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Evaluation of immunocontraception as a publicly acceptable form of vertebrate pest species control: the introduced grey squirrel in Britain as an example.

We sent out a targeted questionnaire to organizations and private individuals across the UK that have expressed an interest in squirrel management and conservation. Respondents were asked to evaluate shooting, trapping, poisoning and immunocontraception (IMC), according to their perceived efficacy, cost efficiency, and whether they were considered to be humane. The majority of both professionals and enthusiasts indicated support for grey squirrel control to help conserve red squirrels and to reduce economic damage to timber crops. Respondents\' comparative evaluations of current forms of control showed that trapping is the most acceptable method. When IMC is compared with the other methods, it was considered to be more humane and acceptable. In contrast, poisoning was seen as humane or acceptable by the fewest respondents. Furthermore, poisoning elicited the greatest difference in opinion between the professional and enthusiast groups. This difference (34\%) may be interpreted partly as concern over the type of death that results from poisoning and partly as due to the possibility of poisoning nontarget species. Our findings indicate a need for more public information regarding secondary poisoning hazards to other species and their predators. Interest and concern about squirrel control correlated with the overlap between contemporary areas of distribution of the two species. It is these areas where consultation and education programs about control methods should be targeted. This research indicates that there would be support for grey squirrel control using IMC from both lay and professional interest groups. It demonstrates the existence of a sound basis for constructive dialog that can lead to the design and implementation of acceptable and efficient control strategies.

Animals↗

The effect of active immunity against placental proteins on pregnancy in monkeys.

Squirrel monkeys were actively immunized against human placental lactogen (HPL) and/or an extract of human placental tissue, or squirrel monkey placenta. Only half of the monkeys immunized with HPL developed a detectable antibody titer to HPL prior to the subsequent mating season. During the subsequent breeding season, the pregnancy rate was cut in half. The titer developed against HPL did not seem to be related to their ability to become pregnant. When the group of animals which had previously been immunized with HPL were challenged with extracts of human placenta, all animals developed detectable circulating antibody. There was no increase in the effectiveness of this immunization as compared to HPL in pregnancy prevention, however. A third group of monkeys were immunized with extracts from their own placental extracts, and all developed some degree of antibody. The titer obtained did not seem to be related to whether or not they became pregnant. In this group, however, the pregnancy rate was decreased to one fourth of what it had been before immunization.

Animals↗

Analysis of menstrual records of women immunized with anti-hCG vaccines inducing antibodies partially cross-reactive with hLH.

Menstrual data of 13 control subjects and 88 subjects immunized with three beta-hCG-based vaccine formulations were analysed. Immunization did not change the menstrual regularity; bleeding days were normal (3-7 days) and 89% of the menstrual cycles were within the normal range of 22-35 days. Irregular (short or long) cycles were observed in both immunized and control groups. These were, however, unrelated to prevailing anti-hCG antibody titres or to cross-reactivity of antibodies with hLH.

Chorionic Gonadotropin↗

Phase I clinical trials with three formulations of anti-human chorionic gonadotropin vaccine.

Comparative phase I clinical trials were carried out in 5 centres with three formulations of beta-hCG-based vaccines inducing antibodies against human chorionic gonadotropin. The objectives of these trials were to determine their relative immunogenicity, duration, reversibility and safety. A total of 116 tubal ligated women volunteers were enrolled in the study and 101 subjects were followed-up for one year or more until the antibody titres declined to near zero levels. Every woman receiving the vaccine produced anti-hCG and anti-tetanus antibodies. Clinical examination carried out at intervals of 4-6 weeks revealed no abnormality. No serious side effects or adverse reactions were reported with any of the formulations during primary immunization with three monthly injections of the vaccine. Eleven women, however, demonstrated hypersensitivity to test dose at the time of the booster injection. The reaction was to tetanus toxoid; gonadotropin subunits conjugated to another carrier did not evoke any such reaction. Progesterone in bleeds taken at midluteal phase, as well as complete progesterone and estradiol done in two immunized women, indicated normal ovulatory cycles. Immunization with these formulations had no significant effect on haematological, clinical chemistry and other metabolic parameters. In summary, the results indicate that none of the three beta-hCG-based contraceptive vaccines had any adverse effects clinically, on endocrine status and metabolic parameters. Formulations A and B induced comparatively higher anti-hCG titres than M. Thus, further work can be undertaken to study the efficacy of these vaccines in humans for preventing pregnancy.

Adult↗

Antifertility effects in female rats immunized with human seminal plasma inhibin.

Adult female rats were actively immunized with sperm-coating protein (10.4 KDa) human seminal plasma inhibin (HSPI). Following 8 weeks of immunization at 2.5 micrograms and 5 micrograms doses of HSPI with Freund's complete adjuvant, antifertility effects were observed in 63% (5/8) and 88% (7/8) of animals, respectively. Estrous cycle was not affected in experimental group. Thus, the present study suggests the potential use of HSPI for immunocontraception in females.

Animals↗

Characterizations of anti-oLH beta antibodies acting as contraceptives in rhesus monkeys. II. In vivo neutralizing ability for gonadotropic hormones.

63 female rhesus monkeys were actively immunized against the beta-subunit of ovine luteinizing hormone (oLH beta). 75% of these monkeys developed antibodies which were sufficiently high to induce infertility. We report here the in vivo neutralizing potencies of anti-oLH beta antisera from 14 monkeys and the correlation between in vitro and in vivo properties of the antibodies. Anti-oLH beta sera neutralized the biological activity (stimulation of testosterone production in male mice) of both hCG and rhCG. The correlation between antibody titers and neutralizing potency of hCG action of the antisera was significant. rhCG-induced testosterone production was inhibited more effectively than that induced by hCG. There was no correlation between binding affinity to hCG in vitro and neutralizing activity. Neutralizing activities correlated well with binding capacities, suggesting that the number of binding sites of the antibody population may play an important role in the mechanism of neutralization. In rhesus monkeys that were effectively immunized against pregnancy circulating oLH beta antisera did induce shortened luteal phases and impaired luteal function. Successful application of oLH beta as an antigen for contraception in humans may depend on relative cross-reactions of hLH and hCG with anti-oLH beta sera and on a neutralizing capability very similar to the one found in rhesus monkeys.

Animals↗

A monoclonal antibody, EC-1, derived from a syngeneically multiparous mouse alters in vitro fertilization and development.

A monoclonal antibody designated 'EC-1' was derived from a fusion of myeloma cells with lymphoid tissue from a syngeneically multiparous, but otherwise unimmunized, mouse and was selected by screening for reactivity with teratocarcinoma cells. The IgM antibody binds to the cell surface of ova, zygotes, and 2-cell embryos. Binding is not detected on the 4- or 8-cell embryo but reappears on the morula and blastocyst. EC-1 binds to the trophoblast but not to the inner cell mass of in vitro attached blastocysts and the ectoplacental cone of the peri-implantation embryo. In adult tissues, EC-1 binds to the follicular cells of the ovary, the lining epithelium of the pregnant uterus, the interstitial region of the testes and to epididymal but not testicular sperm. In nongonadal tissues EC-1 binds to an epitope located in some, but not all, regions of connective tissues associated with basement membrane. The antigen detected by EC-1, as expressed on teratocarcinoma-derived cell line PYS-2, is a large glycoprotein which is sensitive to reduction. EC-1 inhibits in vitro fertilization and partially inhibits in vitro development of in vitro fertilized ova. The possible implications of EC-1 binding and activity are discussed.

Animals↗

Functional morphology of testis and its excurrent ducts in rats immunized with synthetic luteinizing hormone releasing hormone conjugated to tetanus toxoid.

The effects of active immunization against luteinizing hormone releasing hormone (LHRH) on the testis and its excurrent ducts in rats were studied using a synthetic LHRH peptide conjugated to tetanus toxoid (TT), with or without an adjuvant. The study demonstrated that LHRH-TT given alone did not elicit an appreciable immune response and that its immunogenicity could be enhanced by administration with an adjuvant, such as species-specific IgG or SPLPS. The anti-LHRH antibody response showed a corresponding regressive effect on the morphology of the testis and its excurrent ducts. The functional involution of the testis was reflected in the form of a block in spermatogenesis and the lowering of testosterone production; the basic structural organization of the testicular components was, however, maintained. Epididymal ducts displayed rather drastic atrophic changes, indicating that prolonged immunization may result in irreversible damage to the excurrent ducts and could, therefore, adversely affect the restoration of fertility even after the return of testicular function following decline in the immune response against LHRH. This could be an important consideration if LHRH immunization is to be used for male contraception.

Animals↗

Sperm immobilizing and fertilization-blocking monoclonal antibody 2C6 to human seminal plasma antigen and characterization of the antigen epitope corresponding to the monoclonal antibody.

A monoclonal antibody (Mab 2C6) with strong sperm immobilizing and agglutinating activities was generated by cell fusion between spleen cells from a mouse immunized with human seminal plasma (HSP) and mouse myeloma cells. It also showed a strong inhibitory effect on human sperm-egg interaction. The corresponding antigen was present on the whole surface of ejaculated spermatozoa. In male genital organs, immunostaining with Mab 2C6 was observed in epididymis and seminal vesicle but not in testis. By Western blotting, immunostaining with Mab 2C6 was detected around the 15-25 kDa region under both reducing and non-reducing conditions. The antigen corresponding to Mab 2C6 was susceptible to treatment with periodate or trifluoromethanesulfonic acid. The antigenic activities were slightly increased by treatment with neuraminidase but reduced by further treatment with glycosidases. Enzymatic digestions with pronase and papain also reduced the antigenic activities. The antigen molecules exhibited a strong binding affinity to RCA lectin. These results indicated that Mab 2C6 recognized one of the components which might be secreted from epididymis or seminal vesicle and bind to ejaculated spermatozoa as a sperm coating antigen. The corresponding antigen seems to be a glycoprotein and its carbohydrate moiety has an important role in the conformation of the antigen epitope.

Animals↗

Human spermatozoal FA-1 binds with ZP3 of porcine zona pellucida.

Experiments were carried out to study the binding of the major porcine zona pellucida glycoprotein ZP3 (Mr = 55,000) with human FA-1, a fertilization-related antigen isolated from human male germ cell plasma membranes and with GA-1, a germ cell antigen isolated from murine male germ cell plasma membranes. FA-1 showed a dose-dependent binding with ZP3 in an enzyme-linked immunosorbent assay (ELISA) and by the Western blot procedure. FA-1 also bioneutralized the sperm ligand activity of ZP3 in a boar sperm/porcine zona pellucida attachment assay. In contrast, GA-1 displayed no reactivity for ZP3 using any of these procedures. These results indicate that FA-1 but not GA-1 has zona pellucida binding activity in the porcine system.

Antigens↗

Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters.

Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.

Animals↗

Antifertility effects of antisperm cell-mediated immunity in mice.

C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u. protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u. sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a significant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.

Animals↗

Antibodies to sperm-specific human FA-1 inhibit in vitro fertilization in rhesus monkeys: development of a simian model for testing of anti-FA-1 contraceptive vaccine.

The feasibility of using the rhesus monkey as a non-human primate model for testing the efficacy of a contraceptive vaccine based on FA-1 antigen was evaluated. Affinity-purified anti-FA-1 polyclonal antibodies (Fab' fragments) and anti-FA-1 monoclonal antibody were used as probes in these studies. Anti-FA-1 antibodies (polyclonal Fab' as well as monoclonal IgG) predominantly reacted with the postacrosomal, mid-piece and tail regions of rhesus monkey sperm, as with human sperm, by an indirect immunofluorescence technique (IFT). These antibodies also specifically recognized a single protein band of 51 +/- 2 kDa, corresponding to the dimeric form of FA-1 antigen, on a Western blot of lithium diiodosalicylate (LIS)-solubilized monkey sperm. Anti-FA-1 antibodies, when present in the insemination mixture, inhibited the in vitro fertilization (IVF) of monkey oocytes. These results indicate that FA-1 antigen in rhesus monkey sperm is similar in subcellular localization, molecular identity and function to that in human sperm, and that the rhesus monkey represents a permissible non-human primate model in which the efficacy of a contraceptive vaccine based on FA-1 antigen can be tested.

Animals↗

Identification and characterization of a 27 kDa acrosome protein of human sperm defined by a monoclonal antibody with fertilization-blocking effect.

A monoclonal antibody, MAb 1G1, possessing a strong fertilization-blocking activity was prepared by immunizing a BALB/c mouse with capacitated human sperm in order to study the molecular nature of sperm antigens relevant to fertilization. MAb 1G1 inhibited human sperm penetration into zona-free hamster eggs. It reacted to the apical portion of acrosome-reacted human sperm, but did not react to live sperm before the acrosome reaction as demonstrated by immunofluorescence staining. In paraffin-embedded testis sections, the round spermatids, spermatocytes and spermatozoa were stained with MAb 1G1, but the spermatogonia were not stained. Neither Sertoli cells, Leydig cells nor other somatic tissues were stained. The sperm of Japanese monkey, bull, boar, hamster and mouse were not stained. Therefore, the staining of sperm was species specific. The antigen corresponding to MAb 1G1 showed a band at 27 kDa by immunoblotting. The reactivity of the antigenic component was not destroyed by periodic acid treatment. From the results obtained, it was postulated that the antigenic molecule might be a polypeptide. These results indicated that this MAb might be a useful tool for studying the mechanism of human sperm-egg fertilization and the development of a contraceptive vaccine.

Acrosome↗

Potential contraceptive use of epididymal proteins: evidence for the participation of specific antibodies against rat epididymal protein DE in male and female fertility inhibition.

Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.

Animals↗