The contraceptive potential of ZP3 and ZP3 peptides in a primate model.

It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.     

Molecular basis of sperm–egg interaction and the prospects for immunocontraception

As the molecular basis of sperm–egg interaction is resolved, so new opportunities are created for the development of immunological approaches to disrupt the process of conception. Thus, realisation that the zona glycoprotein, ZP3, serves as a specific receptor for spermatozoa, has prompted a detailed examination of its contraceptive potential. In primate models, recombinant ZP3 has been shown to suppress fertility very efficiently, however this efficacy is tempered by the appearance of adverse side-effects involving accelerated primordial follicle depletion and a lymphocytic infiltration of the ovarian stroma. Synthetic peptides encoding B-cell epitopes have been found to circumvent the lymphocyte response although the effectiveness of such reagents in preventing the loss of primordial follicles has not yet been determined. The induction of active immunity against sperm-specific antigens has also been shown to generate long term infertility in both males and females. Molecular and immunological techniques are now being used to produce a rapidly expanding list of unique sperm antigens which are currently being evaluated to determine their potential contribution to the development of safe, effective, contraceptive vaccines.     

Fertility reduced by immunization with p84: A human sperm-coating antigen in the mouse

Background : p84 is an 84-kDa protein with the ABO (H) antigenic epitope, and it is also a member of the family of sperm-coating antigens. In order to investigate whether the antibodies against p84 reduce fertility in mice, p84 and lactotransferrin (Lf), whose N-terminal sequence is similar to that of p84, were injected into female mice.
Results : When p84 was used as the immunogen, the fertilization ratio was 37.5% lower than that of female mice immunized with no antigen (control group; P  < 0.01). When Lf was used as the immunogen, there was no significant difference in the fertilization ratio between the Lf and control groups. Sera from female mice immunized with these antigens showed no agglutination against human erythrocytes, suggesting that the ABO(H) determinant site on p84 is not the factor reducing fertility. Although the titer of antibody (IgG) against p84 in serum from female mice immunized with p84 was generally higher than that against Lf, the titer was not related to the reduction of fertility.
Conclusion : We conclude that an injection of p84 can reduce the fertility of female mice by inducing antibodies against p84. Our results suggest that the antibodies may act to inhibit the sperm–egg interaction, and that the binding site is the carbohydrate chain of p84, excluding the ABO (H) antigenic epitope, rather than the p84 protein itself. (Reprod Med Biol 2002; 1 : 35–39)     

卵巢上皮性癌抗独特型抗体6B11T细胞表位的鉴定

目的 鉴定卵巢上皮性癌(卵巢癌)抗独特型抗体6B11的T细胞表位,探讨其诱导抗卵巢癌细胞免疫的分子基础.方法 利用表位预测和12结合力分析实验筛选6B11的互补决定区(CDR)人白细胞抗原(HLA)A0201结合肽.以负载肽的抗原递呈细胞刺激HLA-A2阳性的自体淋巴细胞,获得特异性细胞毒淋巴细胞(CTL),经51Cr释放实验确定6B11的CTL表位.以6B11特异性CTL为反应细胞、负载肽的树突状细胞为刺激细胞,经细胞增殖实验确定6B11的辅助性T细胞(Th)表位.经细胞因子含量测定和干扰素(IFN)γ分泌细胞频数分析,进一步验证获得的表位.结果 6B11轻链可变区CDR3肽(VL CDR3)诱导的CTL能杀伤靶抗原阳性的卵巢癌细胞,该杀伤作用能被抗人主要组织相容性复合物(MHC)Ⅰ类分子抗体阻断,具有MHC-Ⅰ类分子依赖性,为6B11的CTL表位.6B11重链可变区CDR3肽(VH CDR3)能诱导6B11特异性CTL的增殖,该增殖作用大部分能被抗人MHC-Ⅱ类分子抗体阻断,具有MHC-Ⅱ类分子依赖性,为6B11 Th表位.6B11及其表位联合诱导的CTL与卵巢癌细胞共育后均分泌高水平白细胞介素(IL)2(分别为1630、1503 ng/L)、IFN-γ(分别为5620、5421 ng/L)和低水平IL-4(分别为253、274 ng/L),且存在特异性IFN-γ分泌细胞,细胞频数分别为196个/1 ×106个T细胞和184个/1×106个T细胞.结论 6B11具有模拟卵巢癌抗原的CTL和Th表位,对于抗独特型抗体作为抗肿瘤疫苗应用具有重要的理论和实践价值.     

Detection of epitopes on follicle-stimulating hormone and FSH-antiserum-induced suppression of bioactivity of follicle-stimulating hormone and luteinizing hormone

There are currently two major approaches to hormonal male contraception. One relies on testosterone (analogs) either alone or in combination with gonadotropin releasing hormone (GnRH) (analogs or immunizations), the other on immunizations against follicle-stimulating hormone (FSH). Theoretically, the latter method will suppress spermatogenesis whilst not interfering with libido. An absolute requirement is, however, that an anti-FSH vaccine does not induce anti-luteinizing hormone (LH) antibodies (LH being responsible for the induction of testosterone which is necessary-to maintain libido). In this report we show that when whole FSH is used for vaccination, in most cases in addition to biological activity against FSH, anti-LH activity is also induced. By systematic analysis of the antisera raised with FSH using systematic epitope scanning (PEPSCAN) we found differences between the FSH-specific and FSH-nonspecific sera. Only the FSH-specific antiserum contained antibodies that recognized amino acid sequence 37–55 on the β-subunit in a linear manner. Because antibodies against this epitope have not been found in the cross-reactive sera this epitope forms a prime candidate for an anti-FSH contraceptive vaccine     

Interests and limits of immunocontraception

Recent studies from WHO indicated that a large proportion of human contraceptive needs cannot be covered by the already existing means for different reasons (medical, economical, political, and cultural). Therefore, development of new effective methods targeting birth control methods affordable by under-privileged populations turns out to be necessary. Over the last 20 years, a large number of strategies have been used for contraceptive vaccines and thus multiple antigens have been identified as potential targets for immunocontraception. Nowadays, the most acute researches are based on suppression of the secretion and the activity of gonadotropic hormones (GnRH, LH/hCG, FSH) or the targeting of antibodies specific to sperm surface (RSA-1, SP10, SP17, TCLe-1, PH-20) and oocyte antigens (ZP1, ZP2, ZP3). We developed a contraceptive vaccine against FSH receptor. Adult male monkeys (Macaca radiata) were immunized with filamentous phages displaying at their surface N-terminal peptides of the FSH receptor. Long term male contraception has been achieved without any alteration of circulating testosterone levels, sexual behaviour or of any other discernable metabolic changes. Interruption of vaccination resulted in full recovery of sperm production and male fertility. Contraceptive vaccines are aimed to block an essential step in the reproductive process. From this point of view, efforts have to be focused on the challenge to raise is to ally our knowledge on reproductive physiology and protein biochemistry for a better understanding of the target antigen's function.     

Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans

Follicle-stimulating hormone (FSH), a dimeric glycoprotein synthesized in the anterior pituitary gland, is important for the production of sex steroids and gametes. FSH-beta (FSH beta) and FSH receptor (FSHR) knockout mice display impaired ovarian follicular development and infertility in females and small testes, oligospermia, and fertility in males. Humans with FSH beta gene mutations tend to have a more severe phenotype than those with FSHR gene mutations, although infertility and varying degrees of impaired sex steroid production occur in both types of mutations. Data from human and mouse mutations in the FSH beta and FSHR genes suggest that FSH is necessary for normal pubertal development and fertility in males and females.     

Antibodies to two ZP3 B cell epitopes affect zona pellucida assembly

Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335-342) and ZP3 (171-180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.     

Apparent primary follicle-stimulating hormone deficiency is a rare cause of treatable male infertility

OBJECTIVE: To find the underlying defect in a case of primary FSH deficiency and to estimate the beneficial effect of FSH treatment. DESIGN: Case report. SETTING: University hospital fertility clinic. PATIENT(S): Normal, healthy, 37-year-old male patient with severe oligoteratozoospermia. INTERVENTION(S): Levels of FSH, LH, LHRH provocation test, karyotyping, genomic analysis on the Y-chromosomal AZF region and sequencing of the FSHB gene, FSH treatment. MAIN OUTCOME MEASURE(S): We compiled detailed clinical and molecular data on four pregnancies. We compare this case with a similar case published recently. RESULT(S): There were detectable but very low FSH levels after LHRH provocation; the LH response was not entirely normal, and no genomic abnormalities were found in the FSHB gene. The FSH treatment resulted in four pregnancies, two of which ended in abortion; the other two resulted in the birth of two healthy children. Both our case and the published case had detectable but abnormally low FSH levels on some occasions, but normal or highly normal inhibin B levels that differed from the expected low levels. Both patients had a normal male phenotype and no detectable mutation in the FSHB gene. The published case differed from our patient in that the published case was azoospermic whereas ours was extremely oligoteratozoospermic. The beneficial effect of FSH treatment was only shown in our patient. CONCLUSION(S): The published case and ours may have a common, as yet unidentified, underlying defect. The dramatic and immediate effect of FSH treatment on our patient's fertility was clearly demonstrated.     

Antibodies to two ZP3 B cell epitopes affect zona pellucida assembly

Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335–342) and ZP3 (171–180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12 h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.     

卵巢癌抗独特型抗体6B11特异性表位肽在体内诱导抗原特异性体液免疫应答

目的探讨卵巢癌抗独特型抗体6B11表位多肽与诱导免疫应答之间的关系。方法采用化学合成的方法，合成抗独特型抗体6B11轻重链可变区的六条CDR区，分别以三种不同浓度免疫BALB／c小鼠。采用ELISA的方法检测免疫鼠血清，进行体内免疫学功能研究。结果6B11抗独特型抗体的六条CDR肽分别加入佐剂免疫小鼠后，其中有两条肽可以诱导出Ab3，分别为重链的CDR2区和轻链的CDR2区。结论这两条肽作为后选肽有可能是6B11抗独特型抗体特异性的CTL表位。     

Effect of inhibin-like substance isolated from porcine follicular fluid on the follicle-stimulating hormone (FSH) level in mouse serum and on FSH binding to porcine granulosa cells

An inhibin-like substance, a follicle-stimulating hormone (FSH) inhibitor, was isolated from porcine follicular fluid and purified. Serum FSH levels of unilaterally ovariectomized mice which received injections of the inhibitor were lower than those of unilaterally ovariectomized mice which received injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this protein is an FSH inhibitor and that it has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells.     

The effects of thymus-derived peptides on hypothalamic LRF and pituitary gonadotropin content in prepubertal congenitally athymic nude mice and their normal heterozygous littermates

Congenitally athymic nude mice have recognized reproductive defects, accompanied by decreased pituitary gonadotropin and hypothalamic LRF concentrations at 3 weeks of age, compared to their normal heterozygous littermates. To determine if these hormonal changes are due to decreased secretion of thymic peptides, we measured pituitary concentrations of LH and FSH and hypothalamic LRF content at 21 days of age in 122 congenitally athymic nude (nu/nu) mice and in 190 heterozygous (nu/+) littermates administered daily injections of a crude thymic extract, semipurified thymosin fraction 5, or a synthetic thymic protein preparation. All animals receiving crude thymic protein extract, and some receiving either thymosin or synthetic thymic factor, had reduced levels of hypothalamic LRF. The changes in concentrations of gonadotropins were more variable. All mice receiving crude thymic extract (with reduced hypothalamic LRF content) appeared to have increased pituitary concentrations of LH, FSH, or both when compared to control animals, although statistical significance was not achieved in all groups. A similar but less marked trend toward increased pituitary gonadotropins was also noted in mice receiving thymic factor. These preliminary results suggest that thymus-derived peptides can affect hypothalamic-pituitary function and support our hypothesis that thymic hormones may play a role in programming the hypothalamic-pituitary axis in the prepubertal period in rodents.     

Effect of incubation with different concentrations and durations of FSH for in-vitro maturation of murine oocytes

The purpose of the study was to evaluate whether a lower concentration of FSH or 2-h incubation with FSH would improve the outcome of in-vitro maturation of oocytes. The immature oocytes were obtained from FVB mice, and were allocated to four groups and incubated in the maturation media for 24 h. The maturation media were supplemented with 10 mIU/ml FSH for 24 h (group 1), 10 mIU/ml FSH for 2 h (group 2), 75 mIU/ml FSH for 24 h (group 3) or 75 mIU/ml FSH for 2 h (group 4). In each group, half of the in-vitro-matured oocytes were fertilized and cultured to blastocysts and the remaining matured oocytes were analysed for growth differentiation factor (GDF)-9 and bone morphogenetic protein (BMP)-15 mRNA to assess the oocyte quality. The maturation rates and oocyte BMP-15 mRNA concentrations were similar among the four groups. The GDF-9 mRNA concentrations were similar in group 2 and group 4. The fertilization and blastocyst rates were higher in groups 2 and 4 than in groups 1 and 3. It is concluded that 2-h incubation with FSH is better than 24-h incubation in terms of the fertilization rate and blastocyst development.     

Active immunization with follicle-stimulating hormone for fertility control: a 4 1/2-year study in male rhesus monkeys

Active immunization of four adult rhesus monkeys with highly purified ovine follicle-stimulating hormone (FSH) over 4 1/2 years resulted in the production of specific FSH antibodies. While luteinizing hormone and testosterone secretion were not affected, sperm counts were in most instances reduced to or below the lower normal range. On a few occasions, azoospermia or high sperm counts were observed. Although the antibodies produced neutralized the biologic activity of FSH throughout, spermatogenesis gradually returned, as evidenced by testicular histologic characteristics. However, the diameter of the seminiferous tubules remained decreased, and the germinal epithelium was depleted. No adverse side effects could be demonstrated in the immunized animals, e.g., immune complexes in either serum or tissues and resultant tissue damage. These results show that although active immunization with FSH may not result in an effective method of male fertility control, long-term immunization against a circulating hormone may not result in deleterious side effects.     

一种多价精子抗原表位肽的设计与合成

目的 :开发可供两性使用的多价合成嵌合肽疫苗。方法 :选择小鼠精子 /睾丸特异性抗原 SP1 7、Cyritestin中特异性氨基酸序列与牛核糖核酸酶非限制性 T细胞表位作为骨架 ,按一定连接方式设计、合成新抗原 3 5肽 ,并与等量弗氏佐剂混悬后免疫雌性 BALB/c小鼠。结果 :在 43 0 A固相自动肽合成仪上成功地合成了 3 5肽 ,并经 HPLC纯化、质谱鉴定证实其纯度达 95 %以上 ;新抗原免疫小鼠后获得的特异性抗血清可识别小鼠、大鼠及人睾丸组织蛋白提取物中相应分子量的蛋白质 ,且血清中特异性 Ig G抗体滴度最高达 1∶ 6 0 0 0 ,阴道粘膜冲洗液中特异性 Ig A抗体滴度最高达 1∶ 3 0 0。结论 :由含有两种特异性精子抗原、具有特异性氨基酸序列的新抗原所制备的抗血清可识别两种天然抗原 ,并在小鼠体内激发出较理想的特异性体液免疫 ,从而为研制适宜于两性的多价抗生育疫苗提供了实验基础     

HER-2/neu胞外区的基因工程表达及其免疫避孕作用

目的:研究HER-2/neu胞外区部分片段重组蛋白疫苗在免疫避孕中的作用。方法:构建原核表达重组体pET-hECDu,在大肠杆菌中表达人HER-2/neu胞外区部分片段,经Hi-Trap亲和层析柱纯化得到重组蛋白(hECDu),以ELISA法检测其结合活性后免疫雌性小鼠。用ELISA法检测抗HER-2/neu体液免疫应答,3H-TdR掺入法检测细胞免疫应答。结果:经原核表达系统成功纯化得到与理论分子量77 ku(1 u=1 Da)相符的hECDu,并证实其可与抗HER-2/neu胞外区特异性单克隆抗体结合。该重组蛋白疫苗可诱导雌鼠产生较高水平体液及细胞免疫应答。其生育力(平均产仔数4.0±1.8)与对照组(7.8±1.3)相比明显降低,但免疫鼠产后4 d子宫及卵巢均无明显病理改变。结论:经原核表达系统成功纯化得到HER-2/neu胞外区部分片段重组蛋白,该蛋白疫苗可在小鼠中诱导产生免疫避孕效应。     

Mild ovarian stimulation

Purpose To evaluate the efficacy of using only mild ovarian stimulation protocols for in vitro fertilization. Both groups with decreased and normal ovarian reserve were evaluated. Methods Three different mild stimulation protocols were evaluated: natural with no exogenous follicle stimulation hormone (FSH) drugs at all, natural with a boost of low dose FSH to complete follicular maturation, and minimal stimulation with low dose (75-150IU) FSH from day 3–5 of the menstrual cycle. Ethinyl estradiol was sometimes used to lower high day 3 serum FSH. Results Good pregnancy and implantation rates were found even in those women with elevated serum FSH that could only generate one follicle. Conclusions Mild ovarian stimulation seems preferable to high dose FSH regimens in women with elevated day 3 serum FSH based on previous poor reported pregnancy rates with the latter protocols. Comparable pregnancy rates to high dose regimen were seen in women with normal egg reserve at much lower risk and cost. Capsule Regimens using low dosage of FSH are very effective for in vitro fertilization especially but not restricted to women with diminished egg reserve.     

Immune recognition of the 60kD heat shock protein: implications for subsequent fertility

The 60kD heat shock protein (hsp60) is a highly conserved protein and a dominant antigen of most pathogenic bacteria. In some women, chronic or repeated upper genital tract infections with Chlamydia trachomatis, and possibly with other microorganisms, induces immune sensitization to epitopes of hsp60 that are present in both the microbial and human hsp60. Once a woman becomes sensitized to these conserved epitpes, any subsequent induction of human or bacterial hsp60 expression will reactivate hsp60-sensitized lymphocytes and initiate a pro-inflammatory immune response. Hsp60 is expressed during the early stages of pregnancy, by both the embryo and the maternal decidua. We examined, therefore, whether women who were sensitized to hsp60 experienced less successful pregnancy outcomes compared to women who were not sensitized to this antigen. In women undergoing in vitro fertilization (IVF), the presence of cervical IgA antibodies reactive with the C. trachomatis hsp60 correlated with implantation failure after embryo transfer. Further analysis revealed that an immunodominant epitope for these IgA antibodies was an hsp60 epitope shared between C. trachomatis and man. In subsequent studies of women not undergoing IVF, cervical IgA antibodies to the human hsp60 were identified in 13 of 91 reproductive age women. This antibody was most prevalent in those women with a history of primary infertility (p = 0.003). In addition, cervical anti-hsp60 IgA correlated with the detection of the pro-inflammatory cytokines interferon-gamma (p = 0.001) and tumor necrosis factor-alpha (p = 0.02) in the cervix. Conversely, women with proven fertility had the highest prevalence of the anti-inflammatory cytokine, interleukin 10, in their cervices (p = 0.001). In an analysis of serum samples in a third study, women with a history of two or more consecutive first trimester spontaneous abortions had a higher prevalence (p = 0.01) of IgG antibodies to the human hsp60 (36.8%) than did age matched fertile women (11.1%) or women with primary infertility (11.8%). Immune sensitization to epitopes expressed by the human hsp60 may reduce the probability of a successful pregnancy outcome due to reactivation of hsp60-reactive lymphocytes, induction of a pro-inflammatory cytokine response and interference with early embryo development and/or implantation.     

Native zona pellucida reactivity and in-vitro effect on human sperm-egg binding with antisera against bonnet monkey ZP1 and ZP3 synthetic peptides

In mammals, zona pellucida glycoprotein-3 (ZP3) is the putative ligand for primary sperm binding and induces the acrosome reaction. Recent evidence suggests that zona pellucida glycoprotein-1 (ZP1) also play an important role, in some species, during fertilization. In order to identify synthetic peptide immunogens capable of inducing antibodies reactive with native zona and inhibiting sperm-oocyte interaction, peptide encompassing the amino acid (aa) residues 334-343 of bonnet monkey ZP3 (bmZP3) was synthesized co-linearly with a 'promiscuous' T-cell epitope of circumsporozoite protein (CSP, 378-398 aa) of Plasmodium falciparum. In addition, four peptides corresponding to bonnet monkey ZP1 (bmZP1((58-79 aa)), bmZP1((136-153 aa)), bmZP1((212-228 aa)) and bmZP1((251-273 aa))) were synthesized. The synthetic peptides corresponding to bmZP1 were conjugated with diphtheria toxoid. Immunization of female BALB/cJ mice with the above conjugates and CSP-bmZP3((334-343 aa)) peptide led to the generation of an adequate antibody response against the respective zona peptide. Antibodies against bmZP1((251-273 aa)) and CSP-bmZP3((334-343 aa)) recognized bonnet monkey and human zona pellucida in an indirect immunofluorescence assay. Further, these antibodies when tested independently or in combination also significantly inhibited the binding of human spermatozoa to zona pellucida in a hemizona assay. These studies will further help in the design of synthetic peptide immunogens comprising of multiple B cell epitope from different zona proteins for better immunocontraceptive efficacy.