Human ZP3 Research Articles

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

Identification and in silico Characterization of Deleterious Single Nucleotide Variations in Human ZP2 Gene.

ZP2, an important component of the zona matrix, surrounds mammalian oocytes and facilitates fertilization. Recently, some studies have documented the association of mutations in genes encoding the zona matrix with the infertile status of human females. Single nucleotide polymorphisms are the most common type of genetic variations observed in a population and as per the dbSNP database, around 5,152 SNPs are reported to exist in the human ZP2 (hZP2) gene. Although a wide range of computational tools are publicly available, yet no computational studies have been done to date to identify and analyze structural and functional effects of deleterious SNPs on hZP2. In this study, we conducted a comprehensive in silico analysis of all the SNPs found in hZP2. Six different computational tools including SIFT and PolyPhen-2 predicted 18 common nsSNPs as deleterious of which 12 were predicted to most likely affect the structure/functional properties. These were either present in the N-term region crucial for sperm-zona interaction or in the zona domain. 31 additional SNPs in both coding and non-coding regions were also identified. Interestingly, some of these SNPs have been found to be present in infertile females in some recent studies.

Human Zona Pellucida Glycoproteins: Binding Characteristics With Human Spermatozoa and Induction of Acrosome Reaction.

Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes encoding human ZP glycoproteins are one of the causative factors leading to abnormal ZP matrix and infertility in women. Relevance of the human ZP glycoproteins in ‘sperm–oocyte’ binding has been delineated by using either transgenic animal models expressing human zona proteins or purified native/recombinant human zona proteins. Studies based on the purified native/recombinant human zona proteins revealed that ZP1, ZP3, and ZP4 primarily bind to the capacitated acrosome-intact human spermatozoa whereas ZP2 binds to acrosome-reacted spermatozoa. On the contrary, human spermatozoa binds to the eggs obtained from transgenic mouse lines expressing human ZP2 but not to those expressing human ZP1, ZP3, and ZP4 suggesting that ZP2 has an important role in human ‘sperm–oocyte’ binding. Further studies using transgenic mouse lines showed that the N-terminus of human ZP2 mediate the taxon-specific human sperm–oocyte binding. Both glycans and protein-protein interactions have a role in human gamete interaction. Further studies have revealed that the purified native/recombinant human ZP1, ZP3, and ZP4 are competent to induce acrosome reaction. Human sperm binds to the mouse transgenic eggs expressing human ZP1-4 instead of mouse ZP1-3 proteins, penetrated the ZP matrix and accumulated in the perivitelline space, which were acrosome-reacted suggesting that human ZP2 in transgenic mouse model also induce acrosome reaction. In humans N-linked glycosylation of zona proteins have been shown to play an important role in induction of the acrosome reaction. Hence in humans, based on studies using transgenic mouse model as well as purified native/recombinant zona proteins, it is likely that more than one zona protein is involved in the ‘sperm–oocyte’ binding and induction of the acrosome reaction.

Heterozygous mutations in ZP1 and ZP3 cause formation disorder of ZP and female infertility in human.

The human zona pellucida (ZP) is a highly organized glycoprotein matrix that encircles oocytes and plays an essential role in successful reproduction. Previous studies have reported that mutations in human ZP1, ZP2 and ZP3 influence their functions and result in a lack of ZP or in an abnormal oocytes and empty follicle syndrome, which leads to female infertility. Here, we performed whole‐exome sequencing in two probands with primary infertility whose oocytes lacked a ZP, and we identified a heterozygous mutation in ZP1 (NM_207341:c.326G>A p.Arg109His), which is situated in the N‐terminus, and a heterozygous mutation in ZP3 (NM_001110354:c.400G>A p.Ala134Thr), which is situated in the ZP domain. The effects of the mutations were investigated through structure prediction and in vitro studies in HeLa cells. The results, which were in line with the phenotype, suggested that these mutations might impede the function of cross‐linking and secretion of ZP proteins. Our study showed that the two mutations in ZP1 and ZP3 influenced the formation of the ZP, causing female infertility. Meanwhile, these data highlight the importance of the ZP1 N‐terminus in addition to the conserved domains for ZP1 function and ZP formation. Additionally, the patient with the ZP1 mutation delivered a baby following intracytoplasmic sperm injection (ICSI); thus, we suggest the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve the success rate of assisted reproductive technology (ART) treatments.

Molecular basis of egg coat cross-linking sheds light on ZP1-associated female infertility

Mammalian fertilisation begins when sperm interacts with the egg zona pellucida (ZP), whose ZP1 subunit is important for fertility by covalently cross-linking ZP filaments into a three-dimensional matrix. Like ZP4, a structurally-related component absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Here we report a characterisation of ZP1 proteins carrying mutations from infertile patients, which suggests that, in human, filament cross-linking by ZP1 is crucial to form a stable ZP. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not in human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception.

Structural and molecular analysis of the cancer prostate cell line PC3: Oocyte zona pellucida glycoproteins.

The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention.

Effect of human ZP3 monoclonal antibody on expression of GDF-9 and number of theca cells in ovary of mice (Mus musculus).

ObjectivesThis study investigated the effects of a human ZP3 monoclonal antibody (mAb hZP3) on the expression of growth differentiation factor-9 (GDF-9) and number of theca cells in the ovaries of mice (Mus musculus).MethodsOur study employed a true experiment posttest-only control group design of 48 mice that were divided into the control and three mAb hZP3-treatment groups (20, 40, and 60 μg). Mice in each group were terminated on days 10, 15, and 20. GDF-9 expression was measured by immunohistochemistry and the number of theca cells was counted.ResultsAnalysis of the effects of mAb hZP3 (at 20–60 μg) on the expression of GDF-9 and amount of theca cells did not show significant differences. Similar findings were observed throughout the study period (at 10–20 days). Therefore, mAb hZP3 had no effect on GDF-9 expression and theca cells.ConclusionThis study showed that mAb hZP3 can be considered to be an effective and safe immunocontraception.

The Human Egg's Zona Pellucida.

Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. All four zona proteins share common structural elements such as signal peptide, "ZP domain," consensus furin cleavage site, transmembrane-like domain, and short cytoplasmic tail. In addition, ZP1 and ZP4 also have "Trefoil domain." Recombinant/native human zona proteins have been used to investigate their binding characteristics to the capacitated and/or acrosome-reacted spermatozoa. These investigations revealed that ZP1, ZP3, and ZP4 primarily bind to the head region of the capacitated human spermatozoa, whereas ZP2 binds to the acrosome-reacted sperm. However, using transgenic mice, N-terminal region of human ZP2 has also been shown to play an important role in binding of sperm to the egg. ZP1, ZP3, and ZP4 lead to dose-dependent increase in acrosome reaction, suggesting that in humans more than one ZP glycoprotein is responsible for induction of acrosome reaction. Glycosylation of these proteins, in particular, N-linked glycosylation as well as sialyl-Lewisx, is essential for inducing acrosome reaction. Studies delineating downstream signaling events associated with induction of acrosome reaction reveal subtle differences between ZP3 and ZP1/ZP4 with respect to activation of Gi protein-coupled receptor and protein kinase A. The role of mutations in the zona proteins and ZP autoantibodies leading to infertility in women is suggestive and needs more rigorous experimentations for confirming their role in female infertility. The above-mentioned aspects of the human ZP glycoproteins have been discussed in this review.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP-N and ZP-C. Here, we have synthesized six 'aggregation-prone' peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy indicate that these peptides self-assemble forming fibrils with distinct amyloid-like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self-assembly mechanism of human ZP proteins.

Bovine And Human Zona Pellucida 3 Gene Glycans Site Prediction Using In Silico Analysis

Zona pellucida is one of the protective layer of the egg cell and has a function as an intermediary species-specific fertilization. Glycoproteins of human and bovine zona pellucida is composed of three types, namely ZP1, ZP2 and ZP3. ZP3 gene has amino acid sequence homology with other mammals. Oligosaccharides components of the zona pellucida glycans are composed from units of asparagine residues (N -linked) and serine/threonine (O -linked). The aims of this study was to analyze the DNA sequences of human and bovine and further predicts glycans site on amino acid sequence of human and bovine ZP3. In this study, ZP3 gene fragments have been isolated from bovine and humans were analyzed in Silico. This work were conducted by comparing the data of DNA sequence from human and bovine PCR product using NCBI BLAST. The results showed that there were similarities at amino acid positions number 23-38. bZP3 sequence had three glycans site (Asn-X-Thr/Ser) and one site on hZP3 glycans. One of the sites was conserved between the two species.

A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans.

The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1-4), but which protein binds sperm remains controversial. A defining characteristic of an essential zona ligand is sterility after genetic ablation. We have established transgenic mice expressing human ZP4 that form zonae pellucidae in the absence of mouse or human ZP2. Neither mouse nor human sperm bound to these ovulated eggs, and these female mice were sterile after in vivo insemination or natural mating. The same phenotype was observed with truncated ZP2 that lacks a restricted domain within ZP2(51-149). Chimeric human/mouse ZP2 isoforms expressed in transgenic mice and recombinant peptide bead assays confirmed that this region accounts for the taxon specificity observed in human-mouse gamete recognition. These observations in transgenic mice document that the ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.

Human fertilization: epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

The molecular basis of gamete recognition in mice and humans

Successful fertilization heralds the onset of development and requires both gamete recognition and a definitive block to polyspermy. Sperm initially bind and penetrate the extracellular zona pellucida (ZP) that surrounds ovulated eggs, but are unable to bind the zona surrounding preimplantation embryos. The ZP of humans is composed of four (ZP1-4) and that of mouse three (ZP1-3) glycoproteins. Models for gamete recognition developed in mice had proposed that sperm bind to ZP3 glycans. However, phenotypes observed in genetically engineered mice are not consistent with this widely accepted model. More recently, taking advantage of the observation that human sperm do not bind to mouse eggs, human ZP2 was defined as the zona ligand in transgenic mouse models using gain-of-function assays. The sperm-binding site is an N-terminal domain of ZP2 that is cleaved by ovastacin, a metalloendoprotease released from egg cortical granules following fertilization. Proteolysis of this docking site provides a definitive block to polyspermy as sperm bind to uncleaved, but not cleaved ZP2 even after fertilization and cortical granule exocytosis. While progress has been made in defining the ZP ligand, less headway has been made in identifying the cognate sperm receptor. Although a number of sperm receptor candidates have been documented to interact with specific proteins in the ZP in vitro, continued fertility after genetic ablation of the cognate gene indicates that none are essential for gamete recognition. These on-going investigations inform reproductive medicine and suggest new therapies to improve fertility and/or provide contraception, thus expanding reproductive choices for human couples.

Rabbit zona pellucida composition: A molecular, proteomic and phylogenetic approach

The zona pellucida (ZP) participates in sperm-egg interactions during the first steps of fertilization. Recent studies have shown that the ZP matrix of oocytes in several species is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, rather than the three described in mouse, pig and cow. In this study, investigations were carried out to unveil a fourth glycoprotein in the rabbit (Oryctolagus cuniculus) ZP. Using total RNA isolated from rabbit ovaries, the complementary deoxyribonucleic acid (cDNA) encoding rabbit ZP1 was amplified by reverse transcribed polymerase chain reaction (RT-PCR). The ZP1 cDNA contains an open reading frame of 1825 nucleotides encoding a polypeptide of 608 amino acid residues. The deduced amino acid sequence of rabbit ZP1 showed high identity with other species: 70% identity with human and horse ZP1, and 67% identity with mouse and rat ZP1. At the proteomic level, peptides corresponding to the four proteins were detected by mass spectrometry. In addition, a molecular phylogenetic analysis of ZP1 showed that pseudogenization of this gene has occurred at least four times during the evolution of mammals. The data presented in this manuscript provide evidence, for the first time, that the rabbit ZP is composed of four glycoproteins.

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1-3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm-egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patient's hormonal status, embryo quality, and IVF outcomes. Prospective study. University-affiliated IVF clinic. Couples undergoing IVF-ET cycles. Zona measurements, clinical data collection. Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 μm. No significant correlation was observed between the ZPT and the patient's age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 μm vs. 16.36 ± 2.57 μm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. The thickness of the human ZP of day 3 embryos is not influenced by women's age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Mammalian zona pellucida glycoproteins: structure and function during fertilization

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis(x) sequence present on both N- and O-glycans of human ZP play an important role in human sperm-egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the Gi protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.

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

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

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.

Human ZP4 is not sufficient for taxon-specific sperm recognition of the zona pellucida in transgenic mice

The molecular basis of human fertilization remains enigmatic. Mouse models are often used to study sperm-egg recognition, but the mouse zona pellucida surrounding ovulated eggs contains three proteins (ZP1, ZP2, and ZP3) whereas the human zona contains four (ZP1, ZP2, ZP3, and ZP4). Human sperm are fastidious and recognize human but not mouse eggs. Transgenic mouse lines were established to ascertain whether human ZP4 is the sole determinant of human sperm binding. Human ZP4 expressed in transgenic mice had a molecular mass similar to the range of native protein isoforms and was incorporated into the extracellular zona matrix. Transgenic females were fertile with normal litter sizes. Mouse sperm readily recognized transgenic ovulated eggs, but human sperm did not. We conclude that human ZP4 is not sufficient to support human sperm binding to the zona pellucida in transgenic mice and that other zona proteins may be needed for human gamete recognition.