Journal of Reproductive Immunology, 22 (1992) 197-210
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Elsevier Scientific Publishers Ireland Ltd.
JRI 00783
Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters Akiko Hasegawa, Koji Koyama, Miyuki Inoue, Tadashi Takemura and Shinzo Isojima Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya-shi, Hyogo, 663 (Japan) (Accepted for publication 6 April 1992)
Summary Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine. Key words: porcine zona pellucida antigen," ZP4 glycoprotein family; active immunization," contraception
Correspondence to." Professor Shinzo Isojima, Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya-shi, Hyogo, 663, Japan. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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Introduction
Sacco (1977) found that porcine zona pellucida (ZP) had strong crossreactive antigens to human ZP and inhibition of fertilization by active immunization with porcine ZP has been reported in many different mammalian species, including rabbits (Wood et al., 1983), bitches (Mahi-Brown et al., 1982), mares (Liu et al., 1989) and monkeys (Gulyas et al., 1983; Sacco et al., 1983). Since then, the antigens associated with porcine ZP have attracted considerable interest in relation to the development of a contraceptive vaccine (Isojima et al., 1986; Sacco, 1987; Henderson et al., 1988). The molecular composition of ZP has been well characterized in two-dimensional gel electrophoresis (Dunbar et al., 1981; Subramanian et al., 1981; Hedrick et al., 1986). We have previously shown that heat solubilized porcine ZP (s-PZP) is composed of four glycoprotein families (ZP1, ZP2, ZP3, ZP4) consisting of many isomers with heterogeneous charges (Koyama et al., 1991a). Some mouse antisera raised to each isolated glycoprotein family of s-PZP showed a strong blocking effect on human sperm-ZP interaction in vitro (Hasegawa et al., 1991). In Western blot analysis, anti-ZP1, anti-ZP2 and anti-ZP3 antisera showed a similar staining pattern in which they reacted mainly with the ZP3 glycoprotein family, while anti-ZP4 antiserum reacted only with ZP4 and ZP1. By active heteroimmunization of animals with s-PZP or purified ZP3 from s-PZP, temporary infertility was induced in parallel with the rise in serum antibody titers, but ovarian dysfunction also occurred (Sacco et al., 1983; Skinner et al., 1984; Mahi-Brown et al., 1985; Upadhyay et al., 1989). There are no studies on the immunogenicity and contraceptive effects of ZP4. Although ZP4 is a minor component in s-PZP, we found that some monoclonal antibodies raised to ZP4 blocked human sperm binding to ZP in vitro (Koyama et al., 1991b). ZP4 was also found to possess cross-reactive antigens not only with human ZP but also with other species (rabbit, dog, cat and hamster). The present study was designed to investigate the antifertility effect and any influence on ovarian function by active immunization of hamsters with purified ZP4. Materials and Methods
Preparation of s-PZP and ZP4 Porcine ZP was isolated from minced ovaries by passing through nylon meshes of different sizes and solubilized in 50 mM sodium carbonate-bicarbonate buffer (pH 9.6) by heating at 70°C for 30 rain as described previously (Isojima et al., 1984). Solubilized porcine ZP (s-PZP) was subjected to preparative sodium dodecylsulfate polyacrylamide gel
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electrophoresis (SDS-PAGE) following the method of Laemmli (1970); proteins which migrated to the region corresponding to molecular weight (MW) of 20 000-30 000 were electroeluted from the gel in 2.5 m M Tris-19.2 mM glycine buffer containing 0.01% SDS at 4°C for 2 h. The isolated proteins were confirmed to correspond to the ZP4 glycoprotein family by two dimensional electrophoresis (O'Farrell, 1974). Protein quantities were determined by BCA protein assay kits (Pierse).
Immunization schedule A sample of 20/xg of s-PZP or isolated ZP4 glycoproteins was injected into a golden hamster with complete Freund's adjuvant. Three weeks later a second injection was given with incomplete Freund's adjuvant and followed by 3 booster injections of the same material in saline every other day after a further 2 weeks. Eight hamsters in each group were immunized with s-PZP or purified ZP4. Four from each group of immunized hamsters were examined for ovarian histopathology at different times after immunization. The other four in each group were mated to proven fertile males from 6 weeks after the first immunization to check their fertility. Two-dimensional electrophoresis and Western blot analysis Two-dimensional electrophoresis was performed by a slight modification of the method of O'Farrell (1974) as described previously (Koyama et al., 1991a). A sample of 100 #g of s-PZP was applied to a first dimensional rod gel for isoelectric focusing (IEF) and electrophoresis was carried out in electrode solutions of 0.01 N NaOH for the upper cathode and 0.01 M H3PO 4 for the bottom anode. The pH gradient was adjusted to 3-7. The proteins which migrated into the gel were transblotted to a polyvinylidene difluoride (PVDF) membrane (Millipore) in 3-cyclohexylaminopropane-sulfonic acid (CAPS) blotting buffer (pH 11.0) by electrophoresis at 7 V for 15 h. For immunostaining, the membrane was incubated in a blocking solution of 20 mM Tris-HC1 buffered saline pH 7.2 (TBS) containing 3% bovine serum albumin (BSA) and 10% horse serum at room temperature for 30 min and followed by incubation with the first antibody of a hamster antiserum diluted 1:500 at room temperature for 2 h. After washing with TBS, the membrane was made to react further with the second antibody of peroxidase conjugated anti-hamster IgG (H&L) (Jackson) diluted 1:1000 with the blocking buffer for 1 h. The product of peroxidase reaction was developed with 4-chloro1-naphthol (0.5 mg/1) and 0.01% H202 in TBS. Enzyme-linked immunosorbent assay ( ELISA ) Antibodies produced to s-PZP or ZP4 were determined by ELISA at 2week intervals from 3 weeks after the first injection. For ELISA, s-PZP or
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ZP4 (10/~g/100 #1) in 50 m M carbonate-bicarbonate buffer (pH 9.6) was coated on plates (Falcon No. 3912) by incubating at 4°C overnight. After blocking the protein binding activity of the plates with 0.1%0 BSA solution containing 0.02% NAN3, test samples of 100/~1 of serially diluted antisera from immunized hamsters were put in the wells and incubated at room temperature for 2 h. After extensive washings with PBS, horseradish peroxidase-conjugated rabbit anti-hamster IgG (1:I000 dilution) (Jackson) was added and incubated for 1 h at room temperature. The peroxidase activity bound to the wells was measured by adding 100 ~1 of o-phenylenediamine solution (0.2 mg/ml 0.15 M citrate-phosphate buffer, pH 5.0) with 0.02% HzOz. The plates were kept for 10 min at room temperature. The reaction was stopped by adding 50 tA 10% H2SO 4 to the wells and optical density was measured at 492 nm by a microplate photometer (Bio-Rad, Model 450). Antibody titers were shown as the maximum dilutions of antisera in which the maximum absorbance at 492 nm was given.
Immunofluorescence staining The procedures were carried out following a previous method (Koyama et al., 1985). Briefly, cumulus-free oocytes were incubated in 10 ~1 of antisera diluted 1:100 with PBS for 1 h at room temperature and then washed with PBS. The washed oocytes were transferred to a droplet of fluoresceinisothiocyanate (FITC) conjugated F(ab')2 fragment of IgG-prepared from goat anti-hamster -r-globulin (Cappel No. 1208-0081) diluted 1:1000 with PBS and incubated for 1 h at room temperature. After washing with PBS, oocytes were examined under a UV microscope.
Immunohistochemical staining Specimens sliced from porcine ovary to a thickness of 5 mm were fixed in 2% paraformaldehyde containing 0.01 M NalO4 and 0.075 M lysine by incubating at 4°C overnight. The fixed materials were washed with PBS containing 10% sucrose, followed by successive washings with PBS containing 15% and 20% sucrose. Each step was carried out at 4°C overnight. They were further incubated in PBS containing 20% sucrose and 5% glycerin at 4°C overnight and frozen immediately using dry-ice and ethanol in OCT compound (Miles). Serial cryostat sections of 6/zm thickness were incubated in PBS containing 10%0 normal hamster serum for 15 min at room temperature to block non-specific bindings of hamster globulin. They were first made to react with hamster anti-ZP4 (1:20 dilution) or anti-s-PZP (1:100 dilution) sera for 1 h at room temperature. After washing, peroxidase conjugated anti-hamster IgG (Jackson) diluted at 1:200 was made to react as a second antibody. Peroxidase reaction was developed in 0.05% 3,3'diaminobenzidine and 0.01%0 H202 solution for 5 min.
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Fig. 1. Silver staining of s-PZP and isolated ZP4 in two dimensional electrophoresis, s-PZP was separated into four glycoprotein families (A). ZP4 isolated from s-PZP by preparative SDS-PAGE was confirmed to have a similar migration pattern to ZP4 in s-PZP (B).
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Fig. 2. Changes in antibody titers and estradiol levels in hamsters following primary immunization with s-PZP and ZP4. Antibody titers were determined by ELISA against s-PZP (0) or isolated ZP4 (&) used for immunization. Estradiol (E2) levels were estimated by radioimmunoassay at estrus or estrusequivalent day of the cycle. Note that the increase in antibody titers was accompanied by a decrease in E 2 levels in hamsters immunized both with s-PZP (11) and ZP4 (1~), but the E 2 level of ZP4-immunized hamsters returned to an almost normal level at 12 weeks after immunization. ( [] ) shows E z level of nonimmunized hamster at estrus day. Each value is mean of three animals.
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Histological examination Oophorectomy was performed at 3, 7 and 12 weeks after the first injection. The excised ovaries were fixed in 10% formalin for several days and were processed for paraffin embedding. Tissue sections (4/xm) were prepared and stained with hematoxylin and eosin. Hormone assay Serum from each immunized hamster was taken for estradiol (E2) assay periodically on the day corresponding to the day of estrus following the first immunization. Estrus cycles were assessed by vaginal secretions and serum E2 levels were measured by radioimmunoassay kits (CIS Diagnostic). Results
Solubilized porcine ZP (s-PZP) was separated into four glycoprotein families (ZP1, ZP2, ZP3, ZP4) under reducing conditions as shown in Fig. 1A. Each family consisted of many isomers with similar molecular sizes but different isoelectric points. The average molecular weights of ZP1, ZP2, ZP3 and ZP4 were estimated as 92 000, 69 000, 55 000 and 23 000, respectively. The glycoprotein which migrated to the region corresponding to 20 000-30 000 in one dimensional preparative SDS-PAGE of s-PZP was isolated. A two dimensional SDS-PAGE of the isolated glycoprotein was
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Fig. 3. Western blot analysis of s-PZP in two dimensional SDS-PAGE. Antiserum from a hamster immunized with s-PZP reacted mainly with ZP3 glycoprotein family (A). Antiserum from a hamster immunized with ZP4 reacted mainly with ZP4 glycoprotein family (B).
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performed and a similar migration pattern to ZP4 glycoprotein family (Fig. 1B) was confirmed. This preparation was used as ZP4 for active immunization of hamsters. Figure 2 illustrates the antibody titers determined by ELISA and the estradiol (E2) levels in sera of the hamsters immunized with s-PZP and purified ZP4 during the period of 12 weeks after the initial immunization. Antibody titers began to increase from 3 weeks after immunization and reached maximal level at 5 weeks in both immunization groups. The antibody titer of s-PZP immunized hamsters was much higher than that of ZP4 immunized hamsters throughout the whole observation period. In hamsters immunized with s-PZP, the serum E2 levels gradually decreased from 3 to 7 weeks after immunization and the decreased levels persisted to the 12th week. In hamsters immunized with ZP4, the E2 levels began to drop 5-7 weeks after immunization and then increased to about 60% of normal levels after 12 weeks. Antibody titers correlated well with declining E2 levels in both groups. Four hamsters immunized with ZP4 were mated with fertile males from 6 weeks after the first immunization.
A
C
B
D
Fig. 4. Indirect immunofluorescence staining of normal porcine and hamster oocytes with antisera from hamsters immunized with s-PZP and ZP4. Porcine oocytes reacted with hamster anti-s-PZP (A) and antiZP4 (B) antiscra. Hamster oocytes reacted with hamster anti-s-PZP (C) and anti-ZP4 (D) sera.
Fig. 5. Photomicrographs of ovarian sections from hamsters immunized with s-PZP and ZP4. (A) A control section from a Freund's adjuvant-injected hamster. IB-E) Sections o f s-PZP-immunized hamster ovaries which were taken at 5 (B), 7 (C) and 12 (D,E) weeks after the first injection. Note that many cell clusters ~re present in section E. ( F - H ) Sections o f ZP4-immunized hamster ovaries which were taken at 5 (F), 7 (G) and 12 (H) weeks after the first injection. Magnification x 40, except for E ( x 100).
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Spermatozoa were first recognized in vaginal smears of three hamsters at 13 weeks and they were delivered of 3, 3 and 5 babies, respectively (3.67 ± 0.94, n = 3). Although the litter size was much smaller than that of normal hamsters (9.0 + 0.82, n = 3), these three females partially recovered their fertility. Characteristic properties of antisera were studied by Western blot analysis and immunofluorescent staining of pig and hamster oocytes. Figure 3 shows Western blotting of s-PZP using sera from hamsters immunized with s-PZP and ZP4. Anti-s-PZP serum reacted mainly with ZP3 family, which is the most dominant component in s-PZP. Anti-ZP4 serum reacted strongly with ZP4 but also showed weak staining with ZPI. Thus the staining patterns in Western blotting with these two antisera were quite different. Both antisera produced to s-PZP and ZP4 reacted not only with porcine oocytes but also with hamster oocytes in immunofluorescent staining (Fig. 4). The highest antibody titers determined by the immunofluorescence test are shown in Table 1. Antibody titers of anti-s-PZP and anti ZP4 antisera against porcine ZP were strikingly different, i.e. 1:10000 vs. 1:2000, while those against hamster ZP were the same, 1:200 dilutions in both antisera. Ovarian sections from immunized hamsters taken at 3, 7 and 12 weeks after the initial immunization were stained with hematoxylin and eosin (Fig. 5). There were no remarkable differences in morphology between the control hamster (Fig. 5A) injected only with Freund's adjuvant and the hamsters immunized with s-PZP or ZP4 (Fig. 5B, F) until 3 weeks after immunization, except for a mild desquamation of granulosa cell layers of antral follicles in the hamster immunized with s-PZP. However, considerable morphological changes were observed in the ovaries of s-PZP immunized hamsters at 7 weeks after immunization as compared to the control (Fig. 5C). Ovarian sections showed atretic and degenerative changes of growing follicles with marked reduction of normal antral follicles, though the changes
TABLE 1 Antibody titers of anti-s-PZP and anti-ZP4 antisera against porcine and hamster oocytes. Antibody titers were expressed as m a x i m u m dilution of antisera in which a significant immunofluorescent staining was recognized on ZP. Antisera
Anti-s-PZP Anti-ZP4
Antibody titers against Porcine ZP
Hamster ZP
1: 10 000 1:2000
1:200 1:200
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Fig. 6. lmmunohistochemical staining of porcine ovarian sections with hamster anti-s-PZP and anti-ZP4 antisera. Paraformaldehyde-fixed frozen sections were treated with anti-s-PZP (1:100) or anti-ZP4 (1:20) hamster sera followed by peroxidase-conjugated anti-hamster IgG. Note that ZP in antral follicles are intensely stained with antisera both of anti-s-PZP (A) and anti-ZP4 (B), while ZP in growing and pre-antral follicles are stained only with anti-s-PZP antiserum (C) (arrows) but not stained with anti-ZP4 antiserum (D), Original magnification × 100
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appeared to be milder in a hamster immunized with ZP4 (Fig. 5G) as compared to that immunized with s-PZP. No significant inflammatory reaction was observed. At 12 weeks after immunization, the ovaries of a hamster immunized with s-PZP became markedly atrophic and sclerotic and the degenerative changes of the follicles were further advanced. Follicular structures were completely destroyed and no follicles remained in the ovaries. Only numerous follicle-like cell clusters were observed (Fig. 5D,E). In contrast, the ovaries of hamster immunized with ZP4 were of normal size and several growing follicles were observed in the tissue sections (Fig. 5H). Immunohistochemical staining of porcine ovarian sections was carried out to examine the presence of antigens corresponding to anti-s-PZP and antiZP4 antisera at different stages of follicular development. Both antisera reacted strongly to ZP in antral follicles (Fig. 6A,B), but only anti-s-PZP antiserum reacted to ZP in growing or pre-antral follicles (Fig. 6C). AntiZP4 antiserum did not showed any staining to ZP in a serial section of the same pre-antral follicles (Fig. 6D). Discussion The present study demonstrated that female hamsters could respond well to porcine ZP antigens and produce antibodies autoreactive to their own ZP. All immunized animals developed temporary or permanent infertility depending on the antigen preparations used for the immunization. ZP4 immunization induced temporary infertility, while s-PZP immunization induced permanent infertility. Anti-s-PZP hamster serum reacted most strongly with ZP3 glycoprotein family, while anti-ZP4 hamster serum reacted strongly with ZP4 glycoprotein family. Although the antibody titers determined by ELISA or immunofluorescence staining against porcine ZP were higher in anti-s-PZP antiserum than in anti-ZP4 antiserum, those determined by immunofluorescent staining against hamster ZP were the same levels in both antisera. These results suggest that the difference in the antifertility effect in hamsters immunized with the two antigen preparations might be due to qualitative rather than quantitative differences in the immune response to each antigen. In both hamsters immunized with two different antigen preparations of porcine ZP, the histological changes in the ovaries were associated with a marked reduction of serum E2 levels. Most follicles except very small ones were degenerate and showed oocyte loss. In the hamsters immunized with s-PZP, the degenerative changes were progressive even after stopping further immunization and no follicles remained in the ovaries. Only follicular cell-like remnants were observed. In contrast, in the hamsters immunized with ZP4, the degenerative changes were reversible and small to
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medium size growing follicles appeared in the ovaries after the antibody titers dropped. Three animals in the latter group became pregnant when they were mated with a fertile male. The main cause of infertility in hamsters immunized with porcine ZP antigens appeared to be failure to ovulate and follicular destruction by the antibodies induced to auto-antigens of ZP and/or oocyte components, though an inhibitory effect at the level of fertilization cannot be ruled out. The mechanism for the destruction of follicles by the antibodies was not clear but the same phenomenon has been observed in many species of experimental animals (Skinner et al., 1984; Mahi-Brown et al., 1988; Dunbar et al., 1989). Although a cellular immune response to the follicular components was not completely ruled out, for the reason that the histological studies did not show any inflammatory cell infiltration of degenerative follicles, complementdependent cytotoxic antibodies bound to ZP or oocytes seem to be more causative factors for the destruction of follicles in the ovaries immunized with porcine ZP antigens. That immunization with s-PZP induced irreversible follicular destruction suggests that the antigens corresponding to the antibodies induced by immunization with s-PZP might be expressed at an earlier stage of folliculogenesis, so that even small follicles were affected with cytotoxic antibodies, resulting in the extinction of follicles in the ovaries. On the other hand, the reversibility of folliculogenesis in hamsters immunized with ZP4 indicates that the antigens corresponding to the antibodies induced by ZP4 immunization are expressed at a later stage of folliculogenesis, so that early stage follicles might be left intact and resume development after the antibody titers dropped. If this is true, ZP4 glycoproteins would be ideal candidates for developing a contraceptive vaccine using ZP antigens. We are now analyzing the antigenic structures of ZP4 glycoprotein molecules. References Dunbar, B.S., Liu, C. and Sammons, D.W. (1981) Identification of the three major proteins of porcine and rabbit zonae pellucidae by high resolution two-dimensional gel electrophoresis: Comparison with serum, follicle fluid and ovarian cell proteins. Biol. Reprod. 24, 1111-1124. Dunbar, B.D., Lo, C., Powell, J. and Stevens, V.C. (1989) Use of a synthetic peptide adjuvant for the immunization of glycoproteins. Fertil. Steril. 52, 311-318. Gulyas B.J., Gwatkin, R.B.L. and Yuan, L.C. (1983) Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zona pellucida. Gamete Res. 4, 299-307. Hasegawa, A., Koyama, K. and Isojima, S. (1991) Isolation of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family. Acta Obst. Gynaecol. Jpn. 43, 221-226. Hedrick, J.L. and Wardrip, N.J. (1986) Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Anal. Biochem. 157, 63-70. Henderson, C.J., Hulme, M.J. and Aitken, R.J. (1988) Contraceptive potential of antibodies to the zona pellucida. J. Reprod. Fertil. 83, 325-343. Isojima, S., Koyama, K., Hasegawa, A., Tsunoda, Y. and Hanada, A. (1984) Monoclonal antibodies to
210 porcine zona pellucida antigens and their inhibitory effects on fertilization. J. Reprod. Immunol. 6, 77-87. Isojima, S., Koyama, K., Takeda, Y., Shigeta, M., Tsuji, Y. and Hasegawa, A. (1986) The development of a contraceptive vaccine by purification of antigens from gametes. Am. J. Reprod. lmmunol, Microbiol. 10, 90-92. Koyama, K., Hasegawa, A. and Isojima, S. (1991 a) Further characterization of the porcine zona pellucida antigen corresponding t o monoclonal antibody (3A4-2G1) exclusively cross-reactive with porcine and human zonae pellucidae. J. Reprod. Immunol. 19, 131-148. Koyama, K., Hasegawa, A., Inoue, M. and Isojima, S. (1991b) 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., Tsuji, Y. and Isojima, S. (1985) Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae. J. Reprod. Immunol. 7, 187-198. Laemmli, U.K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Liu, I.K.M., Bernoco, M. and Feldman, M. (1989) Contraception in mares heteroimmunized with pig zonae pellucidae. J. Reprod. Fertil. 85, 19-29. Mahi-Brown, C.A., Huang, T.T.F., Jr. and Yanagimachi, R. (1982) Infertility in bitches induced by active immunization with porcine zonae pellucidae. J, Exp. Zool. 222, 89-95. Mahi-Brown, C.A., Yanagimachi, R., Hoffman, J.C. and Huang, T.F.H., Jr. (1985) Fertility control in bitch by active immunization with porcine zona pellucida: Use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles. Biol. Reprod. 32, 761-772. Mahi-Brown. C.A., Yanagimachi, R., Nelson, M.L., Yanagimachi, H. and Palumbo, N. (1988) Ovarian histopathology of bitches immunized with porcine zonae pellucidae. Am. J. Reprod. Immunol. Microbiol. 18, 94-103. O'Farrell, P.H. (1974) High resolution two-dimensional electrophoresis of protein. J. Biol. Chem. 250, 4007 -4021. Sacco, A.G. (1987) Zona pellucida: Current status as a candidate to antigen for contraceptive vaccine development. Am. J. Reprod. Immunol. Microbiol. 15, 122-130. Sacco, A.G., Yurewicz, E.C., DeMato, F.J. and Dukelow, W.R. (1983) Heteroimmunization of squirrel monkey (Saimiri sciureus) with a purified porcine zona antigen (PPZA): Immune response and biologic activity of antiserum. Fertil. Steril. 39, 350-358. Sacco, A.G., Pierce, D.L., Subramanian, M.G., Yurewicz, D.C. and Dukelow, W.R. (1987) Ovaries remain functional in squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida 55,000 macromoleeule. Biol. Reprod. 36, 481-490. Skinner, S.M., Mills, T., Kirchick, N.J. and Dunbar, B.S. (1984) Immunization with zona proteins results in abnormal ovarian follicular differentiation and inhibition of gonadotropin induced steroid secretion. Endocrinology 115, 2418-3432. Subramanian, M.G., Yurewicz, E.C. and Sacco, A.G. (1981) Specific radioimmunoassay for the detection of a purified porcine zona pellucida antigen (PPZA). Biol. Reprod. 24, 933-943. Upadhyay, S.N., Thillaikoothan, P., Bamezai, A., Jayaraman, S. and Talwar, G.P. (1989) Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkey: A morphological study. Biol. Reprod. 41,665-673. Wood, D.M., Liu, C. and Dunbar, B.S. (1981) Effect of alloimmunization and heteroimmunization with zonae pellucidae on fertility in rabbits. Biol. Reprod. 25, 439-450.
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Elsevier Scientific Publishers Ireland Ltd.
JRI 00783
Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters Akiko Hasegawa, Koji Koyama, Miyuki Inoue, Tadashi Takemura and Shinzo Isojima Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya-shi, Hyogo, 663 (Japan) (Accepted for publication 6 April 1992)
Summary Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine. Key words: porcine zona pellucida antigen," ZP4 glycoprotein family; active immunization," contraception
Correspondence to." Professor Shinzo Isojima, Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya-shi, Hyogo, 663, Japan. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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Introduction
Sacco (1977) found that porcine zona pellucida (ZP) had strong crossreactive antigens to human ZP and inhibition of fertilization by active immunization with porcine ZP has been reported in many different mammalian species, including rabbits (Wood et al., 1983), bitches (Mahi-Brown et al., 1982), mares (Liu et al., 1989) and monkeys (Gulyas et al., 1983; Sacco et al., 1983). Since then, the antigens associated with porcine ZP have attracted considerable interest in relation to the development of a contraceptive vaccine (Isojima et al., 1986; Sacco, 1987; Henderson et al., 1988). The molecular composition of ZP has been well characterized in two-dimensional gel electrophoresis (Dunbar et al., 1981; Subramanian et al., 1981; Hedrick et al., 1986). We have previously shown that heat solubilized porcine ZP (s-PZP) is composed of four glycoprotein families (ZP1, ZP2, ZP3, ZP4) consisting of many isomers with heterogeneous charges (Koyama et al., 1991a). Some mouse antisera raised to each isolated glycoprotein family of s-PZP showed a strong blocking effect on human sperm-ZP interaction in vitro (Hasegawa et al., 1991). In Western blot analysis, anti-ZP1, anti-ZP2 and anti-ZP3 antisera showed a similar staining pattern in which they reacted mainly with the ZP3 glycoprotein family, while anti-ZP4 antiserum reacted only with ZP4 and ZP1. By active heteroimmunization of animals with s-PZP or purified ZP3 from s-PZP, temporary infertility was induced in parallel with the rise in serum antibody titers, but ovarian dysfunction also occurred (Sacco et al., 1983; Skinner et al., 1984; Mahi-Brown et al., 1985; Upadhyay et al., 1989). There are no studies on the immunogenicity and contraceptive effects of ZP4. Although ZP4 is a minor component in s-PZP, we found that some monoclonal antibodies raised to ZP4 blocked human sperm binding to ZP in vitro (Koyama et al., 1991b). ZP4 was also found to possess cross-reactive antigens not only with human ZP but also with other species (rabbit, dog, cat and hamster). The present study was designed to investigate the antifertility effect and any influence on ovarian function by active immunization of hamsters with purified ZP4. Materials and Methods
Preparation of s-PZP and ZP4 Porcine ZP was isolated from minced ovaries by passing through nylon meshes of different sizes and solubilized in 50 mM sodium carbonate-bicarbonate buffer (pH 9.6) by heating at 70°C for 30 rain as described previously (Isojima et al., 1984). Solubilized porcine ZP (s-PZP) was subjected to preparative sodium dodecylsulfate polyacrylamide gel
199
electrophoresis (SDS-PAGE) following the method of Laemmli (1970); proteins which migrated to the region corresponding to molecular weight (MW) of 20 000-30 000 were electroeluted from the gel in 2.5 m M Tris-19.2 mM glycine buffer containing 0.01% SDS at 4°C for 2 h. The isolated proteins were confirmed to correspond to the ZP4 glycoprotein family by two dimensional electrophoresis (O'Farrell, 1974). Protein quantities were determined by BCA protein assay kits (Pierse).
Immunization schedule A sample of 20/xg of s-PZP or isolated ZP4 glycoproteins was injected into a golden hamster with complete Freund's adjuvant. Three weeks later a second injection was given with incomplete Freund's adjuvant and followed by 3 booster injections of the same material in saline every other day after a further 2 weeks. Eight hamsters in each group were immunized with s-PZP or purified ZP4. Four from each group of immunized hamsters were examined for ovarian histopathology at different times after immunization. The other four in each group were mated to proven fertile males from 6 weeks after the first immunization to check their fertility. Two-dimensional electrophoresis and Western blot analysis Two-dimensional electrophoresis was performed by a slight modification of the method of O'Farrell (1974) as described previously (Koyama et al., 1991a). A sample of 100 #g of s-PZP was applied to a first dimensional rod gel for isoelectric focusing (IEF) and electrophoresis was carried out in electrode solutions of 0.01 N NaOH for the upper cathode and 0.01 M H3PO 4 for the bottom anode. The pH gradient was adjusted to 3-7. The proteins which migrated into the gel were transblotted to a polyvinylidene difluoride (PVDF) membrane (Millipore) in 3-cyclohexylaminopropane-sulfonic acid (CAPS) blotting buffer (pH 11.0) by electrophoresis at 7 V for 15 h. For immunostaining, the membrane was incubated in a blocking solution of 20 mM Tris-HC1 buffered saline pH 7.2 (TBS) containing 3% bovine serum albumin (BSA) and 10% horse serum at room temperature for 30 min and followed by incubation with the first antibody of a hamster antiserum diluted 1:500 at room temperature for 2 h. After washing with TBS, the membrane was made to react further with the second antibody of peroxidase conjugated anti-hamster IgG (H&L) (Jackson) diluted 1:1000 with the blocking buffer for 1 h. The product of peroxidase reaction was developed with 4-chloro1-naphthol (0.5 mg/1) and 0.01% H202 in TBS. Enzyme-linked immunosorbent assay ( ELISA ) Antibodies produced to s-PZP or ZP4 were determined by ELISA at 2week intervals from 3 weeks after the first injection. For ELISA, s-PZP or
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ZP4 (10/~g/100 #1) in 50 m M carbonate-bicarbonate buffer (pH 9.6) was coated on plates (Falcon No. 3912) by incubating at 4°C overnight. After blocking the protein binding activity of the plates with 0.1%0 BSA solution containing 0.02% NAN3, test samples of 100/~1 of serially diluted antisera from immunized hamsters were put in the wells and incubated at room temperature for 2 h. After extensive washings with PBS, horseradish peroxidase-conjugated rabbit anti-hamster IgG (1:I000 dilution) (Jackson) was added and incubated for 1 h at room temperature. The peroxidase activity bound to the wells was measured by adding 100 ~1 of o-phenylenediamine solution (0.2 mg/ml 0.15 M citrate-phosphate buffer, pH 5.0) with 0.02% HzOz. The plates were kept for 10 min at room temperature. The reaction was stopped by adding 50 tA 10% H2SO 4 to the wells and optical density was measured at 492 nm by a microplate photometer (Bio-Rad, Model 450). Antibody titers were shown as the maximum dilutions of antisera in which the maximum absorbance at 492 nm was given.
Immunofluorescence staining The procedures were carried out following a previous method (Koyama et al., 1985). Briefly, cumulus-free oocytes were incubated in 10 ~1 of antisera diluted 1:100 with PBS for 1 h at room temperature and then washed with PBS. The washed oocytes were transferred to a droplet of fluoresceinisothiocyanate (FITC) conjugated F(ab')2 fragment of IgG-prepared from goat anti-hamster -r-globulin (Cappel No. 1208-0081) diluted 1:1000 with PBS and incubated for 1 h at room temperature. After washing with PBS, oocytes were examined under a UV microscope.
Immunohistochemical staining Specimens sliced from porcine ovary to a thickness of 5 mm were fixed in 2% paraformaldehyde containing 0.01 M NalO4 and 0.075 M lysine by incubating at 4°C overnight. The fixed materials were washed with PBS containing 10% sucrose, followed by successive washings with PBS containing 15% and 20% sucrose. Each step was carried out at 4°C overnight. They were further incubated in PBS containing 20% sucrose and 5% glycerin at 4°C overnight and frozen immediately using dry-ice and ethanol in OCT compound (Miles). Serial cryostat sections of 6/zm thickness were incubated in PBS containing 10%0 normal hamster serum for 15 min at room temperature to block non-specific bindings of hamster globulin. They were first made to react with hamster anti-ZP4 (1:20 dilution) or anti-s-PZP (1:100 dilution) sera for 1 h at room temperature. After washing, peroxidase conjugated anti-hamster IgG (Jackson) diluted at 1:200 was made to react as a second antibody. Peroxidase reaction was developed in 0.05% 3,3'diaminobenzidine and 0.01%0 H202 solution for 5 min.
201
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Fig. 1. Silver staining of s-PZP and isolated ZP4 in two dimensional electrophoresis, s-PZP was separated into four glycoprotein families (A). ZP4 isolated from s-PZP by preparative SDS-PAGE was confirmed to have a similar migration pattern to ZP4 in s-PZP (B).
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Fig. 2. Changes in antibody titers and estradiol levels in hamsters following primary immunization with s-PZP and ZP4. Antibody titers were determined by ELISA against s-PZP (0) or isolated ZP4 (&) used for immunization. Estradiol (E2) levels were estimated by radioimmunoassay at estrus or estrusequivalent day of the cycle. Note that the increase in antibody titers was accompanied by a decrease in E 2 levels in hamsters immunized both with s-PZP (11) and ZP4 (1~), but the E 2 level of ZP4-immunized hamsters returned to an almost normal level at 12 weeks after immunization. ( [] ) shows E z level of nonimmunized hamster at estrus day. Each value is mean of three animals.
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Histological examination Oophorectomy was performed at 3, 7 and 12 weeks after the first injection. The excised ovaries were fixed in 10% formalin for several days and were processed for paraffin embedding. Tissue sections (4/xm) were prepared and stained with hematoxylin and eosin. Hormone assay Serum from each immunized hamster was taken for estradiol (E2) assay periodically on the day corresponding to the day of estrus following the first immunization. Estrus cycles were assessed by vaginal secretions and serum E2 levels were measured by radioimmunoassay kits (CIS Diagnostic). Results
Solubilized porcine ZP (s-PZP) was separated into four glycoprotein families (ZP1, ZP2, ZP3, ZP4) under reducing conditions as shown in Fig. 1A. Each family consisted of many isomers with similar molecular sizes but different isoelectric points. The average molecular weights of ZP1, ZP2, ZP3 and ZP4 were estimated as 92 000, 69 000, 55 000 and 23 000, respectively. The glycoprotein which migrated to the region corresponding to 20 000-30 000 in one dimensional preparative SDS-PAGE of s-PZP was isolated. A two dimensional SDS-PAGE of the isolated glycoprotein was
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Fig. 3. Western blot analysis of s-PZP in two dimensional SDS-PAGE. Antiserum from a hamster immunized with s-PZP reacted mainly with ZP3 glycoprotein family (A). Antiserum from a hamster immunized with ZP4 reacted mainly with ZP4 glycoprotein family (B).
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performed and a similar migration pattern to ZP4 glycoprotein family (Fig. 1B) was confirmed. This preparation was used as ZP4 for active immunization of hamsters. Figure 2 illustrates the antibody titers determined by ELISA and the estradiol (E2) levels in sera of the hamsters immunized with s-PZP and purified ZP4 during the period of 12 weeks after the initial immunization. Antibody titers began to increase from 3 weeks after immunization and reached maximal level at 5 weeks in both immunization groups. The antibody titer of s-PZP immunized hamsters was much higher than that of ZP4 immunized hamsters throughout the whole observation period. In hamsters immunized with s-PZP, the serum E2 levels gradually decreased from 3 to 7 weeks after immunization and the decreased levels persisted to the 12th week. In hamsters immunized with ZP4, the E2 levels began to drop 5-7 weeks after immunization and then increased to about 60% of normal levels after 12 weeks. Antibody titers correlated well with declining E2 levels in both groups. Four hamsters immunized with ZP4 were mated with fertile males from 6 weeks after the first immunization.
A
C
B
D
Fig. 4. Indirect immunofluorescence staining of normal porcine and hamster oocytes with antisera from hamsters immunized with s-PZP and ZP4. Porcine oocytes reacted with hamster anti-s-PZP (A) and antiZP4 (B) antiscra. Hamster oocytes reacted with hamster anti-s-PZP (C) and anti-ZP4 (D) sera.
Fig. 5. Photomicrographs of ovarian sections from hamsters immunized with s-PZP and ZP4. (A) A control section from a Freund's adjuvant-injected hamster. IB-E) Sections o f s-PZP-immunized hamster ovaries which were taken at 5 (B), 7 (C) and 12 (D,E) weeks after the first injection. Note that many cell clusters ~re present in section E. ( F - H ) Sections o f ZP4-immunized hamster ovaries which were taken at 5 (F), 7 (G) and 12 (H) weeks after the first injection. Magnification x 40, except for E ( x 100).
.h
206
Spermatozoa were first recognized in vaginal smears of three hamsters at 13 weeks and they were delivered of 3, 3 and 5 babies, respectively (3.67 ± 0.94, n = 3). Although the litter size was much smaller than that of normal hamsters (9.0 + 0.82, n = 3), these three females partially recovered their fertility. Characteristic properties of antisera were studied by Western blot analysis and immunofluorescent staining of pig and hamster oocytes. Figure 3 shows Western blotting of s-PZP using sera from hamsters immunized with s-PZP and ZP4. Anti-s-PZP serum reacted mainly with ZP3 family, which is the most dominant component in s-PZP. Anti-ZP4 serum reacted strongly with ZP4 but also showed weak staining with ZPI. Thus the staining patterns in Western blotting with these two antisera were quite different. Both antisera produced to s-PZP and ZP4 reacted not only with porcine oocytes but also with hamster oocytes in immunofluorescent staining (Fig. 4). The highest antibody titers determined by the immunofluorescence test are shown in Table 1. Antibody titers of anti-s-PZP and anti ZP4 antisera against porcine ZP were strikingly different, i.e. 1:10000 vs. 1:2000, while those against hamster ZP were the same, 1:200 dilutions in both antisera. Ovarian sections from immunized hamsters taken at 3, 7 and 12 weeks after the initial immunization were stained with hematoxylin and eosin (Fig. 5). There were no remarkable differences in morphology between the control hamster (Fig. 5A) injected only with Freund's adjuvant and the hamsters immunized with s-PZP or ZP4 (Fig. 5B, F) until 3 weeks after immunization, except for a mild desquamation of granulosa cell layers of antral follicles in the hamster immunized with s-PZP. However, considerable morphological changes were observed in the ovaries of s-PZP immunized hamsters at 7 weeks after immunization as compared to the control (Fig. 5C). Ovarian sections showed atretic and degenerative changes of growing follicles with marked reduction of normal antral follicles, though the changes
TABLE 1 Antibody titers of anti-s-PZP and anti-ZP4 antisera against porcine and hamster oocytes. Antibody titers were expressed as m a x i m u m dilution of antisera in which a significant immunofluorescent staining was recognized on ZP. Antisera
Anti-s-PZP Anti-ZP4
Antibody titers against Porcine ZP
Hamster ZP
1: 10 000 1:2000
1:200 1:200
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Fig. 6. lmmunohistochemical staining of porcine ovarian sections with hamster anti-s-PZP and anti-ZP4 antisera. Paraformaldehyde-fixed frozen sections were treated with anti-s-PZP (1:100) or anti-ZP4 (1:20) hamster sera followed by peroxidase-conjugated anti-hamster IgG. Note that ZP in antral follicles are intensely stained with antisera both of anti-s-PZP (A) and anti-ZP4 (B), while ZP in growing and pre-antral follicles are stained only with anti-s-PZP antiserum (C) (arrows) but not stained with anti-ZP4 antiserum (D), Original magnification × 100
208
appeared to be milder in a hamster immunized with ZP4 (Fig. 5G) as compared to that immunized with s-PZP. No significant inflammatory reaction was observed. At 12 weeks after immunization, the ovaries of a hamster immunized with s-PZP became markedly atrophic and sclerotic and the degenerative changes of the follicles were further advanced. Follicular structures were completely destroyed and no follicles remained in the ovaries. Only numerous follicle-like cell clusters were observed (Fig. 5D,E). In contrast, the ovaries of hamster immunized with ZP4 were of normal size and several growing follicles were observed in the tissue sections (Fig. 5H). Immunohistochemical staining of porcine ovarian sections was carried out to examine the presence of antigens corresponding to anti-s-PZP and antiZP4 antisera at different stages of follicular development. Both antisera reacted strongly to ZP in antral follicles (Fig. 6A,B), but only anti-s-PZP antiserum reacted to ZP in growing or pre-antral follicles (Fig. 6C). AntiZP4 antiserum did not showed any staining to ZP in a serial section of the same pre-antral follicles (Fig. 6D). Discussion The present study demonstrated that female hamsters could respond well to porcine ZP antigens and produce antibodies autoreactive to their own ZP. All immunized animals developed temporary or permanent infertility depending on the antigen preparations used for the immunization. ZP4 immunization induced temporary infertility, while s-PZP immunization induced permanent infertility. Anti-s-PZP hamster serum reacted most strongly with ZP3 glycoprotein family, while anti-ZP4 hamster serum reacted strongly with ZP4 glycoprotein family. Although the antibody titers determined by ELISA or immunofluorescence staining against porcine ZP were higher in anti-s-PZP antiserum than in anti-ZP4 antiserum, those determined by immunofluorescent staining against hamster ZP were the same levels in both antisera. These results suggest that the difference in the antifertility effect in hamsters immunized with the two antigen preparations might be due to qualitative rather than quantitative differences in the immune response to each antigen. In both hamsters immunized with two different antigen preparations of porcine ZP, the histological changes in the ovaries were associated with a marked reduction of serum E2 levels. Most follicles except very small ones were degenerate and showed oocyte loss. In the hamsters immunized with s-PZP, the degenerative changes were progressive even after stopping further immunization and no follicles remained in the ovaries. Only follicular cell-like remnants were observed. In contrast, in the hamsters immunized with ZP4, the degenerative changes were reversible and small to
209
medium size growing follicles appeared in the ovaries after the antibody titers dropped. Three animals in the latter group became pregnant when they were mated with a fertile male. The main cause of infertility in hamsters immunized with porcine ZP antigens appeared to be failure to ovulate and follicular destruction by the antibodies induced to auto-antigens of ZP and/or oocyte components, though an inhibitory effect at the level of fertilization cannot be ruled out. The mechanism for the destruction of follicles by the antibodies was not clear but the same phenomenon has been observed in many species of experimental animals (Skinner et al., 1984; Mahi-Brown et al., 1988; Dunbar et al., 1989). Although a cellular immune response to the follicular components was not completely ruled out, for the reason that the histological studies did not show any inflammatory cell infiltration of degenerative follicles, complementdependent cytotoxic antibodies bound to ZP or oocytes seem to be more causative factors for the destruction of follicles in the ovaries immunized with porcine ZP antigens. That immunization with s-PZP induced irreversible follicular destruction suggests that the antigens corresponding to the antibodies induced by immunization with s-PZP might be expressed at an earlier stage of folliculogenesis, so that even small follicles were affected with cytotoxic antibodies, resulting in the extinction of follicles in the ovaries. On the other hand, the reversibility of folliculogenesis in hamsters immunized with ZP4 indicates that the antigens corresponding to the antibodies induced by ZP4 immunization are expressed at a later stage of folliculogenesis, so that early stage follicles might be left intact and resume development after the antibody titers dropped. If this is true, ZP4 glycoproteins would be ideal candidates for developing a contraceptive vaccine using ZP antigens. We are now analyzing the antigenic structures of ZP4 glycoprotein molecules. References Dunbar, B.S., Liu, C. and Sammons, D.W. (1981) Identification of the three major proteins of porcine and rabbit zonae pellucidae by high resolution two-dimensional gel electrophoresis: Comparison with serum, follicle fluid and ovarian cell proteins. Biol. Reprod. 24, 1111-1124. Dunbar, B.D., Lo, C., Powell, J. and Stevens, V.C. (1989) Use of a synthetic peptide adjuvant for the immunization of glycoproteins. Fertil. Steril. 52, 311-318. Gulyas B.J., Gwatkin, R.B.L. and Yuan, L.C. (1983) Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zona pellucida. Gamete Res. 4, 299-307. Hasegawa, A., Koyama, K. and Isojima, S. (1991) Isolation of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family. Acta Obst. Gynaecol. Jpn. 43, 221-226. Hedrick, J.L. and Wardrip, N.J. (1986) Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Anal. Biochem. 157, 63-70. Henderson, C.J., Hulme, M.J. and Aitken, R.J. (1988) Contraceptive potential of antibodies to the zona pellucida. J. Reprod. Fertil. 83, 325-343. Isojima, S., Koyama, K., Hasegawa, A., Tsunoda, Y. and Hanada, A. (1984) Monoclonal antibodies to
210 porcine zona pellucida antigens and their inhibitory effects on fertilization. J. Reprod. Immunol. 6, 77-87. Isojima, S., Koyama, K., Takeda, Y., Shigeta, M., Tsuji, Y. and Hasegawa, A. (1986) The development of a contraceptive vaccine by purification of antigens from gametes. Am. J. Reprod. lmmunol, Microbiol. 10, 90-92. Koyama, K., Hasegawa, A. and Isojima, S. (1991 a) Further characterization of the porcine zona pellucida antigen corresponding t o monoclonal antibody (3A4-2G1) exclusively cross-reactive with porcine and human zonae pellucidae. J. Reprod. Immunol. 19, 131-148. Koyama, K., Hasegawa, A., Inoue, M. and Isojima, S. (1991b) 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., Tsuji, Y. and Isojima, S. (1985) Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae. J. Reprod. Immunol. 7, 187-198. Laemmli, U.K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Liu, I.K.M., Bernoco, M. and Feldman, M. (1989) Contraception in mares heteroimmunized with pig zonae pellucidae. J. Reprod. Fertil. 85, 19-29. Mahi-Brown, C.A., Huang, T.T.F., Jr. and Yanagimachi, R. (1982) Infertility in bitches induced by active immunization with porcine zonae pellucidae. J, Exp. Zool. 222, 89-95. Mahi-Brown, C.A., Yanagimachi, R., Hoffman, J.C. and Huang, T.F.H., Jr. (1985) Fertility control in bitch by active immunization with porcine zona pellucida: Use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles. Biol. Reprod. 32, 761-772. Mahi-Brown. C.A., Yanagimachi, R., Nelson, M.L., Yanagimachi, H. and Palumbo, N. (1988) Ovarian histopathology of bitches immunized with porcine zonae pellucidae. Am. J. Reprod. Immunol. Microbiol. 18, 94-103. O'Farrell, P.H. (1974) High resolution two-dimensional electrophoresis of protein. J. Biol. Chem. 250, 4007 -4021. Sacco, A.G. (1987) Zona pellucida: Current status as a candidate to antigen for contraceptive vaccine development. Am. J. Reprod. Immunol. Microbiol. 15, 122-130. Sacco, A.G., Yurewicz, E.C., DeMato, F.J. and Dukelow, W.R. (1983) Heteroimmunization of squirrel monkey (Saimiri sciureus) with a purified porcine zona antigen (PPZA): Immune response and biologic activity of antiserum. Fertil. Steril. 39, 350-358. Sacco, A.G., Pierce, D.L., Subramanian, M.G., Yurewicz, D.C. and Dukelow, W.R. (1987) Ovaries remain functional in squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida 55,000 macromoleeule. Biol. Reprod. 36, 481-490. Skinner, S.M., Mills, T., Kirchick, N.J. and Dunbar, B.S. (1984) Immunization with zona proteins results in abnormal ovarian follicular differentiation and inhibition of gonadotropin induced steroid secretion. Endocrinology 115, 2418-3432. Subramanian, M.G., Yurewicz, E.C. and Sacco, A.G. (1981) Specific radioimmunoassay for the detection of a purified porcine zona pellucida antigen (PPZA). Biol. Reprod. 24, 933-943. Upadhyay, S.N., Thillaikoothan, P., Bamezai, A., Jayaraman, S. and Talwar, G.P. (1989) Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkey: A morphological study. Biol. Reprod. 41,665-673. Wood, D.M., Liu, C. and Dunbar, B.S. (1981) Effect of alloimmunization and heteroimmunization with zonae pellucidae on fertility in rabbits. Biol. Reprod. 25, 439-450.
