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Background Gonadotropin‐releasing hormone (GnRH) plays a pivotal role in regulating the reproductive endocrine system. Objective An immunocontraception vaccine aimed at inhibiting the functions of GnRH is tested as a potential tool for controlling animal populations. Methods We developed a recombinant immunocontraceptive vaccine composed of GnRH‐I and GnRH‐II (GnRH I+II), which was conjugated with Salmonella typhimurium flagellin. Forty‐eight BALB/c mice aged 4 weeks were divided into four groups (each group had n = 12): non‐vaccinated male (NVM), non‐vaccinated female (NVF), vaccinated male (VM), and vaccinated female (VF). Mice in the vaccinated groups were vaccinated twice by intramuscular injection at 0 and 2 weeks with 300 μg of the recombinant GnRH protein complex per mouse. Mice in the non‐vaccinated groups were injected with saline and served as the unimmunized controls. Twenty‐four pairs of male and female mice were mated for 10–12 weeks after initial immunization in four groups: 6 NVF × 6 NVM, 6 VF × 6 NVM, 6 NVF × 6 VM, and 6 VF × 6 VM. Results: An increase (p, We developed a new form of immunocontraceptive vaccine composed of GnRH‐I and ‐II complex conjugated with STF‐2. We provide the vaccine efficacies with higher anti‐GnRH antibodies in mice. The vaccine containing the GnRH‐I + II peptide linked to STF‐2 induced the suppression of fertility by inhibiting testicular and ovarian functions and reducing sex hormone levels in both male and female mice. In addition, the effects of fertility reduction were confirmed through mating experiments .

Background Approximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception. Methods Heavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb [“Human Contraception Antibody” (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model. Findings HCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue. Interpretation HCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception. Funding This research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

The small Indian mongoose (Herpestes auropunctatus) was introduced to Japanese islands and has impacted on the island’s biodiversity. Population control has been attempted through capturing but its efficiency has rapidly declined. Therefore, new additional control methods are required. Our focus has been on the immunocontraceptive vaccines, which act in an especially species-specific manner. The amino-acid sequence of the mongoose ovum zona pellucida protein 3 (ZP3) was decoded and two types of synthetic peptides (A and B) were produced. In this study, these peptides were administered to mongooses (each n=3) and the sera were collected to verify immunogenicity using ELISA and IHC. Treated mongoose sera showed an increasing of antibody titer according to immunizations and the antigen-antibody reactions against the endogenous mongoose ZP. In addition, IHC revealed that immune sera absorbed with each peptide showed a marked reduction in reactivity, which indicated the specificity of induced antibodies. These reactions were marked in peptide A treated mongoose sera, and the antibody titer of one of them lasted for at least 21 weeks. These results indicated that peptide A was a potential antigen, inducing autoantibody generation. Moreover, immunized rabbit antibodies recognized mongoose ZP species-specifically. However, the induction of robust immune memory was not observed. Also, the actual sterility effects of peptides remain unknown, it should be verified as a next step. In any case, this study verified synthetic peptides we developed are useful as the antigen candidates for immunocontraception of mongooses.

Immunocastration (vaccination against boar taint) is an alternative method to prevent boar taint without the need for surgical castration. This study investigates the evolution of boar taint compounds in serum and fat, serum steroid compounds as well as behavior in immunocastrated pigs from 3 sire lines: 15 stress positive Belgian Piétrain (BP), 20 stress negative French Piétrain (FP), and 20 stress negative Canadian Duroc (CD). Hormone and boar taint compounds in serum were determined at 4 time points; boar taint compounds in fat were determined at 3 time points. Behavior, skin lesions, animal and pen fouling were also recorded before the first vaccination (V2). Aggressiveness, eating and drinking and general activity behavior declined from V2 for all sire lines. Pigs from BP were cleaner than FP and CD pigs. Even though immunocastration was effective in general (reduced testosterone, estradiol as well as androstenone in serum) for all sire lines, some individual pigs showed either androstenone or skatole levels in fat above cutoff values. While the immunocastration mechanism works as intended for androstenone, and also for skatole for the three sire lines, the risk of carcasses with boar taint compounds above cutoff levels (respectively 1.9 and 3.7%) still remains to some extent. ispartof: RESEARCH IN VETERINARY SCIENCE vol:124 pages:293-302 ispartof: location:England status: published

The feral pig populations of many countries continue to increase. Scientific studies on population size are scarce, while the numbers of reported observations on presence of and damage caused by feral pigs are increasing. Feral pigs can carry and spread several diseases (including zoonotic), but African Swine Fever (ASF) is of most concern. It is a highly transmissible viral disease associated with an extremely high mortality rate. Since 2009 ASF has appeared in several European countries, with cases being identified first among local feral pigs and consequently in domestic pig production units, indicating a clear linkage with the movement of the feral pig population and the spread of the disease across national boundaries. Control of feral pig populations is currently under discussion. Because massive culling raises questions of animal welfare and ethics, fertility control could represent an important and effective means to control feral pig populations. Contraceptive vaccines have been used with some degree of success in many wild species because they are able to provide a long-term effect without any consequent health problems. However, extensive and efficacious use of vaccines to control feral pig populations is not simple. The aim of this article was to review the progress in immunocontraception use in feral pigs, providing an account of the current status and future perspectives.

An important determinant in the selection of any contraceptive agent is the impact on ovarian function, both in the short and longer term. In this study, ovarian activity was monitored in mares immunised with one of the following vaccine formulations; native porcine zona pellucida (pZP), recombinant zona pellucida proteins ZP3 and ZP4 (reZP), pZP and reZP combined or a commercially available anti-GnRH vaccine. The ZP antigens were prepared in an adjuvant formulation consisting of 6% polymeric adjuvant (Montanide™ PetGel A, Seppic, France) and 500 μg polyinosinic-polycytidylic acid - TLR3-agonist (Poly(I:C) HMW VacciGrade™, Invivogen, USA). A vehicle-only control group was administered the adjuvant formulation without antigen. Ovarian activity was monitored using clinical observations (transrectal palpation and ultrasonography of the reproductive tract) in addition to blood sampling for serum progesterone and anti-Müllerian hormone (AMH) concentrations while employing a low sampling frequency. Treatments and measurements were initiated in December (southern hemisphere summer) and subsequent data collection was performed in January, February, March and May. Both reZP and anti-GnRH vaccination were associated with clinically evident ovarian suppression in the short term. Ovarian activity in mares administered a reZP or anti-GnRH vaccine was significantly different to adjuvant control and pZP treated mares. Serum AMH concentrations were different between pZP and anti-GnRH treated mares 3.5 months after the final vaccination. Serum AMH concentrations were significantly correlated with mare age, serum progesterone and ovarian volume.

Background Stray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis. To control the overpopulation of stray dogs, zona pellucida 3 (ZP3), a primary receptor for sperm, is a potential antigen for developing contraceptive vaccines. To enhance the immune responses and contraceptive effects of canine ZP3 (cZP3), dog gonadotropin-releasing hormone (GnRH) and a T cell epitope of chicken ovalbumin (OVA) were selected to construct two fusion proteins with cZP3, ovalbumin-GnRH-ZP3 (OGZ) and ovalbumin-ZP3 (OZ), and their contraceptive effects were evaluated in mice. Methods The synthesized DNA sequences of OGZ and OZ were cloned into plasmid pET-28a respectively. The fusion proteins OGZ and OZ were identified by SDS-PAGE and Western blot. Mice were immunized with OGZ, OZ and cZP3, and the infertility rates were monitored. Mice immunized with mouse ZP3 (mZP3) or adjuvant alone were used as positive control and negative control, respectively. cZP3- and GnRH-specific antibodies (Abs) were detected by ELISA. The bindings of the Abs to oocytes were detected by indirect immunofluorescence assay. The paraffin sections of mice ovaries were observed under microscope for analyzing pathological characteristics. Results SDS-PAGE and Western blot analyses showed that the two fusion proteins OGZ and OZ were correctly expressed. ELISA results showed that OGZ vaccine induced both cZP3- and GnRH-specific Abs, and OZ vaccine induced cZP3-specific Ab, which lasted for up to 168 days. The levels of follicle stimulating hormone (FSH) and estradiol (E2) in sera were significantly decreased in OGZ immunized mice. Indirect immunofluorescence results showed that Abs induced by cZP3 and mZP3 could bind to the mouse ZP and dog ZP each other. Compared with the adjuvant group, all vaccine immunized groups significantly decreased the fertility rate and mean litter size. Interestingly, the fertility rate in OGZ-immunized group is the lowest, and only 1 mouse out of 10 mice is fertile. Histological analysis of murine ovarian sections indicated that most of the infertile mice in the immunized groups lacked mature follicles as well as accompanied by inflammatory infiltration. Meanwhile, immunization with OGZ decreased the number of corpora lutea in the infertile mice. Conclusions The fusion protein OGZ resulted in the lowest fertility rate and the least mean litter size in the immunized mice. OGZ might be a promising antigen for developing a new contraceptive vaccine for stray dog controlling.

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.

Circulating anti-Müllerian hormone concentration (AMH) is positively correlated to the number of small growing follicles in the mare and may reflect ovarian function. Dynamics of AMH during immunocontraception have not previously been investigated. This study aimed to compare serum AMH in mares following treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 (reZP) or gonadotrophin releasing hormone (GnRH) vaccines, and saline-treated controls. Stored sera collected during two previous studies examining ovarian activity in mares during zona pellucida (ZP) or GnRH immunocontraception were analysed for serum AMH. Data were compared among treatment groups using mixed-effects linear regression and one-way ANOVA with post hoc testing. Correlations between AMH and previously reported clinical variables were estimated using Spearman's rho. Mares immunized against GnRH showed variable but detectable AMH throughout successive breeding and non-breeding seasons that were not significantly different to unvaccinated control mares. Mares treated with pZP demonstrated marked, reversible suppression of AMH. Mares immunized using reZP showed an intermediate effect. In the ZP study, AMH was positively correlated to serum progesterone concentrations, mean ovarian volumes and antral follicle counts, whereas no correlations between AMH and serum progesterone concentrations, mean ovarian volumes, or the presence of one or more follicles ≥20 mm in diameter were detected in the GnRH study. In conclusion, marked suppression of AMH during pZP immunocontraception, but not during GnRH immunocontraception, suggested enhanced suppression of ovarian follicular development and, or follicular function during pZP immunocontraception. Serum AMH concentrations may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception.

The rapidly increasing global population has bowed the attention of family planning and associated reproductive health programmes in the direction of providing a safe and reliable method which can be used to limit family size. The world population is estimated to exceed a phenomenal 10 billion by the year 2050 A.D., thus presenting a real jeopardy of overpopulation with severe implications for the future. Despite the availability of contraceptive methods, there are over one million elective abortions globally each year due to unintended pregnancies, having devastating impact on reproductive health of women worldwide. This highlights the need for the development of newer and improved contraceptive methods. A novel contraceptive approach that is gaining substantial attention is “immunocontraception” targeting gamete production, gamete outcome, or gamete function. Amongst these, use of sperm antigens (gamete function) seems to be an exciting and feasible approach. However, the variability of immune response and time lag to attain titer among vaccinated individuals after active immunization has highlighted the potential relevance of preformed antibodies in this league. This review is an attempt to analyze the current status and progress of immunocontraceptive approaches with respect to their establishment as a future fertility control agent.

The objective of this study was to evaluate the effectiveness of recombinant LHRH fusion protein, Ovalbumin-LHRH-7 (OL), using a single-dose vaccination protocol in combination with different adjuvants in suppressing reproductive functions in buck kids. For this purpose, either a mixture of free OL antigen and encapsulated OL antigen, or encapsulated OL antigen was used. Thirty-nine native buck kids at 12 weeks of age were divided into control (n = 7) and treatment groups (n = 8 bucks/group). The four treatment groups were formed according to the different vaccine formulations: Group CpG received 0.5 mg free OL protein together with 1.0 mg of encapsulated protein with CpG adjuvant. Group mFCA received 0.5 mg free OL protein together with 1.0 mg of encapsulated protein with modified Freund's complete adjuvant. Group IS received 1.5 mg encapsulated OL protein with a mix of inulin and saponin adjuvants. Group ISmFCA received 1.5 mg encapsulated OL protein with a mix of inulin, saponin and modified Freund's complete adjuvants. Scrotal circumference in CpG and mFCA groups were significantly smaller than that of Control, IS and ISmFCA groups (P < 0.05). Numbers and percentage of bucks having spermatozoa in their ejaculate were significantly lower in CpG and mFCA groups (P < 0.05). OL immunization completely suppressed sperm production, except one buck, in CpG and mFCA groups (P < 0.05). These results imply that it is possible to use OL protein in a single injection protocol for the purpose of immunocastration. Further investigation with a larger number of animals should be carried out to determine the longevity of response to a single injection. (C) 2013 Elsevier B.V. All rights reserved.

The producer of vaccine against GnRH recommends that immunocastrated pigs are to be slaughtered within 4 to 6 weeks after the second vaccination (V2). The objective of the study was to examine the effect of shorter or longer delay on steroid hormones, boar taint compounds, and morphologic and histologic traits of reproductive organs. Forty male pigs (individually housed and fed a commercial diet) were assigned within litter to four treatment groups, 10 pigs were left entire (EM27) and the others were vaccinated against GnRH (Improvac, Pfizer Animal Health) at the age of 12 and 19 weeks. Pigs were slaughtered at 21 (IC21), 24 (IC24), and 27 (IC27 and EM27) weeks of age. Two EM27 pigs died during the experiment, one IC21 pig was excluded because of illness, one IC27 pig was a nonresponder, and two pigs (IC24 and IC27) were hermaphrodites. To assess the effect on steroid hormones, blood was taken at 12, 15, 19, 21, and 24 weeks of age. Subcutaneous fat and reproductive organs were sampled after slaughter for determination of androstenone, skatole, morphologic, and histologic measurements. Immmunocastration interrupted the rise of estrogen and caused a substantial fall of testosterone in IC21, IC24, and IC27 pigs. As a result, androstenone and skatole levels were successfully reduced regardless of the time elapsed from V2. The weight of the reproductive organs was also drastically reduced, the shrinkage being proportional to the length of the interval between V2 and slaughter and was the most evident for vesicular glands, followed by bulbourethral glands, and testes. Corresponding changes were observed also on a histologic level with a progressive decrease in the size and number of Leydig cells, a diminishing immunoreactivity of 3β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase, and luteinizing hormone receptor, along with a shrinkage of tubuli seminiferi, atrophy of seminiferous epithelium, and a loss of germ cells, indicating a disruption in testicular spermatogenetic function. Regression of the glandular tissue with a decreasing amount of secreta was also observed for bulbourethral and vesicular glands. The investigated physiologic, morphologic, and histologic traits were progressive with the increasing delay to slaughter (clearly seen already 2 weeks after V2), though no signs of functional or morphological restoration was observed within 8 weeks after V2.

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. Fil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Bluguermann, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Busso, Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

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. Fil: García, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Veiga, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lustig, Livia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Veaute, Carolina Melania Isabel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina

Background Although the physiological effects of immunocontraceptive treatment with porcine zona pellucida (PZP) have been well studied, little is known about PZP's effects on the scheduling of reproductive cycling. Recent behavioral research has suggested that recipients of PZP extend the receptive breeding period into what is normally the non-breeding season. Methodology/Principal Findings To determine if this is the case, we compiled foaling data from wild horses (Equus caballus) living on Shackleford Banks, North Carolina for 4 years pre- and 8 years post-contraception management with PZP (pre-contraception, n = 65 births from 45 mares; post-contraception, n = 97 births from 46 mares). Gestation lasts approximately 11–12 months in wild horses, placing conception at approximately 11.5 months prior to birth. Since the contraception program began in January 2000, foaling has occurred over a significantly broader range than it had before the contraception program. Foaling in PZP recipients (n = 45 births from 27 mares) has consistently occurred over a broader range than has foaling in non-recipients (n = 52 births from 19 mares). In addition, current recipients of PZP foaled later in the year than did prior recipient and non-recipient mares. Females receiving more consecutive PZP applications gave birth later in the season than did females receiving fewer applications. Finally, the efficacy of PZP declined with increasing consecutive applications before reaching 100% after five consecutive applications. Conclusions/Significance For a gregarious species such as the horse, the extension of reproductive cycling into the fall months has important social consequences, including decreased group stability and the extension of male reproductive behavior. In addition, reproductive cycling into the fall months could have long-term effects on foal survivorship. Managers should consider these factors before enacting immunocontraceptive programs in new populations. We suggest minor alterations to management strategies to help alleviate such unintended effects in new populations.

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP(393-408) (htNASP(393-408)), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized with the synthesized peptide was reversible. Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

International audience; In fish, gonadotropin hormones FSH-GTH1 and LH-GTH2 are less specific for their cognate receptors than in mammals. The respective reproductive functions of fish LH and FSH are thus difficult to establish. We aimed to study the effect of specific antagonists of the two gonadotropin receptors on trout sexual maturation in both sexes by targeting specific regions of LH and FSH receptors, Lhr and Fshr. Filamentous phages displaying Lhr specific or Fshr specific decapeptides from the extracellular hormone binding domain were engineered. Recombinant phages were used as receptor-specific antagonistic vaccines. Male and female trouts were immunized with anti-LHR, anti-FSHR, anti-FSHR + LHR or adjuvant alone, through multiple injections over 8-24 weeks, starting at different stages of sexual maturation. The consequences of immunization on gonadal development were evaluated by determining gonad growth, by histological analysis of testis and ovaries at the end of the vaccination period and by measuring blood plasma sex steroids using radioimmunoassay. We show for the first time in fish that the anti-receptor vaccinations could have specific antagonistic effects on the development of the reproductive functions; while the anti-FSHR affected the sexual maturation of prepubertal males and delayed sperm production, the anti-LHR blocked vitellogenesis in females. In maturing males, the combined anti-FSHR+LHR vaccine inhibited spermatogenesis and affected steroidogenesis. In that case, the effects of the vaccine on spermatogenesis were transient and reversible when immunization was stopped. Such an immunological strategy to specifically and transiently inhibit a receptor provides a promising approach for discovering their specific functions; it could also lead to a new technology for controlling the onset of puberty in aquaculture species.

International audience; In response to the need to efficiently control mammal pest populations while avoiding unnecessary suffering, applied and theoretical ecologists have recently focused on virus-vectored immunocontraception (VVIC). So far, modellers have only considered a non-sexual approach (models of sexually reproducing populations without explicitly discerning between the sexes), which appears dubious in view of the sex-specificity of VVIC agents. In this paper, we derive and compare predictions of non-sexual and two-sex models of the spread of a VVIC agent in a host population in order to assess the adequacy of non-sexual models in this context. Our results show that predictions of non-sexual and two-sex models generally diverge and that non-sexual models often fail to predict the control impact of VVIC. We thus recommend using two-sex models, especially if the mating system and life history of the target species are known. Our analysis also shows that female-specific viruses generally give better results than male-specific ones, and suggests that virus choice should focus more on its sterilizing power rather than transmission efficiency. (c) 2007 Elsevier Ltd. All rights reserved.

SUMMARY 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.

Sixteen native ram lambs weaned at 10 wk of age were divided into two groups. Eight animals were immunized against LHRH with a mixture of two fusion proteins: ovalbumin-LHRH-7 and thioredoxin-LHRH-7. The immunized lambs received a primary immunization plus two booster immunizations at 4 and 12 wks. Animals in the control group (n = 8) were not treated. Scrotal measurements and blood samples were taken at 2-week intervals. Beginning at 25 wk of age, semen was collected and sexual behaviour was evaluated on a weekly basis. At 35 and 37 wk of age testes and accessory glands of all animals were subjected to ultrasound scanning. At 37 wk of age animals were slaughtered and testes were evaluated histologically. Serum LHRH antibodies (P < 0.01) were detected in animals of the immunized group which had reduced serum testosterone concentrations (P < 0.01). Testicular development was suppressed in the immunized animals (P < 0.01). Immunized animals exhibited mounting activity 5 wks later than control animals. No mature spermatozoa containing ejaculates were collected from immunized animals. Control animals had moderately echogenic ultrasonographic appearance at 37 wk age, whereas immunized animals had hypoechogenic images. Mean seminiferous tubule diameter in immunized lambs was significantly smaller than that in control lambs. Basal membrane was thickened and hyalinized; there was an increase in peritubular connective tissue. No proliferating spermatogonia or mature spermatozoa were present in the tubules in these animals. There were no differences in the ultrasonographic appearance of prostate and vesicular gland between control and immunized animals. The LHRH recombinant fusion proteins were effective in immunological castration in ram lambs when started at 10 wk of age as noted by differences in serum testosterone, testicular histology and ultrasonographic appearance of testis and weight of accessory sex glands. Determining the effects of immunization on ultrasonographic appearance of the testis related to time after immunization requires further investigations. © 2004 Elsevier B.V. All rights reserved., 2001-ZF-043, This study was supported by Yüzüncü Yıl University Research Fund (Project no.: 2001-ZF-043). The authors wish to thank Dr. Serhat Arslan for statistical advising and Jennifer Hernandez for editing the manuscript.

Various forms of birth control have been developed for women; however, there are currently few options for men. The development of male contraceptives that are effective, safe, and reversible is desired for family planning throughout the world. We now report contraception of male nonhuman primates ( Macaca radiata ) immunized with Eppin, a testis/epididymis-specific protein. Seven out of nine males (78%) developed high titers to Eppin, and all of these high-titer monkeys were infertile. Five out of seven (71%) high–anti-Eppin titer males recovered fertility when immunization was stopped. This study demonstrates that effective and reversible male immunocontraception is an attainable goal. This method of immunocontraception may be extended to humans.

Antisperm antibodies (ASA) may be a reason of infertility in some individuals. They may affect pre- as well as post-fertilization stages of the reproductive process. There is ongoing progress in the identification of sperm antigens related to fertilization. The employed methods for this purpose include recombinant DNA technology and the most advanced proteomic analysis. This paper enlists the different approaches undertaken in order to identify and characterize the immunoreactive sperm antigens. We have mainly focused on those, which have been already studied in regard of their immunocontraceptive potential, although it has been impossible to include all published data concerning the topic in a single article. Few novel sperm auto- and isoantigens, discovered recently, have also been reviewed even if their role in fertilization has not been yet established.

The epitopic core sequences recognized by three monoclonal antibodies raised to chicken riboflavin carrier protein (RCP) were mapped to the C-terminal tail-end of the protein using the pepscan method A 21-residue synthetic peptide corresponding to residues 200-219 of the protein and comprising the regions corresponding to the antibodies was synthesized. Administration of polyclonal antibodies specific to this peptide led to termination of early pregnancy in mice. Also, active immunization of rats with the peptide-purified protein derivative conjugate inhibited establishment of pregnancy. These results demonstrate the functional importance of the C-terminal 200-219 region of chicken RCP. Copyright (C) 1996 Elsevier Science Ltd.

The demand for an effective and low cost means of fertility control of domestic animals has raised interest in the development of contraceptive vaccines. A promising candidate for a vaccine component is the brain peptide gonadotropin releasing hormone (GnRH), which plays a central role in the regulation of reproductive functions in vertebrates. Neutralization of GnRH by vaccine-induced antibodies is expected to prevent the reproductive activity in a wide range of species. A GnRH-protein conjugate was prepared by means of recombinant DNA technology. The oligonucleotides encoding GnRH were inserted in hypervariable regions of the subunit gene of P-fimbriae of Escherichia coli. Hybrid fimbriae encoded by the manipulated fimbrial gene clusters were expressed efficiently on the cell surface of Escherichia coli. Vaccination of female rats and bull calves with purified hybrid GnRH-fimbriae strongly affected the reproductive characteristics. We conclude that P-fimbriae represent a very attractive carrier system for application of GnRH in a new type of vaccine.