Targets For Immunocontraception Research Articles

Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility

Regulatory T cells (Tregs) modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, we show that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. We found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of antisperm antibodies (ASA). In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects. Deciphering these immunoregulatory mechanisms may help to design new strategies to treat male infertility, as well as to identify potential targets for immunocontraception.

Region-specific transcriptomic and functional signatures of mononuclear phagocytes in the epididymis.

In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these 'intraepithelial' MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput-corpus and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in 'dendritic cells' or 'macrophages'. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.

Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins’ functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 β subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen “M6” displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Urokinase-type plasminogen activator: a new target for male contraception?

Urokinase-type plasminogen activator (uPA) is closely related to male reproduction. With the aim of investigating the possibility for uPA as a potential contraceptive target, in the present work, Kunming male mice were immunized by human uPA subcutaneous injection at three separate doses for 3 times. Then the potency of the anti-human uPA antibody in serum was analyzed, and mouse fertility was evaluated. Serum antibody titers for human uPA in immunized groups all reached 1:10,240 or higher levels by enzyme linked immunosorbent assay, and mating experiments revealed that pregnancy rates and the mean number of embryos implanted after mating declined obviously (P < 0.05) when compared with control groups. However, the mating capacity and reproductive organ weights had no obvious change, and histological analysis of the testes and epididymides also showed normal morphology for immunized male mice. Sperm function tests suggested that the sperm concentration, sperm viability, sperm motility, and in vitro fertilization rate for the cauda epididymis sperm in uPA-immunized groups were lower than those in the controls (P < 0.05). Together, these observations indicated that subcutaneous injection human uPA to the male mice could effectively reduce their fertility, and uPA could become a new target for immunocontraception in male contraceptive development.

Suppression of fertility in adult cats.

Cats are animals with highly efficient reproduction, clearly pointing to a need for suppression of fertility. Although surgical contraception is highly effective, it is not always the method of choice. This is predominantly because it is cost-intensive, time-consuming and irreversible, with the latter being of major importance for cat breeders. This article reviews the use of progestins, scleroting agents, immunocontraception, melatonin, GnRH antagonists and finally, GnRH agonists, in adult male and female cats in detail, according to the present state of the art. By now, various scientific and clinical options are available for the suppression of fertility in adult cats and the decision as to which should be chosen - independent of the legal registration of any state - depends on different facts: (i) feral or privately owned animal? (ii) temporary or permanent suppression of fertility wanted/needed? (iii) sex of the animal? New effective and available methods for hormonal contraception include melatonin implants for short-term post ponement of oestrus in adult queens and slow-release GnRH-agonist implants containing deslorelin (Suprelorin(®) ) for short- and long-term contraception in male and female companion and breeding cats.

Evaluation of Testicular Sperm CRISP2 as a Potential Target for Contraception

Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.

Immunization of male mice with B-cell epitopes in transmembrane domains of CatSper1 inhibits fertility

To evaluate the contraceptive ability of two B-cell epitopes in CatSper1. In vivo study with mice. University laboratory animal service center. BALB/c mice. Two predicted B-cell epitopes in the extracellular part of transmembrane domains and pore region of CatSper1 were synthesized to immunize male mice. Fertility, epididymal sperm function, and the presence of specific antibody in immunized males were investigated. Significant reduction of fertility was observed in mating trial with no evident systemic illness or abnormal mating behavior. Epididymal sperm of epitope-immunized males exhibited impaired ability to fertilize eggs in vitro, and showed sperm agglutination in some animals, while presenting no changes in sperm viability or progressive motility. High titer of antibodies was induced in the sera, and the antibodies' specificity was confirmed. The binding of the antibodies to epididymal sperm of epitope-immunized males was observed. CatSper members could be the effective and viable targets for immunocontraception. These two epitopes in CatSper1 share high identity between mouse and human and may be effective for fertility regulation in humans.

Birth control vaccine targeting leukemia inhibitory factor

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin-6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF-receptor (LIF-R) that span the regions involved in ligand-receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF-R-mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long-lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF-R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF-R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF-R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF-R is also important for implantation and pregnancy in women.

Contraceptive Vaccines Targeting Factors Involved in Establishment of Pregnancy

Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and pre-implantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.

Distribution of Eppin in mouse and human testis

The human Eppin (hEppin) (SPINLW1; Epididymal protease inhibitor) gene was first described and sequenced in 2001, and later identified as an immunocontraceptive target for males in 2004. The expression and function of the mouse eppin (mEppin) gene was first described in 2003, and recent studies have shown that mEppin protein has a similar male contraceptive effect in mice. In this study, we designed a probe to detect mEppin mRNA in frozen sections from the testes of 60-day-old mice as well as in the GC-1, GC-2 and MLTC-1 cell lines, using a hyperbiotinylated oligonucleotide DNA probe. In addition to Sertoli cells, the expression of mEppin mRNA was localized to the spermatogonia, spermatocytes and Leydig cells. Subsequent RT-PCR and Western blot analysis confirmed the results of the in situ hybridization. Immunohistochemistry was performed on corresponding sections of mouse and human testis using an anti-c-kit antibody to detect spermatogonia and an anti-eppin antibody to detect Eppin protein. The expression of mEppin protein was detected in A1-A4 spermatogonia and the expression of hEppin was detected in typeA spermatogonia as well as in the earliest preleptotene spermatocytes. The present study demonstrates that the expression of hEppin is similar to that of mEppin, and is localized to differentiated spermatogonia. Moreover, the findings support the hypothesis that mEppin protein is secreted from Sertoli cells and taken up by spermatogonia.

Identification of novel immunodominant epididymal sperm proteins using combinatorial approach

Functionally immature spermatozoa leave the testis mature during epididymal transit. This process of maturation involves either addition of new proteins or modification of existing proteins onto the sperm domains that are responsible for domain-specific functions. Epididymal proteins are preferred targets for immunocontraception. In an attempt to identify epididymis-specific sperm proteins, we used a novel combinatorial approach comprising subtractive immunization (SI) followed by proteomics. Following SI, sera of mice were used for immunoproteomics, which led to the identification of 30 proteins, of which four proteins namely sperm head protein 1, sperm flagella protein 2 (SFP2), SFP3, and SFP4 are being reported for the first time on sperm. Another group of four proteins namely collagen alpha-2 (I) chain precursor, homeodomain-interacting protein kinase 1, GTP-binding protein Rab1, and ubiquinol cytochrome c reductase core protein II although reported earlier in testis are being reported for the first time in epididymal sperm. Furthermore, seven out of these eight novel proteins could be validated using peptide ELISA. These data are a useful repository, which could be exploited to develop targets for post-testicular immunocontraception or biomarkers for infertility diagnosis and management.

Inhibition of human sperm function and mouse fertilization in vitro by an antibody against cation channel of sperm 1: the contraceptive potential of its transmembrane domains and pore region

To explore the contraceptive potential of the CatSper1 transmembrane domains and pore region in vitro. In vitro study with human sperm and mouse fertilization. Andrology laboratory of an academic research center. Normozoospermia and viripotent BALB/c mice. The specific binding of an anti-CatSper1 IgG antibody (H-300) to CatSper1 was confirmed by Western blot and immunofluorescence. Sperm from humans and mice were incubated with H-300. The effects of H-300 on human sperm progressive motility, abnormal acrosome, hyperactivated motility, and mouse in vitro fertilization rates were analyzed. A significant decline in sperm progressive motility was observed after 1, 2, and 4 hours of incubation with H-300; the change was mainly ascribed to the decline of fast progressive motility. Significant inhibition of the hyperactivated motility was observed after 5 hours of incubation with H-300. The incubation of mouse sperm with H-300 before insemination reduced the in vitro fertilization rate to 28% of control levels (72% inhibition). CatSper1 may be a potential target for immunocontraception, and the antibody may be a tool to study the function of ion channels in sperm in which relatively fewer methods can be applied.

Vesicle-associated protein 1: a novel ovarian immunocontraceptive target in the common brushtail possum, Trichosurus vulpecula

Ovarian-based immunological research is currently restricted to proteins of the zona pellucida. This study examined the immunocontraceptive potential of a novel vesicle-associated protein, VAP1, previously isolated from the vesicle-rich hemisphere of the brushtail possum oocyte. Seven female possums were immunized against recombinant glutathione S-transferase-VAP1 fusion protein. Control animals (n=3) received antigen-free vaccinations. Following immunization, regular blood sampling determined the level and duration of immune response. Animals were monitored daily, pre- and post-immunization, to determine estrous cycling activity and the percentage of reproductive cycles yielding viable young. The reproductive tracts and somatic organs of VAP1-immunized (n=7), control-immunized (n=3) and non-immunized (n=5) animals were collected and examined by histology and transmission electron microscopy. VAP1 immunization caused a strong and sustained immune response. Elevated levels of VAP1 antibody binding were detected in sera following initial injections, and immune titers rose as boosters were administered. Immunization had no adverse effect upon animal behavior or body condition. Immunized females demonstrated no major change in annual estrous cycling activity; however, the percentage of reproductive cycles resulting in pouch young decreased significantly (P<0.05) by 40%. Histological and ultrastructural analyses revealed an abundance of lipid-like degradation bodies within the ooplasm of developing oocytes and the cytoplasm of failing uterine zygotes. Active macrophage invasion of enlarged endometrial glands was observed in the uteri of two females. Reproductive tract changes are discussed in relation to observed fertility decline. The results of this study indicate that VAP1 has exciting potential as an immunocontraceptive target for possum control in New Zealand.

The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated

CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

To investigate the distribution of cation channel of sperm 1 (CATSPER1) protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Human ejaculated sperm from normozoospermic donors (n = 12) and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors (n = 12) were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 microg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.

Evaluation of the Contraceptive Potential of brZPCp Vaccine in BALB/c Mice: Their Safety and Efficacy

In this study we have examined the potential ability of Microtus branditi partial ZPC (brZPCp) cDNA sequence (436-1150 nt) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in BALB/c mice using recombinant construction pCR3.1-brZPC(p). ELISA outcome indicated that antibodies could be generated by immunized mice, and IgG titer was increased compared to the control. Immunohistochemistry outcome indicated that antibodies could recognize native ZP in vivo, which in turn, prevented the binding of sperm to ovulated eggs. Antibodies could also recognize recombinant protein expressed by BL21 in vitro, which was confirmed by SDS-PAGE and Western blot. Fertility rate was reduced by 45% compared to the control immunized with pCR3.1. Meanwhile, there was no incidence of significant ovarian pathology in treated mice. This experiment indicates that this vaccine can elicit the specific antibody which binds exactly to the corresponding ZPC. This construction is proved to be immunogenic, and can reduce fertility without obvious oophoritis. The result in this study suggests a potentially important method for controlling population for its safety and easy production.

Intérêts et limites de l’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.

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.

Sperm antigenicity shared in five vertebrate classes

The antigens of spermatozoa are of special interest because of their involvement in immunoinfertility and because they are candidate targets for immunocontraception. Conserved sperm epitopes from divergent species may present a suitable source and an animal model for the investigation of the above factors. We compared spermatozoa from six species of vertebrates belonging to five different classes, in order to demonstrate the existence of shared antigens. We used whole fixed male germ cells from trout, frog, turtle, rooster, rabbit and human in the enzyme-linked immunosorbent assay with rabbit polyclonal sperm-specific antisera. After absorption of the antisera with non-gametic (liver powder) and gametic tissue preparations, we established significant cross-reactions among the spermatozoa of all tested species. The antigenic similarities were the strongest between spermatozoa of trout and frog, trout and human, rabbit and turtle, rabbit and rooster. These data demonstrate the existence of highly conserved epitopes in spermatozoa from divergent vertebrate species belonging to different animal classes, in contrast to structures of the female gametes (zona pellucida) for which antigenicity was found to be limited to the animal class.

Analysis of recombinant mouse zona pellucida protein 2 (ZP2) constructs for immunocontraception

In this study we have examined the potential of recombinant mouse zona pellucida glycoprotein 2 (ZP2) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in outbred Swiss–Webster mice using four ZP2 constructs: Val 35–Gly 200 (ZP2 V35–G200), Val 35–Leu 331 (ZP2 V35–L331), Pro 325–Ala 637 (ZP2 P325–A637), and Val 35–Ala 637 (ZP2 V35–A637). A significant antibody response occurred to three of the four immunogens, however antibodies capable of recognizing native ZP occurred only after immunization with ZP2 V35–A637 and ZP2 P325–A637. Only immunization with ZP2 V35–A637 correlated with a reduction in fertility. Examination of the physiological basis for infertility revealed that: (1) passive transfer of ZP2 antiserum induced infertility in non-immune mice; (2) ovaries of infertile mice appeared histologically normal; (3) infertile mice produced normal numbers of eggs and (4) ZP of ovulated eggs from infertile mice demonstrated a significant reduction in the number of sperm bound compared to eggs from adjuvant controls. Infertility can be caused entirely by ZP2 antibodies without the incidence of significant ovarian pathology. This study also demonstrated that immunization with the bioactive (sperm binding) region of ZP2, recombinant ZP2 V35–G200, did not result in a significant immune response that recognized native ZP or inhibited fertility. Consequently we designed a ZP2–sperm antigen construct, replacing the C-terminal region of ZP2 with Sp17. This construct proved to be immunogenic and reduce fertility while directing the immune response to the Val 35–Gly 200 region of ZP2.