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.     

A specific radioimmunoassay for evaluation of serum antibodies to zona pellucida antigens

A radioimmunoassay procedure has been developed for detecting antibodies to porcine zonae pellucidae antigens using Staphylococcus aureus Protein A cells (Pansorbin) as the immunoadsorbent. This method offers a rapid and reproducible procedure for detecting specific antibodies to zona antigens. The zona antigens detected by antibodies in this assay were found not to cross-react with antigens in 11 other tissues. Immune serum produced against a variety of other antigens, including protein hormones, steroid hormones, porcine serum and red blood cells, did not bind to any zonae components in this assay. This assay is compared with other methods which have been used to detect antibodies to zona antigens and has been found to be more specific than immunofluorescence methods and more sensitive than either immunofluorescence or immunoelectrophoresis methods.     

Detection and significance of serum zona pellucida antibodies in sterile females

Sera of 100 infertile women and of 100 patients with early pregnancy as control group were investigated for antibodies to zona pellucida by the indirect immunofluorescence. Zonae of oocytes from pigs were used for the procedure. A positive control serum with a high titer was produced by immunization of rabbits with an antigen from pig zonae. The frequency of antibodies to zona pellucida was 29% for infertile patients and 18% in the control group. The titers of these autoantibodies were higher in infertile than in fertile women. There was no increase of these antibodies if the age increased and no relation to the menarche and the ovulation. Antibodies to zona pellucida were demonstrated frequently in patients with disturbances of cycle, free Fallopian tubes, secondary infertility, and ovarian and unknown origin of infertility.     

Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effects on fertilization

Five hybridomas which produce monoclonal antibodies (Mabs) to porcine zona pellucida (ZP) were established. The immunoglobulin classes were IgG2a from hybridoma B11C8 and IgM from the other four. Using immunofluorescent staining, the Mab from B11C8 stained only porcine oocytes, the Mab from G10G5 stained oocytes of pigs and hamsters but Mabs from C6H1, D3H4, G10F9 stained oocytes of pigs, humans, hamsters, rats and mice. Mabs from B11C8 and G10F9 strongly blocked boar sperm binding to porcine ZP but other Mabs produced only slight blocking. However, no Mabs could block sperm penetration during in vitro fertilization (IVF) of hamster oocytes. When a goat antibody (IgG) to mouse serum gamma-globulin was applied after each Mab as a second antibody, only Mab from G10G5 impaired IVF of hamster oocytes.     

Inhibition of sperm penetration through human zona pellucida by antisperm antibodies

An in vitro penetration test using human spermatozoa, sera, and eggs stored in a highly concentrated salt solution was designed for examination of the effect of antisperm antibodies on the process of fertilization. Spermatozoa from a healthy fertile donor incubated in modified Biggers, Whiiten and Whittingham (BWW) medium containing 7.5% antisperm-antibody-negative serum, could penetrate through the zonae pellucidae of the stored eggs, but not when the spermatozoa from the same donor had been incubated in modified BWW medium containing 7.5% antisperm-antibody-positive serum. After the antisperm-antibody-positive serum was absorbed with washed spermatozoa, the sperm penetration was not blocked. Therefore, antisperm antibodies appear to block human sperm penetration through the human zona pellucida.     

Effects on fertility of immunizing mice with anti-idiotypic antibodies to porcine zona pellucida antigen

OBJECTIVE: To evaluate the effects on fertility by immunization with anti-idiotypic antibodies to porcine zona pellucida (PZP) antigen. METHOD: Anti-idiotypic antibodies (Ab(2)) were produced in New Zealand rabbits immunized with 17D3 monoclonal antibodies (mAbs) (IgG, Ab(1)) to PZP antigen. The antisera were first passed through immuno-affinity chromatography column linked to normal mouse IgG so as to remove the antibody bound to normal mouse IgG The passing elute was then purified by immuno-affinity chromatography using 17D3 mAbs to get the Ab(2). Female BALB/c mice, 5-week-old, were grouped and immunized with the Ab(2), PZP antigen, target antigen of the Ab(1) and normal rabbit IgG, respectively. The treated female mice were mated with male BALB/c mice and sacrificed at gestation day 10. Analyses included ELISA measurement of anti-ZP antibody titer, fetal number determination and evaluation of ovarian histomorphology. RESULTS: The Ab(2) appeared as a single protein band by SDS-PAGE. Shown by a competitive inhibition ELISA, the Ab(2) specifically bound to the variable region of the 17D3 Ab(1). Compared with controls, the female mice immunized with Ab(2) showed a decreased pregnancy rate and a statistically significant reduction in fetal numbers. Histological examination of ovaries demonstrated that Ab(2) exposure interfered less with follicular development than did exposure to PZP. CONCLUSION: Immunization of female mice with Ab(2) to PZP mAbs suppresses fertility and fetal numbers with minimal ovarian pathology.     

Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women.     

Immunogenicity of deglycosylated zona pellucida antigens and their inhibitory effects on fertility in rabbits.

Influence of carbohydrates on the immunogenicity and immunocontraceptive potential of zona pellucida glycoproteins has been investigated in rabbits. Porcine zonae pellucidae, following deglycosylation with trifluoromethane-sulfonic acid, retained significant immunogenic potential, as shown by the ability to generate antibodies which cross-reacted with the heterologous antigen. Antibody response, however, was much stronger against the native zona glycoproteins, thereby suggesting that both carbohydrate and protein moieties contribute to the overall immunogenicity of the zona pellucida antigens. Contraceptive efficacy of active immunization with the deglycosylated zona antigens, when evaluated by mating experiments, demonstrated inhibition of fertility in all immunized rabbits. Normal ovarian functions were disrupted in these animals, as revealed by the reduction in ovarian weights and gross impairment of folliculogenesis. Flushing of the oviducts of the immunized animals yielded a markedly reduced number of ova ovulated in response to hCG administration, none of which were fertilizable. Results collectively suggest that active heteroimmunization with deglycosylated zona pellucida antigens is effective in reducing fertility in rabbits.     

Status of autoantibodies to zona pellucida in human reproduction

The sera of 80 patients were tested by the indirect immunofluorescence technique to evaluate the presence of autoantibodies to zona pellucida in human reproduction. The incidence of positive anti-zona activity was 71.4% (25/35) in the infertile women, 40.0% (6/15) in the normal, nonpregnant fertile women, 20.0% (3/15) in the normal pregnant women and 26.7% (4/15) in the normal fertile men, when unabsorbed sera were tested. To overcome false positive reactions due to nonspecific serum components, all the positive sera were absorbed with formalinized porcine red blood cells and retested. However, after absorption, anti-zona activity was lost in all the positive sera from non-pregnant fertile women, pregnant women and fertile men; but it was retained in 51.4% (18/35) of the infertile women. These positive sera (18) were further absorbed with zona-coated eggs and retested. Fluorescence was lost in all the positive sera, thus demonstrating the presence of antibodies specific to zona antigen in the infertile women. The study also revealed that autoantibodies to zona were seen more often in patients of greater age (26 to 40 years) and in those who had been infertile for a greater period of time (greater than 6 years).     

Studies on incidence and characterization of antibodies to zona pellucida (Z.P.) in human sera

Incidence and characterization of antibodies to zona pellucida (Z.P.) in human sera were studied and the following results were obtained. Some human sera showed positive immunofluorescent staining of porcine Z.P. after absorption with porcine red blood cells, liver and kidney. The incidence of immunofluorescence positive sera in infertile women was not significantly different from pregnant women and healthy men after absorption with porcine red blood cells, liver and kidney. Among sixteen sera with positive staining of porcine Z.P. after absorption, only two showed positive staining of human oocytes. The gammaglobulin fraction from the above two sera also stained human Z.P. but neither precipitated on the surface of Z.P. nor blocked human spermatozoa to penetrate into Z.P. of human oocytes. There was no correlation between the incidences of antibody to porcine Z.P. and of sperm immobilizing antibody in human sera. Some human sera blocked monoclonal antibodies to porcine Z.P. with different specificities to bind the solubilized porcine Z.P. antigens in an enzyme linked immunosorbent assay. The incidences of the positive sera were not significantly different among the groups of infertile women, pregnant women and healthy men. These results clearly indicate that anti Z.P. antibodies detected by the immunofluorescent staining of porcine Z.P. had no correlation with infertility. The presence of such antibodies, even in the sera of men, suggests that they might be the antibodies produced to some foreign antigens cross-reactive to porcine Z. P. but not autoantibodies produced to human Z.P.     

A novel homozygous variant in ZP2 causes abnormal zona pellucida formation and female infertility

PurposeWe aimed to identify pathogenic variants in two infertile sisters in a family with a thin zona pellucida (ZP) phenotype.MethodsWhole-exome sequencing was performed in the two affected sisters, and Sanger sequencing was used to confirm the identified variants. The effects of the identified variant were further investigated in mouse oocytes and Chinese hamster ovary (CHO) cells.ResultsWe identified a novel homozygous frameshift variant in ZP2 (c.1235_1236del, p.Q412Rfs*17) in the two affected individuals. Immunoblotting demonstrated that the variant produced a truncated ZP2 protein that was expressed at low levels in CHO cells. Immunofluorescence in mouse oocytes confirmed the decreased protein level of mutant ZP2, although the subcellular localization was not affected. In addition, immunoprecipitation showed that the pathogenic variant reduced the interaction between ZP2 and ZP3.ConclusionThis study identified a novel pathogenic variant in ZP2 that produces a truncated ZP2 protein. The variant might disrupt the assembly of ZP2-ZP3 dimers, thus resulting in a thin ZP and female infertility.     

Evaluation of immunofluorescence on pig zona pellucida for detection of anti-zona antibodies in human sera

Using immunofluorescence tests, antibodies reacting with pig zona pellucida could be detected in 31% of 103 sera from patients in different clinically defined categories, mainly fertile males and females and patients from couples with unexplained infertility. The antibodies could in all cases be absorbed by means of pig erythrocytes — in most sera with all erythrocyte suspensions tested, but in some cases only with erythrocytes from certain animals — indicating that antibodies both to generally occurring pig antigens and alloantigens may be present in human sera. Staining of pig zonae was recorded equally frequently with unabsorbed sera from infertile and fertile males and females, respectively, but titres of 1 : 16 or more occurred more frequently among infertile than among fertile females (P = 0.053).     

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.     

Defining bioassay conditions to evaluate sperm/zona interaction: Inhibition of zona binding mediated by solubilized human zona pellucida

Objective: Our goal was to study the influence of solubilized human zonae pellucidae on zona binding potential and acrosome reaction. Materials and Methods: Zona pellucida (ZP) solutions were prepared by dissolving zona in acidic buffer, NaH₂PO₄ (pH 2.5), to obtain 0.1 and 0.5 zona pellucida/µl. Zona binding capacity was evaluated by the addition of oocytes (10-fold) to sperm/zona pellucida solution droplets. The number of sperm bound to each oocyte was recorded. Zona pellucida-mediated acrosome activity was evaluated after 60 min of coincubation of sperm and 0.5 ZP/µl. Results: The mean (±SE) number of sperm bound for control, 0.1 ZP/µl, and 0.5 ZP/µl was 181.2±12, 79.6±5, and 38.8±3, respectively. Zona pellucida-exposed sperm populations showed significant more acrosome-reacted sperm compared to control sperm, namely, 78 versus 32%, respectively (P=0.001). Conclusions: The observed zona binding inhibition might be ascribed to zona receptor blocking on the sperm surface or the inability of acrosome-reacted sperm to bind to the zona pellucida.     

Egg yolk enhances the receptiveness of zona pellucida to sperm binding

Purpose

Our purpose was to test whether treatment of zona pellucida with egg yolk enhances its receptiveness to sperm binding. Hams F-10 supplemented with 0.06 g% freeze-dried egg yolk (TM) was evaluated using the hemizona assay. Ham's F-10 supplemented with 7.5% human serum served as the control medium (CM). Salt-stored zona pellucida were bisected and each matched hemizona processed in either TM or CM for the preincubation study or both hemizona processed in CM for the coincubation study. TEST yolk-treated sperm from 10 ejaculates were then incubated with the hemizona preincubated in TM and CM but coincubated in CM or coincubated with hemizona in TM and CM.

Results

A significantly (P<0.02) higher number of tightly bound sperm and hemizona index for the hemizona either preincubated or coincubated in TM compared to the respective hemizonae in CM were observed.     

Characterization of human sperm binding to homologous recombinant zona pellucida proteins

The union between mammalian gametes begins with the sperm binding to the zona pellucida (ZP). We studied the interaction between human sperm and ZP by using recombinant human ZP proteins (rhZP). The cDNAs coding for human ZP2, ZP3, and ZP4 were expressed in Sf9 cells and proteins were characterized to determine their competence for sperm binding. Capacitated human sperm binding abilities were analyzed using immobilized rhZP and a well-characterized antihuman sperm antiserum. The results demonstrated that all rhZP proteins were structurally similar to their native counterparts and were specifically recognized, in a dose and time dependent manner, by human sperm. The rhZP4 was the main sperm binder followed by rhZP3 and rhZP2, although combinations of rhZP proteins enhanced sperm adhesion. Moreover, this experimental approach may represent a useful model to study sperm-ZP interactions for research and clinical purposes.     

Hemizona assay: Use of fresh versus salt-stored human oocytes to evaluate sperm binding potential to the zona pellucida

Salt-stored human oocytes (pH 7.0) showed sperm binding ability equal to that of fresh, living oocytes under hemizona assay (HZA) conditions.     

Biochemical and functional characterization of the human zona pellucida

A successful interaction between spermatozoa and the zona pellucida is critical for fertilization. This biological step reflects multiple sperm functions, including the acquisition and completion of capacitation, recognition and binding to specific zona pellucida receptors, and induction of the physiological acrosome reaction. The recognition of carbohydrate sequences by complimentary receptors has been demonstrated in gamete interaction in different animal species. It has been proposed that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human spermatozoa to the homologous zona pellucida) and advanced methods of carbohydrate analysis have been used to test this hypothesis. Compelling evidence exists to demonstrate that oligosaccharide recognition is also required for specific, tight human gamete binding. The induction of the acrosome reaction using the physiological inducers, i.e. the zona pellucida and progesterone, was also examined. It has also been demonstrated that there is a priming effect of the steroid on the acrosome reaction inducing capacity of the zona pellucida. These studies may allow for a better understanding of human gamete interaction in physiological and pathological situations.     

Genetic and physiological study of morphologically abnormal human zona pellucida

Objective

To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance.

Study design

The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed.

Results

No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3.

Conclusions

The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.