Hum. Reprod. Advance Access originally published online on September 7, 2006
Human Reproduction 2006 21(11):2894-2900; doi:10.1093/humrep/del068
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Immunogenicity study of recombinant human sperm-associated antigen 9 in bonnet macaque (Macaca radiata)
Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India
1 To whom correspondence should be addressed at: Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. E-mail: anil{at}nii.res.in
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Abstract |
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BACKGROUND: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction. METHODS AND RESULTS: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes. CONCLUSION: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.
Key words: bonnet macaque/human sperm/immunogenicity/sperm-associated antigen 9/sperm–egg interaction
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Introduction |
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Around the world, human population is experiencing an unprecedented growth reaching 6.4 billion in 2004, and another 3 billion individuals may be added to the global population over the next half-century. Modern biotechnologies are trying to make intervention into the domain of human reproduction possible through the development of a variety of new methods and products to control fertility. Whereas conventional methods, such as the intrauterine device, the oral contraceptive pill, hormone injections and implants are being used worldwide, proponents of anti-fertility vaccines claim that they offer a wider choice of family planning methods. The development of a contraceptive vaccine is generally based on the principle that antibodies generated against a particular antigen impair the fertilizing capacity of sperm, acting on its motility, cervical mucus penetration and at the level of in vitro gamete interaction. In the preliminary studies involving rodent models, mouse-specific immunocontraceptive peptides have been identified in mouse sperm proteins with key roles in reproduction from sequence comparisons with other species and tested for efficacy as immunocontraceptive antigens. Fertility of female BALB/c mice was reduced after immunization with several peptides conjugated to a carrier protein or in the form of recombinant polyepitopes (Hardy et al., 2004a
). Similarly, immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide encompassing peptides of multiple zona pellucida glycoproteins led to generation of antibodies that reacted with the chimeric peptide, suggesting promising candidate antigen for designing immunocontraceptive (Sivapurapu et al., 2004). Immunization of female cynomolgus monkeys and of female baboons with recombinant human zona pellucida protein induced an immune response that caused infertility in both species without permanent loss of the estrous cycle (Martinez and Harris, 2000). More recently, immunization of male cynomolgus monkeys with human recombinant epididymal protease inhibitor (Eppin), a testis-/epididymis-specific protein, induced an immune response that caused infertility in macaques (O’Rand et al., 2004).
This investigation describes a strong humoral response against rhSPAG9 in female macaques, which resulted in inhibition of sperm–egg interaction. Our previous studies in rodent model also elaborated antibody response to immunization with the pcDNA-hSPAG9 plasmid and rhSPAG9 protein (Jagadish et al., 2006). Furthermore, anti-rhSPAG9 antibodies raised in rats employing alum as an adjuvant revealed the localization of SPAG9 in acrosomal compartment and its relocalization on equatorial segment after acrosome reaction which underlines the credential of SPAG9 to be used as a contraceptive vaccinogen (Jagadish et al., 2005a). In addition, inhibition of human sperm adherence or penetration in zona-free hamster oocyte assay and 100% block of sperm binding with human oocytes was also documented (Jagadish et al., 2005a). These results, therefore, suggest the possible use of alum as an adjuvant to explore the humoral response against rhSPAG9 protein in non-human primates. Non-human primates demonstrate distinct similarities to humans in almost all aspects of their anatomy (Vagtborg, 1963), endocrinology and physiology (Hainsey et al., 1993; Harewood et al., 1999). Moreover, macaques are genetically related to humans, which is evident from the chromosomes and DNA homologies between primates and humans. The significant similarity in genome between these phylogenetically related species testifies to the commonality of the genetic material, and these species are therefore widely employed as animal models for human vaccine development (Sibal and Samson, 2001). In our laboratory, we have characterized SPAG9, which is conserved in macaque (Jagadish et al., 2005b), baboon (Shankar et al., 2004) and human (Shankar et al., 1998). Macaque SPAG9 encodes 712 amino acids having 100% homology to human SPAG9 protein in the N-terminus region from 1 to 610 amino acids and having conserved functional domains, namely JNK binding domain, leucine zipper motif, two coiled-coil domains and a transmembrane domain (Jagadish et al., 2005a,b). The regions of significant homology thus provide important information for the rational design of rhSPAG9 contraceptive vaccine formulations. These studies revealed the conservation of SPAG9 protein in human and non-human primates (Shankar et al., 1998, 2004; Jagadish et al., 2005b) predicting the evolutionary relationships between genomes. Amalgamating the results from previous studies, this investigation was undertaken to evaluate the immunogenicity of rhSPAG9 with alum in female macaques. The results thus obtained from this study would lay the foundation for the future fertility trials in non-human primates.
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Materials and methods |
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Cloning, expression, purification and immunization of hSPAG9
An expression plasmid comprising the prokaryotic expression vector pET28b (+) (Novagen, Madison, WI, USA) and a cDNA encoding a complete open-reading frame (comparable with 111–2410 bp of the published hSPAG9, amino acid residues from 1 to 766) of hSPAG9 were constructed as described earlier (Jagadish et al., 2005a
). The recombinant construct (pET28b-hSPAG9) was expressed in Escherichia coli and purified using Ni-NTA resin [nickel-nitrilo triacetic acid (Qiagen, Hilden, Germany)] according to the manufacturer’s instruction. Before immunization, the identity of the expressed recombinant protein was confirmed by microsequencing with tandem mass spectrometry. For active immunization studies, female bonnet monkeys (n = 4), 7–10 years of age, reared at the Primate Research Centre, National Institute of Immunology, New Delhi, India, were employed for this study as per the guidelines and approval of the Institutional Animal Ethics Committee. Primary immunization in female monkeys was done with rhSPAG9 (500-µg dose) adsorbed on alum along with 1 mg of adjuvant sodium pthalyl derivative of lipopolysaccharide (SP-LPS) followed by two booster immunizations on weeks 4 and 8. Subsequent booster doses were given based on enzyme-linked immunosorbent assay (ELISA) titres. Booster immunizations were done only with rhSPAG9 adsorbed on alum. Blood samples were collected at 15-day intervals for a month after primary and booster immunizations. The collection of blood samples was continued thereafter at monthly intervals until the 3 months. Serum was recovered by centrifugation at 1250 g for 10 min at 4°C and frozen at –20°C until used.
Gel electrophoresis and immunoblotting
For western blotting, protein samples from macaque and human sperm extracts or purified rhSPAG9 were resolved on denatured 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel under reducing conditions and transferred onto nitrocellulose membrane (Immobilon-NC, Millipore) according to the procedure described earlier (Jagadish et al., 2006). Briefly, sperm from human and monkey were isolated from liquefied (37°C, 30 min) semen and subjected to swim-up procedure (Suri et al., 1996) to isolate highly motile sperm populations without contamination of immature germ cells and leukocytes. For preparation of sperm extracts, sperm were washed (X2) with phosphate-buffered saline (PBS) containing 1 mM phenyl methylsulphonylfluoride (PMSF), and the washed sperm pellet was solubilized overnight at 4°C in lysis buffer (20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 µL of PMSF and 10 µg/ml of leupeptine). The lysate was centrifuged, and the supernatant (sperm extract) was collected, aliquoted and stored at –20°C until used. SDS–PAGE gel (10%) was run with 20 µg of each sperm extract and 0.5 µg of rhSPAG9 protein. Then, sperm proteins and recombinant protein were electrophoresed and transferred onto nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane having rhSPAG9 and sperm extracts were incubated with monkey anti-rhSPAG9 antibodies (1:1000 dilution, previously adsorbed with BL21 cells) for 2 h and subsequently with secondary antibody, horseradish peroxidase-conjugated goat anti-monkey IgG (1:5000 dilution, KPL, Gaithersburg, MD, USA) for 1 h at room temperature. Immunoreactive bands were visualized using chromogen 0.05% 3,3-diaminobenzidine (DAB). Neutralization experiment was performed to determine specificity of anti-rhSPAG9 antibody for native SPAG9 protein including rhSPAG9 protein (15 µg/ml) in the incubation with the primary antibody in immunoblotting procedure. The protein content of the sperm extracts and rhSPAG9 was determined by the bicinchoninic acid (BCA) method as described in manufacture’s protocol (Sigma, BCA-1 kit for protein determination) or by UV absorption at 280 nm.
Assay of antibodies
Serum IgG levels were determined by ELISA in microtitration plates (Nunc, Rosaklide, Denmark). The rhSPAG9 was suspended in 50 mM PBS, pH 7.4 and added to microtitration plates (200 ng per well) and incubated at 37°C for 1 h and then at 4°C overnight. After the incubation, wells were washed with PBS. Non-specific binding was prevented by incubation with 3% bovine serum albumin (BSA) in PBS for 2 h at 37°C. After blocking, the plates were incubated with serial dilutions of the macaque serum samples followed by goat anti-monkey immunoglobulins conjugated to horseradish peroxidase (KPL, Gaithersburg) at an optimized dilution of 1:5000 in PBS. Enzyme activation was carried out with 0.05% orthophenylenediamine in 50 mM citrate phosphate buffer, pH 5.0, with 0.06% hydrogen peroxide as the substrate. The reaction was stopped with 50 µL of 5 N H2SO4, and then the absorbance was read at 492 nm with 620 nm as reference filter. Results for serum dilution (1:250) were accepted with the estimated ELISA titres of above the mean (+2SD) of pre-immune titres. The antibody response generated in immunized macaques was represented as the geometric mean of the absorbance of the sera of the individual animals.
Immunoreactivity of anti-rhSPAG9 antibodies
Approximately 20 x 106 sperm from macaque and human were fixed with methanol as described previously (Jagadish et al., 2005a). After fixation, sperm were incubated for 2 h at room temperature with monkey anti-rhSPAG9 antibodies (1:500 dilution). Samples were washed and incubated with goat anti-monkey IgG fluorescein isothiocyanate (FITC) (KPL) for 1 h at room temperature at a dilution of 1:2500. Control samples were prepared by incubating sperm samples with pre-immune or neutralized serum [neutralization experiment was performed by including rhSPAG9 (15 µg/ml) in the incubation with the primary antibody] at the same dilution of 1:500. After washing the sperm samples with PBS, the slides were mounted in glycerol : PBS (9:1) and observed under ECLIPSE, E 400 Nikon microscope (Nikon, Fukok, Japan).
Hamster sperm–oocyte penetration assay
The sperm penetration assay (SPA) test was performed according to the method described by Jagadish et al. (2006). Briefly, swim-up sperm (20 x 106) were pretreated with 100 µg/ml of IgG from monkey anti-rhSPAG9 antibodies (serum dilution equivalent to 1:6000) for 15 min at 37°C in 5% CO2 in air. One hundred microgram/ml of pre-immune IgG was used as negative control. Zona-free oocytes were obtained through digestion with a 0.1% trypsin solution followed by five washes with PBS–HSA. Hamster zona-free oocytes were then placed into a drop of spermatozoa and incubated for 3 h at 37°C in 5% CO2 in air. Following gamete co-incubation, the oocytes were washed with fresh Biggers-Whitten-Whittingham (BWW-HSA medium supplemented with 30 mg/ml HSA) to remove the sperm loosely bound to the zona pellucida, by repeated pipetteing. To quantitate sperm adherent, oocytes were placed between a microscope slide and an elevated cover slip, and the number of sperm bound per oocyte was recorded using fluorescent microscopy (TE 300, Nikon). The assay was repeated four times for each treatment group. Experimental and control group averages were reported as means ± standard error of the mean (SEM).
Statistical analysis
The statistical differences between the antibody titres in different weeks in macaques immunized with rhSPAG9 protein were compared and analysed using unpaired and paired Student’s t-test. A P-value of <0.05 was considered significant. Student’s t- test was used to identify differences in the mean numbers of adherent or penetrated spermatozoa.
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Specific antibody response in macaque immunized with rhSPAG9
Earlier, we reported cloning and expression of hSPAG9 protein (Jagadish et al., 2005a
) Recombinant hSPAG9 protein was subjected to microsequencing and the amino acid sequence was confirmed. Immunization of female macaques with rhSPAG9 adsorbed onto alum elicited specific antibodies against rhSPAG9. The antibody titres of individual female macaque are presented in Figure 1. Elevated antibodies titres were observed after the first booster, which subsequently increased after second booster. The comparative analysis of antibody titres revealed that titres from week 8–12 were significantly higher than titres estimated from week 1–4 (Table I; P < 0.001). Lower titres were observed on week 16 compared to week 8–12. Subsequent booster immunization on week 16 resulted in higher antibody response from week 18–24 (Table I). After 28 weeks, the antibody titres revealed declined trends, which were not significantly different to the antibody responses observed between weeks 6–16. A booster immunization was given on week 36 which resulted again in high antibodies titres. The antibody titres remained high and consistent from week 38–52.
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Antibodies generated in response to recombinant protein recognize native SPAG9 in macaque and human sperm
Western blot analysis revealed that serum from immunized monkeys showed reactivity with macaque and human sperm protein of
170 kDa (Figure 2, lane 1, 2), whereas no reactivity was observed with pooled pre-immune serum from immunized macaques (Figure 2, lane 3, 4). The specific reactivity of antibodies was demonstrated by carrying out neutralization experiment including rhSPAG9 protein in the incubation with the primary antibody from immunized macaques. The immunoreactivity with native SPAG9 was completely inhibited in macaque and human sperm protein extracts in the presence of rhSPAG9 protein as shown in the Figure 2, lane 5 and 6. In addition, E. coli expressed rhSPAG9 protein also showed immunoreactivity with the anti-rhSPAG9 antibodies from immunized macaque (Figure 2, lane 7). The pooled pre-immune serum failed to show any reactivity with rhSPAG9 protein (Figure 2, lane 8).
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Localization of SPAG9 on macaque and human sperm using anti-rhSPAG9 antibodies
Indirect immunofluorescence analysis of macaque and human sperm using anti-rhSPAG9 antibodies showed strong reactivity with the acrosomal compartment of the sperm head respectively (Figure 3A and E). The pre-immune serum failed to show any reactivity with the monkey and human sperm (Figure 3C and G). The inhibition of immunofluorescence in the sperm treated with neutralized serum (data not shown) further indicates the specificity of anti-rhSPAG9 antibody with macaque and human sperm.
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Immunobiological effects of anti-rhSPAG9 antibodies
To examine the effect of anti-rhSPAG9 antibodies in sperm–oocyte interaction, we tested the ability of anti-rhSPAG9 antibodies to block adherence or penetration of sperm to zona-free hamster oocytes (Figure 4). The anti-rhSPAG9 antibodies inhibited the human sperm adherence to or penetration in zona-free hamster oocytes in comparison with pooled pre-immune serum. The sperm penetration rate was 72.25 ± 1.60% in pooled-pre-immune serum group. However, the rates decreased significantly after the prior incubation of sperm with anti-rhSPAG9 antibodies from immunized group (3.55 ± 2.11%). Comparing the rhSPAG9 immunized group with pooled pre-immune sera further revealed extremely significant (P < 0.0001) decline in the number of oocytes found positive for sperm adherence or penetration (Figure 4A).
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In addition, the number of sperm adhering to or penetrating zona-free hamster oocytes was also examined, which revealed 13.48 ± 1.11 in pre-immune pooled serum group. A significant block (P < 0.0001) of sperm adhering to or penetrating the oocytes was observed with anti-rhSPAG9 antibodies from immunized animals (1.05 ± 0.25, Figure 4B).
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Discussion |
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Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm–oocyte binding and fertilization, offers an attractive approach to the growing global problem of overpopulation. A number of sperm antigens have been evaluated as targets for contraceptive vaccines in laboratory animals with varying degree of success rates (Suri, 2004
, 2005a,b). Studies on native PH-20 as an immunocontraceptive vaccinogen demonstrated fertility suppression in the female guinea pigs (Primakoff et al., 1988), which, however, failed in mice and rabbit fertility trials (Pomering et al., 2002; Hardy et al., 2004b). Similarly, LDH-C4 showed promising results in mouse but failed to show any contraceptive effect in non-human primates by an independent fertility trial in macaques (Tollner et al., 2002). These studies indicate that it is not necessary that a potential candidate sperm antigen in rodents will generate the similar immunocontraceptive effects in primates. These studies thereby suggest the importance of undertaking immunogenic studies employing those sperm proteins, which demonstrate high homology between its human and non-human primate’s counterparts. For example, the study employing 
subunit of HCG as a contraceptive vaccinogen in macaque encountered difficulties because of limited cross-reactivity between antibodies raised against HCG and other non-human primate HCGs because of differences in the subunit of the hormone (Talwar, 1997), suggesting the importance of sequence homology of vaccinogen counterparts from different biological sources in carrying out fertility trials. In this context, GenBank database searches revealed 100% homology with human protein highly expressed in testis (PHET) (Yasuoka et al., 2003), 94% homology with macaque SPAG9 (Jagadish et al., 2005b), 92.2% with baboon SPAG9 (Shankar et al., 2004), 50% with Mus musculus JIP3 (JSAP1) (Yasuda et al., 1999), 36% with Drosophila melanogaster-Jun/SAPK Sunday driver 1 (Bowman et al., 2000) and 30% with Caenorhabditis elegans coiled-coil protein (Byrd et al., 2001). The sequence homology suggests that SPAG9 gene is conserved through evolution (Jagadish et al., 2005a). Our studies have distinctly indicated that SPAG9 from macaque testis exhibits considerable homology with human SPAG9 (hSPAG9) at DNA and amino acid level (Jagadish et al., 2005b). The similarities in amino acid sequence (Jagadish et al., 2005b,c) and thus potentially conserved function of SPAG9 protein from human and macaque suggest the possible use of macaque as a non-human primate model for assessing the immunogenicity of rhSPAG9 and for future fertility trials.
In this investigation, we demonstrated that the rhSPAG9 vaccinogen was immunogenic in all four monkeys. The antibody response generated against rhSPAG9 was assessed using ELISA. High antibody titres were observed after primary immunization, which further increased consequent to each booster immunization. As evident from the results, 4th and 5th booster maintained the level of antibodies in the serum for longer periods of time indicating the persistence of immunological response against rhSPAG9. Various studies are reported in the literature that demonstrated varying degree of immunogenicity of different sperm proteins based on the nature of antigen, adjuvant, dose and immunization periods. To evaluate the immunogenicity of human LDH-C antigen, female baboons were immunized with 2 mg of LDH-C conjugated to diphtheria toxoid. Booster immunizations administered in weeks 5, 9 and 22 consisted of 1 mg of antigen and 100 µg of adjuvant (O’Hern et al., 1995). The immune response resulted in development of generally very low and highly variable serum antibody titres within the group against human LDH-C. Furthermore, the immunogenicity of LDH-C was evaluated in female macaque by using synthetic peptide (bC5–19) conjugated with tetanus toxoid as a chimeric immunogen (Tollner et al., 2002), which generated moderate to high antibody titres. Another study reported high and sustained antibody titres up to 6 months in the serum as well as in oviductal fluid from female macaques when immunized with 2 mg of recombinant SP-10 in monthly intervals (Kurth et al., 1997). Recently, a study reported sustained high anti-Eppin titres in monkeys immunized with 100 µg of Eppin in squalene (up to 775 days) or complete Freund’s adjuvant (up to 481 days). Subsequent boosters (100 µg) were given on approximately every 3 weeks until day 691 and 488 for both groups, respectively (O’Rand et al., 2004). However, this study demonstrates a considerable merit of inducing a strong immune response against rhSPAG9 (500 µg) adsorbed on alum, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates. The sustenance of high antibody titres for several weeks after 4th and 5th booster immunizations indicates that rhSPAG9 exhibits a considerable amount of immunogenicity with its further possible use in developing contraceptive vaccine.
Another important aspect for evaluating humoral response is antibody reactivity with native protein. The reactivity of immune sera with native SPAG9 was confirmed in Western blot analysis employing macaque and human sperm. As revealed by the Western blotting, anti-rhSPAG9 antibodies reacted specifically with 170 kDa protein band of rhSPAG9 and with native SPAG9 in macaque and human sperm extract. Since the deduced molecular weight of SPAG9 is 83.9 kDa, the 170 kDa molecular mass is apparently because of anomalous mobility of protein as a result of higher aggregates or dimerization of protein. Mass spectrometry analysis further confirmed the amino acid sequence analysed from high-order aggregates of recombinant protein and monoisotopic mass to 83.9 kDa (Jagadish et al., 2005a). In this regard, our earlier studies demonstrated that aggregation tendency in SPAG9 appears to be quite strong even in 8 M urea-PAGE, SPAG9 showed an apparent mobility of 170 kDa (Jagadish et al., 2005a). Subsequently, the specificity of immunoreactivity of antibodies was confirmed by neutralization experiment revealing inhibition of immunoreactivity with native SPAG9 in the presence of rhSPAG9.
In addition, the immunoreactivity of the antibodies was also confirmed in an indirect immunofluorescence assay with macaque and human sperm. The antibodies reacted strongly with the acrosomal compartment of both macaque and human sperm. In functional assay, the zona-free hamster oocyte penetration test demonstrated that monkey anti-rhSPAG9 antibodies significantly inhibited both the adherence and penetration of human sperm in zona-free hamster oocytes. Our earlier studies also demonstrated that the antibodies raised in rats against rhSPAG9 not only recognized acrosomal compartment in human sperm but also significantly inhibited, in vitro, the adherence of human spermatozoa to human oocyte (Jagadish et al., 2005a). Similar observations were observed with the antibodies generated against pcDNA-hSPAG9 plasmid DNA in mice (Jagadish et al., 2006). It is well known that the region of the sperm membrane that makes the initial contact with the oocyte plasma membrane is the inner acrosomal membrane, followed by the equatorial/posterior head region of the membrane (Yanagimachi, 1994). The fusion begins with the sperm head plasma membrane, but only the equatorial/posterior head region actually fuses with the oocyte plasma membrane. Considering that the SPAG9 was associated with anterior acrosomal compartment and to the equatorial region post-acrosome reaction, it is not surprising that anti-SPAG9 antibodies inhibited sperm adherence and penetration.
In conclusion, characterization of immune response to SPAG9 associated with in vitro induction of infertility is essential for making decisions as how best to obtain appropriate contraceptive immune response. On the basis of our results, we have demonstrated that rhSPAG9 is immunogenic and appears to be suitable for use in contraceptive vaccine for human. Active immunization with rhSPAG9 in female bonnet macaque of proven fertility is in progress to study the efficacy of fertility regulation.
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This work was supported by grants from Department of Biotechnology, Government of India and Mellon foundation and CONRAD, USA.
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References |
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Bowman AB, Kamal A, Ritchings BW, Philp AV, McGrail M, Gindhart JG, Goldstein LSB. (2000) Kinesin-dependent axonal transport is mediated by the Sunday Driver (SYD) Protein. Cell 103:583–594.[CrossRef][Web of Science][Medline]
Byrd DT, Kawasaki M, Walcoff M, Hisamoto N, Matsumoto K, Jin Y. (2001) UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport C. elegans. Neuron 32:787–800.[CrossRef][Web of Science][Medline]
Hainsey B, Hubbard G, Leland M, Brasky K. (1993) Clinical parameters of the normal baboons and chimpanzees. Lab Anim Sci 43:236–243.[Web of Science][Medline]
Hardy CM, Clydesdale G, Mobbs KJ, Pekin J, Lloyd ML, Sweet C, Shellam GR, Lawson MA. (2004a) Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20. Reproduction 127:325–334.
Hardy CM, Clydesdale G, Mobbs KJ, Pekin J, Lloyd ML, Sweet C, Shellam GR, Lawson MA. (2004b) Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20. Reproduction 127:325–334.
Harewood WJ, Gillin A, Hennessy A, Armistead J, Horvath JS, Tiller DJ. (1999) Biochemistry and haematology values for the baboon(Papio hamadryas): the effects of sex, growth, development and age. J Med Primatol 28:19–31.[Web of Science][Medline]
Jagadish N, Rana R, Selvi R, Mishra D, Garg M, Yadav S, Herr JC, Okumura K, Hasegawa A, Koyama K, et al. (2005a) Characterization of a novel human sperm associated antigen 9 (SPAG9) having structural homology with c-Jun NH2-terminal kinase interacting protein. Biochem J 389:73–82.[CrossRef][Web of Science][Medline]
Jagadish N, Rana R, Selvi R, Mishra D, Shankar S, Mohapatra B, Suri A. (2005b) Molecular cloning and characterization of a haploid germ cell specific sperm associated antigen 9 SPAG9) from the macaque. Mol Reprod Dev 71:58–66.[CrossRef][Web of Science][Medline]
Jagadish N, Rana R, Mishra D, Kumar M, Ramasamy S, Suri A. (2005c) Sperm associated antigen 9 (SPAG9): a new member of c-Jun NH 2-terminal Kinase (JNK) interacting protein exclusively expressed in testis. Keio J Med 54:66–71.[CrossRef][Medline]
Jagadish N, Rana R, Mishra D, Garg M, Selvi R, Suri A. (2006) Characterization of immune response in mice to plasmid DNA encoding human sperm associated antigen 9 (SPAG9). Vaccine 24:3695–3703.[CrossRef][Web of Science][Medline]
Kurth BE, Weston C, Reddi PP, Bryant D, Bhattacharya R, Flickinger CJ, Herr JC. (1997) Oviductal antibody response to a defined recombinant sperm antigen in macques. Biol Reprod 57:981–989.[Abstract]
Martinez ML and Harris JD. (2000) Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen. J Reprod Fertil 120:19–32.[Abstract]
O’Hern PA, Bambra CS, Isahakia M, Goldberg E. (1995) Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase. Biol Reprod 52:331–339.[Abstract]
O’Rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha Rao A. (2004) Reversible immunocontraception in male monkeys immunized with Eppin. Science 306:1189–1190.
Pomering M, Jones RC, Holland MK, Blake AE, Beagley KW. (2002) Restricted entry of IgG into male and female rabbit reproductive ducts following immunization with recombinant rabbit PH-20. Am J Reprod Immunol 47:174–182.
Primakoff P, Lathrop W, Woolman L, Cowan A, Myles D. (1988) Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 335:543–547.[CrossRef][Medline]
Shankar S, Mohapatra B, Suri A. (1998) Cloning of a novel human testis mRNA specifically expressed in testicular haploid germ cells, having unique palindromic sequences and encoding a leucine zipper dimerization motif. Biochem Biophys Res Commun 243:561–565.[CrossRef][Web of Science][Medline]
Shankar S, Mohapatra B, Verma S, Selvi R, Jagadish N, Suri A. (2004) Isolation and characterization of a haploid germ cell specific sperm associated antigen 9 (SPAG9) from the baboon. Mol Reprod Dev 69:186–193.[CrossRef][Web of Science][Medline]
Sibal LR and Samson KJ. (2001) Nonhuman primates: a critical role in current disease research. ILAR J 42:74–84.[Medline]
Sivapurapu N, Hasegawa A, Gahlay GK, Koyama K, Gupta SK. (2004) Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding. Mol Reprod Dev 70:247–254.[CrossRef]
Suri A. (2004) Sperm specific proteins-potential candidate molecules for fertility control. Reprod Biol Endocrinol 2:10–15.[CrossRef][Medline]
Suri A. (2005a) Contraceptive vaccines targeting sperm. Expert Opin Biol Ther 5:381–392.[CrossRef][Web of Science][Medline]
Suri A. (2005b) Sperm-based contraceptive vaccines: current status, merits and development. Expert Rev Mol Med 7:1–16.[Medline]
Suri A, Chhabra S, Upadhyay S. (1996) Identification of human sperm antigen recognized by serum of an immunoinfertile woman: a candidate for immunocontraception. Am J Reprod Immunol 36:317–326.
Talwar GP. (1997) Fertility regulating and immunotherapeutic vaccines reaching human trials stage. Hum Reprod Update 3:301–310.
Tollner TL, Overstreet JW, Branciforte D, Primakoff PD. (2002) Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titres but does not reduce fertility. Mol Reprod Dev 62:257–264.[CrossRef][Web of Science][Medline]
Vagtborg H. (1963) The Baboon in Medical Research(University of Texas Press, Austin.).
Yanagimachi R. (1994) The Physiology of Reproduction 2nd edn (Raven Press, New York) Mammalian fertilization. In pp. 189–317.
Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma M, Davis RJ. (1999) The JIP group of mitogen-activated protein kinase scaffold proteins. Mol Cell Biol 19:7245–7254.
Yasuoka H, Ihn H, Medsger TA, Hirakata M, Kawakami Y, Ikeda Y, Kuwana M. (2003) A novel protein highly expressed in testis is overexpressed in systemic sclerosis fibroblasts and targeted by autoantibodies. J Immunol 171:6883–6890.
Submitted on October 18, 2005; resubmitted on January 6, 2006; accepted on February 10, 2006.
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  M. Garg, D. Kanojia, A. Khosla, N. Dudha, S. Sati, D. Chaurasiya, N. Jagadish, A. Seth, R. Kumar, S. Gupta, et al. Sperm-Associated Antigen 9 Is Associated With Tumor Growth, Migration, and Invasion in Renal Cell Carcinoma Cancer Res., October 15, 2008; 68(20): 8240 - 8248. [Abstract] [Full Text] [PDF]
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 M. Garg, D. Chaurasiya, R. Rana, N. Jagadish, D. Kanojia, N. Dudha, N. Kamran, S. Salhan, A. Bhatnagar, S. Suri, et al. Sperm-Associated Antigen 9, a Novel Cancer Testis Antigen, Is a Potential Target for Immunotherapy in Epithelial Ovarian Cancer Clin. Cancer Res., March 1, 2007; 13(5): 1421 - 1428. [Abstract] [Full Text] [PDF]
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