IMMUNIZATION OF MICE WITH HEAT-SOLUBILIZED HAMSTER ZONAE: PRODUCTION OF ANTI-ZONA ANTIBODY AND INHIBITION OF FERTILITY

Transcription

1 FERTILITY AND STERILITY C~pyright ' 1977 The American Fertility Society Vol. 28, No.8, August 1977 Printed in U.s.A. IMMUNIZATION OF MICE WITH HEAT-SOLUBILIZED HAMSTER ZONAE: PRODUCTION OF ANTI-ZONA ANTIBODY AND INHIBITION OF FERTILITY RALPH B. L. GWATKIN, PH.D.* DORSEY T. WILUAMS, B.S. DENNIS J. CARLO, PH.D. Merck Institute for Therapeutic Research, Rahway, New Jersey Immunization of female mice with heat-solubilized hamster zonae pellucidae resulted in the production of antibody specific for the zona pellucida of several species, including that of primates. Complete, although temporary, infertility was produced in the mice. The zonae of ovulated eggs from the immune mice were found to contain antibody and complement. Many also appeared abnormal, but no apparent effect was observed on either vitelli or any of the other tissues that were examined. These results suggest the feasibility of developing a zona vaccine for contraception. Previous studies 1,2 have shown that when hamster zonae pellucidae are dissolved by heating them in an aqueous buffer the resulting solution retains receptor-like activity, since after addition of the solution to capacitated spermatozoa their ability to associate with eggs is impaired and fertilization is prevented. Following the zona reaction, induced by fertilization or artificially by electrical stimulation, this activity is lost. When a solution of solubilized zonae was prepared from mouse eggs it exhibited significant (but lesser) activity, as would be predicted from the limited cross-binding between mouse and hamster gametes. 2 In this communication we report the results of immunizing female mice against heat-solubilized hamster zonae. Such immunization with pure zona material was shown to elicit the production of antibody specific for the zonae of several species and to produce complete, although temporary, infertility. MATERIALS AND METHODS Hamster zonae pellucidae were collected and solubilized by heating them to 65 C in phosphate- Received January 31, 1977; revised February 24, 1977; accepted March 8,1977. *Reprint requests: Dr. Ralph Gwatkin, Building 80A 54N, Merck Institute for Therapeutic Research, P.O. Box 2000, Rahway, N. J buffered saline (PBS) as described previously. 2 Zona solutions (2000 zonae/ml) were combined with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.), and 0.3-ml aliquots (300 solubilized zonae) were injected intramuscularly into 6- to 8-week-old female Swiss mice. At 10-day intervals these injections were repeated, using incomplete adjuvant, until a total of four injections had been given. Control animals received buffer and adjuvant only. The development of anti-zona antibody was determined at intervals by bleeding the animals from the orbital sinus. The 0.5 to 1.0 ml of blood obtained from each animal was allowed to clot at room temperature for 1 hour, was kept at 5 C for 1 hour, and then was centrifuged at 1000 x g for 5 minutes to obtain the serum. The serum was assayed by two procedures: inhibition of in vitro fertilization and the indirect fluorescence method. Antibody could also be demonstrated by inhibition of zona digestion with pronase. The in vitro fertilization assay was carried out by exposing hamster eggs, from which the cumulus had been removed by treatment with hyaluronidase,3 to various dilutions of serum for 30 minutes. The eggs were then washed by transferring them through three 0.5-ml volumes of medium 199M2,4 and groups of10 eggs were added to 40-J.tl drops of cumulus-capacitated sperm (approximately 5 x 10 6 sperm/m!) under equilibrated mineral oil,3 After incubation of the eggs

2 872 GWATKIN ET AL. August 1977 TABLE 1. Development of Infertility following Immunization of Mice with Solubilized Hamster Zonae Experiment I" Experiment 3" Experiment 2": 4 injections Treatment 3 Injections 4 Injections 4 Injections 3 Mo later Serum titer" No. of Serum titer" No. of Serum titer" No. of Serum titer" No. of Serum titer" No. of young young young young young Immunized >1:10 0 >1:4, <1:16 O >1:2, <1:20 0 >1:4, <1:16 0 <1:4 3 >1:2, <1:10 5 >1:4, <1:16 0 >1:2, <1:20 0 >1:4, <1:16 0 <1:4 7 >1:10 6 >1:16 0 >1:2, <1:20 0 >1:4, <1:16 0 <1:4 0 >1:2, <1:10 0 >1:16 0 >1:2, <1:20 0 >1:4, <1:16 0 <1:4 8 >1:2, <1:10 6 >1:16 6 >1:2, <1:20 0 Adjuvant only "Each line of data within each experiment represents data on a single animal. btiter (at approximately 10 days after the last injection) was the dilution required to produce complete inhibition of sperm penetration in vitro. and sperm on a rocker (five to six oscillations per minute) for 90 minutes at 37.5' C in an atmosphere of 5% CO 2 in air, the unbound sperm were removed by transferring them again through three 0.5-ml volumes of medium 199M2. The eggs were then mounted in a 1- to 2-/.d drop of medium on a microscope slide and examined for sperm penetration under a phase-contrast microscope at a magnification of x 600. The indirect fluorescence assay was carried out by placing eggs in the serum for 30 minutes and washing them in 1 ml of PBS + 1% polyvinylpyrrolidone four times, allowing the eggs to soak in each wash for 5 minutes. The washed eggs were placed in 1 ml of fluorescein-conjugated goat antimouse immunoglobulin G (IgG) (heavy and light chains, Cappel Laboratories, Cochranville, Pa), diluted 1:20 in PBS, for 30 minutes at 37" C. They were then washed six times, allowing 5 minutes between each wash, in PBS + 1% polyvinylpyrrolidone, and mounted on a slide for examination under a fluorescence microscope. Tests for the presence of antibody and complement on mouse eggs employed fluorescein-conjugated rabbit anti-mouse IgG (heavy and light chains) and rabbit anti-mouse complement C3, followed by fluorescein-conjugated goat antirabbit IgG. These reagents were also obtained from Cappel Laboratories. RESULTS Immunization of Mice with Solubilized Hamster Zonae. Table 1 shows the results obtained when female mice were immunized with solubilized hamster zonae. Antibody which blocked the in vitro fertilization of hamster eggs developed following the third injection, and 10 days after the fourth injection the titer rose again, but rarely exceeded 1:32. When such immunized animals were mated, almost all of them were found to be infertile asjudged by their failure to deliver young. Control animals that received injections of adjuvant alone did not develop antibody, and all delivered normal litters. Three months after the last injection (experiment 3, Table 1) the serum titers had declined to < 1:4, and after mating three of the four animals became pregnant and delivered apparently normal young. When immunized animals were killed on the morning after mating and their oviducts were flushed, many of the eggs obtained were found to be unpenetrated by spermatozoa (Table 2). It was also observed that the zonae of many eggs were fragile (easily distorted and broken with a pipette) and that many appeared irregular and had multiple perforations (Fig. 1). However, such TABLE 2. Effect of Immunizing Mice with Solubilized Hamster Zonae: Proportion of Eggs Fertilized in Vivo and the Condition of Their Zonae" Pror.:,rtion Condition Treatment Serum titer' of zonae penetr:tedgf~ sperm Immunized >1:2, <1:4 9/9 Fragile >1:4, <1:16 0/5 Normal >1:16 0/9 Fragile >1:4, >1: Fragile Adjuvant only 0 10/10 Normal Normal 0 15/15 Normal Normal "Animals were mated approximately 10 days after each had received four injections of 300 solubilized zonae. btiter (at approximately 10 days after the last injection) was the dilution required to produce complete inhibition of sperm penetration in vitro.

3 Vol. 28, No.8 IMMUNIZATION OF MICE WITH HAMSTER ZONAE 873 FIG. 1. Eggs and zonae of mice immunized with solubilized hamster zonae. Upper left, Normal egg from nonimmunized animal; upper right: egg with abnormal, irregular, zona; lower left, egg with irregular zona so fragile it was broken open by gentle pipetting; lower right, fragile zona fragment showing patchy appearance due to multiple perforations. TABLE 3. Pre-exposure of Hamster Eggs in Vitro to Mouse Anti-Hamster Zona Serum: Effect on Time Required for Digestion of Their Zonae by Pronase" Pretreatment Normal mouse serum (1:4) Antiserum (1:4) Time required for zonae to dissolve Experiment 1 20 min >24 hr Experiment 2 15 min >24 hr "Hamster eggs were incubated at 31' C in 20-ILI drops of antiserum diluted 1:4 in medium 199M2, washed three times, and incubated in a solution of pronase (5 ILg/ml of PBS). nique, employing fluorescein-conjugated rabbit anti-mouse IgG (Fig. 2). Their zonae fluoresced brightly at a serum dilution of approximately 1:1000 (Table 4), whereas controls exposed to a 1:20 dilution of normal mouse serum showed no fluorescence. When mouse eggs were tested with the same sera, a bright fluorescence was obtained only up to a dilution of 1:40. Thus, there was significant, but limited, cross-reaction between the anti-hamster zona serum and mouse zonae, which could account for the infertility produced by actively immunizing mice with solubilized hamster zonae. Cross-reaction was also observed with the zonae of rhesus monkey eggs (Fig. 3) and squirrel monkey eggs, although the limited abnormalities were not necessary for the induction of infertility, since in one animal sperm entry was blocked without this change. To determine whether immunization with solubilized hamster zonae had affected other tissues of the mice, animals were killed at 1 week and at 10 weeks after the final (fourth) injection. Pieces of the liver, spleen, kidneys, uterus, oviducts, and ovaries were fixed, sectioned, and sta~ned with hematoxylin and eosin. Germinal centers were seen in the spleen, indicating active immunization, but there were no signs of cellular infiltration or of other changes in these tissues. Assay of Mouse Anti-Hamster Serum by Other Methods. When hamster eggs were pre-exposed to normal serum their zonae dissolved after exposure to dilute pronase for 15 to 20 minutes. However, when anti-hamster zona serum was used, pronase failed to dissolve the pre-exposed eggs even after 24 hours (Table 3). Mouse anti-hamster zona sera were also assayed on hamster eggs by the indirect fluorescence tech- FIG. 2. Hamster eggs exposed to mouse anti-hamster zona serum (diluted 1:100) and then to fluorescein-conjugated antimouse IgG. Note the bright fluorescence of zonae. Controls exposed to normal mouse serum produced no detectable fluorescence (x 900).

4 874 GWATKIN ET AL. August 1977 TABLE 4. Reaction of Mouse Anti-Zona Serum with the Zonae of Various Species Type of egg Hamster Mouse Rhesus monkey Squirrel monkey Highest dilution giving bright fluorescence of zonae 1:1000 1:40 >1:40 >1:20 number of eggs available did not permit further quantitation. Mouse anti-hamster zona serum also reacted with the zonae of hamster eggs which had undergone a zona reaction (induced by electrical stimulation) but not with vitelli, cumulus cells, spermatozoa, cornea, or with the vitelline membrane of the chicken egg (Table 5). Detection of Antibody and Complement on the Zonae of Immunized Mice. Mice which had been immunized against solubilized hamster zonae were induced to ovulate with 10 IU of pregnant mare's serum gonadotropin, followed 48 hours later by 10 IU of human chorionic gonadotropin. The eggs were flushed from the oviduct 20 hours later and freed of cumulus by treatment with hyaluronidase. They were then tested for the presence of mouse antibody by exposure to fluorescein-conjugated rabbit anti-mouse IgG and for TABLE 5. Specificity of Mouse Anti-Hamster Zona Serum" Cell or cell product Hamster zonae Hamster zonae from zona-reacted eggs Hamster vitelli Hamster cumulus cells Hamster cornea Vitelline membrane of chicken egg Relative fluorescence Experiment 1 Experiment 2 o "Cells and cell products were incubated in antiserum for 30 minutes, washed, and exposed to rabbit anti-mouse IgG; washed again, and examined under a fluorescence microscope. The zona reaction was induced by electrical stimulation (150 volts, 1 msecond).5 complement by exposure to rabbit anti-mouse complement followed by fluorescein-conjugated goat anti-rabbit IgG. All of these reagents were diluted 1:20. Bright fluorescence was obtained with both tests (Figs. 4 and 5) in the immunized animals, but none was observed with eggs obtained from animals which had received adjuvant alone. Isoimmunization. When mice were immunized in the same manner as above but with the solubilized zonae of their own species, the antibody o FIG. 3. Rhesus monkey egg exposed to mouse anti-hamster zona serum diluted 1:20 and then to fluorescein-conjugated anti-mouse IgG (x 675). FiG. 4. Eggs of mice immunized against solubilized hamster zonae. The eggs were incubated with rabbit anti-mouse complement followed by fluorescein-conjugated anti-rabbit IgG to reveal the presence of complement in the zona pellucida (x 675).

5 Vol. 28, No.8 IMMUNIZATION OF MICE WITH HAMSTER ZONAE 875 FIG. 5. Egg of mouse immunized against solubilized hamster zonae. The egg was incubated with fluorescein-conjugated anti-mouse IgG to reveal the presence of mouse IgG in the zona pellucida (x 675). response, as measured by indirect immunofluorescence, was only 1:20, i.e., only 2% of that obtained with heterologous zonae. The undiluted sera of hamsters immunized with solubilized hamster zonae when applied to hamster eggs had no effect on hamster egg fertilization in vitro; i.e., there was no antibody response detectable by this assay, compared with complete inhibition at dilutions of up to 1:16 and 1:32 of sera from mice immunized with solubilized hamster zonae. DISCUSSION These observations show that, although isoimmunization with heat-solubilized zonae may produce little or no antibody response, immunization with heterologous zona material elicits a high titer of anti-zona antibody. Mice immunized with hamster zonae not only produced antibody against hamster zona (a titer of approximately 1:1000 by indirect immunofluorescence) but antibody that cross-reacted with mouse zonae at a dilution of approximately 1:40. This degree of cross-reaction was sufficient to result in a complete loss of fertility in the immunized animals. The antiserum also cross-reacted with the zonae of primate eggs, but cross-reaction did not extend to the vitelline membrane of the chicken egg, which may be the avian counterpart of the zona pellucida. To our knowledge this is the first time that animals have been immunized with pure zona substance and that a complete loss of fertility has been produced by active immunization directed against the zona pellucida. However, a number of other investigators have published reports showing that the zona pellucida is highly antigenic and that immunization with extracts of whole ovaries can give rise to anti-zona antibodies. The serum of rabbits immunized with homogenates of whole mouse eggs, including the cumulus oophorus, has been shown to react strongly with mouse zonae. 6 Rabbit antisera prepared against saline extracts of hamster ovary homogenates and made relatively specific by absorption with hamster intestine and lung were observed to form a precipitate on hamster eggs, preventing their dissolution by trypsin 7 8 -an observation similar to the one made by us in this study. An antiserum coating of the zona was also observed to block fertilization of hamster eggs in vitro.9 More recently, passive immunization of mice, 10 hamsters,l1 and rats12 with heteroantisera prepared in rabbits against the supernatants of whole ovary homogenates was reported to inhibit fertilization in vivo. IgG prepared in rabbits against hamster ovaries, labeled with 1311, and injected into hamsters disappeared from the lungs, kidneys, spleen, and uterus but persisted in the ovaries during the period of infertility. 13 In accord with our observations which indicate that zona antibodies are not species-specific, Tsunoda and Chang14 observed that rabbit anti-hamster ovary serum inhibited fertilization in rats and mice. They also observed that rabbit anti-rat ovary serum inhibited fertilization in mice and, to a lesser extent, in hamsters.12 We were also able for the first time to demonstrate that in mice actively immunized with solubilized hamster zonae, complement and antibody are present in the zonae of ovulated eggs. The joint action of these agents may be responsible for the softening and multiple perforation observed in the zonae of eggs ovulated by immunized animals. Despite these alterations of the zona pellucida, the vitelli within them appeared to be completely normal. In addition, no pathology was noted in the other tissues that were

6 876 GWATKIN ET AL. August 1977 examined, so that the zona pellucida should be considered highly, or even completely, specific immunologically. The production of infertility in mice by immunization with solubilized hamster zonae may be the result of several actions of the antibody. First, it probably coats the eggs, thereby preventing fertilization. Eventually, destruction of the zonae through the joint action of antibody and complement could lead to a failure of normal egg transport, which is known to depend on an intact zona. 15 Finally, coating of eggs with antibody may prevent them from attaching to the endometrium of the uterus or prevent the emergence of the blastocyst from the zona, thereby blocking embryo implantation. Dudkiewicz et al. 16 have observed that hamster embryos in the morula and blastocyst stages, when treated with rabbit anti-ovary serum and transferred to the uterus, fail to shed their zonae or to implant. It is significant that, although the zona pellucida is a strong heteroantigen, its isoantigenic activity is slight. This probably accounts for the recent failure of Tsunoda and Chang l7 to inhibit significantly the fertility of rats by isoimmunizing them with ovarian homogenates. With mice there was a significant reduction but not a complete suppression of fertility. Low isoantigenicity of the zona pellucida may also account for our observation that the infertility of immunized mice disappears with time, there being insufficient circulating antigen to sustain high antibody levels. A number of questions remain to be answered. It is not known whether the antibody acts only on the ovulated egg or passes into the follicles to react with the egg prior to ovulation. It would also be helpful to know the nature of the antigenic determinants on the zona antigen, which is probably a glycoprotein. 18-2o Since anti-zona sera were shown by immunofluorescence to react with the zona after it had undergone a zona reaction, these determinants must include others in addition to those responsible for binding the sperm to the egg. The cross-reaction of mouse anti-hamster zona serum with the zonae of primate eggs and the reversibility of the infertility produced by active immunization suggest that it may be possible to use zona matefial isolated from slaughterhouse animals, e.g., cow or sow, as a vaccine for human contraception, as has been proposed by Shivers. 21 Acknowledgment. We would like to thank Dr. H. E. Carter for assistance with the histologic procedures. REFERENCES 1. Gwatkin RBL, Williams DT: Receptor activity of the solubilized zona pellucida (abstr 7). In Abstracts of the Ninth Annual Meeting of the Society for the Study of Reproduction, Philadelphia, 1976, p Gwatkin RBL, Williams DT: Receptor activity of the hamster and mouse solubilized zona pellucida before and after the zona reaction. J Reprod Fertil 49:55, Gwatkin RBL, Andersen OF, Hutchison CF: Capacitation of hamster spermatozoa in vitro: the role of cumulus components. J Reprod Fertil 30:389, Gwatkin RBL, Andersen OF: Effect of glycosidase inhibitors on the capacitation of hamster spermatozoa by cumulus cells in vitro. J Reprod Fertil 35:565, Gwatkin RBL, Williams DT, Hartmann JF, Kniazuk M: The zona reaction of hamster and mouse eggs: production in vitro by a trypsin-like protease from cortical granules. J Reprod Fertil32:259, Glass LE, Hanson JE: An immunological approach to contraception: localization of antiembryo and antizona pellucida serum during mouse pre implantation development. Fertil Steril 25:484, Ownby CL, Shivers CA: Antigens of the hamster ovary and effects of anti-ovary serum on eggs. BioI Reprod 6: 310, Dudkiewicz AB, Shivers CA, Williams WL: Ultrastructure of hamster zona pellucida treated with zona-precipitating antibody. BioI Reprod 14:175, Shivers CA, Dudkiewicz AB, Franklin LE, Fussell EN: Inhibition of sperm-egg interaction by specific antibody. Science 178:1211, Jilek F, Pavlok A: Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse. J Reprod Fertil 42:377, Oikawa T, Yanagimachi R: Block of hamster fertilization by anti-ovary antibody. J Reprod Fertil45:487, Tsunoda Y, Chang MC: Effect of anti-rat ovary serum on the fertilization of rat, mouse and hamster eggs in vivo and in vitro. BioI Reprod 14:354, Yanagimachi R, Winkelhake J, Nicolson GL: Immunological block to mammalian fertilization: survival and organ distribution of immunoglobulin which inhibits fertilization in vivo. Proc Natl Acad Sci USA 73:2405, Tsunoda Y, Chang MC: In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum. J Exp ZooI195:409, Modlinski JA: The role of the zona pellucida in the development of mouse eggs in vivo. J Embryol Exp Morphol 23:539, Dudkiewicz AB, Noske IG, Shivers CA: Inhibition of implantation in the golden hamster by zona-precipitating antibody. Fertil Steril 26:686, Tsunoda Y, Chang MC: Reproduction in rats and mice isoimmunized with homogenates of ovary or testes with epididymis, or sperm suspensions. J Reprod Fertil46:379, Austin CR: Ultrastructure of Fertilization. New York, Holt, Rhinehart, Winston, Inoue M, WolfCP: Comparative solubility properties ofthe zonae pellucidae of unfertilized and fertilized mouse ova. BioI Reprod 11:558, 1974

7 Vol. 28, No.8 IMMUNIZATION OF MICE WITH HAMSTER ZONAE Oikawa T, Nicolson GL, Yanagimachi R: Inhibition of hamster fertilization by phytoagglutinins. Exp Cell Res 83:239, Shivers CA: Antigens of the ovum as a potential basis for the development of contraceptive vaccine. In Development of Vaccines for Fertility Regulation. WHO Session, Third International Symposium on Immunology of Reproduction, Varna, Bulgaria. Copenhagen, Scriptor, 1975, p 81