Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs and preimplantation embryos. Fertilization results in transformation of the zona pellucida (“zona reaction”), such that additional sperm are unable to bind to the zona pellucida of fertilized eggs and embryos, and sperm that had partially penetrated the zona pellucida of eggs prior to fertilization are prevented from further penetration after fertilization. The failure of sperm to bind to fertilized mouse eggs and embryos is attributable to modification of the sperm receptor, ZP3, an 83,000-molecular weight glycoprotein present in zonae pellucidae isolated from both eggs and embryos [Bleil, J. D., and Wassarman, P. M. (1980). Cell, 20, 873–882]. In this investigation, ZP2, the major glycoprotein found in mouse zonae pellucidae [Bleil, J. D., and Wassarman, P. M. (1980). Develop. Biol., 76, 185–202] was analyzed by gel electrophoresis under a variety of conditions in order to determine whether or not it undergoes modification as a result of fertilization. Under nonreducing conditions, ZP2 present in solubilized zonae pellucidae that were isolated individually from mouse oocytes, eggs, and embryos migrates on SDS-polyacrylamide gels with an apparent molecular weight of 120,000. However, under reducing conditions, ZP2 from embryos, but not from oocytes or unfertilized eggs, migrates with an apparent molecular weight of 90,000 and has been designated ZP2_f. The evidence presented suggests that modification of ZP2 following fertilization involves proteolysis of the glycoprotein, but that intramolecular disulfide bonds prevent the release of peptide fragments. It is shown that the same change in ZP2 can be generated in vitro by artificial activation of unfertilized mouse eggs with the calcium ionophore A23187, thus eliminating the possibility that a sperm component is responsible for the modification of ZP2 following fertilization. These results suggest that some of the changes in the biochemical and biological properties of zonae pellucidae, observed following fertilization or activation of mouse eggs, result from modification of the major zona pellucida glycoprotein, ZP2.     

Sperm selection capacity of the human zona pellucida.

Previous hemizona assay (HZA) results have illustrated a positive and significant correlation between the percentage of morphologically normal spermatozoa in the semen and the number of spermatozoa tightly bound to the zona pellucida. The present study was designed to evaluate the morphologic features using strict criteria of spermatozoa tightly bound to the zona pellucida. Semen samples of 4 normozoospermic and 11 teratozoospermic men were used to compare the percentage of normal spermatozoa in the semen with that found 1) after swim-up separation and 2) bound to the zona under HZA conditions. The mean (+/- SEM) % normal forms for normozoospermic men in semen, after swim-up and zona-bound spermatozoa were 21.5 +/- 1.6, 27.5 +/- 2.9, and 44.8 +/- 3.4, respectively. A significantly higher % of normal forms were found among zona-bound sperm compared to swim-up forms (p = 0.02) and seminal sperm (p = 0.02). The mean % of normal sperm forms present in semen, after swim-up and zona pellucida-binding for teratozoospermic men, were 3.7 +/- 0.9, 5.8 +/- 1.6 and 15.6 +/- 3.1, respectively. Significant differences existed between the % of normal sperm forms found in the swim-up and zona-bound spermatozoa (P = less than 0.01 and P = less than 0.0003, respectively) compared to the original ejaculates. Results indicate that a selective process against abnormal spermatozoa occurs at the site of the zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida

An assay to determine the binding of pig spermatozoa to the zona pellucida (ZP) of pig oocytes was developed using conditions compatible with in-vitro fertilization of pig eggs and with pig sperm penetration of zona-free hamster ova. These conditions were used to define which of the pig oocyte ZP components were involved in sperm binding by a competitive inhibition approach. Assay variables that were optimized included: the method of sperm preparation; sperm preincubation time; sperm-oocyte coincubation time; sperm concentration and temperature; and methods for the separation of free from oocyte-bound spermatozoa. Inclusion of solubilized ZP in the sperm preincubation medium inhibited sperm binding approximately 50%. Both the 55K and 90K components inhibited sperm binding although the 55K component was more effective. The two polypeptides derived from chemical deglycosylation of the 55K families did not inhibit sperm binding. Of several monoclonal antibodies to the ZP components tested, only one directed against the 55K alpha glycoprotein family inhibited sperm binding. Sperm binding to pig oocyte ZP is therefore dependent on the carbohydrate moiety of the glycoproteins and appears to involve more than a single ZP glycoprotein.     

Structure and function of the mammalian egg zona pellucida.

The zona pellucida is a thick extracellular coat that surrounds all mammalian eggs and preimplantation embryos. The zona pellucida supports communication between oocytes and follicle cells during oogenesis; protects oocytes, eggs, and embryos during development, and regulates interactions between ovulated eggs and free-swimming sperm during and following fertilization. Mutant females that produce eggs that lack a zona pellucida are infertile. The functions of the zona pellucida during fertilization now can be ascribed to certain of its glycoproteins. Here we describe some aspects of zona pellucida structure and function as they relate to mammalian fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:251-258, 1999.     

A thermodynamic study of sperm-egg interaction.

We have studied the binding of spermatozoa to the receptor sites on the vitelline coat (VC) of glycerol-treated eggs (ghost eggs) of the Ascidian, Ciona intestinalis (Protochordate). Glycerol treatment cytolyses the egg without affecting the ability of the VC to bind spermatozoa in a species-specific manner; however, in this system binding is not followed by the acrosome reaction. The ghost eggs are metabolically inert. As a base line for our analysis, we have studied the concentration-dependent heat evolved and oxygen consumption of spermatozoa when diluted in sea water. The process has been analyzed on the basis of equations derived by Liquori and Tripiciano to describe cell growth. Upon binding to the ghost eggs, the spermatozoa produce an explosive heat evolution (excess heat) which is not accompanied by oxygen consumption. The excess heat produced plotted against sperm concentration (at constant egg concentrations) gives an asymmetric bell-shaped curve. This is interpreted as being due to the competitive effect of sperm agglutination at a high sperm concentration. It is concluded that only spermatozoa that attach singly (monomeric spermatozoa) to the egg undergo metabolic activation.     

The specificity of human spermatozoa/zona pellucida interaction under hemizona assay conditions

The objective of this prospective study was to evaluate the specificity of human sperm/zona pellucida interaction under hemizona assay (HZA) conditions in experiments with gametes from the same and different species. Human, cynomolgus monkey and hamster oocytes were used after salt-storage. Oocytes were bisected into matching hemizonae by micromanipulation and used in the HZA. Semen was obtained from healthy men (donors) and male cynomolgus monkeys and prepared by wash and swim-up. Sperm binding to matching hemizonae was assessed (tight binding) after 4-h coincubation in the HZA in homologous and interspecies experiments. Acrosome reaction was evaluated in the sperm droplets using FITC-PSA and on the hemizonae using the T-6 monoclonal antibody. On human hemizonae, the number of tightly bound sperm for human and monkey were 93.2 ± 15.8 and 3.9 ± 1.3, respectively (P<0.001). On monkey hemizonae, the number of tightly bound sperm for monkey and human were 126.0 ± 34.8 and 2.8 ± 1.6, (P = 0.02) respectively. On hamster hemizonae, there was negligible binding of human and monkey sperm. There was a significantly higher incidence of acrosome reacted sperm on the zona pellucida in homologous compared to heterologous experiments. These results demonstrate a high species-specificity of human gamete functions under HZA conditions, providing further support for the use of this bioassay in infertility and contraception testing. © 1993 Wiley-Liss, Inc.     

Maturation-associated changes in the rat zona pellucida

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical properties of zona pellucida hardening

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Cross-reacting antigens of the mammalian ovular zona pellucida

Antigens of zona pellucida (ZP) of different mammalian species, including the man, were studied by means of various immunochemical methods. The analysis was carried out using rabbit antisera to the pig, mouse, guenon and Java macaque eggs. After immunoadsorption (by blood serum and tissue extracts) these anti-ZP-sera reacted with water-insoluble ZP components only (in immunofluorescence). The adsorbed anti-ZP-sera were specific not only to homologous ZP, but also to ZP of other mammalian species. The spectrum of antigens of the ape ZP was most closely related to that of human ZP. The pig eggs contained antigens common with the human ZP as well. The antiserum to the mouse ZP did not react with ZP of man, ape, pig, cow, rabbit and other species under study. The blood of sterile women contained the antibodies reacting (in immunofluorescence) with ZP of mouse, pig, guinea pig, sheep and man (very weakly). The data obtained suggest the complexity of antigenic mosaic of the mammalian ZP including both "cross-reacting" (common) antigens and species specific ones.