characterization of equine and

Characterization of equine zona pellucida glycoproteins bypolyacrylamide gel electrophoresis and immunological

techniquesC. C. Miller, R. A. Fayrer-Hosken, T. M. Timmons, V. H. Lee,

A. B. Caudle and B. S. DunbarDepartment of1 Physiology and Pharmacology and 2Large Animal Medicine, College of Veterinary

Medicine, University of Georgia, Athens, GA 30602, USA; and 3 Department of Cell Biology,Baylor College of Medicine, Houston, TX 77030, USA

Summary. This study was designed to explore the composition of the equine zona pellu-cida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpableon frozen\p=n-\thawedequine ovaries were aspirated with a needle and syringe, and theresultant follicular fluid, cellular material and oocytes were pooled. Oocytes were

placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate\x=req-\buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from thesecollected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silverstained 2D-PAGE of the EZP revealed the presence of three EZP glycoproteinfamilies of apparent molecular mass ranges of 93\p=n-\120kDa, 73\p=n-\90kDa and 45\p=n-\80kDa.Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit anti-sera against pig zonae pellucidae (R\g=a\HSPZ)confirmed the presence of three EZPglycoprotein families and established the existence of common epitopes between equineand porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separatedglycoproteins illustrated that the 45\p=n-\80kDa family is recognized by the monoclonalantibody R5, developed against the porcine ZP glycoprotein of molecular mass 55\p=n-\120 kDa. Guinea-pig antiserum against endo-\g=b\-galactosidase-treatedrabbit ZP 55 kDaglycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with thelowest molecular mass, also recognized the EZP 45\p=n-\80kDa glycoprotein family.Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP(GP\g=a\HSRZ)recognized the 73\p=n-\90kDa EZP glycoprotein family exclusively. Afterheat solubilization and treatment of EZP with endo-\g=b\-galactosidaseto remove poly-lactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of threeglycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partiallydeglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the mono-clonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP\g=a\HSRZ;and allthree EZP glycoproteins separated by 1D-PAGE are recognized by R\g=a\HSPZ.Thesedata add further support to the concept of cross-species zona pellucida glycoproteinantigenicity.Keywords: horse; zona pellucida; polyacrylamide gel electrophoresis; antigenicity; glycoproteins

Introduction

The zona pellucida (ZP), a complex glycoprotein matrix that surrounds the mammalian oocyte, isof interest because of its biological importance in the processes of folliculogenesis (Wolgemuth et

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al., 1984; Léveillé et al., 1987; Dunbar et ai, 1989), fertilization (for reviews see Dunbar, 1983;O'Rand et ai, 1986; Yanagimachi, 1988) and early embryonic development (Modlinski, 1970). TheZP glycoproteins of several mammalian species including mice, pigs, hamsters, rabbits and humanshave been studied using a variety of histological, immunochemical and electrophoretic methods(for reviews see Timmons & Dunbar, 1988; Dunbar et al, 1991). Methods that have been used tocharacterize mammalian ZP include (i) one- and two-dimensional polyacrylamide gel electro¬phoresis (ID-PAGE, 2D-PAGE) of reduced or nonreduced zona proteins in the presence orabsence of sodium dodecyl sulfate (SDS), (ii) column chromatography, (iii) lectin binding and (iv)compositional analysis of protein and carbohydrate content (for review see Nakano, 1989).Of these techniques, ID- and 2D-PAGE have been used extensively to study the antigenic andbiochemical properties of ZP. These methods, as well as epitope analysis using monoclonal anti¬bodies, have been used to demonstrate that ZP glycoproteins of several species have antigenicdeterminants that are shared, as well as others that are unique (Dunbar et ai, 1981; Sacco et al.,1981; Drell & Dunbar, 1984; Wolgemuth et al., 1984; Maresh & Dunbar, 1987; Timmons et ai,1987).

The morphological characteristics of the equine ZP (EZP) have been evaluated by lightmicroscopy (Betteridge et ai, 1982) as well as by scanning and transmission electron microscopy(Enders et al., 1987; Vogelsang et ai, 1987). The mature EZP has a thickness of 7-10 µ (Vogelsang et al., 1987), which is comparable to that of pig and cow ZP, and the surface mor¬

phology as visualized by scanning electron microscopy demonstrates a pattern typical of other ZPstudied to date (see review by Dunbar et al., 1991).

Zona pellucida proteins have become of increasing interest since it has been clearly demon¬strated that immunization of several species with heterologous ZP preparations will result intemporary infertility or permanent sterilization (see reviews by Sacco, 1987; Skinner et al.,1990; Paterson & Aitken, 1990). Studies have implied that contraceptive vaccines may be practicalfor use in domestic animals as well as women. Previous work demonstrated that porcine ZP (PZP)and EZP share common antigens because autoantibodies to EZP isolated from the serum of infer¬tile mares can inhibit pig sperm-egg interaction (Liu & Shivers, 1982; Shivers & Liu, 1982).Additionally, indirect immunofluorescence has proven that equine, porcine and bovine ZP sharecommon antigens (Bousquet et al., 1987). Finally, it is known that contraception can be achieved inmares heteroimmunized with pig ZP (Liu et ai, 1989; Kirkpatrick et al., 1990). Ultimately, it willbe necessary to identify and characterize the EZP antigenic domains if a large-scale, effective con¬

traceptive vaccine is to be developed for horses. The paucity of information on the biochemicalcomposition of the EZP is probably a result of the difficulty encountered when retrieving oocytes orZP from equine ovaries in sufficient quantities to carry out detailed biochemical or immuno¬chemical studies. We have developed methods to isolate sufficient EZP and have modified existingmethods of analysis to study the constituent glycoproteins. The aim of this study was to determinethe number of glycoproteins and antigens that are associated with the EZP and to evaluate theircharge heterogeneity and molecular mass. The availability of a bank of monoclonal and polyclonalantibodies to the ZP of other mammalian species has also allowed studies to begin to map theantigenic domains associated with the EZP glycoproteins.

Materials and Methods

Chemicals

Sodium dodecyl sulfate (SDS), ammonium persulfate JV,/V,/V',/V-tetramethylethylenediamine (TEMED),bromophenol blue, ß-mercaptoethanol, urea, acrylamide and bis-acrylamide were obtained from BioRad (Richmond,CA, USA). Phosphoric acid and glycerol were obtained from Fisher Scientific (Springfield, NJ, USA). Ampholines(pH 3-10) were obtained from LKB (Hicksville, NY, USA). Nonidet P-40 was obtained from Accurate Chemical(Westbury, NY, USA). Endo-ß-galactosidase (EßGD) was obtained from Seikagaku Kogyo (Tokyo, Japan). Allother reagents were obtained from Sigma Chemical Co. (St Louis, MO, USA).


Zona-intact oocyte collectionZona-intact oocytes were collected from frozen-thawed equine ovaries by aspirating palpable antral follicles with

a 12 ml syringe and an 18 gauge needle. The follicular fluid was pooled in 50 ml tissue culture flasks and the partícula tematerial allowed to settle. The lower 20-30 ml of this suspension was transferred to scored Petri dishes and the oocyteswere located using a stereomicroscope. A narrow-bore pipette was used to move the oocytes to 50 µ droplets of PBSunder silicone oil. Cumulus cells were removed mechanically by repeated washing through fresh droplets of PBS usinga glass pipette with internal bore slightly less than that of the egg and its zona. Oocytes were stored in pools of 25 inPBS at -20°C until further use.

Heat solubilization and enzyme treatment of EZPFor ID-PAGE, 100 equine oocytes were pooled in 25 µ PBS. The ZP was heat-solubilized by adding 100 µ of

distilled H20 adjusted to pH 9-5 with Na2C03 to these pooled oocytes and incubating at 68°C until the ZP could no

longer be visualized by microscope examination (approximately 1 h). Samples were centrifuged at 12 000 g for 10 min,then the supernatant, which contained the heat solubilized equine zonal material, was transferred to a fresh micro-centrifuge tube to which 100 µ] of 01 mol sodium acetate buffer l"1 (pH 5-8) and 1·5µ1 EßGD (01 units in 250 µ )were then added. Overnight (16 h) incubation at 37°C was used to remove polylactosaminoglycans from the heatsolubilized equine zonae. The samples were speed-vacuumed to a volume of 20-30 µ to stop the enzyme activity andbriefly held on ice before being subjected to 1D-PAGE.

One-dimensional polyacrylamide gel electrophoresis (ID-PAGE)The BioRad modular Mini-Protean II system (BioRad, Richmond, CA, USA) was used to carry out ID-PAGE as

described by Laemmli (1970). The EßGD-treated heat-solubilized zonae of 50 equine oocytes was suspended in an

equal volume of buffer (00625 mol Tris-HCl 1" ', 20% SDS, 100% glycerol, 10 µ ß-mercaptoethanol ml"1, pH 6-8)and held in boiling water for 15 min. These samples were then centrifuged at 12 000g for 10 min to remove impurities.The ID-PAGE was run on a 10% gel at room temperature for 0-5 h at 200V. Colour-based molecular weight markers(RPN 756, Amersham; Arlington Heights, IL, USA) were used in a separate lane to define the apparent molecularweight (Mr) of the isolated proteins.

High resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)The same BioRad modular Mini-Protean II system was used to carry out 2D-PAGE as described by O'Farrell

(1975). The precise conditions are those described by Dunbar (1987) and Dunbar et al. (1990). Briefly, pools of 100equine ZP-intact oocytes were prepared for isoelectric focusing by adding 5 µ of SDS solubilization buffer (005 mol2-(V-cyclohexyl-amino) ethane sulphonic acid I"1, 2% SDS, 10% glycerol, 0-2% ß-mercaptoethanol) and incubatingat room temperature with intermittent agitation for 2h, then heating the solution for 10 min by suspending it inboiling water. Nuclear material and impurities were removed by centrifuging the samples at 164000 g (BeckmanL8-M Ultracentrifuge with Ti-42-2 Beckman Rotor) for 1 h at 20°C. The isoelectric focusing was carried out at room

temperature at 500 V for 10 min, then at 1500 V for 1-75 h. The gels were removed from the tubes into plastic vials andheld at

—

70°C. Second-dimensional slab gels of 15% acrylamide were prepared as previously described for the Mini-Protean II system (Dunbar et al., 1990). After addition of equilibration buffer (0-6 ml; 0· 125 mol Trizma base 1

~', 2%

SDS, 10% glycerol, 0-2-0-8% ß-mercaptoethanol), each frozen gel was equilibrated at room temperature for 5 min.Electrophoresis was carried out for 1 h at 200 V. Colour-based molecular weight markers were used in a separate laneto define the apparent Mr of the isolated proteins.

Protein detection by silver stainingA silver stain method of protein detection was used to determine the number, apparent Mr and isoelectric points of

the EZP glycoprotein families (Spiering, 1985). Gels were fixed overnight in 50% methanol and 01% acetic acid,briefly washed in water, then incubated in an aqueous solution of 001 g dithiothreitol 1_1. The dithiothreitol was

poured off and silver nitrate (2 g 1~ ') added for a 30 min incubation. The gels were rinsed three times with fresh water.The colour was developed to the desired intensity by shaking the gels in an aqueous solution of 30 g Na2C03 and0-5 ml 37% formaldehyde per litre of distilled water. To stop development, 20 mol citric acid l"1 was added to thedeveloping solution until the bubbling, which occurs upon acid addition, ceased. The gels were rinsed in water andprepared for photography. This procedure produces protein patterns in shades of brown on a transparentbackground.

Antibody preparationPolyclonal antibodies to total or to 2D-PAGE-purified glycosylated or deglycosylated proteins were prepared as

previously described (Wood et ai, 1981 ; Skinner et al, 1984; Dunbar et al., 1984). Additional polyclonal antisera were


prepared by immunizing female guinea-pigs with rabbit ZP glycoproteins that had been separated by 2D-PAGEafter they had been deglycosylated with EßGD, as described above. Monoclonal antibodies were prepared usingmethods outlined in Drell & Dunbar (1984) and Timmons et al. (1987). All antibodies were evaluated for theirspecificity using ID- and 2D-PAGE and immunoblot analysis. A summary of the antisera used in these studies ispresented (Table 1).

Table 1. Summary of antibodies used in ZP immunoblottingAntibody source Immunogen Abbreviation Reference

Rabbit antiserum

Guinea-pig antiserum



Monoclonal PSI (mouse)

Monoclonal R5 (mouse)

Heat-solubilized porcine ZP RuHSPZglycoproteins2D-PAGE purified, heat-solubilized, R55KEßGD-deglycosylated, 55 kDa rabbitZP glycoprotein*2D-PAGE purified, heat-solubilized, R75KEßGD-deglycosylated, 75 kDa rabbitZP glycoproteinHeat-solubilized rabbit ZP GPoHSRZglycoproteinsPorcine ZP glycoprotein I spot from PSI2D-PAGEHeat-solubilized rabbit ZP R5glycoproteins

Skinner et al., 1984

Lee et al, 1991

Lee étal, 1991

Lee et al, 1991

Timmons et al, 1987

Drell & Dunbar, 1981

*Protein deglycosylated with endo-ß-galactosidase as described in Materials and Methods.

ImmunoblottingTo characterize further the EZP in terms of the antigenic determinants present, the glycoproteins separated by 1D-

and 2D-PAGE on unstained, unfixed gels were transferred to Immobilon polyvinylidene difluoride membrane(PVDF: Millipore; Bedford, MA, USA) in the presence of buffer (10 mmol 3-[cyclohexylamino]-l-propanesulfonicacid l"1, pH 100 plus 10% methanol) using a TE-70 SemiPhor semi-dry electroblotter (Hoefer; San Francisco, CA,USA) for 70 min at 7 V. All gels were stained after transfer to check for complete transfer of proteins. The blot was

blocked overnight with shaking in blocking buffer (0-01 mol Tris-HCl I"1, 0-9% NaCl, 0-02% sodium azide, 5%(w/v) Carnation Instant Non-fat Dry Milk, pH 7-2-7-4). The following day the blots were washed twice in blockingbuffer. Then primary antibody, suspended in blocking buffer was added and the blots were incubated at room tem¬perature with shaking for 1-5-3-Oh. The blots were washed free of unbound primary antibody using three washes ofblocking buffer. In those situations where a second antibody bridge was necessary, it was suspended in blocking bufferand incubated with the blot for 1 h, then washed as before. Finally, 3-5 µ 125I-labelled protein A (0-966 mCi ml"1)was suspended in blocking buffer, added to the transfer membrane, and incubated with shaking at room temperaturefor 0-75-3 h. The blots were dried and processed by autoradiography (Timmons & Dunbar, 1990).

Antibodies that recognize only a single EZP glycoprotein family were used first. In some instances the membranewas reprobed with an antibody that recognizes a different EZP family. As a control, all membranes were ultimatelyreprobed with an antibody that recognizes all three EZP glycoproteins to ensure that PAGE separation andelectroblotting had been successful.

Results

Equine oocyte collection

The equine ovary is anatomically distinct from other mammalian ovaries in that the corticaland medullary tissue layers are reversed. Thus, use of conventional ZP isolation methods estab¬lished for other species was not successful. Aspiration of follicular fluid from frozen-thawed equineovaries resulted in an average recovery rate of 1-14 oocytes per ovary (772 oocytes from 680ovaries). Mechanical removal of adherent cumulus cells was easily accomplished on thoseoocytes that had a compact cumulus. After thawing, the oocytes and surrounding ZP appearedmorphologically normal upon observation using a stereomicroscope.


Fig. 1. The equine zona pellucida as characterized by mini two-dimensional polyacrylamide gelelectrophoresis (PAGE) and silver staining techniques. Isoelectric focusing (IEF) in the firstdimension (+ = pH 40;

—

= pH 80). Second dimension sodium dodecyl sulfate-PAGE in15% gel. Three major glycoproteins are noted with apparent Mr of 93-120 kDa (1), 73-90 kDa(2), and 45-80 kDa (3), respectively.

Fig. 2. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat-solubilized equine zone pellucida (HSEZ) glycoproteins before and after treatment with endo-ß-galactosidase (EßGD) to remove polylactosaminoglycans. Lane A: molecular weight markers;lane B: EßGD-HSEZ; three protein bands are noted: 90 kDa (1), 75 kDa (2) and 60 kDa (3);lane C: HSEZ, open arrows are ß-mercaptoethanol artifacts. Techniques used did not allowresolution of non-enzyme treated EZP on one-dimensional-PAGE. HSEZ: heat solubilizedequine zonae.

Characterization of equine ZP proteinsAlthough no other EZP preparations have previously been reported for comparison, the pro¬

tein pattern is consistent with other mammalian ZP patterns previously reported (porcine:Downloaded from Bioscientifica.com at 12/24/2021 03:39:11AM

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Fig. 3. Immunoblot patterns of equine zonae pellucidae (EZP) proteins separated by two-dimensional polyacrylamide gel electrophoresis. (a) Incubation with the monoclonal antibodyR5 prepared against heat-solubilized rabbit ZP glycoproteins for 13 h resulted in recognition ofthe 45-80 kDa family (3). (b) Reprobe of the same membrane with rabbit antibodies preparedagainst heat-solubilized porcine ZP glycoproteins for 48 h proved that all three glycoproteinfamilies (1,2 and 3) had been transferred.

Timmons et al, 1987; human: Shabanowitz & O'Rand, 1988; rabbit: Wolgemuth et ai, 1984). Theextensive charge and molecular weight heterogeneity could be attributed to variations in post-translational modification, including glycosylation and sulfation, typical of ZP glycoproteins, ashas been determined in other species. Three major EZP glycoprotein families were noted withapparent Mr of 93-120 kDa, 73-90 kDa and 45-80 kDa, respectively (Fig. 1).

The apparent Mr of the three EZP glycoproteins after EßGD treatment and ID-PAGE were 90,75 and 60 kDa (Fig. 2, lane B). It was of interest that the non-deglycosylated proteins did notappear to go into the gel using the methodology described.

Identification of antigenic determinants with monoclonal and polyclonal antibodies

Immunoblotting procedures were used to determine which antigens were associated with eachglycoprotein. Rabbit polyclonal antisera developed against total heat-solubilized porcine ZP(RaHSPZ) consistently recognized all three EZP glycoproteins (Figs 3b, 4c and 5c). This antibodywas therefore used as a final reprobe in each immunoblotting series to ensure that the glycoproteinfamilies identified by silver staining techniques had been adequately transferred to the PVDFmembrane.

The monoclonal antibody R5 (Drell & Dunbar, 1984) recognizes only the lowest Mr EZP glyco¬protein (Figs 3a and 5a, lane A). This antibody recognizes the lowest MT major glycoproteins of the


Fig. 4. Immunoblot pattern of equine zonae pellucidae (EZP) proteins separated by two-dimensional polyacrylamide gel electrophoresis. (a) Incubation with guinea-pig antibodiesprepared against the 55 kDa rabbit ZP glycoprotein for 7 days. The pattern is consistent withrecognition of the 45-80 kDa family (3) exclusively, (b) Reprobe of the same membrane withguinea-pig antibodies prepared against total heat-solubilized rabbit ZP glycoproteins for48 h. Here, only the 73-90 kDa family (2) is recognized, (e) Reprobe of the same membranewith rabbit antibodies prepared against heat-solubilized porcine ZP glycoproteins for 18 h.As in Fig. 3, this final probe proved that all three glycoprotein families (1,2 and 3) had beenappropriately separated and transferred.

pig and rabbit ZP on PAGE analysis (Drell & Dunbar, 1984) which is equivalent to the ZP3aprotein described by Yurewicz et al. (1987).

R55K and R75K were developed against the rabbit ZP 55 kDa and 75 kDa glycoproteinspurified by 2D-PAGE (Lee et al., 1991). The R75K glycoprotein did not recognize any EZP glyco¬protein by immunoblot procedures, but R55K recognized the lowest MT EZP glycoprotein exclus¬ively after 7 days of exposure (Fig. 4a). A 48 h reprobe of this same blot with guinea-pig polyclonalantisera developed against total heat-solubilized rabbit ZP (GPaHSRZ; Fig. 4b) revealed that the73-90 kDa EZP glycoprotein could be individually recognized. Similarly, the 75 kDa protein bandwas recognized by GPaHSRZ (Fig. 5b). A final 18 h reprobe with RaHSPZ showed that all threeproteins were appropriately transferred to the PVDF membrane (Figs 4c and 5c).

The monoclonal antibody PSI recognizes a carbohydrate antigenic domain and detects themost acidic forms of all three porcine ZP glycoprotein families separated by 2D-PAGE (Timmonset ai, 1987). This antibody did not detect EZP glycoproteins separated by 2D-PAGE (data not

shown).Downloaded from Bioscientifica.com at 12/24/2021 03:39:11AM

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Fig. 5. Immunoblot pattern of deglycosylated heat-solubilized equine zonae pellucidae(EßGD-HSEZ), deglycosylated, heat-solubilized rabbit zonae pellucidae (EßGD-HSRZ) andglycosylated, heat-solubilized rabbit zonae pellucidae (HSRZ) separated by one-dimensionalpolyacrylamide gel electrophoresis. (a) Incubated with the monoclonal antibody R5 preparedagainst HSRZ glycoproteins for 48 h. Lane A: EßGD-HSEZ 60 kDa glycoprotein (3) corres¬

ponding to (3) in Fig. 2. Lane B: EßGD-HSRZ 55 kDa glycoprotein and a higher weightaggregate of the same. Lane C: HSRZ smear of Mr 65-130 kDa. (b) Reprobe of the EßGD-HSEZ western blot with a guinea-pig polyclonal antisera raised against HSRZ (GPaHSRZ) for48 h. Recognition of the 75 kDa equine zona glycoprotein (2) is apparent and corresponds to(2) in Fig 2. Recognition of the 60 kDa equine zona pellucida glycoprotein (EZP) is attributedto the radioactivity remaining from the previous R5 probe, (e) Reprobe of the EßGD-HSEZwestern blot with rabbit antisera raised against heat-solubilized porcine zonae for 72 h. Allthree EZP glycoproteins (1,2 and 3) are recognized.

Discussion

Previous authors have suggested that the equine ZP has antigenic properties similar to thosefound in other species (Liu & Shivers, 1982; Shivers & Liu, 1982; Bousquet et al., 1987).However, the data presented in this report are the first to directly prove this hypothesis and tocharacterize the EZP biochemically. The difficulty encountered in obtaining adequate numbers ofequine oocytes to process (3-4 oocytes per ovary: Liu & Shivers, 1982; 1-4 oocytes per ovary:Okolski et al., 1987) has been a primary obstacle to studies such as this. Our recovery rate (1T4oocytes per ovary) is similar to previously reported rates, but by using mini-techniques the numberof oocytes required for analysis was drastically reduced and allowed the investigation to proceed.

The EZP is composed of three major glycoprotein families. This is consistent with othermammalian species investigated that exhibit significant ZP heterogeneity when analysed by PAGEprocedures. The glycoprotein families of the EZP do not contain the more acidic charged forms seen

on 2D-PAGE analysis of the rabbit or pig ZP (Dunbar et al., 1981; Drell & Dunbar, 1984). In thisrespect, the EZP is more similar to human ZP patterns (B. S. Dunbar, unpublished data). Thisdifference may, in part, reflect the stage of the oocytes used. Human and horse zonae acquisitiontechniques (aspiration of follicles) recover oocytes that are more mature than zonae recovered fromother species by techniques that usually involve mincing the ovary and thus result in the recovery ofimmature, as well as mature, zonae pellucidae.


The presence of the acidic charge forms generally reflects the addition of post-translationalmodifications such as sialation and sulfation. It is expected that mature ZP would have undergonemore extensive alteration during cellular processing. It is therefore interesting that the mono¬clonal antibody PSI does not recognize EZP glycoproteins when 2D-PAGE and immunoblottingtechniques are used. Timmons et al. (1987) demonstrated that PSI recognizes the most acidiccharge forms of the PZP glycoproteins and that it recognizes carbohydrate moieties. Theseobservations have recently been confirmed (Timmons et al., 1990). It is possible that the post-translational modifications of the EZP differ from those of pigs. Alternatively, although a 3-5%solution ofnonfat dry milk does efficiently block most nonspecific binding sites for immunoglobulins(Tovey & Baldo, 1987), the carbohydrates contained in such a preparation may interfere withbinding of an antibody that recognizes a carbohydrate determinant (Timmons & Dunbar, 1990).This dilemma may be resolved by future investigations into the composition and sequence of PZPand EZP carbohydrates.

Use of antibodies developed against the zona pellucida of the rabbit and pig makes it apparentthat the EZP has antigenic domains that are similar to those of the porcine and rabbit ZP. Themonoclonal antibody R5 or the polyclonal antibody R55K can be used to identify specifically theEZP glycoprotein family of molecular mass 45-80 kDa. GPaHSRZ uniquely recognizes the 73-90 kDa EZP glycoprotein family. All three EZP glycoprotein families are recognized by RaHSPZ.Since these antibodies recognize all of the charged species within a ZP glycoprotein family, thedeterminants recognized are believed to be proteins (Drell & Dunbar, 1984).

It is well known that disturbing ZP function by active immunization with heterologous ZPantigens alters fertility in rabbits (Gwatkin & Williams, 1978; Gwatkin et ai, 1980; Wood et al.,1981), dogs (Mahi-Brown et ai, 1982), primates (Gulyas et ai, 1983; Sacco et ai, 1989), horses (Liuet al., 1989; Kirkpatrick et ai, 1990) and mice (Gwatkin et al., 1977). Clinical application of thisknowledge to provide methods of immunocontraception for domestic and feral equine populationswill ultimately rely upon recombinant techniques (Tyndale-Biscoe, 1991). Porcine and rabbit zonae

pellucidae are more readily available than are equine ZP. Knowing that the EZP has epitopes incommon with the rabbit and pig will promote the development of such vaccines for use in horses.

In summary, the isolation, separation techniques and immunological properties of the EZPpresented here provide a basis for future studies on the molecular mechanisms of fertilization andinfertility in the horse.

This work was supported, in part, by a grant from the University of Georgia, VeterinaryMedical Experiment Station (VMES Grant 29-26-GR207-002). We thank L. Allison for her ableassistance in oocyte collection and C. Lo for her expert technical assistance.

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Received 2 December 1991


characterization of equine and

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