binding by solubilized components of the zona pellucida

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Sperm binding t the pig zna pellucida and inhibitin f binding by slubilized cmpnents f the zna pellucida Trish Berger, Arlene Davis, N. J. Wardrip and Jerry L.

1 Sperm binding t the pig zna pellucida and inhibitin f binding by slubilized cmpnents f the zna pellucida Trish Berger, Arlene Davis, N. J. Wardrip and Jerry L. Hedrick Departments f * Animal Science and fbichemistry and Biphysics, University f Califrnia, Davis, Califrnia 95616, USA Summary. An assay t determine the binding f pig spermatza t the zna pellucida (ZP) f pig cytes was develped using cnditins cmpatible with in-vitr fertilizatin f pig eggs and with pig sperm penetratin f zna-free hamster va. These cnditins were used t define which f the pig cyte ZP cmpnents were invlved in sperm binding by a cmpetitive inhibitin apprach. Assay variables that were ptimized included: the methd f sperm preparatin; sperm preincubatin time; sperm\p=n-\cyte cincubatin time, sperm cncentratin and temperature; and methds fr the separatin f free frm cyte-bund spermatza. Inclusin f slubilized ZP in the sperm preincubatin medium inhibited sperm binding apprximately 50%. Bth the 55K and 90K cmpnents inhibited sperm binding althugh the 55K cmpnent was mre effective. The tw plypeptides derived frm chemical deglycsylatin f the 55K families did nt inhibit sperm binding. f several mnclnal antibdies t the ZP cmpnents tested, nly ne directed against the 55K\g=a\glycprtein family inhibited sperm binding. Sperm binding t pig cyte ZP is therefre dependent n the carbhydrate miety f the glycprteins and appears t invlve mre than a single ZP glycprtein. Keywrds: zna pellucida; sperm binding; fertilizatin; pig Intrductin Binding f spermatza t the zna pellucida (ZP) is an initial step in the fertilizatin prcess. Uncapacitated pig spermatza can bind t the ZP in vitr (Petersn et al, 1984). Spermatza cntinue t bind t the pig ZP after in-viv fertilizatin (Hunter, 1974). We d nt yet knw whether inefficient sperm-zp binding is a significant prblem in fertilizatin failure r whether variatin in sperm-zp binding cntributes t quantitative differences in fertility amng fertile males. A zna-free hamster va biassay fr pig spermatza which examines steps in the fertiliz atin prcess after penetratin f the ZP has been develped (Berger & Hrtn, 1988). Althugh a successful technique fr in-vitr fertilizatin in the pig has been develped (Cheng, 1985), large numbers f fertile pig va are nt readily available. The ZP cmpsitin f cytes is very similar t that f va (Hedrick et al, 1987) and cytes are readily available. Hence, we chse t develp a sperm-zp binding assay using cytes that was cmpatible with sperm preparatin fr the zna-free hamster va biassay. The pig ZP cnsists f three glycsylated plypeptide chains which are referred t by a nmenclature based n apparent mlecular weight as the 55, 55 ß, and 90K families (Hedrick & Wardrip, 1986, 1987). Under cnditins in which disulphide bnds are reduced, a 25K cmpnent and a 65K cmpnent are derived frm a prtin f the 90K family. We als wished t examine which f the pig ZP glycprtein families were invlved in sperm binding.

Materials and Methds Pig ZP were prepared as previusly described (Hedrick & Wardrip, 1986). Briefly, pig varies were btained frm the slaughter huse and stred frzen until use.

2 Materials and Methds Pig ZP were prepared as previusly described (Hedrick & Wardrip, 1986). Briefly, pig varies were btained frm the slaughter huse and stred frzen until use. The fllicles were ruptured using a ganged razr blade apparatus. Zna pellucida-intact cytes were separated frm debris by multiple sievings thrugh nyln screens f different mesh sizes. These cytes were stred at 4 C in PBS with azide fr up t 10 days befre use. n the day f use, cytes were rinsed thrugh 4 1-ml vlumes f Tris-buffered medium (1131 mm-nacl, 3mM-KCl, 20 mm-tris, 11 mm-glucse, 5 mmsdium pyruvate, and 50 pg gentamycin sulphate/ml; Berger & Hrtn, 1988) cntaining 40mM-CaCl2 and 5 mg bvine serum albumin/ml (BSA; #A7906, Sigma, St Luis, M, USA). Any cytes with a damaged ZP were remved. Ejaculated spermatza frm the sperm-rich fractin were cllected and prcessed as previusly described (Berger & Hrtn, 1988) using Tris-buffered medium cntaining 40mM-CaCl2 and 5 mg BSA/ml. Prcessed spermatza were preincubated in 2 ml vlumes (2 I6 spermatza/ml at 39 C fr 1 h in 5% C02 in air. The sperm suspensin was then diluted t 2 I5 spermatza/ml and 100 µ were used t inseminate a 100-µ vlume f medium cntain ing 100 cytes. Unless therwise stated, gametes were cincubated fr 3 h at 39 C in 5% C02 in air. cytes were separated frm the unbund spermatza using a mdificatin f the stp-fix prcedure described by Saling et a! (1978). A dextran gradient was prepared in a 1-0-ml plystyrene centrifuge tube (Fisher) by layering 0-35 ml f 1-8% dextran (average mlecular weight ) in Tris-buffered medium cntaining BSA abve 0-35 ml f 2-25% dextran in the same medium. The cytes in 100 µ medium were then layered n tp and centrifuged fr 3 min at 160 g. All but 100 µ ~ was aspirated and the cytes resuspended in 1 ml 0-625% glutaraldehyde. They were allwed t settle, the first glutaraldehyde slutin aspirated and an additinal 1 ml glutaraldehyde slutin was used t transfer cytes t a dish. A 1-ml syringe fitted with a 26-gauge needle and filled with a mixture f vaseline:paraffin wax (8:1 w/w) was used t frm a ribbn alng each lng edge f a cleaned micrscpe slide. The cytes were placed between the 2 ribbns and slightly cmpressed with a cverslip. Excess fluid was remved and the cverslip sealed with munting medium. Spermatza bund t hemispheres f each f 30 cytes were cunted using differential interference cntrast ptics at 400 magnificatin. When indicated, heat-slubilized pig ZP r islated ZP cmpnents were added t the spermatza during pre incubatin. ZP and ZP cmpnents were islated by preparative tw-dimensinal plyacrylamide gel electrphresis as previusly described (Hedrick & Wardrip, 1986,1987). Sdium ddecyl sulphate (SDS) was extracted frm islated cmpnents r frm BSA previusly mixed with SDS using Extracti-Gel D (Pierce Chemical C., Rckfrd, IL, USA). A ttal f 12 mnclnal antibdies t zna pellucida cmpnents were evaluated fr their effect n sperm-cyte ZP binding (Betancurt et ai, 1984, 1985; Hedrick & Wardrip, 1987). cytes were preincubated at 39 C fr 45 min with each mnclnal antibdy at a 1:100 dilutin f the ascites fluid, rinsed in three 10 ml vlumes f medium t remve free antibdy, and cincubated with spermatza. T develp the assay cnditins, 3-5 ejaculates frm a minimum f 3 bars were used. A minimum f 1 ejaculate frm each f 3 bars was used in subsequent experiments. Analyses f variance and regressin were perfrmed n mean numbers f spermatza bund per cyte using the BMDP statistical package (BMDP Statistical Sftware, Ine, Ls Angeles, CA, USA). Differences amng means were evaluated with Student-Newman-Keul's sequential range test. Percentage mtilities were analysed bth befre and after transfrmatin t the arcsin square rt and statistical significance resulting frm the transfrmatin is reprted. Results ptimizatin f the ZP-binding assay cnditins Varying the sperm preincubatin time frm 1 t 4 h and the sperm-cyte cincubatin time frm 1 t 3 h had little effect n the number f spermatza bund per cyte (P = 0-33). Hwever, very shrt incubatin times (0 h f sperm preincubatin fllwed by 1 h f sperm-cyte cincubatin) appeared insufficient fr maximum sperm binding (P 0-15). Increasing the = sperm preincubatin time frm 3 h t 5 r 7 h decreased the number f spermatza bund (24, 16 and 7 spermatza/cyte, respectively; < 005). As expected, the number f spermatza bund per cyte increased with increasing sperm cncentratin (15, 35, 42 and 130 fr 4 104, 1 I5, 4 I5 and 1 6 spermatza/ml; < 0005). Sperm-cyte cincubatin at 39 C cmpared t rm temperature increased the number f spermatza bund per cyte regardless f whether the spermatza were preincubated fr 1 h at 39 C (60 vs 22 spermatza/cyte, s.e.m. 5; = < 0-01) r nt preincubated (53 ví 32 spermatza/cyte, s.e.m. 5, = < 005). Sperm mtility ranged frm 86 t 94% at the end f the cincubatin perids in these experiments and did nt differ amng treatments. Preincubatin and cincubatin at 2-10 C reduced binding even further

(1 spermatzn/cyte vs 54 and 34 spermatza/cyte fr cincubatin at 39 C and rm temperature, respectively; < 005). In this case, hwever, mtility was reduced t 22% after incubatin at 2-10 C.

3 (1 spermatzn/cyte vs 54 and 34 spermatza/cyte fr cincubatin at 39 C and rm temperature, respectively; < 005). In this case, hwever, mtility was reduced t 22% after incubatin at 2-10 C. Washing spermatza by dilutin and centrifugatin cmpared t washing n the Percli gradient befre preincubatin and/r cincubatin f gametes reduced the number f bund spermatza by 30% (P < 005). Cincubatin f gametes in the absence f BSA greatly reduced sperm binding regardless f sperm washing prcedures (3, 3, 35 and 57 spermatza/cyte fr samples washed by dilutin r n a Percli gradient and cincubated withut BSA and fr samples washed by dilutin r n a Percli gradient and cincubated with BSA; < 0-01). Hwever, mtility was greatly reduced after incubatin in the absence f BSA (27% and 15% fr sper matza washed n a Percli gradient and spermatza washed by dilutin vs 87% and 73% fr spermatza prepared by the same prcedures and incubated with BSA). Preincubatin and cincubatin with 5 mg plyvinyl alchl/ml (PVA, average ml. wt ; Sigma, St Luis, M, USA) als supprted binding f spermatza t the ZP (74 and 65 spermatza/cyte fr BSA and PVA, respectively). Separatin f cytes and bund spermatza frm unbund spermatza n a dextran gradient was equivalent t separatin by centrifugatin f cytes thrugh 5% Percli (85 and 90 spermatza/cyte, s.e.m. 9). Hwever, the number f bund spermatza = was greatly reduced when cytes were layered n 5% Ficll-Paque (Pharmacia, Piscataway, NJ, USA; 42 spermatza/cyte, < 005). cyte recvery was als reduced t <50% n the Ficll-Paque. The length f centrifugatin n the dextran gradient did nt affect the number f spermatza bund (56, 62 and 54 spermatza/cyte fr 2, 3 and 4 min; s.e.m. 5). The number f bund = spermatza did nt differ when cytes were washed free f unbund spermatza in 3 drps f medium using a micrpipette (as described by Petersn et al, 1980) cmpared t the dextran gradi ent (75 and 82 spermatza/cyte). The dextran gradient is therefre cmparable t washing thrugh 3 drps in measuring bund spermatza, nt attached spermatza (Hartmann et al, 1972). Hwever, since the micrpipette methd is pipette-dependent, i.e. the shear frce is a functin f the pipette bre, the dextran gradient centrifugatin methd is ultimately mre reprducible. The variability f sperm binding in 3-6 ejaculates frm each f 6 previusly fertile bars is shwn in Fig. 1. This variability was nt due t age f cytes used nr was it primarily due t dayt-day variability in assay cnditins since variability amng ejaculates frm different bars analysed n the same day was as great as the day-t-day variability in ejaculates frm the same bar. Effect fslubilized ZP and islated ZP cmpnents n sperm-cyte ZP binding Preincubatin f spermatza fr 1 h with 25, 50 r 100µg slubilized ZP/ml significantly reduced sperm-cyte ZP binding cmpared t preincubatin withut slubilized ZP r with 15 µg ZP/ml (35, 32 and 28 vs 65 and 66 spermatza/cyte, s.e.m. = 6, < 0001). Regressin analysis indicated that spermatza frm the 6 bars examined differed in their dse respnse (P < 005). Sperm binding was reduced frm 52 spermatza/cyte (mean f 6 ejaculates) t 34 and 26 spermatza/cyte when spermatza were preincubated with disulphide bnd-reduced r nnreduced 55K cmpnents at 30µg/ml (s.e.m. = 4; < 0001). Preincubatin with 30 µg f SDStreated BSA/ml (as described in 'Materials and Methds') did nt affect subsequent binding (58 spermatza/cyte). Preincubatin f spermatza frm 6 ejaculates with 5 µg 25K cmpnents/ ml r 10 µg 65K cmpnents/ml did nt inhibit sperm binding (75 and 62 vs 70 spermatza/cyte in the absence f ZP cmpnents, s.e.m. = 6). Hwever, preincubatin with 10µg/ml f the 90K family had a slight effect n binding (53 spermatza/cyte, = 0-06 in the abve experiment and 64 vs 48 spermatza/cyte in the absence and presence f 10 µg/ml f 90K family, s.e.m. = 4,

4 " C 80r 60 r 40 -y 8 S Fig. 1. Variability in sperm-cyte ZP binding amng ejaculates frm fertile bars. Sperm atza were washed n a Percli gradient, preincubated fr 1 h at 39 C, and cincubated with ZP-intact cytes fr 3 h. Each pint represents the mean number f spermatza/cyte fr spermatza frm ne ejaculate. Each bar represents the mean number f spermatza/cyte fr spermatza frm 3-6 ejaculates frm an individual bar. Bar Table 1. Effect f ZP mlecules n sperm-zp binding Effect n sperm-zp binding ZP mlecule % f cntrl sperm-zp binding pg ZP/ml pg 55K (reduced)/ml pg 55K (nn-reduced)/ml 50 10pg90K/ml pg65K/ml 89 5 pg 25K/ml pg ZP plypeptide (M, 37000)/ml pg ZP plypeptide (Mr40 000)/ml 100, nt significant. Significance < 0001 < 0001 < 0001 < 005 < 005, in a separate experiment invlving spermatza frm 10 ejaculates). The cncentratins f individual ZP cmpnents used were in apprximate prprtin t their cmpsitin in the ZP. Preincubatin f spermatza with 15 µg/ml f the Mt and plypeptides prepared by chemical deglycsylatin f the 55K glycprteins did nt affect binding (47, 52 and 47 spermatza/cyte fr cntrl, Mr and Mr vs 31 spermatza/cyte after preincu batin with 30 µg 55K cmpnents/ml fr spermatza frm 10 ejaculates, s.e.m. = 4, = 002).

5 - - In this experiment, mtility ranged frm 90 t 92% at inseminatin and frm 81 cincubatin and did nt differ amng treatments. t 87% after Effect fglucsaminidase n sperm-cyte ZP binding Preincubatin f cytes with ß-TV-acetylglucsaminidase (Sigma, St Luis, M, USA; #A2264 frm jack bean) eliminated the ability f spermatza t bind t the ZP (8 spermatza/cyte fr cytes preincubated fr 2 h with 6 IU ß-7V-acetylglucsaminidase/ml and rinsed in 3 drps f medium befre inseminatin vs 49 spermatza/cyte fr cytes preincubated in medium alne, mean f 4 ejaculates, s.e.m. = 6; < 0005). Preincubatin f cytes with 6 IU ß-galactsidase/ ml (Sigma; #G6512 frm E. cli) did nt affect binding (59 spermatza/cyte). Effect fmnclnal antibdies t ZP n sperm-cyte ZP binding ne mnclnal antibdy t the peptide miety f the 55Ka glycprtein significantly inhibited sperm-cyte ZP binding (40 ví 80 spermatza/cyte in the absence f mnclnal antibdies in the first experiment utilizing an ejaculate frm each f 3 bars, s.e.m. = 8; and 31 vä 56 spermatza/cyte in the secnd trial using ne ejaculate frm each f 5 bars, s.e.m. = 7). The ther mnclnal antibdies (2 t the peptide miety f the 55Kp glycprtein, 1 t the peptide miety f the 25K cmpnent derived frm the 90K family, 2 t the peptide miety f the 65K cmpnent derived frm the 90K family, 2 which react with carbhydrate f all families, 3 which are cnfrmatin-dependent and react nly with intact r heat-slubilized ZP but nt with SDSdissciated ZP, and an additinal mnclnal antibdy t the peptide miety f the 55Ka glycprtein) did nt significantly inhibit sperm binding. The mnclnal antibdy t the 55Ka glycprtein had a dse-respnse effect n sperm binding (Fig. 2). Dse-respnse studies (extend ing frm a 1:5 t 1:100 dilutins f the mnclnal antibdies in ascites fluid) indicated n effect cmpared t cntrl ascites fluid dilutins fr tw additinal mnclnal antibdies, ne specific t the peptide miety f the 55Kp glycprtein and ne specific t the carbhydrate miety f all cmpnents. 35 rd * ^ Lg (1/dilutin) Fig. 2. Sperm-cyte ZP binding after preincubatin f cytes with varius cncentratins f a mnclnal antibdy t 55Ka. cytes were preincubated fr 45 min with an IgGi mn clnal antibdy (123-7) and rinsed 3 times befre cincubatin with spermatza., Mean n. f spermatza/cyte fr cytes preincubated with the indicated cncentratin f anti bdy; D, mean n. f spermatza/cyte fr cytes preincubated with medium alne.

6 Discussin Petersn et al. (1984) reprted binding by washed uncapacitated pig spermatza. Since the gamete interactin ccurred in the presence f BSA at rm temperature, BSA may have prmted partial capacitatin in the previus study. In this study, n substantial binding ccurred when gametes were incubated in the absence f BSA r plyvinyl alchl but this was prbably due t the decrease in mtility. Plyvinyl alchl was a satisfactry substitute fr BSA in permitting binding. Hwever, spermatza incubated with plyvinyl alchl are nt sufficiently capacitated t penetrate znafree hamster va (Berger & Parker, 1989). Hence, ur bservatins are in agreement with the previus cnclusin that uncapacitated pig spermatza bind t the ZP. Seminal plasma prteins reprtedly inhibit binding f pig spermatza t ZP (Petersn et al, 1984). Remval f such pr teins prbably ccurs during capacitatin (Brackett & liphant, 1975). Remval f adsrbed seminal plasma prteins during the washing prcedure n the Percli gradient culd explain why such spermatza bund t a greater extent than did spermatza washed by dilutin. ne might als describe spermatza washed n the Percli gradient as partly capacitated since such spermatza will penetrate zna-free hamster va whereas spermatza washed by dilutin and incubated under the same cnditins have a reduced ability t penetrate zna-free hamster va (Berger & Hrtn, 1988). Prbably, binding f partly capacitated spermatza and uncapacitated spermatza ccurs in the pig as in the hamster. Day-t-day variability in the numbers f spermatza bund t the ZP has been bserved in the pig and the muse (Petersn et al, 1980; Flrman et al, 1984). The day-t-day variability we bserved was nt due t the age f the ZP but may have been at least partly due t day-t-day fluctuatins in the ejaculates. Slubilized ZP strngly inhibited sperm-cyte binding. The slubilized 55K cmpnent als strngly inhibited sperm binding, indicating that at least ne f the 55K families is invlved in sperm binding. Inhibitin studies with mnclnal antibdies suggest that the 55K0 family is a majr ZP ligand fr spermatza. The 55K cmpnent crrespnds t the 58K PPZA described by Sacc et al. (1984) wh reprted inhibitin f ZP binding by spermatza frm 2 ejaculates. Hw ever, ther cmpnents f the ZP were nt examined in their previus study. In this study, the 25K and 65K cmpnents, which under nn-reducing cnditins cmigrate with the 90K family and are derived frm this family, had n effect n sperm-cyte ZP binding. The 90K cmpnent had a small but significant effect. The 55K cmpnent f the pig ZP is cmpsed f 2 glycprtein families which have been termed 55Ka and 55Kp (Hedrick & Wardrip, 1987). The stichimetric cmpsitin f the 55Ka, 55Kp, and 90K ZP glycprtein families is apprximately 1:1:1. Each f the glycprtein families pssesses extreme micrhetergeneity primarily due t variatin in the carbhydrate miety (Dunbar et al, 1980; Sacc et al, 1981). Hwever, we d nt yet knw if the variatin in ligsaccharide cmpsitin affects sperm binding. Inhibitin f sperm-cyte ZP binding by a mn clnal antibdy t the 55Ka glycprtein and lack f inhibitin by ther mnclnal antibdies suggests that the 55Ka family is a majr ZP ligand fr spermatza. The plypeptides f Mt and derived frm the 55Ka and 55Kp families had n inhibitry activity n sperm-cyte ZP binding. The lack f effect f the plypeptides suggests that the carbhydrate prtin is essential fr sperm-cyte ZP binding in the pig; this is cnsistent with what has been reprted fr the muse (Flrman & Wassarman, 1985). Further evidence indicating the imprtance f the carbhydrate prtin is the bservatin that preincubatin f the ZP-intact cytes with ß-/V-acetylglucsaminidase, which presumably is remving terminal N-acetylglucsamine residues, almst cmpletely ablished sperm-cyte ZP binding. Inhibitin f binding by a mnclnal antibdy t the peptide prtin f the 55Ka family is cmpatible with a rle fr carbhydrate in the sperm binding prcess since binding f a relatively large (MT ) antibdy in the immediate vicinity f the sperm-binding site culd sterically hinder sperm binding t the carbhydrate miety.

T understand further sperm-cyte ZP binding in the pig, additinal binding studies using islated glycprteins f the 55Ka, 55K? and 90K families and their derived ligsaccharides mieties are needed.

7 T understand further sperm-cyte ZP binding in the pig, additinal binding studies using islated glycprteins f the 55Ka, 55K? and 90K families and their derived ligsaccharides mieties are needed. We thank Mary Hrtn, Susan Dixn and D. Gldhawk fr technical assistance. Research was supprted in part by USDA grant 85-CRCR References Berger, T. & Hrtn, M.B. (1988) Evaluatin f assay cnditins fr the zna-free hamster va biassay f bar sperm fertility. Gamete Res. 19, Berger, T. & Parker,. (1989) Mdificatin f the znafree hamster va biassay f bar sperm fertility and crrelatin with in viv fertility. Gamete Res. 22, (in press). Betancurt, M., Rdriguez, F., Serran, H., Wardrip, N. & Hedrick, J.L. (1984) Mnclnal antibdies against the 55K glycprteins islated frm the pig zna pellucida. J cell Bil. 99, 395a, abstr. Betancurt, VI.. Rdriguez, 11., Serran, H., Wardrip, N. & Hedrick, J.L. (1985) El us de anticuerps mnclnales para estudiar las funcines de la zna pelúcida de óvuls de mamífers. Salud Publica Med. 27, Bracke«, B.G. & liphant, G. (1975) Capacitatin f rabbit spermatza in vitr. Bi! Reprd. 12, Cheng, W.T.K. (1985) Test-tube piglets: studies n fer tilizatin f pig cytes in vitr. J Taiwan Livest. Res. 18, Dunbar, B.S., Wardrip, N.J. & Hedrick, J.L. (1980) Is latin, physichemical prperties, and macrmlecular cmpsitin f zna pellucida frm prcine cytes. Bichemistry, NY2, Flrman, H.M. & Wassarman, P.M. ( 1985) -linked ligsaccharides f muse egg ZP3 accunt fr its sperm receptr activity. Cell 41, Flrman, H.M., Bechtl, K.B. & Wassarman, P.M. (1984) Enzymatic dissectin f the functins f the muse egg's receptr fr sperm. Devi Bil. 106, Hartmann, J.F., Gwatkin, R.B. & Hutchisn, CF. (1972) Early cntact interactins between mammalian gametes in vitr: evidence that the vitellus influences adherence between sperm and zna pellucida. Prc. natn. Acad. Sci USA 69, Hedrick, J.L. & Wardrip, N.J. (1986) Islatin f the zna pellucida and purificatin f its glycprtein families frm pig cytes. Analyt. Bichem. 157, Hedrick, J.L. & Wardrip, N.J. (1987) n the macrmlecular cmpsitin f the zna pellucida frm prcine cytes. Devi Bil. 121, Hedrick, J.L., Wardrip, N.J. & Berger, T. (1987) Differ ences in the macrmlecular cmpsitin f the zna pellucida islated frm pig cytes, eggs, and zygtes. J. exp. Z! 241, Hunter, R.H.F. (1974) Chrnlgical and cytlgical details f fertilizatin and early embrynic develp ment in the dmestic pig, Sus scrfa. Anat. Ree. 178, Petersn, R.N., Russell, L., Bundman, D. & Freund, M. (1980) Sperm-egg interactin: evidence fr bar sperm plasma membrane receptrs fr prcine zna pellucida. Science, NY 207, Petersn, R.N., RusseU, L.D. & Hunt, W.P. (1984) Evi dence fr specific binding f uncapacitated bar spermatza t prcine znae pellucidae in vitr. J. exp. Z! 231, Sacc, A.G., Yurewicz, E.C, Subramanian, M.G. & DeMay, F.J. (1981) Zna pellucida cmpsitin: species crss reactivity and cntraceptive ptential f antiserum t a purified pig zna antigen (PPZA). Bi! Reprd.2S,991-lS. Sacc, A.G., Subramanian, M.G. & Yurewicz, E.C. (1984) Assciatin f sperm receptr activity with a purified pig zna antigen (PPZA). J. Reprd. Immunl. 6, Saling, P.M., Strey, B.T. & Wlf, D.P. (1978) Calciumdependent binding f muse epididymal spermatza t the zna pellucida. Devi Bil. 65, Received 10 ctber 1988

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