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