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 method for specific detection of autoantibodies to the zona pellucida in infertile women.

Human antibodies to porcine erythrocytes were found to bind to porcine but not to human zonae pellucidae, whereas human isohemagglutinins bound to human but not to porcine zonae. On the basis of the binding behavior of agglutinins, it was found that the immunofluorescence of porcine zonae produced by selected sera from infertile women was due to an antibody of different specifity from that which agglutinated pig red blood cells. However, no serum component other than heteroagglutinin was contained in selected sera from control subjects which fluoresced porcine zonae. Since the component was present in the immunoglobulin G fraction, bound to human zonae, and was reactive with zona-specific antigen(s), it was judged an autoantibody to zonae. Thus a method for specific detection of autoantibodies to zonae has been developed by indirect membrane immunofluorescence using porcine ova as targets.     

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.     

Immunosuppression supports implantation of zona pellucida dissected human embryos

The effect of low dose immunosuppression with methylprednisolone during the first 4 days after oocyte retrieval on potential immune cell invasion of partially zona dissected embryos in utero was investigated in alternate in vitro fertilization patients (n = 32). The incidence of pregnancy was significantly higher in patients receiving methylprednisolone (7 of 18, 39%) than in control patients (1 of 14, 7%). Twenty-eight percent (11 of 39) of the embryos replaced in the corticosteroid treated patients implanted, whereas only 7% (2 of 31) of embryos in control patients had a fetal heart beat. There were no side effects reported in any of the patients receiving corticosteroids. It can be concluded that methylprednisolone supports implantation of embryos with small holes in their zonae. However, the actual mechanisms of corticosteroid support on the interaction between immune cells and micromanipulated embryos are not well understood.     

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.     

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.     

Recombinant human zona pellucida protein C produced in Chinese hamster ovary cells binds to human spermatozoa and inhibits sperm-zona pellucida interaction

Recombinant human zona pellucida protein C expressed in Chinese hamster ovary cells associates to the acrosomal region of human spermatozoa and inhibits sperm-zona pellucida interaction in the hemizona assay. Recombinant human zona pellucida protein C may be a useful tool toward the development of diagnostic methods for male factor infertility and the elucidation of the molecular basis of fertilization.     

Involvement of carbohydrate molecules on zona pellucida in human fertilization.

Although the involvement of several receptors and ligand molecules in sperm-zona interaction in many species have been proposed, there has been a little analysis of the kinetics between these molecules during the interaction. In the present study, we applied the detection method using surface plasmon resonance (SPR) by a BIAcore apparatus for the analysis of the putative receptor-ligand interaction of sperm-egg binding. Mannose-BSA or [man](5)-[GlcNAc](2)-Asp was immobilized on the surface of a sensor chip. When concanavalin A (Con A) was delivered to each of two different sensor chips to evaluate their usefulness, the resonance signal after sample injection onto a [man](5)-[GlcNAc](2)-Asp-fixed chip decreased rapidly than the mannose-BSA-fixed chip. However, the amount of binding for Con A during the injection onto the [man](5)-[GlcNAc](2)-Asp-fixed chip was high. When acid sperm extracts (acid extracts) and fractions through a CM column, containing protease activity (protease fractions), were delivered to the mannose-BSA-fixed chip, the SPR signal during the injection was not obviously changed compared with that of the control. However, when sperm samples were delivered to the [man](5)-[GlcNAc](2)-Asp-fixed chip, the SPR response during the injection was enormous. These results suggest that the [man](5)-[GlcNAc](2)-Asp-fixed chip is more useful than the mannose-BSA-fixed chip for investigating the interactions with sperm extracts and that the sensitive method using SPR by a BIAcore apparatus is applicable for the analysis of the putative receptor-ligand interaction of sperm-egg binding.     

Enzymatic characterization of zona pellucida hardening in human eggs and embryos

Purpose To characterize possible hardening of the human zona pellucida (ZP) and evaluate the effect of culture duration, patient age, and ZP thickness, ZP of unfertilized eggs (experiment 1, n =367; experiment 2, n =174) and abnormal embryos (experiment 1, n=52) were randomly designated for -chymotrypsin treatment after 0, 24, 48, 72, 96, 120 h (experiment 1) and 48 h, 72 h, and 1 week (experiment 2) of in vitroculture in HTF medium supplemented with 0.5% human serum albumin. Mean ZP thickness was predetermined in experiment 2.Methods The dispersion of the ZP glycoproteins was assessed, and the duration of time for complete digestion was recorded as an index of ZP hardness.Results In experiment 1, enzyme digestion duration increased (P<0.05) in the first 24 h in vitrofrom 18.0±2.0 to 34.6±2.5 min, and tended to decrease over the next 4 days in culture (25.2±1.3, 29.4±0.9, 27.3±0.6, 26.6+1.1, and 20.7±1.5 min on Day 2– 6 ZP, respectively). Zona hardening of fertilized eggs was revealed by a longer (P<0.01) digestion time (32.2±1.8 vs 25.8±0.6 min).Conclusions There were significant patient-to-patient variations (16.4±0.7 to 39.6±2.2 min); however, age was not correlated to enzyme digestion duration. In experiment 2 we determined that ZP thickness (range 8.4–21.6 m; mean 14.6±0.2 m) was not correlated (r=0.09) to the digestion interval (mean 24.3 ± 0.8 min). Based on our enzymatic ZP digestion measurements, it is apparent that spontaneous zona hardening does occur within 24 h of in vitroculture, similar to levels achieved postfertilization. The data do not support, however, the concept that additional, abnormal hardening of the ZP occurs during extended culturing.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

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.     

Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration

Partial zona dissection (PZD), a method using mechanical force to open the human zona pellucida, and zona drilling, which uses acidic Tyrode's (AT) medium, were compared in 1-day-old oocytes prior to reinsemination. The incidences of monospermy and polyspermy were 13/54 (24%) and 14/54 (26%) following PZD and 6/46 (13%) and 8/46 (17%) following the use of AT medium. This compared favorably with conventional reinsemination: 15/161 (9%) monospermy and 4/161 (3%) polyspermy. Three of the 27 PZD embryos became blastocysts, while none of the AT-exposed embryos developed satisfactorily. Eleven male-factor couples had some of their oocytes randomly treated with PZD prior to insemination; each of the patients had non-micromanipulated control oocytes. Monospermic fertilization and cleavage (23/34; 68%) doubled (P less than 0.05) when PZD was compared with the control oocytes (10/30; 33%). Replacing two PZD and a single control embryo in two patients resulted in twin pregnancies. A third twin pregnancy was established following replacement of only micromanipulated embryos.     

In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction

This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.     

Horse and marmoset monkey sperm bind to the zona pellucida of salt-stored human oocytes

The present study demonstrates that horse and marmoset monkey sperm can bind to the human zona of salt-stored oocytes that failed to fertilize in vitro. Marmoset monkey sperm are also able to penetrate the salt-stored human zona. In contrast, human sperm do not bind to the zona of either horse or marmoset monkey oocytes. These results suggest that human sperm binding to the zona pellucida is more strictly species-specific than it is for horse and marmoset monkey sperm. In contrast, horse and marmoset monkey sperm contain receptors recognized by the human zona.     

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.     

Antibody reactivity with porcine zona pellucida

A comparative study on anti-zona antibody activities in the sera from clinically defined categories of patients registered at the WHO Reference Bank for Reproductive Immunology was performed at five different laboratories with different detection methods. Considerably higher incidences of positive reactions were detected by immunofluorescence on porcine zonae in infertile women (16.3%) than in control subjects (7.1%). A similar proportion of positives was found by radioimmuno-binding assay (RIBA) using porcine zona antigen preparation in the infertile group (13.0%) but not in the female control group (0%), giving an indication of the specificity of this test. It is noteworthy that high incidences of positives were observed by RIBA with sera from male subjects with unexplained sterility, vasectomy and aspermatogenesis. A test system of passive hemagglutination reaction (PHAR) using purified porcine zona substance as antigen gave a low but slightly higher incidence of positives in infertile (3.1%) than in control sera (0.9%). No positive reactions were observed with infertile and control sera by another PHAR or by radioimmunoassay using an antigen preparation common to the two test systems. Anti-zona activities in these sera were therefore seen to vary, depending largely upon the detection systems and the antigen preparations.