Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

A novel treatment strategy for ovarian cancer based on immunization against zona pellucida protein (ZP) 3

We tested the principle of treating malignant ovarian tumors by vaccination against their ectopically expressed protein, zona pellucida glycoprotein (ZP) 3, using as the experimental model the granulosa cell tumors that develop in transgenic mice expressing the simian virus 40 T-antigen under the inhibin-α promoter (inhα/Tag). We found high ZP3 expression in granulosa cell tumors of the transgenic mice, in human surface ovarian cancer and granulosa cell lines, and in human granulosa cell tumors and their metastases. Early preventive immunization (between 2 and 5.5 mo of age) of transgenic mice with recombinant human (rh) ZP3 prevented ovarian tumorigenesis, and delayed therapeutic immunization (between 4.5 and 7 mo) reduced weights of existing tumors by 86 and 75%, respectively (P<0.001), compared to vehicle-treated control mice. No objective side effects of the immunizations were observed. Liver metastases were found in nontreated/vehicle-treated controls (n=7/39), but none following active rhZP3 immunizations (n=0/36; P<0.05). Immunization with rhZP3 was highly effective, as demonstrated by the induction of anti-ZP3 antibodies, as well as proliferative responses to the ZP3 antigen. These results signal rhZP3 immunization as a novel strategy to be developed for the immunotherapy of ovarian granulosa cell tumors, as well as for that of other malignancies that may express ZP3.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1)

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.     

A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.

Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

重组人卵透明带蛋白(rhZP3)的生物活性研究

为了研究毕赤酵母表达的重组人卵透明带蛋白(rhZP3)的生物活性,分别用空白培养液,含孕酮或rhZP3的培养液对人精子进行顶体诱发实验,用考马斯亮蓝染色法对顶体状态进行评价;用不同浓度的rhZP3以及空白培养液分别处理精子,然后再与卵子进行结合实验,观察经过不同处理的精子在精卵结合中的情况;用抗rhZP3抗血清与阴性血清分别处理卵子,再与精子进行结合实验,观察经过不同处理后的卵子在精卵结合中的情况。rhZP3诱发顶体反应实验结果显示,rhZP3处理组与空白对照组之间差异显著(P<0.01);精卵结合实验结果显示,各实验组和对照组之间存在显著性差异(P<0.01),rhZP3、抗rhZP3抗体均能抑制精卵结合。实验结果表明,rhZP3具有天然人卵透明带蛋白相似的活性。     

Long-term contraceptive efficacy of vaccine of ovine follicle-stimulating hormone in male bonnet monkeys (Macaca radiata).

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.     

重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein.

Three peptide segments (YAL-198, YAL-201 and YAL-212) corresponding to the extracellular domain of a human sperm protein designated as YWK-II antigen were synthesized as multiple antigen peptide (MAP). Male and female rats were immunized with the YWK-II-MAPs and fertility determined. In a group of 12 female rats immunized with YAL-198, seven animals were infertile and two animals were subfertile. When immunized with YAL-201 and YAL-212, 4 and 2 animals were infertile, respectively. In a group of 15 males immunized with YAL-198, 2 animals were infertile and 6 were subfertile. Two animals immunized with YAL-201 were subfertile. All control male and female rats immunized with bovine serum albumin and adjuvant were fertile. Sera obtained from infertile rats immunized with YAL-198 contained higher titers of antibodies compared to those obtained from fertile animals. The present study shows that immunization with synthetic peptide segments of a sperm protein can effectively reduce fertility.     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titers but does not reduce fertility

Previous studies have reported reduced fertility in female baboons immunized with a synthetic peptide derived from the sperm-specific isozyme of lactate dehydrogenase (LDH-C). In this study, a similar approach was used to immunize female cynomolgus macaques with the same peptide sequence (bC5-19) conjugated to a T-cell epitope from tetanus toxin (TT). Twelve female monkeys were immunized with bC5-19:TT delivered with Ribi MPL adjuvant vehicle, and 10 control female monkeys were injected with the adjuvant vehicle only. All 12 females in the treatment group developed LDH-C-specific serum antibodies as measured by ELISA, but anti-LDH-C antibodies were not detected in vaginal fluids of the immunized animals. After 4 months of timed mating immediately following the immunizations, five of the ten immunized females became pregnant, as did six of the ten control females. Anti-sera from both pregnant and nonpregnant bC5-19:TT-immunized females recognize a single band at 35 kDa on Western blots of whole sperm extracts, and purified Igs from the same sera localize along the principle piece of the flagellum of permeabilized sperm.     

Evaluation of bendectin embryotoxicity in nonhuman primates: I. Ventricular septal defects in prenatal macaques and baboon

Cynomolgus monkeys, rhesus monkeys and baboons were administered 10 to 40 times the human dose equivalent of Bendectin throughout the major period of organogenesis (22(+/-3)-50 days of gestation). In animals examined prenatally (100 +/- 2 days gestation) the total incidence of ventricular septal defects (VSD) was 40% in cynomolgus monkeys, 18% in rhesus monkeys, and 23% in baboons. The majority of VSD involved the muscular portion of the septum. No dose response was evident and there were no other cardiac or extracardiac defects found except for one baboon fetus with multiple defects. No defects were observed in cynomolgus monkeys administered Bendectin for 4-day periods between 22 and 41 days of gestation. There was no association of Bendectin treatment with any noncardiac defect. In cynomolgus and rhesus monkeys examined at term there was one mitral valve defect and no incidence of VSD. The increased incidence of VSD observed prenatally in all three species and the absence of defects in macaques at term suggests a delay in closure of the ventricular septum in treated animals. The Bendectin-treated monkey may be a suitable model for the study of the pathogenesis of VSD and the mechanism of spontaneous closure of the defect.     

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

Novel hyperprolactinemia and hyperprolactinemic anovulation model using the cynomolgus monkey (Macaca fascicularis)

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.     

Ovaries remain functional in squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida 55,000 macromolecule

Fifty female squirrel monkeys were each immunized with 200 micrograms of a purified preparation of the 55,000 macromolecule (ZP3) from porcine zona pellucida. The fertility status of these immunized monkeys, as well as the effect of ZP3 antibodies on ovarian function, was monitored. High anti-ZP3 titers were achieved (greater than 75% binding levels as determined by radioimmunoassay) and remained high (approximately 67% binding level) for the duration of this study. Hormonal evaluations indicated initial disturbances in normal ovarian steroid secretion and function that were confirmed by laparoscopic observation and oocyte production data. Histological examination of ovaries at 6-7 mo post-injection suggested an interference in folliculogenesis. No pregnancies were observed in the immunized monkeys during this period. By 10-15 mo post-immunization, hormonal and laparoscopic data indicated that ovarian function was recovering in injected monkeys despite the continued presence of high titers to ZP3. Collectively, these results demonstrate that although immunization with ZP3 initially produces disturbances in normal ovarian function that inhibit fertility, these effects are reversible. Such findings encourage the continued intensive investigation of purified porcine zona macromolecules for immunocontraceptive purposes.     

A nicotinic acetylcholine receptor is involved in the arosome reaction of human sperm initiated by recombinant human ZP3

One of the essential steps in mammalian fertilization is the acrosome reaction (AR), a modified exocytotic event in the sperm head that occurs upon contact with the glycoprotein matrix of the zona pellucida (ZP) surrounding the oocyte. Acetylcholine (ACh) at concentrations of 10-250 micro M and nicotine at 10-250 nM significantly initiate the AR of capacitated human sperm. Preincubation with three antagonists of the nicotinic acetylcholine receptor (nAChR), alpha-bungarotoxin (alpha-BTX, 100 nM), alpha-conotoxin IMI (alpha-CTX IMI, 250 nM and 25 nM), and methyllycaconitine (MLA, 100 nM and 10 nM), significantly blocked AR initiation by ACh. alpha-BTX is an anatagonist of several nAChRs, including the alpha7 nAChR, and alpha-CTX IMI and MLA are highly specific antagonists of alpha7 subunit-containing AChRs. The sperm nAChR plays a role in the AR initiated in vitro by a purified recombinant human ZP protein (rhZP3). Previously, rhZP3 was able to stimulate the AR by mechanisms similar to those seen with native ZP. Preincubation of human sperm with alpha-BTX (from 10 micro M to 100 nM), alpha-CTX IMI (250 and 100 nM), or MLA (100 nM and 10 nM) caused a significant inhibition in the rhZP3-initated AR. The inhibition of the ACh-initiated and rhZP3-initiated AR by these nAChR antagonists strongly suggests the involvement of an alpha7 subunit-containing nAChR in the AR initiated by both ligands. AR initiation by progesterone was not inhibited by MLA or alpha-BTX, suggesting that this particularnAChR is not involved in the AR initiated by that ligand. In vitro results show for the first time that ACh can initiate the human sperm AR and strongly suggest that a human sperm alpha7 subunit-containing nAChR plays a role in the rhZP3-initiated AR. This nAChR ligand-gated ion channel may be important to the signal transduction events of ZP-initiated AR in vivo.     

Synergism between psychosocial and metabolic stressors: impact on reproductive function in cynomolgus monkeys

The role of energy imbalance versus psychosocial stress in the pathogenesis of female reproductive dysfunction characterized by anovulation and amenorrhea remains controversial. In women, functional hypothalamic amenorrhea can develop in the absence of significant weight loss, excessive exercise, or profound psychosocial disruption. We posited, therefore, that commonplace, seemingly minor stressors that alone would have minimal impact upon reproductive function might interact synergistically such that combinations of stressors would cause a greater impairment of the reproductive axis than any single stressor alone. We then developed a monkey model to test this hypothesis. Adult female cynomolgus monkeys with normal menstrual cycles were randomized into three experimental groups and studied over four menstrual cycles. The groups were: low-level psychosocial stress (i.e., moving to a new housing environment; Move, n = 8), moderate energy imbalance (Exercise + Diet, n = 9); and all stressors in combination (Move + Exercise + Diet, n = 10). Food intake, body weight, menstrual cyclicity, and reproductive hormones were assessed for two control menstrual cycles followed by two experimental cycles during which the monkeys experienced the stressors. Abnormal cycles were considered to be abnormally long or anovulatory cycles. Few abnormal cycles occurred in the Move group (1 of 8 monkeys) and in the Exercise + Diet group (1 of 9 monkeys). In contrast, 7 of 10 monkeys in the Move + Exercise + Diet group displayed at least one abnormal cycle (chi(2) = 9.61, P = 0.008). These findings suggest that infertility due to hypothalamic hypogonadism can result from the combination of commonplace, seemingly minor stressors that often escape clinical attention.     

Laminin immunized monkeys develop sera toxic to cultured rat embryos and fail to reproduce

In a previous study antilaminin antibodies in a monkey with a poor reproductive history were found to be the cause of serum toxicity to cultured rat embryos. In the present study four monkeys were immunized with murine tumor laminin and a fifth with bovine serum albumin. Subsequently, sera from only the laminin immunized monkeys became toxic to cultured rat embryos. This serum toxicity was not mediated by complement but did require the antibody to have a divalent structure. Finally, mating trials were conducted with two of the laminin immunized monkeys that previously had excellent reproductive histories. Based on progesterone levels and observation the monkeys continued to have normal menstrual cycles but failed to initiate a successful pregnancy following immunization in over 2 years of mating trials. These data demonstrated that antibodies against laminin could have prevented conception or could have interrupted pregnancy because of embryotoxicity or failure of implantation.     

Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.