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Manipulation of the fertility of marsupials for conservation of endangered species and control of over-abundant populations.

Marsupials present a dichotomy in population management; the numbers of many Australian marsupial species have declined due to loss of habitat, competition from introduced herbivores and predation by introduced carnivores, but other species have become locally overabundant in Australia or are introduced pests in New Zealand. The manipulation of reproduction offers the means to increase or decrease productivity; however, considerable fundamental research is required before reproductive technologies can be applied to marsupials. Marsupials differ from eutherian mammals in several aspects of their reproduction including sex differentiation, gamete function and endocrinology, as well as in the relative lengths of gestation and lactation. Although these differences present unique problems in the application of reproductive technologies to marsupials, they also present unique opportunities for marsupial-specific fertility control. This paper summarises the assisted breeding technologies currently being applied to marsupials including superovulation, artificial insemination, in vitro fertilization and gene banking; unique marsupial targets for contraceptive intervention including gamete production, sperm capacitation, gamete surface antigens and embryonic development; and some options for the delivery of contraceptive vaccines to marsupial populations.

Animals↗

Unconventional vaccine targets. Immunization for pregnancy, peptic ulcer, gastric cancer, cocaine abuse, and atherosclerosis.

Vaccine technology can be applied to targets of intervention that currently have not been considered preventable by immunization. Targets include some diseases caused by, or related to, infectious agents, and other conditions clearly unassociated with disease pathogens. This article considers vaccines for pregnancy, peptic ulcer disease, gastric cancer, cocaine abuse and atherosclerosis.

Animals↗

[Interests and limits of immunocontraception].

Recent studies from WHO indicated that a large proportion of human contraceptive needs cannot be covered by the already existing means for different reasons (medical, economical, political, and cultural). Therefore, development of new effective methods targeting birth control methods affordable by under-privileged populations turns out to be necessary. Over the last 20 years, a large number of strategies have been used for contraceptive vaccines and thus multiple antigens have been identified as potential targets for immunocontraception. Nowadays, the most acute researches are based on suppression of the secretion and the activity of gonadotropic hormones (GnRH, LH/hCG, FSH) or the targeting of antibodies specific to sperm surface (RSA-1, SP10, SP17, TCLe-1, PH-20) and oocyte antigens (ZP1, ZP2, ZP3). We developed a contraceptive vaccine against FSH receptor. Adult male monkeys (Macaca radiata) were immunized with filamentous phages displaying at their surface N-terminal peptides of the FSH receptor. Long term male contraception has been achieved without any alteration of circulating testosterone levels, sexual behaviour or of any other discernable metabolic changes. Interruption of vaccination resulted in full recovery of sperm production and male fertility. Contraceptive vaccines are aimed to block an essential step in the reproductive process. From this point of view, efforts have to be focused on the challenge to raise is to ally our knowledge on reproductive physiology and protein biochemistry for a better understanding of the target antigen's function.

Animals↗

Can epitope-focused vaccines select advantageous immune responses?

One of the great success stories of preventive medicine is the achievement of protection against many pathogens by the simple procedure of vaccination. The rationale behind vaccination is to generate a protective immune response and an expanded population of memory cells ready to encounter the infectious agent, which will then elicit a potent secondary immune response. However, the development of effective vaccines against many pathogens has, so far, been unsuccessful. Many vaccines in current use fail to direct the immune response towards the epitopes that will ensure optimal protection. In these circumstances, can vaccines be produced that focus the immune system in a calculated, epitope-specific manner?

Animals↗

Transmission of two Australian strains of murine cytomegalovirus (MCMV) in enclosure populations of house mice (Mus domesticus).

To control plagues of free-living mice (Mus domesticus) in Australia, a recombinant murine cytomegalovirus (MCMV) expressing fertility proteins is being developed as an immunocontraceptive agent. Real-time quantitative PCR was used to monitor the transmission of two genetically variable field strains of MCMV through mouse populations after 25% of founding mice were infected with the N1 strain, followed by the G4 strain 6 weeks later. Pathogen-free wild-derived mice were released into outdoor enclosures located in northwestern Victoria (Australia). Of those mice not originally inoculated with virus, N1 DNA was detected in more than 80% of founder mice and a third of their offspring and similarly, G4 DNA was detected in 13% of founder mice and in 3% of their offspring. Thus, prior immunity to N1 did not prevent transmission of G4. This result is promising for successful transmission of an immunocontraceptive vaccine through Australian mouse populations where MCMV infection is endemic.

Animals↗

Evaluation of two sperm antigens, rSMP-B and YWK-II, as targets for immunocontraception.

To determine whether sperm membrane components, rSMP-B and YWK-II, are suitable candidates as immunocontraceptives in humans, antifertility activities of the antibodies to the peptide fragments, rSMP-229 and rSMP-230 of rSMP-B and YAL-198 of YWK-II, were examined. In a previous report, anti-rSMP-230 antibody was shown to immobilise human sperm and to block human fertilisation, and the antigen (rSMP-230) to interact with antisperm antibodies found in sera of infertile women. Antibody to the second synthetic peptide, rSMP-229, corresponding to a different segment of rSMP-B, mimicked the biological activities of the anti-rSMP-230 antibody. Anti-YAL-198 antibody significantly, although weakly, inhibited human fertilisation. In the murine model, the anti-rSMP-B antibodies blocked in vitro fertilisation of mouse eggs but had no influence on embryo growth. Anti-YAL-198 antibody, however, arrested the growth of zygotes. In conclusion, rSMP-B, a human sperm protein, is a promising candidate in the development of an immunocontraceptive for human application. A second sperm protein, YWK-II, is effective as an antifertility immunogen in experimental animals.

Amyloid beta-Protein Precursor↗

Infertility in mice induced by the rhesus monkey chorionic gonadotropin beta-subunit glycoprotein (rmCGbeta) using DNA immunization.

The recombinant eukaryotic expression vector pCMV4-rmCGbeta, inserted full-length cDNA of the beta-subunit of rhesus monkey chorionic gonadotropin (rmCGbeta), as DNA immuno-contraceptive against CGbeta glycoprotein, has previously demonstrated the biological expression of rmCGbeta in vitro and in vivo. The plasmid DNA of pCMV4-rmCGbeta was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCGbeta DNA delivery method for BALB/c mice, and the dose of 10 microg should be enough to elicit immune response. With different doses from 10-50 microg, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCGbeta DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 microg of pCMV4-rmCGbeta DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCGbeta DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of beta-subunit of mouse chorionic gonadotropin (muCGbeta) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCGbeta shares 99.6% homology with rmCGbeta and 90.6% with hCGbeta respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCGbeta contraceptive should be further studied as a CGbeta DNA contraceptive. (Mol Cell Biochem 231: 89-96, 2002)

Amino Acid Sequence↗

Biodegradable delivery system for a birth control vaccine: immunogenicity studies in rats and monkeys.

PURPOSE: The purpose of this study was to develop a single administration delivery system for a model birth control vaccine, in order to reduce the need for multiple injections and enhance immunogenicity. METHODS: The immunogen-loaded microspheres were prepared by solvent evaporation method and characterized for loading levels, size distribution and in vitro release kinetics. The microspheres were immunized intramuscularly in wistar rats and bonnet monkeys, and the antibody response was compared to that obtained with the same total dose of the immunogen on alum given at a monthly interval. RESULTS: Results indicated that a single injection of the immunogen entrapped in the microspheres generated a response comparable to that obtained by the same immunogen on alum injected at a monthly interval. The antibodies generated by the microspheres in the monkeys also had a good bioneutralization capacity indicating immunogen integrity during the microencapsulation process. CONCLUSIONS: Biodegradable microspheres served as an effective delivery system for a model immunogen used in this study to reduce the need for frequent immunizations and enhance immunogenicity.

Animals↗

A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Antibodies, Monoclonal↗

Prospect for immunocontraception using the NH2-terminal recombinant peptide of human zona pellucida protein (hZPA).

PROBLEM: The zona pellucida is a feasible candidate for contraceptive vaccine. Our previous study showed that a human zona pellucida (ZPA) recombinant protein produced antiserum that inhibited human in vitro fertilization in rabbits. The objective of this study is to examine whether the human recombinant protein produce fertilization-blocking antiserum in a non-human primate. METHOD OF STUDY: The amino terminal regions consisting of pig (198 amino acids) and human (206 amino acids) zona pellucida proteins were prepared by Escherichia coli to produce antisera in bonnet monkeys (Macaca radiata). Antibody production was assessed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). Fertilization-blocking test was carried out using human oocytes that had failed in intracytoplasmic sperm injection (ICSI) treatment under patient's informed consent. RESULTS: Both antisera against pig and human recombinant proteins recognized native human zona pellucida by immunofluorescent staining. However, anti-recombinant human ZPA reacted to native pig zona protein more strongly than pig zona pellucida by ELISA. When human oocytes were treated with antisera before insemination, anti-human ZPA antiserum inhibited human sperm binding to human zona pellucida, but anti-pig ZPA antiserum did not. CONCLUSIONS: Bonnet monkey produced fertilization-blocking antibody by immunization with human recombinant ZPA. The homologous or highly similar sequence is effective for developing a contraceptive vaccine when using recombinant peptides.

Animals↗

Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization.

PROBLEM: We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. METHOD OF STUDY: The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay. RESULTS: Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro. CONCLUSIONS: These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.

Acrosome↗

Restricted entry of IgG into male and female rabbit reproductive ducts following immunization with recombinant rabbit PH-20.

PROBLEM: Successful immunocontraception using sperm antigens is dependent on achieving sufficient sperm-specific antibody in the reproductive ducts to prevent fertilization. The blood: luminal barrier of the male and female reproductive ducts must be overcome for this to occur. We have, therefore, investigated the relative titers of antigen-specific immunoglobulin G (IgG) in luminal fluids collected from male and female rabbit reproductive ducts following immunization with recombinant rabbit PH-20 (rPH-20). METHOD OF STUDY: Male and female rabbits were immunized subcutaneously with rPH-20 in Freund's adjuvant. Reproductive tract fluids and plasma were collected and assayed for specific IgG by enzyme-linked immunoabsorbent assay (ELISA). Plasma anti-rPH-20 antibodies were tested for their ability to inhibit in vitro fertilization. RESULTS: Plasma rPH-20-specific IgG titers of > 21 x 106 were induced in bucks. Antibody levels in the rete testis and cauda epididymidis fluids were only 0.026 and 0.168% of plasma levels, respectively. Plasma IgG titers were > 30 x 10(6) in does, but antibody levels in free flow vaginal fluid, free flow uterine fluid and free flow oviduct fluid were only 0.016, 0.078 and 0.072% of plasma levels, respectively. Induction of ovulation by administration of human chorionic gonadotrophin (hCG) significantly increased rPH-20-specific IgG only in free flow vaginal fluids. Plasma antibody from immunized rabbits inhibited in vitro fertilization but conception rates following mating of rPH-20 immunized male to untreated female rabbits were not affected. CONCLUSIONS: The IgG antibody entry into the reproductive ducts of both male and female rabbits is restricted to less than 0.2% of levels induced in plasma following subcutaneous immunization. This finding raises doubts about the suitability of rPH-20 to induce immunocontraception in rabbits using strategies optimized for induction of a serum antibody response. Whether mucosal immunization strategies can achieve this remains to be tested.

Animals↗

Immunology in India.

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Allergy and Immunology↗

Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20.

Immunization of male and female animals with extracts of whole sperm cells is known to cause infertility. Also, men and women who spontaneously produce antisperm antibodies are infertile but otherwise healthy. Although the critical sperm antigens are unknown, these observations have led to the proposal that sperm proteins might be useful in the development of a contraceptive vaccine. The guinea pig sperm surface protein PH-20 is essential in sperm adhesion to the extracellular coat (zona pellucida) of the egg, a necessary initial step in fertilization. Here, we report that 100% effective contraception was obtained in male and female guinea pigs immunized with PH-20. Antisera from immunized females had high titres, specifically recognized PH-20 in sperm extracts, and blocked sperm adhesion to the egg zona pellucida in vitro. The contraceptive effect was long-lasting and reversible: immunized females, mated at intervals of six to fifteen months after immunization, progressively regained fertility.

Animals↗