Molecular and immunological characteristics of sperm antigens involved in egg binding.

Development of a vaccine(s) based on sperm antigens represents a promising approach to contraception. The utility of an antigen in immunocontraception is contingent upon sperm/testis-specificity and its involvement in the fertilization process. Since, the sperm-zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, molecules involved at this site are attractive candidates for immunocontraception. Using hybridoma technology, subtractive hybridization, and differential display technology, our laboratory has delineated several sperm antigens. These antigens have testis-specific expression and have a role in the fertilization process. The cDNAs encoding for the antigens have been cloned and sequenced. Among these, the fertilization antigen (FA-1) is particularly interesting, because it is involved in immuno-infertility in humans. Using the phage peptide display technique, a novel dodecamer sequence of a approximately 72+/-5 kD antigen, designated as YLP(12), that is testis-specific and involved in human sperm-ZP recognition/binding, was identified. A synthetic 12-mer peptide was generated based on this sequence. In the hemizona assay, YLP(12) peptide and its monovalent Fab' antibodies specifically and significantly inhibited human sperm-ZP binding. Furthermore, the presence of specific antibodies reactive with YLP(12) peptide, were identified in the serum and seminal plasma of immuno-infertile men. Thus, FA-1 and YLP(12) are promising target antigens for the development of contraceptive vaccines as well as for specific diagnosis and treatment of male infertility.     

非哺乳动物卵透明带的生殖免疫学研究

从淡水鱼类(鲤、草、鲢、鳙)卵中显微分离出的透明带,经聚丙烯酰胺凝胶圆盘电泳,对蛋白染料氨基黑10B 着染,呈单条蓝黑色带,它和大白鼠卵透明带、细菌粘多糖及唾液粘蛋白有类似的电泳谱及迁移率。用鱼卵透明带经福氏佐剂乳化后免疫家兔,可诱发家兔产生抗鱼卵透明带抗体。被动血凝试验测得抗体滴度为1000,玻片沉淀反应及琼脂凝胶平板双扩散试验表明,此抗血清可以和不同种鱼透明带及大白鼠透明带起交叉反应,猴抗鱼透明带抗血清可以和猪卵透明带起交叉反应。鱼卵透明带可以成功地用放射性同位素~(131)Ⅰ进行标记。用鱼卵透明带主动免疫家兔所诱发抗血清的电泳分析表明,丙种球蛋白区带随免疫时间加长而逐渐加深加宽.~(131)Ⅰ标记的鱼透明带与兔抗鱼卵透明带的抗血清一起混合后再电泳,丙种球蛋白区带的放射性计数率最高。初步认为鱼卵透明带的化学成分及免疫学特异性可能与哺乳动物存在着一定关系,有一定的共同抗原性,这为寻找丰富的异种卵透明带抗原提供了一定的线索。     

Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1)

The zona pellucida (ZP) of mammalian oocytes forms an extracellular matrix composed of three major glycoproteins and plays an important role in sperm-zona interactions. As ZP had a strong organ-specific but species-cross-reactive antigenicity and passive or active immunization with ZP antigens could impair fertilization, the possibility of developing a immunocontraceptive vaccine has been extensively studied. Studies on active immunization with porcine ZP (pZP) that contain B cell epitopes and T cell epitopes demonstrated that a temporary infertility could be induced along with the elevation of antibody titers, but it was always associated with ovarian failure. This could be due to the oophoritis by activation of pathogenic T cell immunity. It is the general consideration that any adverse effects by vaccination should be avoided for an immunocontraception. From this point of view, the analysis of B cell epitopes of pZP protein would be helpful for construction of a safe immunocontraceptive vaccine with zona antigens. We determined the amino acid sequence of the B epitope in the pZP1 protein by using a monoclonal antibody (MAb-5H4) that possesses a fertilization blocking ability. In addition, antiserum raised to the epitope sequence was revealed to block in vitro fertilization of homologous animal species.     

Egg-sperm interactions at fertilization in mammals

Mammalian eggs are surrounded by a zona pellucida (ZP) that regulates egg-sperm teractions during fertilization. The ZP consists of long filaments composed of two glycoproteins, ZP2 and ZP3, that are crosslinked by a third glycoprotein, ZP1. The presence of both ZP2 and ZP3 is essential for assembling a ZP around growing oocytes, as well as for fertility of females. Acrosome-intact sperm recognize and bind to O-linked oligosaccharides linked to Ser residues at the sperm combining-site of ZP3. Structural differences in oligosaccharides on ZP3 from different species may account for whether or not sperm are able to bind to the ZP. Bound sperm undergo the acrosome reaction, penetrate the ZP, and can then fuse with egg plasma membrane. Following fertilization, sperm are unable to bind to either ZP3 or the ZP of one-cell embryos.     

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.     

Infertility and Immunocontraception based on zona pellucida

The zona pellucida (ZP) is an extracellular matrix surrounding ovarian oocytes, ovulated eggs and preimplantation embryos. It plays several important roles at different stages of reproduction. Its constituent glycoproteins are expressed specifically in the ovary. It is thus possible to produce autoantibodies to ZP proteins that interfere with reproductive functions including folliculogenesis, fertilization and implantation. First, this article describes the history of anti-ZP antibodies detected in women with idiopathic infertility. Second, the current relationship between anti-ZP antibodies and infertility is discussed in relation to assisted reproductive medicine. Third, we introduce the latest studies of animal experiments involving the ZP. Finally, immunocontraceptive vaccine development using various ZP antigens is reviewed.     

Human zona pellucida glycoproteins: functional relevance during fertilization

The zona pellucida (ZP), a glycoproteinaceous matrix surrounding the mammalian oocyte plays an important role in species-specific sperm–egg binding, induction of acrosome reaction in the ZP-bound spermatozoa, avoidance of polyspermy and protection of the embryo prior to implantation. In contrast to mouse, human ZP matrix is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4 (Zp4 pseudogene in mouse). Recent studies employing recombinant and immunoaffinity purified human zona proteins revealed that in addition to ZP3, capacitated acrosome-intact spermatozoa also bind ZP4. Human ZP2 primarily binds to the acrosome-reacted spermatozoa, supporting its role as secondary sperm receptor, as delineated in the murine model. For binding of human zona proteins to spermatozoa, glycosylation is not critical. Both human ZP3 and ZP4 induce dose-dependant acrosomal exocytosis in capacitated sperm. In contrast to the murine model, N-linked glycosylation is more critical for the human ZP3/ZP4 mediated induction of acrosomal exocytosis. Subtle differences in the downstream signaling events associated with ZP3 vs. ZP4 mediated induction of acrosomal exocytosis have been observed. To conclude, in humans, ZP3 and ZP4 are involved in binding of the spermatozoa to the egg and subsequent induction of acrosome reaction. The contribution, if any, of human ZP glycoprotein-1 (ZP1) during these stages of fertilization remains to be elucidated.     

Production of monoclonal antibodies to porcine zona pellucida and their inhibition of sperm penetration through human zona pellucida in vitro

Two hybridoma cell lines producing murine monoclonal antibodies to antigens common to the zona pellucida (ZP) of pigs and humans were obtained by immunization of mice with solubilized porcine zona antigen. Indirect immunofluorescence tests showed that both these monoclonal antibodies stained the entire layer of porcine ZP but stained different regions of human ZP, one staining the entire layer and the other only the outer surface. At high concentrations, these two monoclonal antibodies directed against antigens common to porcine and human ZP prevented sperm binding and penetration into human ZP in vitro, whereas a monoclonal antibody directed against an antigen restricted to porcine ZP did not have these inhibitory effects. It is concluded that human and porcine ZP share at least two antigens with different locations in the ZP, and that these influence or are essential for interaction of human sperm with the ZP. These results provide a rationale for using porcine ZP clinically as a vaccine for human immunocontraception.     

The sperm proteasome during sperm capacitation and fertilization

The 26S proteasome is a multi-subunit protease specifically targeting ubiquitinated proteins. A consensus has emerged from studies by multiple laboratories on the role of sperm-borne proteasomes in human, mouse, pig, bovine, ascidian and echinoderm fertilization. Major findings from the studies in various mammalian and non-mammalian fertilization systems are (1) proteasomes are present in the mammalian sperm acrosome and on the acrosomal surface; (2) ubiquitinated proteins are present on the mammalian, ascidian and echinoderm egg coat; (3) proteasomal proteolytic and ubiquitin-deconjugating (deubiquitinating) activities can be detected in viable, motile mammalian spermatozoa; (4) proteasomes remain associated with the sperm head following ZP-induced acrosomal exocytosis; (5) inhibition of ubiquitination and proteasomal proteolysis blocks fertilization in mammals, ascidians and echinoderms; (6) inhibition of proteasomal proteolysis alters the course of mammalian sperm capacitation and acrosomal exocytosis induced by sperm binding to the egg coat, zona pellucida (ZP); (7) depletion of the sperm surface-associated ATP blocks porcine and echinoderm fertilization, most likely by affecting the integrity of sperm proteasomes, of which several subunits are ATPases; (8) inhibition of proteasomal proteolysis blocks sperm–ZP penetration, but does not alter the rate of sperm–ZP binding in mammals, and (9) experimental modification of sperm-associated deubiquitinating activities shifts the balance of monospermic fertilization to polyspermic fertilization in vitro. Altogether, these studies provide evidence for the involvement of the 26S proteasome in multiple steps of animal and human fertilization, offering a novel model of sperm–egg coat interactions, and identifying a range of potential new sperm quality markers and contraceptive targets.     

Tests of human sperm function and fertilization in vitro.

OBJECTIVE: To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in vitro by logistic regression analysis. STUDY SELECTION: Recent studies on the relationship between putative and new tests of human sperm function and fertility in vitro or in vivo are discussed in this review. Some physiological and technical aspects are included. MAIN OUTCOME MEASURES: Fertilization rates in vitro and sperm tests including standard semen analysis, improved morphology assessment, objective assessment of sperm motility and movement characteristics, nuclear maturity, hypo-osmotic swelling, the acrosome and the acrosome reaction, acrosin activity, human sperm-hamster oocyte penetration assay, and sperm-zona pellucida (ZP) and sperm-oolemma binding. RESULTS: The percentages of sperm with normal morphology and a normal intact acrosome, mean linearity, and the number of sperm binding to the ZP were highly significant related to fertilization rates in vitro. Other sperm tests evaluated usually provided no additional information about fertilization rates. The human ZP is highly selective for binding of morphologically normal sperm. Acrosome-reacted human sperm have little or no ability to bind to the ZP. CONCLUSION: Results of in vitro fertilization can be used to evaluate tests of human sperm function. Logistic regression analysis is a powerful method for determining which groups of sperm characteristics are independently related to fertilization rates. Normal morphology, linearity, acrosome status, and sperm-ZP binding are the most important characteristics for fertilization in vitro.     

The zona pellucida: using molecular genetics to study the mammalian egg coat

An extracellular matrix that mediates critical steps in fertilization and early development surrounds all vertebrate eggs. In mice and humans, this matrix is known as the zona pellucida and comprises three glycoproteins: ZP1, ZP2 and ZP3. Homologues of these proteins isolated from other vertebrates have conserved protein motifs that may be important for establishing a common fibrillar structure. However, specific but contradictory biological roles have been assigned to individual egg coat proteins based on assays in vitro in a wide range of species. Mouse lines lacking either ZP1 or ZP3 have been established with abnormal or absent zona matrices and varying degrees of infertility to examine zona structure and function in vivo. By crossing mouse lines lacking individual zona proteins with those expressing human homologues, the structural integrity of the zona matrix can be restored. Because mouse and human spermatozoa exhibit order-specific binding to the zona pellucida, mice with 'humanized' chimaeric zonae may provide an experimental system to elucidate the molecular basis of sperm-zona interaction.     

Zona pellucida-based contraceptive vaccines for human and animal utility

Contraceptive vaccines can be designed to inhibit (i) production of the gametes (sperm and oocyte), (ii) functions of gametes leading to block in fertilization, and (iii) the gamete outcome (pregnancy). The zona pellucida (ZP) glycoproteins have been proposed as candidates for developing contraceptive vaccines by virtue of their critical role in fertilization. Immunization of non-human primates with either native or recombinant ZP proteins leads to curtailment of fertility, which however is invariably associated with ovarian pathology. To avoid oophoritis, immunogens corresponding to mapped B cell epitopes of ZP proteins that are devoid of 'oophoritogenic' T cell epitopes have been proposed. However, ways to overcome the observed oophoritis associated with the ZP-based contraceptive vaccines are yet to be fully defined. This is essential if their use for control of human fertility is to be considered. Nonetheless, contraceptive vaccines based on ZP proteins have shown very promising results in controlling wildlife population such as wild horses, white-tailed deers, elephants, marsupials, grey seals and dogs, where long term infertility or even permanent sterility is desirable.     

A prospective randomized controlled study of the effect of short coincubation of gametes during insemination on zona pellucida thickness.

A prospective, randomized study was conducted to evaluate the thickness, of zona pellucida (ZP) after brief or standard exposure of human oocytes to spermatozoa, and to determine the correlation between ZP thickness, fertilization rate and embryo quality. The mean ZP thickness 48 h after insemination was found to be significantly less in fertilized oocytes than in non-fertilized oocytes in all treated groups (13.72 +/- 3.0 microns and 15.08 +/- 2.5 microns, respectively; p < 0.007). Zona pellucida thickness correlated positively with embryo quality. Brief exposure of gametes was found to influence ZP thickness. The ZP was significantly thinner after brief and intracytoplasmic sperm injection (ICSI) exposure of oocytes to spermatozoa than after standard in vitro fertilization (IVF). The mean ZP thickness 24 and 48 h after fertilization was significantly greater in standard IVF (16.43 +/- 2.8 microns and 15.22 +/- 2.7 microns, respectively) than in either the brief exposure or ICSI groups (12.78 +/- 2.4 microns and 13.01 +/- 3.5 microns vs. 13.46 +/- 2.2 microns and 13.16 +/- 2.4 microns; p < 0.0001).     

Use of immunoaffinity purified antibodies to zona pellucida to compare alloimmunization of male and female rabbits

In order to study the immunogenicity as well as tissue specificity of zona pellucida (ZP) antigens, the present studies have been designed to examine the effects of alloimmunization of male and female rabbits with rabbit zonae pellucidae. These studies are the first to demonstrate that high titers of antibodies to homologous ZP antigens are developed in male rabbits while no detectable antibodies are developed in females. As demonstrated using the ELISA assay, the antibodies from these males immunized with rabbit ZP, have a greater reactivity against rabbit ZP antigens than do antibodies from female rabbits heteroimmunized with porcine ZP. The antibodies from the male rabbits immunized with rabbit ZP also recognize antigenic determinants of porcine ZP. Methods for the immunoaffinity purification of antibodies from serum were developed to determine whether low levels of antibodies against ZP are present in sera of alloimmunized female rabbits. They also allow more detailed analysis of antibodies used to detect antigenic determinants which are cross-reactive between different mammalian species. Although this method was effective in isolating low levels of antibodies from male alloimmunized rabbits or from female rabbits heteroimmunized with porcine ZP proteins, no specific antibodies could be isolated from the serum of females alloimmunized with rabbit ZP. These studies more clearly demonstrate that zona pellucida antigens are specific to the ovary in that female rabbits do not develop significant antibody levels against rabbit ZP antigens, even following active immunization with adjuvant, while male rabbits develop high titers of antibodies.     

Isolation of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family

Porcine zona pellucida (ZP) contains antigens which cross-react with man, and anti porcine ZP antibody was found to exhibit a strong blocking effect on human sperm-ZP interaction. Previously, we found that porcine ZP was composed of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4). In this study, each of the glycoprotein families was isolated by O'Farrell's two-dimensional electrophoresis and injected into mice to obtain antisera to each component. All the mouse antisera had high titers of antibodies to porcine and human ZP in an immunofluorescent staining test. Western blot analysis showed that although the antisera most strongly reacted with the corresponding components of porcine ZP, each glycoprotein family also possessed antigens which cross-reacted with each other. The antisera to ZP1, ZP3 and ZP4 strongly inhibited human sperm-ZP interaction but antiserum to ZP2 had no inhibitory effect on it.     

Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro.

OBJECTIVE: To study whether the results of tests of sperm chromatin and deoxyribonucleic acid (DNA) normality are related to fertilization rates in vitro. DESIGN: Normal morphology, nuclear maturity determined by acidic aniline blue stain, and DNA normality determined by acridine orange fluorescence of sperm in insemination medium and the number of sperm bound to the zona pellucida (ZP) of the oocytes that had failed to fertilize in vitro were determined. The relationship between sperm test results and fertilization rates were analyzed by logistic regression. SETTING: Samples were obtained from patients undergoing in vitro fertilization (IVF) treatment. RESULTS: The number of sperm bound to the ZP, the percentage of sperm with normal morphology, and the percentage of sperm with normal DNA were the most significant factors related to fertilization rates in vitro. In patients with normal morphology > or = 15% or with > 10 sperm bound per ZP, the percentage of sperm with normal DNA, the number of sperm bound to the ZP, and motility grade were significantly related to IVF rates. CONCLUSION: In patients with normal morphology > or = 15%, failure of fertilization may be because of defects of sperm-ZP binding or abnormal DNA. Assessment of DNA normality of motile sperm in the insemination medium may aid prediction of fertilization rates in addition to normal morphology and sperm-ZP binding.     

A prospective randomized controlled study of the effect of short coincubation of gametes during insemination on zona pellucida thickness

A prospective, randomized study was conducted to evaluate the thickness of zona pellucida (ZP) after brief or standard exposure of human oocytes to spermatozoa, and to determine the correlation between ZP thickness, fertilization rate and embryo quality. The mean ZP thickness 48?h after insemination was found to be significantly less in fertilized oocytes than in non-fertilized oocytes in all treated groups (13.72?±?3.0?µm and 15.08?±?2.5?µm, respectively; p?<?0.007). Zona pellucida thickness correlated positively with embryo quality. Brief exposure of gametes was found to influence ZP thickness. The ZP was significantly thinner after brief and intracytoplasmic sperm injection (ICSI) exposure of oocytes to spermatozoa than after standard in vitro fertilization (IVF). The mean ZP thickness 24 and 48?h after fertilization was significantly greater in standard IVF (16.43?±?2.8?µm and 15.22?±?2.7?µm, respectively) than in either the brief exposure or ICSI groups (12.78?±?2.4?µm and 13.01?±?3.5?µm vs. 13.46?±?2.2?µm and 13.16?±?2.4?µm; p?<?0.0001).     

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.     

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.     

Are zona pellucida genes involved in recurrent oocyte lysis observed during in vitro fertilization?

Purpose

Complete oocyte lysis in in vitro fertilization (IVF) is a rare event, but one against which we remain helpless. The recurrence of this phenomenon in some women in each of their IVF attempts, regardless of treatment, together with the results of animal experiments led us to investigate the possible involvement of the genes encoding for the glycoproteins constituting the zona pellucida (ZP).

Patients & methods

Over the last ten years, during which we treated over 500 women each year, three women suffered recurrent oocyte lysis during their IVF attempts in our Centre for Reproductive Biology. For each of these three cases, we sequenced the four genes and promoter sequences encoding the glycoproteins of the ZP. The sequence variations likely to cause a change in protein expression or structure, were investigated in a control group of 35 women who underwent IVF without oocyte lysis and with normal rates of fertilization.

Results & conclusion

We found no mutations in the ZP genes sequenced. Only some polymorphisms present in the control group and in the general population were detected, excluding their specific involvement in the phenotype observed. Thus, although we suspected that complete oocyte lysis was due to a genetic cause, it did not seem possible to directly incriminate the genes encoding the proteins of the ZP in the observed phenotype. Further study of the genes involved in the processing and organization of ZP glycoproteins may allow elucidation of the mechanism underlying recurrent oocyte lysis during in vitro fertilization.