Inhibition of goat sperm-zona binding by monoclonal antibodies
to a glycoprotein family (ZP4) of porcine zona pellucida
Gautam Kaul
*, K.K. Gandhi
Division of Animal Biochemistry, National Dairy Research Institute, Karnal 132001, Haryana, India
Accepted 12 January 1999
Abstract
The zona pellucida (ZP) in a majority of species consists of three antigenic families with wide inter species cross reactivity. The glocoproteins of heat solubilized goat zona pellucida (gZP) were isolated into three families by high performance liquid chromatography on a gel ®ltration column. Their molecular identities were 180, 95 and 75 kDa for gZP1, gZP2 and gZP3 respectively. However under reducing conditions, four zona pellucida fractions of 135, 95, 75 and 42 kDa were observed. The monoclonal antisera Mab-5H4 against porcine ZP4 (a 23 kDa glycoprotein) easily recognized the 180 and 42 kDa gZP fractions. It was also found that the 5H4 antisera strongly inhibited homologous goat sperm-zona binding. Finally, using immuno¯uorescent staining the goat occytes reacted positively with the 5H4 antisera. The ¯uorescence was uniform at 1 : 10 antisera dilution but tended to fade at higher dilution's. Results suggest that the gZP has an antigen epitope correlative to the 5H4 antisera which is involved in sperm-zona interaction and could be a good candidate for the development of a contraceptive vaccine.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Zona-sperm inhibition; Mab-5H4; Goat
1. Introduction
The ZP which is the hard outer transparent encase-ment of the mammalian egg has an important role in species-speci®c recognition between gametes. The mouse zona pellucida consists of three glycoprotein families; ZP1 (185±200 kDa), ZP2 (120±140 kDa) and ZP3 (83 kDa) (Bleil and Wasserman, 1980; Was-sarman, 1990). The zona of horse (Shabanowitz and O'Rand, 1988), rats (Miller et al., 1992), human (Araki et al., 1992) and rabbits (Maresh and Dunar, 1987) also consists of three glycoproteins, whereas the
zona of the cat has only two families of glycoprotein (Maresh and Dunar, 1987). The porcine zona pellucida is highly heterogenous and can be resolved into four glycoprotein families designated as ZP1 (92 kDa), ZP2 (69 kDa), ZP3 (55 kDa) and ZP4 (23 kDa) according to their molecular size (Yurewicz et al., 1987; Hedrick and Wardship, 1987). It has been shown that the 92 kDa (ZP1) component under reducing conditions is split into two smaller molecules of 69 kDa (ZP2) and 23 kDa (ZP4) (Koyama et al., 1991; Hasegawa et al., 1994). More recently (Hase-gawa et al., 1994) the pig zona is also said to be composed of three glycoprotein families pZP1, pZP3a and pZP3b, of 79, 59 and 46 kDa respectively. The pZP2 and pZP4 are derived from pZP1 and
mono-*Corresponding author. Fax :91-184-250042.
E-mail address: [email protected] (G. Kaul).
clonal antibodies to pZP1, 3A4-2G1 is effective in not only inhibiting homologous sperm-zona binding but also strongly inhibits human sperm-ZP interac-tion (Hedrick, 1993; Koyama et al., 1985). A monoclonal antibody against the porcine ZP4, 5H4, also inhibited the binding of human spermatozoa to homologous zona pellucida (Koyama et al., 1991). In fact the pZP4 corresponds to the amino terminal region of pZP1 and the MAb-5H4 reacts not only with human in addition to porcine zona pellucida, but also with that of rabbit, but not with mice zona (Hasegawa et al., 1995). As consequence it has also been suggested that the antigenic epitope corresponding to MAb-5H4 could be a good candidate for development of a contraceptive vaccine (Koyama et al., 1991). In the present study solubilized gZP was fractionated according to size-exclusion on high performance liquid chromatography and the inhibitory effect of MAb-5H4 was studied on goat sperm-zona binding.
2. Materials and methods
Sodium lactate, Tyrode's culture medium-199 (TCM-199), fetal calf serum (FCS), bovine serum albumin (BSA), tetracycline hydrochloride (CTC-HCl), ethylene diamine tetra acetic acid (EDTA), sodium pyruvate, orcein, diaminobenzidine (DAB), dithiotheritol (DTT), phenyl methyl sulphonyl ¯uor-ide (PMSF), goat anti-mouse (Fab0)
2IgG-HRP (Horse
radish peroxidase) conjugate, rabbit anti-mouse F(ab0
)2 IgG-FTTC (¯uorescein isothiocyanate)
con-jugate and ¯uorescamine were purchased from Sigma Chemical Co., St. Louis, USA. Nitrocellulose mem-brane (0.45mm pore size) were obtained in rolls from Schleicher and Schuell, Germany.
2.1. Oocyte collection and maturation
Ovaries were removed from goats at an unknown stage of the reproductive cycle at a local slaughter house and brought to the laboratory in Tris±saline within an hour of the slaughter. Small antral follicles (3±5 mm diameter) were aspirated with a 20 gauge needle and syringe and aspirates collected in a tissue culture dish (FALCON 3001, Becton Dickinson and Company, USA). The petri dish was placed under an
inverted microscope and oocytes were carefully picked up with the aid of very ®ne pasteur pipettes and transferred to TCM-199. The oocytes were again transferred to fresh TCM-199 and washed four times with 5 ml of the medium to remove cell debris and granulosa cells. 10±12 oocytes with compact cumulus mass were selected and transferred to 2 ml TCM-199 containing 10% FCS and sealed with paraf®n oil. Oocytes were then incubated in CO2 incubator at
378C for 32 h until the polar body was extruded in an atmosphere of 5% CO2, 95% air and 99% relative
humidity. At the end of incubation period the cumulus cells were removed completely from the oocytes by pipetting the complexes into and out of a ®nely drawn pipette. Care was taken to avoid the breakage of zona pellucida (Chauhan and Anand, 1991).
2.2. Preparation and solubilization of zona pellucida ghosts
The aspirates from the antral follicles were col-lected in a conical centrifuge tube (FALCON 2097, Becton Dickinson and Company, USA) and suspended in isolation buffer (10 mM sodium phosphate, pH 7.4, 125 mM NaCl, 3 mM sodium citrate, 2 mM EDTA). The cumulus cells were liberated as previously described and the denuded oocytes were removed and gently homogenized in a hand held Potter-Elveh-jem homogenizer (100mm clearance). The contents were then transferred to a culture dish. Zona pellucida ghosts were recovered by a ®nely drawn pasteur pipette and washed with distilled water to remove excess buffer salts (Dunbar and Raynor, 1980). The isolated zona pellucida ghosts were transferred into Eppendorf tubes containing 100ml of ammonium bicarbonate buffer pH 8.0. Solubilization of the dis-aggregated zona pellucida was carried out by placing the tubes in a water bath at 728C for 30 min. There-after, the tubes were centrifuged at 5000 rpm for 10 min to sediment particulate matter. The solubilized zonae were stored atÿ208C until further use (Hedrick and Wardship, 1986).
2.3. Collection, washing and motility measurement of spermatozoa
pre-warmed vessel at 358C and processed within 5 min. Ejaculates showing high initial motility, with a score 3.5±4.5 (0no motility, 5100% motility) were used. Semen was diluted with albumin±saline and freed of seminal plasma by centrifugation at 800 g for 10 min. The sperm pellet was washed once more with albumin±saline. Sperm counts were made with a haemocytometer. Sperm motility was assessed by examining a uniform drop of semen under a coverslip on a warm stage at 378C; the assessment were made independently by two experienced workers using two arbitrary scales of 0±10 and 0±100.
2.4. Sperm capacitation
Ejaculated spermatozoa were capacitated according to the procedure of Kaul et al. (1997) in which modi®ed Krebs±Ringer bicarbonate buffered medium was used as capacitation medium. The composition was (mM): Nacl, 119.37; KCl, 4.78; CaCl2, 1.0;
KH2PO4, 1.19; NaHCO3, 25; sodium lactate, 25;
sodium pyruvate, 1; glucose, 5.56 and bovine serum albumin, 20 mg/ml. The pH was adjusted to 7.4 and before use it was saturated with 5% CO2. For control
experiments, Tris±saline was used having the compo-sition: 20 mM Tris±HCl, pH 7.4 and 130 mM NaCl. Washed pellet of ejaculated goat spermatozoa at a concentration between 1106and 2106sperm/ml was suspended in capacitation medium and Tris± saline medium. It was then distributed in small tubes each containing 0.5 ml sperm suspension and incu-bated at 378C in CO2 incubator with 5% CO2±95%
air. The spermatozoa were assessed using chlo-rtetracycline (CTC) ¯uorescence stain whereby different ¯uorescence for fresh (`F'), capacitated (`B') and acrosome reacted (`AR') spermatozoa are seen under the ¯uorescence microscope (Reichert Jung, Austria).
2.5. Sperm zona binding assay
These were carried out by ®rst incubating various dilution's of the non-immune and monoclonal antisera matured denuded goat oocytes for 1 h at 378C in a CO2
incubator. Homologous capacitated spermatozoa (2107cells/ml) were transferred to the fertilization media having 10 matured oocytes in a ®nal volume of 40ml. The gametes were sealed with paraf®n oil and
incubated at 378C for 2 h in CO2 incubator. The
oocyte±sperm complexes were thereafter rinsed four times with PBS using a narrow bore pasteur pipette to completely remove loosely attached spermatozoa and ®xed and cleared with 25% acetic alcohol (w/v). These complexes were then washed with PBS and placed on slide, slightly compressed with a cover slip and sealed. The number of spermatozoa bound to zona pellucida was counted under an Olympus inverted microscope.
2.6. Size fractionation of solubilized ZP by HPLC
The method followed was essentially that of Nakano et al. (1987), with slight modi®cation. Brie¯y, 50ml DTT (1 mM) and non-DTT treated solubilized zonae (8±10mg protein) and mol. wt. standards were run isocratically through an analytical size-exclusion column, UltroPac TSK-G3000 SW (7.5 mm
300 mm, 16 500 Theoret plates/meter, fractionation range 1000 Dal±3105Dal) on an LKB-Bromma Pharmacia, Sweden, HPLC system operated under 2140±202 WAVESCAN SPECTRAL SEARCH. The column was run at room temperature at a ¯ow rate of 250ml/min with an average pressure of 27 bar. The running buffer was 0.1 M Na2HPO4/NaH2PO4, 0.1 M
NaCl, 0.2 mM PMSF, pH 7.2 and the eluant fractions detected at 206 nm were collected with the help of HeliFrac fraction collector. Each family of proteins was dialyzed extensively against water and lyophi-lized. All the solvents and samples were ®ltered though 0.45mm membrane ®lters (Millipore, USA) and degassed before use.
2.7. Dot-blot immunoassay
wash-ings of 10 min each in TBS, the washed blot was immersed in substrate solution (7±8 mg DAB in 10 ml TBS10ml H2O2). After colour development the
blot was washed with H2O2and then with distilled
H2O (Hasegawa et al., 1995).
2.8. Immunofluorescent staining
A suspension of 30±40 denuded goat oocytes in 50ml PBS were added to an Eppendorf tube contain-ing 0.5 ml monoclonal antisera (MAb-5H4) at 1 : 10, 1 : 100 and 1 : 1000 dilution or control serum and incubated at 378C for 30 min. With the help of a ®ne pasteur pipette oocytes were picked up and washed 3 times with PBS and allowed to settle for 1 h in PBS so as to diffuse away any unbound sera into the medium. Washed oocytes were then transferred to a droplet of ¯uorescein±isothiocyarate (FITC)-conjugated F(ab0
)2
fragment of IgG prepared from rabbit anti-mouse g-globulin serum (1 : 200) and incubated for 1 h at 378C. The oocytes were thereafter washed 3 times with PBS and transferred to microscope slides. Coverslips were supported by Vaseline to prevent breakage of oocytes and were examined under a ¯uorescence microscope equipped with an FITC ®lter unit (Reichert Jung, Austria) (Isojima et al., 1984).
3. Results
3.1. Isolation and solubilization of zona pellucida
In a typical ZP preparation, 100 ovaries yielded 1000 denuded oocytes, which on heat solubilization yielded 30±32mg zona protein (35 ng protein per
zona pellucida) as determined using the ¯uorochrome dye, ¯uorescamine according to Udenfriend et al. (1972) using lysozyme as a standard protein. The zonae isolated were sticky and tended to adhere to surface especially to glass and thus it was necessary to work with plasticware as much as possible. Moreover, the cellular and subcellular purity of the oocytes and zonae, their disaggregation and solubilization was routinely established by light and phase contrast microscopic methods. In order to study the zona composition in terms of its macromolecular constitu-ents, it was essential to solubilize it under conditions where covalent modi®cation was strictly controlled
e.g. reduction of disulphide bond. Disaggregated goat zonae were found to be solubilized at 728C in 30 min and remained so after returning the solution to room temperature.
3.2. Fractionation of zona pellucida glycoproteins
The solubilized zona pellucida (sZP) was ef®ciently fractionated and showed 3 proteins peaks of 180,95 and 75 kDa successively eluted in a size exclusion HPLC column (TSKÿ43 000 SW), which was mon-itored by pre-calibrating it with a mixture of standard proteins having mol. weights ranging from 12 000 to 150 000 Dalton. All the protein peaks emerged as a quite broad single peak, sloping gently on both sides (Fig. 1). On DTT treatment (1 mM) of the sZP four peptide peaks of 135, 95, 75 and 42 kDa were observed. The retention times of the protein families increased in order of 180, 135, 95, 75, and 42 kDa. In rechromatography, each fraction emerged as a single peak at the same retention time as in the ®rst chro-matography. Moreover the 75 kDa peptide fraction represented as much as 50% of the total zona glyco-protein fractions. The individual zona fraction were collected, lyophilized and stored atÿ208C. It is quite
possible that the 180 kDa macromolecule component was split into two smaller peptides of 75 and 42 kDa under the reducing conditions as is the case in porcine.
3.3. Dot-blot immunoassay
glyco-protein and were obtained as consequence of reduc-tion of intermolecular disulphides.
3.4. Sperm-zona binding inhibition by MAb-5H4
It was clearly shown by the sperm-zona binding assay that pretreatment of the oocytes with the mono-clonal antisera 5H4 inhibited the binding of sperms to the eggs (Table 1). The antisera was found to form a well de®ned precipitin ring around the zona pellucida. However when the goat eggs were incubated with the NIS, no visible precipitin ring or inhibition of sperm binding was observed at antisera dilution's as low as 1 : 10. With MAb-5H4 maximum inhibition of bind-ing was observed at 1 : 10 antisera dilution, however
even at 1 : 1000 sera dilution there was a very sig-ni®cant inhibition of binding. Very few of the eggs were found to be penetrated using the Mab-5H4 sera even after 32 h as examined after staining with aceto-orcein.
3.5. Indirect immune localization
Immuno¯uorescent staining was carried out with intact denuded oocytes using Mab-5H4 and NIS. A typical immuno¯uorescence pattern is shown in Fig. 3. The staining was throughout the surface of the oocyte being most intense at 1 : 10 antisera dilution. More-over, comparison of the staining patterns at three antisera dilution's (1 : 10, 1 : 100 and 1 : 1000) Fig. 1. Chromatographic separation of goat zona proteins by HPLC. About 8±10mg of heat-solubilized zona protein was applied to a column of TSK-G3000SW (7.5 mm300 mm) and elution performed with 0.1 M Na2HPO4/Na2PO4, 0.1 M NaCl, 0.2 mM PMSF, pH 7.2 at a flow
showed that at lower antisera dilution the immuno-¯uoresent staining tended to be more uniform, while at higher dilution it was both more feeble and patchy. The NIS did not show any ¯uorescent staining at all, indicating no binding of the antisera to the oocyte. In addition no staining of ZP was detected with oocytes used in experiments without a ®rst antibody.
4. Discussion
In the present study it has been shown that it is quite reasonable to assume that the gZP consists primarily of three glycoprotein families. Moreover, observing their molecular identities there is every possibility that the 135 and 42 kDa peptides are the dissociation products of the 180 kDa protein observed only in unreduced conditions (Fig. 1). It has been reported that the mouse ZP2 (Mr. 120 000) was converted to ZP2f (Mr. 90 000) and a smaller glyco-protein (Mr. 23 000) by the enzymes released fol-lowing activation of eggs (Bleil et al., 1981). In fact the primary sequence of the ZP proteins from a Fig. 2. Representative dot-blot immunoassay of five zona proteins
(ZP) and sZP using MAb-5H4 and sZP with NIS also. For analysis 10ml of the sample was dotted on a nitrocellulose membrane at concentrations of 0.01mg to 1 pg in serial dilution's. The membrane was blocked with 3% BSA containing TBS and was followed by immunological detection with MAb-5H4.
Table 1
Effect of Mab-5H4 on goat sperm-zona binding assay.
Antisera dilution No. of sperm (MeansS.E.M.)a,b
Bound/oocyte Penetrated/oocyte
1. NIS 1 : 10 75.43.98 (21) 1.020.21 (21)
1 : 100 72.34.21 (16) 1.100.23 (16)
1 : 1000 71.22.98 (18) 0.980.19 (18)
2. Mab-5H4 sera 1 : 10 7.22.63 (19) 0.000.00 (19)
1 : 100 14.01.31 (16) 0.000.00 (16)
1 : 1000 19.33.92 (18) 0.210.10 (18)
aFigures in parenthesis represents the numbers of oocytes tested.
bExperimental value was significantly different from the control value (P< 0.01; Student's test).
number of species has shown that ZP are composed of three families of glycoproteins which are related at the genetic level with a 50±97% amino acid homol-ogy (Dunbar et al., 1994; Epifano and Dean, 1995; Harris et al., 1994). However, due to the post transla-tional modi®cations which the zona proteins undergo, the ZP from different species are heterogenous with respect to their physical, chemical, structural and immunological properties (Timmons et al., 1990). Moreover the overall structure of ZP3 mRNA is conserved amongst mammals (Ringnette et al., 1988). Hydropathicity comparison of human and mouse zona protein indicate a high degree of con-servation between them as also the concon-servation of alpha helical secondary structure (Chamberlin and Dean, 1990). The Mab 5H4 sera recognized both 180 and 42 kDa fractions in levels as low as 0.01 ng and 1 pg respectively (Fig. 2), suggesting the shared antigenicity between the two glycoproteins. The dominant component of the solubilized gZP, the 75 kDa fraction also cross reacted but at higher con-centration (5 ng) with the Mab 5H4 antisera. Immu-no¯uorescent staining of denuded intact goat oocytes (Fig. 3) further con®rms the presence of an antigen(s) which successfully recognized the 5H4 antisera. Sev-eral reports have shown such cross reactivity among the different glycoprotein families of mouse and porcine (East and Dean, 1984; Timmons et al., 1987). The 5H4 antisera is against the peptide portion of the ZP4 (Hasegawa et al., 1994) and the inhibition of goat sperm-egg binding by the antisera observed could not be due to any direct inhibition of sperm binding to the receptors by the 5H4 antibodies but due to the steric hindrance of the sperm receptors by the immune complexes. The inhibition could also be due to interference because of a possible peptide function involved in the physical interaction of gametes. More-over, as none of the eggs were penetrated the inter-ference with the process of sperm penetration could also be plausible.
Comparison of the amino acid sequence of pZP4 with available a.a. sequences has shown 39.1% homol-ogy to the amino terminal portion of mouse ZP2 (Liang et al., 1990). Also the 55 kDa rabbit zona protein corresponds to the carboxy terminal region of mouse ZP2 (Schwoebel et al., 1991) but no sig-ni®cant homology between pZP4 and 55 kDa rabbit protein was observed. It has been suggested that pZP4
contains an antigen epitope useful for developing contraceptive vaccine and further its active immuni-zation has shown to induce temporary infertility in hamsters (Hasegawa et al., 1992).
A need has been felt to identify antigenic epitopes that can induce fertilization blocking antibodies but which do not induce harmful cellular or humoral immunities to normal ovarian function. Moreover, the problem of low antisera titers from homologous zona pellucida immunization which resulted from immunological tolerance to self antigens (Wood et al., 1981) could be circumvented by utilizing inter-species cross reactive zona antigens or their epitopes as vaccines. The Mab-5H4 has been shown to react with zona pellucida of humans, dogs, cats, cows and rabbits as well as pigs and now collectively the ®nd-ings of this study clearly indicate the 5H4 antisera recognizes a protein(s) on the goat zona which could be involved in sperm-zona interaction.
Acknowledgements
The author thanks Prof. Koji Koyama of Hyogo Medical College, Japan for so kindly providing the MAb-5H4 antisera and the Indian Council of Agri-cultural Research (ICAR), New Delhi for ®nancial assistance.
References
Araki, Y., Orgebin-Crist, M-C., Tulsiani, D.R.P., 1992. Quantitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates. Biol. Reprod. 46, 912±917. Bleil, J.D., Beall, C.F., Wassarman, P.M., 1981. Mammalian
sperm-egg interaction: fertilization of mouse sperm-eggs triggers modifica-tion of the major zona pellucida glycoprotein ZP2. Dev. Biol. 86, 189±197.
Bleil, J.D., Wasserman, P.M., 1980. Structure and function of the zona pellucida. identification and characterization of the proteins of the mouse oocytes' zona pellucida. Dev. Biol. 76, 185±203.
Chamberlin, E.M., Dean, J., 1990. Human homology of the mouse sperm receptor. Proc. Natl. Acad. Sci. USA 87, 6014±6018. Chauhan, M.S., Anand, S.R., 1991. In vitro maturation and
Dunbar, B.S., Raynor, D., 1980. Characterization of porcine zona pellucida antigens. Biol. Reprod. 22, 941±954.
East, I.J., Dean, J., 1984. Monoclonal antibodies as probes of distribution of ZP2: the major sulfated glycoprotein of the murine ZP. J. Cell Biol. 98, 795±800.
Epifano, O., Dean, J., 1995. Coordinate expression of the three ZP genes during mouse oogenesis. Development 121, 1947±1956. Harris, J.D., Hilbler, D.W., Fontenot, G.K., Hsu, K.T., Yurewicz, E.C., Sacco, A.G., 1994. Cloning and characterization of ZP genes and cDNA's from a variety of mammalian species: The ZPA, ZPB and ZPC gene families. DNA sequence 4, 361±393. Hasegawa, A., Koyama, K., Inoue, M., Takemura, T., Isojima, S., 1992. Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters. J. Reprod. Immunol. 22, 197±210.
Hasegawa, A., Koyama, K., Okazaki, Y., Sugimato, M., Isojima, S., 1994. Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determination by peptide mapping and cDNA cloning. J. Reprod. Fertil. 100, 245±255.
Hasegawa, A., Yamasaki, N., Jone, M., Koyama, K., Isojima, S., 1995. Analysis of an epitope sequence recognized by a monoclonal antibody Mab-5H4 against a porcine zona pellucida glycoprotein (pZp4) that blocks fertilization. J. Reprod. Fertil. 105, 295±302.
Hedrick, J.J., The pig zona pellucida: sperm binding ligands, antigens and sequence homologies. In: Dondero, F., Johnson, P.M. (Eds.), Reproductive Immunology. Raven Press, New York, 1993, pp. 59±65.
Hedrick, J.L., Wardship, N.J., 1986. Isolation of the ZP and purification of its glycoprotein families from pig oocytes. Anal Biochem. 157, 63±70.
Hedrick, J.I., Wardship, N.J., 1987. On the macromolecular composition of the ZP from porcine oocytes. Dev. Biol. 121, 478±488.
Isojima, S., Koyama, K., Hasegawa, A., Tsunoda, Y., Hanada, A., 1984. Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effects on fertilization. J. Reprod. Immunol. 6, 77±87.
Kaul, G., Singh, S., Gandhi, K.K., Anand, S.R., 1997. Calcium requirement and time course of capacitation of goat sperma-tozoa assessed by chlortetracycline assay. Andrologia 296(5), 243±251.
Koyama, K., Hasegawa, A., Inoue, M., Isojima, S., 1991. Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida. Biol. Reprod. 45, 727±735.
Koyama, K., Hasegawa, A., Tsuii, Y., Isojima, S., 1985. Production and characterization of monoclonal antibodies to cross reactive
antigens of human and porcine zona pellucida. J. Reprod. Immunol. 7, 187±198.
Liang, L., Chamow, S.M., Dean, J., 1990. Oocyte specific expression of mouse ZP2: development regulation of the ZP genes. Mol. Cell Biol. 10, 1507±1515.
Maresh, G.A., Dunar, B.S., 1987. Antigenic comparison of five species of mammalian zonae pellucida. J. Exp. Zool. 244, 299± 307.
Miller, C.I., Fayrer-Hosken, R.A., Timmons, T.M., Lee, V.H., Candle, A.B., Dunbar, B.S., 1992. Characterization of equine ZP glycoproteins by polyacrylamide gel electrophoresis and immunological techniques. J. Reprod. Fertil. 96, 815±825. Nakano, M., Hatanaka, Y., Sawai, T., Kobayashi, N., Tobita, T.,
1987. Fractionation of glycoproteins from porcine zonae pellucidae into three families by high-performance liquid chromatography. Biochem. Int. 14, 417±423.
Ringnette, M.S., Chamberlin, M.E., Baur, A.W., Sobieski, D.A., Deab, H., 1988. Molecular analysis of cDNA coding for AP3 a sperm binding protein of the mouse ZP. Dev. Biol. 127, 287± 295.
Schwoebel, E., Prasad, S., Timmons, T.M., Cook, K., Kimura, H., Nio, E., Cheung, P., Skinner, S., Avery, S.E., Wilkins, B., Dunbar, B.S., 1991. Isolation and characterization of a full length cDNA encoding the 55 kDA rabbit ZP protein. J. Biol. Chem. 266, 7214±7219.
Shabanowitz, R.B., O'Rand, M.G., 1988. Characterization of the human ZP from fertilized and unfertilized eggs. J. Reprod. Fertil. 82, 151±161.
Timmons, T.M., Maresh, G.A., Bundman, D.S., Dunbar, B.S., 1987. Use of specific monoclonal and polyclonal antibodies to define distinct antigens of the porcine zonae pellucida. Biol. Reprod. 36, 1275±1287.
Timmons, T.M., Skinner, S.M., Dumbar, B.S., Glycosylation and maturation of the mammalian ZP. In: Alexanda, N., Griffin, D., Waites, G., Spieler, J.(Eds.), Gamete Interaction, Prospects for Immunocontraception. Wiley, New York, 1990, pp. 277±292. Udenfriend, S., Stain, S., Bohlen, P., Dairman, W., Leimgruber, W.,
Weigele, M., 1972. Fluorescamine: A reagent for assay of Amino acids, peptides, protein and primary amines in the picomole range. Science 178, 871±880.
Wassarman, P.M., 1990. Profile of a mammalian sperm receptor. Development 108, 1±17.
Wood, D.M., Liu, C.L., Dunbar, B.S., 1981. Effect of alloimuniza-tion and hetero-immunizaalloimuniza-tion with pellucida on fertility in rabbits. Biol. Reprod. 23, 439±450.
