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a Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo, Ohio 43614
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These data indicate that the spermatozoon has both auto- and iso-antigenic potentials and thus can generate an immune response in both males and females that is capable of inducing infertility. However, the whole spermatozoon per se cannot be employed for the development of a vaccine because of the presence of several antigens, both internal and on the surface, that are likely to be shared with various somatic cells [9, 10]. The utility of a sperm antigen for the development of a contraceptive vaccine is contingent upon its sperm specificity and its involvement in the fertilization process, and upon raising an antibody response sufficient to intercept fertility in a reversible manner. During the past decade, several antigens have been defined that are relevant to fertility, and the antibodies to some of these have shown inhibition of fertilization in vitro (reviewed in [9, 10]). A few of them have also been demonstrated to affect fertility in vivo in actively immunized animals of various species [11, 12].
In these studies, purified/semi-purified cognate antigens were used for immunization to examine the effect on fertility. However, to obtain Food and Drug Administration approval for clinical use and to conduct appropriate multicenter fertility trials in a quality control manner, active immunization studies need to be carried out using purified recombinant or synthetic molecules. Complementary DNAs encoding for a few sperm antigens have been cloned and sequenced, and the recombinant proteins or their peptides expressed by some of the cloned cDNAs have also been examined for their effect on fertility in actively immunized animals [13–15]. However, despite the production of high antibody titers, the effect on fertility has not been encouraging for many of these antigens.
Recently we cloned and sequenced cDNA encoding for a sperm antigen, designated fertilization antigen-1 (FA-1) [16–18], from murine testes [19]. The murine FA-1 cDNA of 649 base pairs has an open reading frame of 164 amino acids, and an extensive computer search in the GenBank/protein database indicated that it is a novel protein. Northern blot analysis/reverse transcription-polymerase chain reaction indicated its testis-specific expression. The recombinant murine FA-1 antigen reacted with a specific component of murine oocyte zona pellucida, namely zona pellucida glycoprotein 3 (ZP3), and its antibodies completely blocked sperm-zona binding in mice in vitro [19]. The present study was conducted to examine the effect of recombinant (r) FA-1 antigen on fertility of actively immunized female mice. The long-term objective of these studies is to investigate the potential utility of rFA-1 antigen in contraceptive vaccine development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Murine rFA-1 antigen was obtained using the glutathione S-transferase gene fusion system (Pharmacia, Kalamazoo, MI) as described elsewhere [19]. FA-1 cDNA isolated from pBluescript vector by EcoRI was subcloned into pGEX-2T vector, and the subcloned insert showed sense orientation and identical sequence. Expression of rFA-1 protein was conducted according to the manufacturer's protocol (Pharmacia) [19]. Bacteria containing the plasmid with the right insert were cultured overnight at room temperature with vigorous shaking. The culture was diluted (1:100), and when the absorbance600nm reached 0-5.2, the target gene expression was induced with isopropyl ß-D-thiogalactoside (0.1 mM final concentration). After the 6-h incubation, the bacteria were centrifuged, washed, and sonicated. The supernatant containing the rFA-1-glutathione S-transferase fusion protein was incubated with glutathione conjugated to Sepharose-4B (Pharmacia) beads. The unbound proteins were washed off the immunocolumn, and rFA-1 protein was released from the fusion protein bound to the beads by treatment with thrombin. The protein was further purified by using an anti-FA-1 monoclonal antibody immunocolumn to remove thrombin [16–19]. The purified protein was analyzed by SDS-PAGE and Western blot procedures for homogeneity and authenticity [19]. The homogeneity was indicated by the presence of only the expected band of ~18 kDa or its polymers corresponding to rFA-1 antigen in the silver-stained SDS gel. The immunoreactivity of the expected band(s) with the FA-1 monoclonal antibody in the Western blot procedure confirmed the authenticity of the protein bands observed in the SDS gel. Only those batches that showed specific bands in SDS-PAGE and Western blot procedure were used in the present study.
Active Immunization and Fertility Trial
Virgin B6D2F1/J 10- to 12-wk-old female mice (Jackson Labs., Bar Harbor, ME) were immunized against the purified rFA-1 antigen (n = 16 in two trials; Table 1) as described elsewhere [20]. Each animal received a total of four injections at 2-wk intervals. Each injection consisted of 100 µl of PBS containing 25 µg of rFA-1 protein emulsified with 100 µl of complete (primary injection) or incomplete (three booster injections) Freund's adjuvant, and the injection volume was equally distributed between two sites, half injected at the s.c. and the other half at the i.m. site. The control animals (n = 22 in two trials; Table 1) were injected similarly except that injections did not have the antigen. Starting 2-3 wk after the last injection, the animals were bled by retro-orbital puncture to collect serum for examination of the antibody titer as described below. The animals were then mated overnight with males of proven fertility (two females with one male in each cage). The next morning, the mating was confirmed by the presence of a vaginal plug, and the mated animals were separated and kept to deliver. The number of babies delivered by each mated animal was counted and the babies were killed by CO2 asphyxiation.
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Two different trials were conducted 5 mo apart using different purified batches of the recombinant antigen. The animals in the second trial were kept for a longer time, up to 283 days, to examine the effect of disappearance of antibody titers on the regain of fertility. Fertility was defined as the mean number of babies borne by the rFA-1-immunized group divided by the mean number of babies borne by the PBS-control group, multiplied by 100.
ELISA
The presence and titers of antibodies were analyzed against rFA-1 antigen by using the ELISA, as described earlier [21]. Each serum was run in duplicate, and the uncoated wells, treated identically, served as controls. The absorbance reading of the uncoated wells was subtracted from the absorbance reading of the antigen-coated wells, and the mean of the resultant values was recorded. The serum samples were run in batches of 10–15, and a known positive was included as quality control in each batch. This positive was antiserum #1002 raised in rabbit against the lithium diiodosalicylate (LIS)-solubilized caudal murine sperm membrane preparation (0.3 M LIS, 0.05 M Tris-HCl, pH 8.0, containing 1 mM PMSF and 5 mM soybean trypsin inhibitor at room temperature for 30 min and then at 4°C for 2 h) aliquoted, and stored at -20°C. The absorbance readings were converted to SD units by the following formula: SD unit = mean (test) - mean (control)/SD of control group [18]. The samples with SD units of 
+2 were considered as having a positive reaction with rFA-1 antigen.
Western blot procedure Antigenic specificity of the antiserum raised after immunization with rFA-1 was examined using the Western blot procedure [16–18]. Briefly, the purified rFA-1 antigen and LIS-solubilized preparation of washed caudal murine sperm were run under nonreducing conditions (50–100 µg protein per lane) in slab SDS-PAGE (5–15% gradient gel) [22], transferred to nitrocellulose paper [23], and reacted with the antiserum (10 µl/ml of the incubation buffer). The reacted antigens were localized by incubating the antibody-reacted strips first with alkaline-phosphatase-conjugated rabbit anti-mouse antibodies (heavy- and light-chain specific; Cooper Biomedical Inc., Malvern, PA) and then with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as a substrate as described elsewhere [16–18]. Sperm extract was prepared by dissolving washed sperm cell membrane proteins in LIS as described above. The serum from PBS-control mice served as control.
Sperm-Zona Interaction and In Vitro Fertilization (IVF) Procedure
The antibodies from the rFA-1-immunized mice that showed a complete block of fertility (e.g., mouse #3: 0 babies born) and the mice that showed a reduction rather than a complete block of fertility (e.g., mouse #12: 4 babies born) were examined for their effects on sperm-zona interaction and IVF in mice. The sperm-zona interaction and murine IVF assays were carried out as described elsewhere [16, 19, 24]. The antibodies were affinity purified from the antiserum using protein A and G immunobeads (Oncogene Sciences, Cambridge, MA).
Unfertilized eggs were obtained from the oviducts of superovulated adult (8–10 wk of age) female CD-1 mice (Charles River Labs, Wilmington, MA) at 10–14 h after hCG injection. Superovulation was induced by i.p. injection of eCG (7 IU; Sigma Chemical Co., St. Louis, MO) and hCG (7 IU; Sigma). The eggs were treated with hyaluronidase (0.2 mg/ml; Sigma) to remove the cumulus. Mouse sperm were collected by flushing caudae epididymides from male CD-1 mice in Ham's F-10 medium supplemented with BSA (3 mg/ml; Sigma). Sperm were washed (twice) by pelleting in a microcentrifuge set at low speed (500 x g). The sperm (1 x 106/ml) were placed in a 100-µl drop under oil and capacitated at 37°C for 1 h in a humidified atmosphere of 5% CO2 in air in the presence of anti-rFA-1 antibodies or control immunoglobulins. Cumulus-free unfertilized eggs (n = 8–12) were transferred to the 100-µl drop containing capacitated sperm and incubated for another 1 h. At the end of the culture period, the eggs with associated sperm were transferred to fresh medium and counted. The number of sperm associated with zonae pellucidae of eggs was determined by counting the number of sperm bound in one plane of focus. The experiment was repeated 3–4 times on different days using sperm and eggs from different mice.
IVF was performed on zona-intact eggs as described previously [16, 19, 24]. Epididymal sperm were collected by flushing cauda epididymidis with 300–500 µl of Toyoda's medium [16, 19, 24). Sperm (4000 motile sperm) in 90 µl of Toyoda's medium were mixed with 10 µl of PBS containing various antibodies, under oil. Eggs in a cumulus mass collected from superovulated CD-1 mice (as described above) were added to the fertilization drop and incubated at 37°C in a humidified atmosphere (5% CO2 in air) for 6–8 h. After the end of incubation period, the cumulus cells were removed with hyaluronidase. The eggs were then fixed in 1% paraformaldehyde at 4°C, stained with lacmoid in 45% acetic acid, destained with 20% acetic acid and 20% glycerol, and mounted. Eggs were considered fertilized if two of the following criteria were met: 1) two pronuclei were present, 2) sperm tail was present in the cytoplasm, or 3) the second polar body with condensed chromatin was present.
Tissue Specificity
Tissue specificity of the rFA-1 antibodies was determined by Western blot procedure. Ten tissues, namely testes, kidney, liver, intestine, spleen, muscle, heart, lung, brain, and ovary, were collected from mice (n = 2-3) and then homogenized in 0.1% Triton X-100 and centrifuged (3000 x g, 30 min). The supernatant was collected and acetone precipitated, and the precipitates were dissolved in PBS, aliquoted, and kept at -70°C until use. The protein extracts (~40 µg/lane) were run in nonreduced slab SDS-PAGE (5-15% gradient gel) and transferred to nitrocellulose membrane; the blot was incubated with the anti-rFA-1 antiserum/control serum (10 µl/ml), and the reacted bands were detected as described above. The procedure was repeated five times using antiserum from five different mice immunized with rFA-1 antigen.
Statistical Analysis
Significance of difference in Tables 1 and 2 was analyzed by using unpaired Student's t-test. The correlation between the antibody titer (SD units) and fertility (number of babies born) was analyzed by linear regression. A p value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the animals immunized with rFA-1 antigen in trial I (n = 8) and trial II (n = 8) developed circulating anti-rFA-1 antibodies (Fig. 2). Some of the animals responded better in trial II in comparison to those in trial 1. The peak antibody titers in both trials were observed at 48-50 days postimmunization. In trial I, the titers varied from SD units 10 to 17, and in trial II the titers varied from 11 to 32. In trial II, in which the animals were kept for a longer time to examine the reversibility of the immune response, the antibody titers started decreasing from the 101st day, and by the 255th day none of the immunized animals demonstrated any positive (> 2 SD units) antibody titer. None of the animals in the control group showed a positive reaction with the antigen, indicating lack of anti-rFA-1 antibodies.
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Active immunization with rFA-1 antigen caused a significant (p < 0.0001) reduction in the fertility of mice in both trials (Table 1). There was an overall 64% reduction in fertility in trial I and a 70% reduction in trial II, compared to values in the respective control groups, using a 25-µg dose of rFA-1 antigen for immunization. All the animals in the immunized group demonstrated some degree of reduction in fertility ranging from 100% to 26% compared to control values. There was a significant (p = 0.025) correlation (r = 0.76) between the reduction in fertility and the circulating rFA-1 antibody titers. However, there were a few animals that had high antibody titer but showed only a partial reduction in fertility (e.g., mouse #12: SD units, 23; babies delivered, 4) in comparison to other animals that had relatively lower antibody titer but showed a complete block of fertility (e.g., mouse #3: SD units, 18; babies delivered, 0). To examine whether the antibodies developed in these animals were equally potent in inhibiting fertility, sperm-zona binding and IVF assays were performed. Ten micrograms of affinity-purified serum antibodies from each of these animals was equally potent (p < 0.001) in inhibiting sperm-zona interaction (Fig. 3) and murine IVF (Table 2); the antibodies from mouse #3 were slightly more effective, but the difference was nonsignificant (p > 0.05). Similar treatment of eggs, instead of sperm, with the antibodies prior to their coincubation did not effect sperm-zona binding and the fertilization rates in IVF, indicating the sperm specificity of the antibodies.
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On the Western blot that had protein extracts from 10 murine tissues, the anti-rFA-1 antibodies from immunized mice (Fig. 4A), and not the immunoglobulins from the PBS-control mice (Fig. 4B), specifically reacted with a protein band of ~47 kDa (dimeric form of cognate FA-1 antigen) only in the testes lane (Fig. 4A, lane a). The rFA-1 antibodies did not recognize this band in any other tissue (Fig. 4A, lanes b–j). The band observed in the testes lane was specific, since it was not recognized by the immunoglobulins from the PBS-control mice (Fig. 4B, lane a). Five different blots were tested with the use of antibodies from five different mice immunized with rFA-1 antigen, and antibody from each of these immunized mice demonstrated similar reactivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant FA-1 antigen was immunogenic in mice and raised an antibody response in all 16 mice tested in the two trials. The circulating immune response was long lasting. By Day 181 postimmunization, all 8 animals in trial II still had positive antibody titers, and by the 255th day the titers had decreased to the levels observed in PBS-control animals. The rFA-1 antibodies were measured using the heavy- and light-chain-specific second antibody that reacts with all the classes of primary antibodies. Thus, the antibody response described here includes all the classes/subclasses of immunoglobulins.
Active immunization with rFA-1 antigen caused a significant reduction in fertility. Using 25 µg of the purified recombinant antigen, the antibody titers raised were enough to cause an overall 64–70% reduction in fertility as compared to that of controls. These findings are in agreement with previous data published using the native FA-1 antigen. Besides demonstrating a reduction/block in fertilization in vitro in various mammalian species including mice, cattle, monkeys, and humans [16–19, 27–29], active immunization with cognate FA-1 antigen inhibits fertility of female rabbits [30]. The immunocontraceptive effect observed with rFA-1 antigen was reversible. When the antibody titers reached control levels at approximately 9 mo postimmunization, these animals delivered healthy babies without any effect on the litter size.
Overall, there was a significant linear correlation (r = 0.76) between the antibody titer and the reduction in fertility, indicating that animals having higher circulating antibody titer bore a lower number of babies. However, the correlation was not perfectly linear. There were animals that despite having high titers showed a lesser reduction in fertility compared to other animals that had relatively lower antibody titers. In the present study, we did not examine the local antibody response in the genital tract, where fertilization takes place. Although systemic immunization with sperm antigens has been shown to induce both the circulating and the local immune response, findings from other studies suggest that it is the local immune response that is more correlative and thus predictive of antifertility effect (reviewed in [9, 10]). The cumulative data from various studies, however, indicate that both systemic and local immune responses are important for immunocontraception [9, 10]. An equal amount (10 µg) of circulating antibodies from animals showing a complete (#3) or partial (#12) inhibition of fertility demonstrated an equally potent effect in sperm-zona binding and IVF assays. These data indicate that there are additional factors besides circulating antibodies, probably local immune response, that are involved in affecting fertility after active immunization with the sperm antigens.
To examine the mechanism(s) of inhibition of fertility in vivo, the antibodies were tested for their effect on sperm-zona binding and within IVF procedure. The antibodies almost completely blocked sperm binding to zona pellucida and fertilization of oocytes in vitro. The antibodies neither agglutinated nor immobilized sperm; thus there was no apparent effect of the antibodies on sperm motility. A previous study [19] showed that the rFA-1 protein recognizes specifically the ZP3 component of the zona pellucida of murine oocytes. These results indicate that the FA-1 antigen is a complementary receptor involved in binding of the sperm cell with the zona pellucida of the oocyte. Similar results have been observed with the native human FA-1 antigen for binding to the ZP3/zona pellucida of the human oocyte [29]. Thus, the antibodies seem to inhibit/block sperm-zona receptor recognition and binding, resulting in fertilization failure.
The immune response generated after immunization with rFA-1 was tissue specific. Anti-rFA-1 antibodies from immunized mice specifically recognized rFA-1 antigen and the cognate FA-1 antigen of 47 kDa (dimeric form) in murine sperm and testes extracts. The antibodies did not recognize the specific band of 47 kDa corresponding to FA-1 antigen in extracts of other tissues tested, indicating that these tissues do not have expression of FA-1 antigen at the protein level. Previously, using multiple-tissue Northern blot and reverse transcription-polymerase chain reaction procedure, it was found that no tissue except the testis showed FA-1 antigen expression at the mRNA level [19]. Similar sperm/testes-specific reaction patterns have been observed using monoclonal and polyclonal antibodies against the native FA-1 antigen [16–18, 30].
In conclusion, our findings indicate that active immunization with rFA-1 antigen induces a testis/sperm-specific antibody response in female mice that causes a reduction in fertility by mechanism(s) involving an inhibition of the fertilization process. The immunocontraceptive effect is long lasting and reversible; with the disappearance of antibodies there was a total regain of fertility. The utility of a sperm antigen in the development of a contraceptive vaccine is contingent upon its sperm specificity, upon involvement in the fertilization process, and upon raising an immune response that is capable of inhibiting fertility in a reversible manner. Recombinant FA-1 antigen fulfills all these criteria. In addition to these parameters, the involvement of rFA-1 antigen in human immunoinfertility makes it an attractive candidate for immunocontraception.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rajesh K. Naz, Division of Research, Department of Obstetrics and Gynecology, Richard D. Ruppert Health Center, 3120 Glendale Avenue, Toledo, OH 43614. FAX: 419 383 4473; rnaz{at}gemini.mco.edu
Accepted: June 24, 1998.
Received: May 7, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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