The mating game: what happens when sperm meets egg.
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Human and pig ovaries were tested by agar gel diffusion and found to contain several cross-reacting (common) antigens. At least one common antigen was located in the zona pellucida as determined by indirect immunofluorescence. Serum samples from 22 infertile women were tested on pig eggs by immunofluorescence, and six of these samples produced strong and nine produced moderate reactions with the zona pellucida. The autoantibodies may be responsible for infertility in these women.
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Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181(Perth) strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors.
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The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy.
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.
A chromatographically purified preparation of gamma globulin produced against isolated zonae pellucidae of mice was used to immunize mice. A single 2.5 mg dose totally inhibited fertility for a minimum of 11 days.
Ten fertile feral mares and 6 domestic horses (4 fertile mares, 1 infertile mare, 1 gelding) were immunized with heat-solubilized pig zonae pellucidae by 4 injections equivalent to 2000 or 5000 zonae each at 2-4-week intervals and a booster injection of 20,000 zonae 6-10 months after the last of the initial inoculations. The immune response was reflected by high antibody levels as measured by an enzyme-linked immunosorbent assay (ELISA) using immobilized pig zona antigen. In-vivo inhibition of fertility occurred in 12 (86%) of the 14 fertile mares studied and persisted for a minimum of 7 months. Repeated mating of the fertile domestic mares resulted in conception when anti-pig zona antibody concentrations had decreased from initial peak absorbance ratios (greater than 1.0) to relatively lower levels (0.64 or less with one exception). An indirect immunofluorescence assay, revealed a considerably lower cross-reactive antibody titre with horse oocytes as compared to pig oocytes. Clinical, endocrinological and histological analyses of the ovaries and their function following regained fertility after immunization revealed no abnormalities. One mare remained infertile.
Rabbit polyvalent antiserum raised against the solubilized cumulus matrix was a powerful inhibitor of human fertilization in vitro, affecting sperm-zona pellucida interaction. Both sperm binding to, and penetration of, the zona pellucida were severely impaired by the anti-cumulus matrix antiserum, whereas no effects of this antiserum on cumulus matrix solubilization or penetration of zona-free human eggs were evident. Moreover, the anti-cumulus antiserum partly neutralized the acrosome reaction-inducing activity of the cumulus matrix. These results warrant further research into the functional specificity of different cumulus matrix components and into the effects of the respective antibodies on reproductive function as a possible lead to a new approach to contraceptive vaccine development.
Mice were passively immunized over 6 weeks with contraceptive doses of an anti-cumulus oophorus antibody preparation. The persistence of oestrus, accompanied by complete inhibition of conception, was observed throughout treatment. After cessation of treatment, fertility was restored within 2 weeks. Histological examination did not reveal any depletion of the ovarian oocyte stock. These results warrant further research into the nature of cumulus antigens and their use in active immunization studies.
Ten feral mares free-roaming in Maryland, USA, were inoculated with porcine zonae pellucidae (PZP) protein before the breeding season for three consecutive years (1988-90). Ovarian function was monitored for 51 days during the peak of the breeding season after the third annual PZP inoculation, in seven of these mares and in four untreated control mares, by means of urinary oestrone conjugates and nonspecific progesterone metabolites. None of the ten inoculated mares became pregnant in 1990, compared with 55% of 20 control mares, which included two of the four monitored for ovarian function. Three of the untreated mares demonstrated apparent normal ovarian activity, characterized by preovulatory oestrogen peaks, concurrent progesterone nadirs at ovulation, breeding activity, and luteal-phase progesterone increases after ovulation. Two of the seven monitored PZP-treated mares demonstrated ovulatory cycles that did not result in conception. One was pregnant as a result of conception in 1989 and demonstrated a normal, late-gestation, endocrine profile. The remaining four PZP-treated mares revealed no evidence of ovulation, and urinary oestrogen concentrations were significantly depressed. The experiments indicated that (i) a third consecutive annual PZP booster inoculation is greater than 90% effective in preventing pregnancies in mares and (ii) three consecutive years of PZP treatment may interfere with normal ovarian function as shown by markedly depressed oestrogen secretion.
Female rabbits (n = 36, 6 per group) were immunized with: (i) solubilized isolated porcine zona pellucida (SIZP), which contains ZP1, 82 kDa; ZP3 alpha, 55 kDa; and ZP3 beta, 55 kDa; (ii) a purified preparation of ZP3 alpha and ZP3 beta (ZP3); (iii) purified endo-beta-galactosidase digested glycoproteins ZP3 alpha-(EBGD) and (iv) ZP3 beta-(EBGD) (each about 30% deglycosylated); (v) chemically deglycosylated core proteins ZP3 alpha-(DG) and (vi) ZP3 beta-DG (each greater than 92% deglycosylated). Rabbits injected with saline (n = 6) or Freund's adjuvant (n = 6) served as controls. Rabbits were bled weekly to monitor titres. Every six weeks two animals from each group (n = 16) were selected for unilateral oophorectomy followed by histological examination. Sections were scored for numbers of primary, secondary and tertiary follicles. Anti-ZP3 titres developed in all treatment groups and correlated with carbohydrate content (peak per cent [125I]-labelled ZP3 binding by radioimmunoassay: SIZP 71.9 +/- 1.2, ZP3 70.0 +/- 2.5, ZP3 alpha-EBGD 60.9 +/- 5.3, ZP3 beta-EBGD 56.4 +/- 5.0, ZP3 alpha-DG 56.4 +/- 4.0, ZP3 beta-DG 53.5 +/- 4.3) (means +/- SEM). Animals immunized with SIZP, ZP3 and ZP3 beta-EBGD showed a statistically significant reduction in the number of primary, secondary and tertiary follicles compared with controls (P less than 0.01, MANOVA), whereas animals immunized with ZP3 alpha-EBGD, ZP3 alpha-DG and ZP3 beta-DG did not (P greater than 0.05, MANOVA). These results demonstrate that immunization with purified ZP3 alpha macromolecules (ZP3 alpha-EBGD, ZP3 alpha-DG) or ZP3 beta-DG does not produce histopathological changes in ovaries. Such deglycosylated ZP macromolecules represent potential target antigens for immunocontraceptive development.
A group of ten healthy fertile adult male bonnet monkeys were actively immunized using procedures acceptable for human use with pure follicle-stimulating hormone (oFSH) isolated from sheep pituitaries. The vaccine elicited an immunogenic response in all ten monkeys; the antibody-binding capacity, determined by Scatchard analysis, varied from 3 to 18 micrograms oFSH ml-1, the binding affinity ranging from 0.13 to 2.0 x 10(10) mol-1. A substantial population of antibodies against oFSH crossreacted with 125I-labelled human (h) FSH, used here as a representative ligand of primate FSH. The bioneutralization activity of the antisera assessed by a specific bioassay in vitro, when the antibody titre was high, was 6.9 +/- 0.18 micrograms hFSH ml-1. Immunization for 4.7-5.7 years did not affect the health and libido of the animals. Concentration of testosterone in serum remained normal throughout the study, but, within 150 days of immunization, there was a marked decrease (75-100%) in the number of spermatozoa in seminal ejaculates. Oligospermic status interspersed with azoospermia was maintained by periodic boosting. The fertility of these animals was monitored between 6 months and 2 years after primary immunization. All the ten animals proved infertile in repeated mating experiments with females of proven fertility. After stopping booster injections, nine of ten animals regained fertility, but the time taken for this depended upon the rate of decline of antibody titres. Re-boosting these monkeys with 100 micrograms oFSH after confirming that recovery had occurred revealed prompt increases in antibody titres followed once again by onset of oligo-azoospermia and infertility, underscoring the specificity of immunization effect. The immunized monkeys, apart from being acutely oligospermic, ejaculated spermatozoa that were markedly deficient in key acrosomal enzymes, such as acrosin and hyaluronidase, and motility as well as in their ability to penetrate a gel in vitro, suggesting that the infertility observed was due to gross reductions in the numbers of spermatozoa that could effectively interact with the oocyte and cause successful fertilization.
The zona pellucida glycoprotein that surrounds the mammalian oocyte has several target antigens that have potential use in the development of a contraceptive vaccine. In the present study, an epitope sequence recognized by a monoclonal antibody to the porcine zona pellucida glycoprotein ZP4 was determined. Three candidate peptides were synthesized, based on an epitope mapping by cDNA and an analysis of chain flexibility of porcine ZP4. Only one synthetic peptide, corresponding to amino acid positions 50-67, reacted with the monoclonal antibody; the other synthetic peptides, corresponding to positions 60-79 and 70-100, did not react. The reactive epitope was identified as CTYVLDPENL, corresponding to positions 50-59 of porcine ZP4. The peptide inhibited the reaction of the monoclonal antibody binding to native ZP4 in a dose-dependent manner. When the synthetic peptide 50-67 was used to immunize mice, the resultant antisera reacted not only with the synthetic peptide but also with native pig zona pellucida. In addition, anti-peptide 50-67 antibody inhibited porcine fertilization in vitro. It is thus concluded that the peptide identified as an epitope for the monoclonal antibody would be a promising candidate for the development of a contraceptive vaccine.
Regulation of local overpopulations of free-roaming feral equids is in demand worldwide for ecological balance and habitat preservation. Contraceptive vaccines have proven effective in feral horses, which breed seasonally, but no data are available for equids such as the burro, which is reproductively active all year round. In the present study, 27 individually identified female feral burros (Equus asinus) roaming free in Virgin Islands National Park (St John, US Virgin Islands; Lesser Antilles) were remotely treated with pig zonae pellucidae (PZP) vaccine. Between January and May, 16 burros were darted with a 1 ml emulsion of PZP plus Freund's adjuvant. Ten to twelve months later each treated burro was given a single booster injection of PZP plus adjuvant to maintain contraception through a second year. Eleven adult untreated jennies served as controls. Beginning one year after initial vaccination, these burros were monitored for pregnancy and foal production. Collection of data to determine treatment effect was not begun until 12 months after initial treatment to ensure that pregnancies existing before vaccination were not included. Pregnancy was assessed using previously validated methods for steroid metabolite measurement in fresh faecal samples. None of the PZP-treated burros produced foals between 0 and 12 months after the last inoculation. One PZP-treated burro tested positive for pregnancy at 10 months after the final inoculation. During this same period, six of 11 untreated burros tested pregnancy-positive, and four were observed with foals. There was no difference in pregnancy rates among treated, control and randomly sampled jennies between 12 and 24 months after the last inoculation. The results demonstrate that, in free-roaming feral burros that are reproductively active all year round: (1) burros can be accessed for remotely delivered PZP vaccination; (2) PZP contraception is effective; (3) PZP contraception is reversible; and (4) pregnancy can be reliably detected by faecal steroid analysis.
Glycosylation is one of the important post-translational modifications of sperm plasma membrane proteins during the maturation of epididymal spermatozoa that results in the development of motility and fertilizing capability. The aim of the present study was to identify and characterize the maturation-dependent asparagine-linked (N-linked) and serine- and threonine-linked (O-linked) glycoproteins of the epididymal spermatozoa of rhesus monkeys. The presence of N- and O-linked glycoproteins was confirmed by treatment of sperm membranes with N-glycosidase F and O-glycosidase. The major maturation-dependent sperm membrane glycoproteins identified on blots of SDS-PAGE-fractionated proteins of purified sperm plasma membranes from five segments of epididymis, probed with biotinylated lectins and Vectastain-ABC reagent included O-linked 170, 150, 86 and 60/58 kDa glycoproteins; N-linked 68, 56, 48 and 38 kDa glycoproteins and N- and O-linked 116 kDa glycoprotein, all of which exhibited marked differences in the degree of glycosylation between immature and mature sperm surfaces. These glycoproteins can be used as markers of sperm maturation in the epididymis of rhesus monkeys, during the screening of antifertility agents acting at the epididymis, or may be developed as potential sperm antigens. The 100% inhibition of fertility in female rats and rabbits immunized with major maturation-dependent 116 kDa glycoprotein showed the significance of glycosylation changes in the maturation status of epididymal spermatozoa. This 116 kDa protein can be used as a marker parameter of sperm maturation in the rhesus monkey, which is often the preferred animal model for preclinical studies. These results will contribute to the identification of an appropriate animal model for the development of male contraceptives in humans.