in sperm adherence Figure 7 is a phase-contrast micrograph of rabbit spermatozoa or boar spermatozoa adhering to the surface of pig zona pellucida after treatment with trypsin. More than 50 spermatozoa were bound to zonae pellucidae in these studies (over 30 zonae pellucidae in each of the triplicate exper¬ iments). This number of spermatozoa was the same regardless of exposure of the zonae pellucidae to trypsin or a-chymotrypsin at different concentrations and incubated at 25°C or 37°C. The sperm binding pattern was identical to those of control zonae pellucidae which were not treated with any enzyme (Swenson & Dunbar, 1982; Drell & Dunbar, 1984). Since Glycoproteins II and III are proteolysed by trypsin or chymotrypsin treatment, these studies suggest that the intact forms of these glycoproteins are not essential for sperm-zona interaction.

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Fig. 6. High-resolution 2D-PAGE analysis of zona pellucida proteins and peptides detected by silver staining after treatment of intact matrices with proteinase for 20 min: (a) pellet of 10 000#, 20 min, 0·04U/200µg zona pellucida protein—zona pellucida matrices from this treatment were swollen but intact; and (b) 0-5 U/85 µg zona pellucida protein, 20 min. These zona pellucida matrices had undergone complete dissolution and were not detectable by light microscopy after this treatment. Asterisk = silver stain artefact; + = pH 3-8; = pH 9.

—

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Table 2. Effect of proteolysis of glycoproteins from intact pig zona pellucida (ZP) matrices Identification of peptides by 2D-PAGE after proteolysisj Dissolution Sperm binding Enzyme ofZP to ZP after Glyco- Glyco- Glyco- Glyco¬ activity matrix* treatmentf protein I protein II protein III protein IV

Buffer control + + Trypsin§ + la, lb a-Chymotrypsin§ + + Protease, S. aureus V8§ N.D. +1 + 11 +r Proteinase (T. alburn)^ N.D.

* + = positive effect which causes visible dissolution of zona pellucida matrix; = no effect on zona pellucida matrix visible by light microscopy. — t+ = 100 spermatozoa bound/zona pellucida; more than 30 zonae pellucidae tested in triplicate experiments. N.D. = not done. ^Relative molecular weights of proteins identified by 2D-PAGE not altered by proteolysis. §Trypsin = 0-1 U^g for 60 min at 37°C; a-Chymotrypsin = 0-8 IO-3 U/pg zona pellucida; V8 protease = 0-8 10~2 U^g zona pellucida; proteinase = 0-6 IO"2 U^g zona pellucida. ^Proteolysis evident only after solubilization with Na2DodS04.

Fig. 7. Isolated pig zonae pellucidae treated with trypsin (37CC, 1 h) washed with Dulbecco's PBS and incubated with (a) rabbit spermatozoa or (b) boar spermatozoa (37°C, 1 h). Zonae pellucidae are completely covered with spermatozoa, typical of all trypsin-, and a- chymotrypsin-treated zonae pellucidae.

Discussion

The results obtained in this study give insight into the complex molecular structure of the pig zona pellucida and the interaction of its specific glycoproteins which aid in the maintenance of the extra¬ cellular matrix while allowing passage of the spermatozoa during the fertilization process. All three Glycoproteins I, II and III exhibit charge and molecular weight heterogeneity when analysed with

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access 2D-PAGE and are resolved as distinct 'families' of glycoproteins. Since the first analysis of pig zona pellucida glycoproteins by 2D-PAGE (Dunbar et al, 1981), the three major glycoproteins described in this study have also been identified by other laboratories (Sacco et al, 1981 ; Hedrick & Wardrip, 1986; Yurewicz et al, 1987). A complete summary of the nomenclature of zona pellucida proteins of different laboratories is given by Timmons & Dunbar (1987). The major pig zona pellucida proteins bind RCA and WGA uniformly (Fig. 1), and only the more acidic species of these proteins are recognized by these lectins after enzymic proteolysis by trypsin. Previous studies have been carried out which demonstrate that the enzyme acrosin isolated from boar spermatozoa will cause partial proteolysis of pig zona pellucida proteins (Dunbar et al, 1985). Other studies have been limited to one-dimensional polyacrylamide gel electrophoresis (Brown, 1986) or to protein detection by Coomassie blue staining (Urch et al, 1985). Because of the sensitivity and resolution of the silver-based colour stain, the present studies have allowed more detailed analysis of the peptides generated by proteolysis resulting from treatment of zona pellucida with enzymes of different specificities. The present studies demonstrate that the two high molecular weight glycoproteins of the native pig zona pellucida (II and III) are accessible to exogenous enzymes in that they are proteolysed differentially by trypsin, a-chymotrypsin, or V8 protease, even though the zona pellucida matrices maintain their suprastructure (Figs 1, 2 & 3; Tables 1 & 2). This observation has been made previously utilizing trypsin and the boar sperm enzyme acrosin (Dunbar et al, 1985). The present studies have been extended to utilize a variety of different enzymes and enzyme concentrations to further demonstrate that Protein III is more sensitive to proteolysis than is Protein II and that this sensitivity is dependent upon enzyme concentration and time of exposure. The differences in sensi¬ tivity to proteolysis could be due to the location of these molecules in the suprastructure of the matrix which makes them more accessible to proteolytic enzymes as well as to differences in amino acid sequence. Alternatively, the differences in the post-translational modification of these proteins (e.g. glycosylation and sulphation) or quaternary structure may account for their differences in sensitivity and proteolytic digestion. This conclusion is supported by the observations shown in Figs 2(b) and 4(b) and Table 1 which demonstrate that heat solubilization or detergent denaturation of zona pellucida matrices dramatically alter the susceptibility of zona pellucida glycoproteins to proteolysis. In all cases, it is apparent that zona pellucida Protein la is the most resistant to proteolysis regardless of the specificity of enzymes used to treat intact zona pellucida. These studies further demonstrate that enzymes of different specificities will generate distinct peptide patterns. It has also been demonstrated that Protein I is composed of two distinct peptides. Yurewicz et al (1987) have used deglycosylated pig zona pellucida proteins to determine that the major zona pellucida glyco¬ protein, which they refer to as ZP3, is composed of two separate peptides, and ß, which can be distinguished by amino acid compositions and antibody binding. These observations correlate with our previous observations (Dunbar et al, 1985) as well as the present finding that the major zona pellucida glycoprotein is composed of two distinct peptides which are differentially susceptible to enzymic digestions. Although it has been proposed that the zona pellucida is a major barrier to interspecies fertiliz¬ ation, several studies have shown that the initial sperm-zona pellucida interaction as well as sperm protein-zona protein interaction displays only limited species specificity (Bedford, 1977; Swenson & Dunbar, 1982; O'Rand et al, 1985). In fact, rabbit spermatozoa bind with equal avidity to the zona pellucida of many species including the pig as is illustrated in Fig. 7. The penetration of the zona pellucida by the spermatozoa appears to be a major factor in the species specificity of fertilization since while rabbit spermatozoa adhere to the surface of pig zonae pellucidae, they do not penetrate this structure in vitro (B. S. Dunbar, unpublished observations). The differences in the suprastructures of zona pellucida matrices of different species are also exemplified by studies which demonstrate that macroscopic dissolution of the rabbit, mouse and hamster zona pellucida matrix occurs by treatment with a-chymotrypsin or trypsin (see review by Dunbar, 1983a). As demonstrated, however, this dissolution does not occur with a-chymotrypsin or trypsin treatment of pig zonae pellucidae.

Downloaded from Bioscientifica.com at 10/10/2021 08:25:58PM via free access Although some of the antigenic determinants associated with the rabbit and pig zona pellucida are immunochemically similar, the glycoproteins appear to have distinct biochemical and immuno- chemical properties (Dunbar et al, 1981; Sacco et al, 1981; Wood & Dunbar, 1981; Drell et al, 1984). It is likely, therefore, that the macromolecular nature of sperm penetration of the zona pellucida of different species will also vary. The present studies demonstrate that specific zona pellucida glycoproteins (11 and III) of the native pig zona pellucida are proteolysed even though the matrix remains morphologically intact. The possibility exists that Glycoproteins II and III are only partly proteolysed, and migrate in the same area as Glycoprotein I. This does not appear to be the case, however, because if the same immunoblots which have been probed with the antibody to purified Glycoprotein I are reprobed with the antisera recognizing all three glycoproteins, no increase in antibody binding was ever observed which would be attributed to additional antigens migrating in that area (B. S. Dunbar, unpublished observations). Acrosin has been shown to be a serine protease and has properties similar to those oftrypsin (Srivastava et al, 1965; Schleuning et al, 1973; McRorie & Williams, 1974; Stanbaugh, 1978), and so it may be involved in the penetration of the zona pellucida in a manner which would cleave specific proteins to allow sperm passage but which would not cause complete dissolution of the matrix (Dunbar et al, 1985). It is therefore possible that differential sensitivity of Glycoproteins II, III and lb in the intact zona pellucida matrix to proteolysis may also be related to these functions. These studies further demonstrate that neither Proteins II nor III need to be intact for sperm- zona pellucida interaction since proteolysis of these by a-chymotrypsin or trypsin does not affect sperm binding to the surface of the zona pellucida. Although these studies would suggest that Proteins II and III do not contain binding sites for spermatozoa, it is possible that the proteolysed peptides of these two proteins may remain bound to Protein I in the intact matrix. These studies confirm a previous report by Sacco et al. (1984) that this major protein, referred to as PPZA, has sperm receptor activity. In summary, the present studies have demonstrated that the pig zona pellucida is a complex matrix which is composed of major glycopeptides which are differentially susceptible to proteolysis by different enzymes. These studies also emphasize the importance ofexamining intact zona pellucida matrices instead of preparations which are treated using acid, base or heat to dissociate the complexes before functional studies.

We thank Ms G. Maresh and Dr T. Timmons for critical reading of the manuscript; Ms Claire Lo for expert technical assistance; and Ms Suzanne Mascóla for secretarial assistance. This work was supported by a grant from the Mellon Foundation and a grant from NICHD (HD-17543) to B.S.D.

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