Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3(tm/tm)) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3(tm/tm) null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.     

Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes

The ZP2 protein is a zona pellucida glycoprotein that plays a major role in fertilization. It mediates secondary binding of spermatozoa and is one of the proteins that are involved in zona 'hardening'. ZP2 proteins were identified in various mammalian zonae pellucidae. Their primary structures are highly conserved as revealed by cDNA cloning. Antisera were used against synthetic peptides generated either against a ZP2 amino acid that is homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or antibodies against a synthetic human ZP2 peptide (AS ZP2-26). Immunoblots showed that antiserum AS ZP2-20 and AS ZP2-26 strongly recognized human ZP2 protein with an apparent molecular mass of about 72 kDa; both antisera reacted with a minor immunoreactive polypeptide at 96 kDa. In human ovary sections, both antisera revealed immunoreactivity to human zonae pellucidae. Immuno-electron microscopy demonstrated an equal distribution of ZP2 throughout the human zona pellucida. Considerable amounts of immunoreactive material were observed in the ooplasm; some ramification-like extensions of zona pellucida antigen were found close to cells surrounding the oocyte. Our results indicate that antisera against synthetic ZP2 peptides can be used as specific markers for the identification of ZP2 protein in human oocytes.     

Gamete recognition in mammals: sperm and zona pellucida interactions

Association of sperm with the acellular protective envelope of the oocyte, the zona pellucida, and their penetration is a determinant step in fertilization process. It is at this stage that species barriers take place to prevent cross fertilizations. This association is dependent upon binding of ZP3 oligosaccharides to specific sperm receptors. Their activation triggers the acrosome reaction via transduction pathways and release of proteolytic enzymes that dissociate the zona pellucida network. Then, secondary binding to zona pellucida components allows sperm to penetrate and cross the zona pellucida barrier. Several sperm surface proteins are putative receptors for zona pellucida partners. Repercussion of these studies on human fertility are discussed.     

Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.     

Protein-carbohydrate interactions during fertilization

Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the zona pellucida (ZP) and carbohydrate binding proteins of the sperm surface. This review focuses on the protein-carbohydrate interactions during the primary binding of the sperm to the ZP in different species. Synthesis, structure and composition of the ZP are summarized. The functional significance of carbohydrate residues of the ZP as sperm receptor is discussed. Sperm surface proteins known to have specific ZP and carbohydrate-binding sites including the mouse beta1, 4-galactosyltransferase and sp56, the rabbit protein Sp17, a human mannose-binding protein and several members of the spermadhesin family are presented.     

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.     

Variation in porcine zona pellucida morphology following pronase treatment

The effects of cell stage and day of embryo collection on the pronase-induced zona pellucida morphology in pig embryos were investigated. Three hundred and seventy-two two- to eight-cell embryos were treated for 3.0 min in 5.0% pronase in Whitten's Medium. Responses in zona pellucida morphology observed after pronase treatment were: (1) absent, (2) stretched and (3) intact. Each pig was coded as to the type of zona pellucida morphology its embryos possessed after enzyme treatment according to the following scale: (1)--only intact, (2)--intact and stretched, (3)--only stretched, (4)--stretched and absent and (5)--only absent. Also, the number of embryos of a particular cell stage was expressed as a fraction of the total number of embryos collected from each pig. The incidence of intact zonae pellucidae was greatest (P less than .05) among two- to three-cell embryos and the incidence of absent zonae pellucidae was greatest (P less than .05) among six-to eight-cell embryos. A significant correlation (r = .79) was observed between day of embryo collection and coded zona pellucida morphology. The results suggest that the pronase-induced zona pellucida morphology observed is dependent on cell stage and day of embryo collection.     

Merkel cell carcinoma of the vulva

The objective of this study was to investigate the requirement for calmodulin in the cell division of the early human preimplantation embryo. Experiments using three agents capable of inhibiting calmodulin activity, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), W7 linked to agarose beads (W7-agarose) and calmodulin antibody, showed that W7 and calmodulin antibody arrested division of embryos in a dose-dependent manner. As W7 is able to penetrate the zona pellucida and enter the cells, calmodulin antibody can traverse the zona but not enter the cells and W7-agarose cannot traverse the zona, we have deduced that the calmodulin which appears relevant to embryo division may be both intracellular and intrazonal but not extrazonal. We conclude that calmodulin is specifically required for cell division in the early human preimplantation embryo and that the concentration of calmodulin surrounding the embryo within the zona is particularly important to embryo development.     

Immunoelectrophoretic analysis of the porcine zona pellucida

Antigens of the porcine zona pellucida were evaluated by 2-dimensional and line immunoelectrophoretic techniques. Zona antigen preparations studied included heat-solubilized isolated zonae pellucidae (SIZP), a purified 60 000 Mr glycoprotein (PPZA, purified pig zona antigen), and two fractions of this 60 000 Mr zona component which had been exposed to SDS (ZP3-E1C and ZP3-E2C). Antisera were raised to intact zonae pellucidae (IZP), SIZP and PPZA. Collectively, electrophoretic data revealed that the porcine zona system is antigenically complex with each zona antiserum tested detecting numerous antigens in the various zona preparations. These antigens, however, all had similar electrophoretic mobilities, and this limited the resolution of these techniques. The 60 000 Mr pig zona macromolecule (ZP3) appeared to be the most immunogenic of the three major pig zona glycoproteins since antisera prepared against IZP or SIZP reacted primarily with this component. However, the 60 000 Mr component does share antigenic determinants with the other major zona glycoproteins as revealed by cross-reactions of the antisera with the various zona preparations. Electrophoretic studies also suggested that the various zona antisera could distinguish, with different degrees of sensitivity, multiple antigenic determinants on the individual zona macromolecules. These studies also indicated that SDS treatment of zona glycoproteins does alter the antigenicity of the macromolecule, both with respect to the total number and individual identity of antigens detected.     

Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro

The mammalian zona pellucida contains several glycoproteins whose oligosaccharide moieties are known to play a key role in the interaction with spermatozoa. Since zona pellucida defects may represent one of the most likely causes of failed fertilization in human in-vitro reproduction, we have studied the carbohydrate composition and distribution over the human zona pellucida by means of lectins. Donated, not inseminated cumulus-oocyte complexes, from cohorts with high fertilization rates, and fertilization-failed oocytes from cohorts inseminated with proven fertile donor semen, were analysed using 11 fluorescein-labelled lectins, on deplasticized semi-thin epoxy sections. Results showed that wheat germ agglutinin (WGA), Maclura pomifera (MPA) and Pisum sativum (PSA) bound to the extracellular matrix bordering the zona pellucida-corona radiata interface of cumulus-oocytes complexes, while the zona pellucida was labelled by WGA, Concanavalin A (ConA) and PSA. WGA labelling and correlative electron microscopy on the cumulus-oocyte complexes demonstrated that this lectin is a useful tool to trace the cortical granule distribution in the human oocyte. Surprisingly, in the failed-fertilized oocytes the zona pellucida was also labelled by MPA and showed three different patterns: (i) labelling of the zona pellucida outer surface; (ii) uniform labelling; (iii) labelling of an outer zona pellucida layer with variable thickness. Comparative analysis of WGA and MPA labelling on single failed-fertilized oocytes demonstrated that MPA zona pellucida patterns are not related to the cortical reaction. The nature and meaning of the MPA pattern of failed-fertilized oocytes were discussed in the light of zona pellucida defects impairing sperm receptivity.     

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.     

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.     

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.     

The P2Y1 receptor is necessary for adenosine 5''-diphosphate-induced platelet aggregation

Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We have tested the concept that immunocontraception can be achieved with a recombinant ectromelia virus expressing an ovary-specific antigen, the mouse zona pellucida 3 (ZP3) glycoprotein. Female mice infected with the recombinant virus produced autoimmune antibodies against ZP3 and were infertile for 5-9 mo after infection. For almost half the infertile mice, immunity to ZP3 was associated with a disruption of ovarian follicular development and the depletion of mature follicles without observable oophoritis. Mice returned to fertility as the anti-ZP3 antibody level in the serum decreased. Reinfection of the mice with the recombinant virus boosted the anti-ZP3 response and restored infertility.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.     

Tubal hydatidiform mole

We report a tubal hydatidiform mole, most likely resulting from dispermic fertilization. Early hatching of the embryo, because of a defective zona pellucida, may have favored tubal implantation.     

Structural and functional differentiation of follicular and oviductal mouse oocytes visualised with FITC-protein conjugates

The fluorescence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida, perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage (GV) ovarian oocytes exhibited a bright, amorphous, mesh-like staining pattern (immature type). In contrast, mature polar body stage (PB) oocytes, either ovarian or oviductal, displayed faint, spotty fluorescence labelling of the ZP (mature type). The perivitelline space (PVS) of mature ovarian oocytes (12 h post-hCG) failed to label, whereas approximately 50% of oviductal oocytes showed PVS labelling. The incidence of PVS staining increased with postovulatory age, possibly as a result of the accumulation of materials secreted by the oviduct. Following in vivo or in vitro fertilisation of oocytes, a characteristic pattern of plasma membrane (PM) labelling was observed. Similar patterns of PM labelling were seen in oocytes parthenogenetically activated with ethanol or ionophore (A23187) but not in control oocytes. The pattern of PM labelling observed with FITC-protein conjugates was strikingly similar to that observed with FITC-labelled lectins, which are thought to interact with glycoconjugates released from cortical granules. Immature type of ZP staining also occurred when GV oocytes were treated with FITC alone or with a variety of FITC-protein conjugates. Thus, protein may not be required for labelling of the ZP by FITC-protein conjugates as previously thought. FITC-conjugated proteins including casein, bovine serum albumin, peroxidase and non-immune immunoglobulin G (IgG), all labelled the PM of activated oocytes; however, FITC-IgG failed to label the PVS. Results demonstrate for the first time that various components of viable mouse oocytes exhibit and undergo characteristic structural and functional changes during periovulatory differentiation as evidenced by their interaction with one or more FITC-protein conjugates and/or FITC. On the basis of these results the intrafollicular and oviductal mechanisms mediating these changes are discussed as is the possibility that the fluorescent molecule attached to conjugates may play a role in oocyte labelling.     

Beta-carotene intake and risk of postmenopausal breast cancer

Improved procedure for efficient cryopreservation of single human spermatozoa in cell-free human zona pellucida is reported. We used a diode laser system for efficient and precise creation of a single hole into the zona pellucida of a degenerated or immature human oocyte. This allowed the extraction of the cytoplasm using a micropipette with a diameter of 10-15 microns. Through the same opening, human spermatozoa were inserted into the empty zona. We used motile and laser immobilized spermatozoa. Immobilized sperm were obtained by a single laser irradiation delivered in the vicinity of the sperm tail prior to insertion. This new immobilization procedure was shown to have no deleterious effect on membrane integrity and sperm viability. Following sperm transfer into the zona, the laser-drilled hole was closed with an oil droplet which was expelled from the micropipette during withdrawal to avoid loss of spermatozoa. This facilitated detection of the otherwise translucent zona during the cryopreservation procedure. After thawing, all cryopreserved zonae (20/20) could be successfully retrieved. Spermatozoa were recovered from the zona pellucida through the hole used for insertion. The rate of sperm recovery for initially motile spermatozoa was 80% vs. 92% for laser immobilized spermatozoa. Sperm viability was 81% and 84%, respectively, detected by a Hoechst stain. This technique makes cryopreservation of single human spermatozoa easy and feasible and appears beneficial for couples with severe male infertility and for those facing repeated surgical sperm extraction.