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Mammalian zona pellucida (ZP) is composed of three to four glycoproteins, which plays an important role during fertilization. Mutations in the genes encoding zona proteins are reported in women with empty follicle syndrome, degenerated oocytes and those with an abnormal or no ZP further emphasizing their relevance during fertility. Immunization with either native or recombinant ZP glycoproteins/proteins leads to curtailment of fertility in various animal species. Observed infertility is frequently associated with ovarian pathology characterized by follicular atresia and degenerative changes in ZP, which may be due to oophoritogenic T cell epitope(s) within ZP glycoproteins. To avoid ovarian dystrophy, B cell epitopes of ZP glycoproteins have been mapped by using bio-effective monoclonal antibodies. Immunization with the immunogens encompassing the mapped B cell epitopes by and large led to amelioration of follicular atresia. However, their use for human application will require more rigorous research to establish their safety and reversibility of the contraceptive effect. Nonetheless, to minimize human-animal conflicts, ZP-based contraceptive vaccines have been used successfully in the population management of free-ranging animal species such as feral horses, white-tailed deer and elephants. To control zoonotic diseases, attempts are also underway to control the population of other animal species including stray dogs, which acts as one of the major vectors for the rabies virus., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Problem: To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E. coli., Method of Study: Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice., Results: Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP., Conclusion: Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine., (© 2013 John Wiley & Sons A/S.)

Problem: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility., Method of Study: Mice received 50 μg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied., Results: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05)., Conclusion: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility., (© 2012 John Wiley & Sons A/S.)

Problem: Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice., Method of Study: Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization., Result: Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility., Conclusion: These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target., (© 2011 John Wiley & Sons A/S.)

Problems: Study on feasibility of pCXN2-mIzumo as a potential immunocontraceptive antigen., Method of Study: Two groups of mice received 100 microg/mouse plasmids of pCXN2-mIzumo and pCXN2 respectively. RT-PCR Immunofluorescence assay and ELISA were performed to observe pCXN2-mIzumo expression and antibody response in the inoculated mice. Sperm penetration assay and animal mating were employed to detect differences of in vitro fertilization (IVF) rate and mean litter size between the experimental and control groups., Results: Izumo cDNA positive bands were detected in sample from mice immunized with pCXN2-mIzumo. IgG response started to rise at 2 weeks after first boost and reached the highest antibody titers at 2 weeks after third boost of immunization with pCXN2-mIzumo in the experimental mice. In vitro fertilization rate in the experimental group (11.57%) was significantly lower than that in control (36.60%). Significant difference of mean litter size between female experimental and control groups was observed, and there was significant negative correlation between individual anti-serum titers and litter size (r = -0.308, P < 0.05)., Conclusion: pCXN2-mIzumo plasmid possesses appreciable anti-fertility potential.

Problem: An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations., Method of Study: Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments., Results: The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine., Conclusion: One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.

Problem: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies., Method of Study: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro., Results: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract., Conclusion: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.

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.

Problem: The present study was conducted to investigate the oocyte membrane protein(s) that is involved in sperm binding in the mouse, and whether or not it can be used for the development of a contraceptive vaccine., Method of Study: The zona-free oocytes were treated with Triton X-100 and the extract was analyzed for homogeneity in the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after staining with silver nitrate. The appropriate band of 50+/-4 kD, designated as OM antigen, was excised from the gel and used for the immunization. The female rabbits were immunized with the excised band per se and the female mice were immunized with the OM antigen after conjugation to keyhole limpet hemocyanin (KLH). The affinity-purified antibodies were analyzed in the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation procedure, western blot procedure, indirect immunofluorescence technique (IFT), and spermoolemma binding assay. Actively-immunized mice were analyzed for in vivo fertility., Results: The Triton X-100 extract of zona-free oocytes predominantly showed a single protein band of 50+/-4 kD in the SDS-PAGE. Active immunization of female rabbits and of female mice with OM antigen raised high antibody titers (ELISA titer > 1:4096) that specifically recognized the OM antigen in the immunoprecipitation and Western blot procedures, and reacted with the oocyte in the IFT. These antibodies demonstrated a significant (P < 0.05) up to a complete block of sperm-oolemma binding in the in vitro binding assay. Binding of both the acrosome-intact and acrosome-reacted sperm was inhibited. Mice actively immunized with OM antigen also showed a significant reduction in vivo fertility as seen by the 9-day implants in uteri. Preliminary data indicate that the antibodies to OM antigen were tissue-specific and did not react with the specific band in any tissue extract in the western blot procedure., Conclusions: These results indicate that the OM antigen is involved in spermoolemma binding and constitute an attractive molecule that needs further investigation for examining its utility in the contraceptive vaccine development.

Problem: An 80 kDa human sperm antigen (80 kDa HSA) has been identified by western blot technique using serum of an immunoinfertile woman as a probe. The 80 kDa HSA has been subsequently purified from sperm extract and investigated for antifertility effects., Method of Study: The purified 80 kDa HSA was used to immunize adult male and female rats. Rabbit anti 80 kDa HSA antibodies were used for immunofluorescent and immunohistochemical staining to demonstrate the presence of 80 kDa HSA on the sperm and to investigate its tissue distribution. The N-terminal sequence of native 80 kDa HSA and the peptides obtained by its endoproteinase Lys-C was determined., Results: Active immunization of male and female rats with 80 kDa HSA caused infertility in all the immunized animals. Immunofluorescent staining showed its localization on the head region of the human and rat spermatozoa. While immunohistochemical studies showed its localization in the testes and epididymis but not in other somatic tissues. Partial amino-acid sequence analysis showed no sequence homology with any of the known protein in the database., Conclusions: 80 kDa HSA is a promising candidate antigen for immunocontraception as it is characterized and found to cause infertility upon active immunization. specific to spermatozoa and is conserved.

Problem: Reduction of excess numbers of white-tailed deer (Odocoileus virginianus) is a prime example of a potential use for immunocontraception as a means of wildlife population management. Oral vaccination appears to be the most pragmatic way to deliver immunocontraceptive vaccines to free-roaming populations of deer, but there was little, if any, prior evidence that oral vaccination is a viable concept in deer., Method of Study: We used live Bacillus Calmette-Guérin (BCG) in a recombinant form (rBCG), which expressed Borrelia burgdorferi outer surface protein A, to test whether deer vaccinated orally with a specific antigen expressed in a live vector produce detectable antibody titers., Results: The data indicate that oral vaccination of deer with an expressed antigen is feasible, as demonstrated by peak antibody titers to the expressed antigen. Also, peak titers measured by enzyme-linked immunosorbent assay were highest in orally vaccinated deer: 1600 in deer vaccinated by injection and 6400 in those vaccinated orally., Conclusions: The results of this study demonstrate that it is feasible to vaccinate deer orally with a live vector.

Problem: Prototype human chorionic gonadotropin (hCG) vaccines based on natural sources are unsuitable for widespread applications due to their complex manufacturing procedures, cost, and carrier-mediated immune suppression., Method of Study: Wistar rats were immunized with alum-adsorbed CG beta (recombinant), CG beta-TT, and nCG beta (native CG beta)-TT, whereas Bonnet monkeys were immunized with only CG beta. The anti-hCG antibody titre in the sera obtained at different time points were quantified by radioimmunoassay. The sera of Wistar rats were characterized in terms of their affinity to hCG, bioneutralization capacity (by inhibition of hCG-induced testosterone production in Leydig cells), and cross-reactivity with human luteinizing hormone, human follicle-stimulating hormone, and human thyroid-stimulating hormone (by direct binding assays)., Results: Antigen-binding capacities of sera obtained upon immunization with CG beta were 3,080 +/- 943 ng/ml (n = 6) and 3,993 +/- 1,292 ng/ml (n = 4), respectively, in rats and monkeys. The analysis of data revealed that immunization of rats with CG beta produced antibodies comparable to that of CG beta-TT and nCG beta-TT., Conclusion: The study opens up the possibility of producing pure and highly immunogenic CG beta by a recombinant DNA route, as a consistent source of antigen for birth control vaccine.

Problem: Integrins belong to a family of cell adhesion molecules that are present on virtually all cells. The temporal and spatial expression of these important proteins on the human endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation., Methods: Using immunohistochemistry, we studied the expression of 12 different integrins in up to 600 samples of human endometrium throughout the menstrual cycle. Intensity and distribution of staining was determined using the semiquantitative HSCORE, with specific focus on the differences between glandular and luminal expression., Results: We noted that the glandular and luminal epithelium undergo independent alterations in integrin expression throughout the menstrual cycle. Specifically, glandular epithelium express certain integrins only during the window of implantation, while luminal epithelium down-regulate certain integrins during this time. The expression of one integrin (the alpha v beta 3 vitronectin receptor) on both luminal and glandular epithelium coincides with the time of embryo attachment; aberrant expression of this integrin is associated with infertility., Conclusion: It appears that the endometrium is a unique tissue with regard to the number of integrins that undergo temporal and spatial changes during the menstrual cycle. These data may offer new directions for the development of a novel contraceptive approach targeted to the endometrium as well as a better understanding of occult causes of infertility in women.

Autoantibodies to ZP3, a major glycoprotein of the zona pellucida (ZP) with sperm receptor function, can block sperm/oocyte interaction. However, only mice of certain major histocompatibility complex (MHC) haplotype respond to the ZP3 peptide. Moreover, ZP3-specific T cells can mediate ovarian autoimmune disease. A chimeric peptide has been designed that induces antibody to native ZP3 regardless of the MHC haplotype of the inbred mice tested. This results in reduction in fertility that is reversible. Infertility correlates well with ZP antibody titer, and the mice do not develop concomitant autoimmune oophoritis. The vaccine contains (1) a promiscuous foreign T-cell peptide capable of eliciting a T-cell response regardless of the animals' MHC haplotype, and (2) a modified native B-cell peptide of ZP3.

Problem: To develop a method to measure a recombinant sperm protein, SP-10, during scale up and purification for a contraceptive vaccine formulation., Method: A quantitative assay method for the human intraacrosomal protein SP-10 was developed utilizing the format of indirect capture enzyme-linked immunosorbent assay (ELISA). A SP-10 specific monoclonal antibody mAb, MHS-10, was used as the capture antibody. Two recognition reagents, a rabbit polyclonal anti-SP-10 antisera (pAb) and a biotin-labeled mAb, MHS-10, were used as the recognition antibodies, respectively. A SP-10 recombinant fusion protein consisting of 125 SP-10 amino acids linked to glutathione transferase was used as a working SP-10 standard. The coefficient of variance for the assay system using the rabbit pAb was in the range of 0.099 to 0.157, and for the assay system using the biotinylated mAb MHS-10 was in the range of 0.081 to 0.084., Results: Employing biotinylated MHS-10 as the recognition antibody, it was found that the native SP-10 molecule had more than one MHS-10 epitope. The concentration of SP-10 in a pool of human sperm extracts was found to be approximately 1% of the total proteins, assayed by both of the recognition antibody systems., Conclusions: The assay system described is useful to monitor the yield of recombinant SP-10 during scale-up production of the SP-10 vaccine.

Problem: The present study was carried out to see if the anti-idiotypic (anti-Id) antibody (Ab2) to progesterone could mimic the immunogenicity of the steroid hormone by giving rise to antiprogesterone antibodies (Ab3) and whether these antiprogesterone antibodies (Ab3) were biologically active., Method: Twenty virgin female Balb/C mice were actively immunized with the anti-Id antibody (Ab2). The antiprogesterone antibody (Ab3) titres in the serum were determined and the animals were used for fertility studies. In the passive immunization studies Balb/C female mice were injected i.p. with 100 micrograms of anti-Id antibody to see its effect on pregnancy., Results: The actively immunized animals when mated showed 80% reduction in their fertility rate. The duration of infertility (20-121 days) in these animals could be directly correlated with the concentration of antiprogesterone antibody (Ab3). The anti-Id antibody (Ab2) blocked pregnancy in 80% of the passively immunized mice., Conclusion: The studies show that anti-Id antibody to progesterone could mimic the immunogenicity of progesterone and give rise to antiprogesterone antibodies (Ab3). The anti-Id antibody successfully blocked pregnancy in mice both after active and passive immunization.

Problem: Immunization with beta subunit of gonadotropin may elicit antibody formation to endogenous heterodimeric gonadotropin and result in reproductive unresponsiveness. The objectives of this study were to determine if antibodies produced in rats following immunization with human follicle stimulating hormone beta-subunit (hFSH-beta) could bind to and immunoneutralize heterodimeric FSH, and to elucidate the immunoneutralizing epitope., Method: Mature female Sprague-Dawley rats received subcutaneous injections of 10 micrograms of hFSH-beta emulsified in complete Freund's adjuvant, while control animals received only adjuvant. Animals received 10 micrograms hFSH-beta booster injections emulsified in incomplete Freund's adjuvant 2, 4, 11, and 21.5 wk after the initial immunization., Results: Immunization with hFSH-beta produced appreciable antibody titers against human FSH (hFSH) as measured in enzyme-linked immunosorbent assays (ELISA) of immunized rat sera. A more modest titer to rat (rFSH) and no antibody response to rat Luteinizing Hormone (rLH) was observed, thus confirming the specificity of the immune response. Titers against hFSH increased throughout the study. Rat anti-hFSH-beta sera was tested to determine its ability to inhibit binding (immunoneutralizing) of 125I-hFSH to the FSH receptor. Continued immunization resulted in all animals producing immunoneutralizing antibodies. Immunization of rats also resulted in disrupted estrous cycles, but only animals with subsequent titers high enough to completely block binding of FSH to its receptor failed to conceive. In order to assess the immunoneutralizing epitope, antisera were tested in a peptide ELISA. Peptides used in the ELISA corresponded to amino acids that spanned the entire hFSH beta sequence. It was found that antibodies from all rats immunized with hFSH beta bound to amino acids within hFSH-beta 33-53., Conclusions: Collectively, these data suggest that amino acids within hFSH-beta 33-53 are necessary but not sufficient to confer immunocontraception. Amino acids within this linear sequence appear in a variety of epitopes of hFSH-beta and hFSH, only some of which are immunoneutralizing. (Am J Reprod Immunol. 1993; 29:000-000.)

Data is presented on the bioneutralization capacity per unit immunoactivity of 30 serum samples of women immunized with the beta-subunit of human chorionic gonadotropin (beta-hCG) or beta hCG associated noncovalently with the alpha-subunit of ovine luteinizing hormone (alpha-oLH), to form a heterospecies dimer (HSD), which were linked to carriers. The bioassays utilized were inhibition of radioiodinated hCG binding to rat testicular receptors in vitro and the inhibition of hCG induced testosterone production in mice. In both assays, antisera of women immunized with the HSD had a bioactivity to immunoactivity ratio that was about 25% higher, on an average, than antisera of women immunized with beta-hCG, suggesting a better bioneutralization capacity of sera raised by the HSD in women.