The zona pellucida: a coat of many colors

The zona pellucida is an extracellular coat that surrounds all mammalian eggs. It is a porous matrix of interconnected filaments that are assembled from glycoproteins synthesized and secreted by growing oocytes. The zona pellucida is responsible both for species-specific binding of sperm to unfertilized eggs and inducing bound sperm to undergo the acrosome reaction. The latter enables sperm to penetrate the extracellular coat and fertilize the egg. The zona pellucida also aids in prevention of polyspermy following fertilization and in protection of preimplantation embryos. In mice, several of these important functions can now be ascribed to specific zona pellucida glycoproteins that have been purified and characterized. Furthermore, the enzyme responsible for hatching of embryos from the zona pellucida, just prior to implantation, has been identified and characterized.     

Zona pellucida glycoproteins

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida, that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse zona pellucida consists of three glycoproteins that are synthesized solely by growing oocytes and assemble into long fibrils that constitute a matrix. Zona pellucida glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing sperm to undergo acrosomal exocytosis, and preventing sperm from binding to fertilized eggs. Many features of mammalian and non-mammalian egg coat polypeptides have been conserved during several hundred million years of evolution.     

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.     

Follicular dysfunction induced by autoimmunity to zona pellucida

The mammalian zona pellucida is an extracellular matrix that occurs in growing oocytes, ovulated eggs and pre-implantation embryos, and is known to be involved in several important events during ovarian folliculogenesis and fertilization. Since the zona pellucida is formed at an early stage of oocyte growth, circulating antibodies against zona pellucida may impair ovarian function. In this article we discuss whether anti-zona antibodies cause ovarian dysfunction and infertility. The discussion is based on clinical examination and animal experiments including the following approaches: 1/ immunological method using solubilized human zona pellucida detected anti-zona antibody with a high frequency in infertile patients, especially premature ovarian failure syndrome; 2/ in vivo experiment using hamsters showed that some, but not all, animals experienced ovarian failure after immunization with hamster recombinant zona proteins; 3/ in vitro experiment using mouse isolated ovarian follicles showed significant inhibitory effects on follicular growth and oocyte development. We concluded that anti-zona antibody may be involved in causing ovarian failure.     

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.     

The effect of partial dissection of the zona pellucida on the fertilization of bovine oocytes in vitro and on the subsequent development of the embryos outside the body

Influence of partial zona dissection (PZD) on fertilization and cleavage of cow oocytes and on pre-implantation development of embryos obtained by this method was investigated. Decreased concentration of spermatozoa in less degree influenced on rates of fertilization and cleavage of oocytes with partial zona dissection than on intact eggs. The embryos obtained by method PZD can develop in vitro to blastocyst stage. However, their development is slowed down and presence of dissection in zona pellucida can result in premature hatching such blastocysts.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

The secretions of the cumulus-oocyte complex in relation to fertilization and early mouse embryonic development: a histochemical study

Summary A study has been made of the histochemical composition of the murine cumulus—oocyte complex and zona pellucida following treatment of immature females with exogenous gonadotrophins. Selected developmental stages were studied in detail, namely (i) the ovulated and unfertilized egg, (ii) the fertilized oocyte and (iii) the preimplantation embryo. In addition, the histochemical features observed in normal fertilized embryos have been compared with those of haploid and diploid parthenogenetic embryos at comparable stages following activation. Shortly after fertilization, glycosaminoglycans, which form a major component of the extracellular matrix surrounding the cumulus cells, become incorporated into the zona pellucida of the fertilized egg. In oocytes with few or no attendant cumulus cells, there appeared to be a diminished uptake of glycosaminoglycans and a reduced intensity of the zona staining reaction to Alcian Blue. In these oocytes, uptake of glycosaminoglycans appeared to be from the secretions lining the oviduct. There was little incorporation of the glycosaminoglycans from the extra-cellular matrix of the surrounding cumulus cells into the zona pellucida in unfertilized or parthenogenetic eggs despite the activation stimulus. After fertilization or activation, the zona pellucida became increasingly PAS-positive. Enzymic studies clearly indicate that the composition of the zona pellucida of the early embryo is histochemically different from the zona that surrounds the oocyte in the preovulatory follicle. These findings are discussed in relation to the decreased viability of embryos from oocytes which have been ovulated.The death of Mrs Carol Grainge is sadly recorded.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg: changes in the secondary structure of bovine zona pellucida proteins during fertilization

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of beta-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. Alpha-helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have beta-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the beta-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.     

Influence of zona pellucida thickness on fertilization, embryo implantation and birth

Defective sperm-zona pellucida binding and penetration are the main causes of IVF failure. The purpose of this study was to evaluate the effect of zona pellucida thickness in fertilization failure and test the influence of zona pellucida thickness on implantation and birth in rabbits. Embryos and oocytes were collected from 72 females on Day 2 post-insemination. A total of 559 normal embryos were recovered; 402 embryos were transferred by laparoscopy and 157 embryos were used to measure the zona pellucida thickness using the ImageJ program. Laparoscopies were also performed on all does at Day 12 of gestation to record the number of implanted embryos. Litter size at birth was recorded. The mean zona pellucida thickness of the 157 embryos and of the 64 control group oocytes (18.3 ± 0.2 and 18.5 ± 0.3 μm, respectively) was significantly less than the zona pellucida thickness of the 74 failed fertilization oocytes (19.2 ± 0.3 μm). The probabilities of the regression coefficient being positive were 0.72 and 0.74 for implantation and birth, respectively, and the subsequent means of the coefficient were 2.92 and 0.03 for implantation and birth, respectively. In conclusion, the zona pellucida thickness has an important influence on in vivo fertilization and implantation processes, but not on birth.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Differences in the rotation spectra of mouse oocytes and zygotes

Rotation spectra of mouse oocytes, zygotes and embryos in the two-cell stage under the influence of high-frequency rotating fields were studied. The characteristic frequency (fc1) of cells isolated from superovulated + mated mice is different from that of oocytes. This was attributed to an increase in the membrane resistance and, less probably, to a change in the zona pellucida conductivity. The rotation spectra can be used to differentiate between non-fertilized and fertilized eggs. A theoretical interpretation of the measured spectra and simulation of the changes caused by fertilization is given.     

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.     

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.     

Profile of a mammalian sperm receptor

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen-thawed mouse oocytes

Frozen-thawed oocytes have a reduced rate of fertilization (48.8%) when compared with unfrozen controls (97%). In this study we have used zona-drilling to bypass the zona pellucida and investigate whether the decreased rate of fertilization is due to freezing-induced changes in the zona pellucida which prevent sperm penetration. After zona drilling the fertilization rate of frozen-thawed oocytes (87.8%) was the same as for zona-intact unfrozen controls (88%), indicating that freeze-thaw-induced changes at the level of the zona pellucida were responsible for the decreased rate of fertilization. To determine whether the changes were occurring during the manipulations before and after freezing or the complete freeze-thaw cycle, oocytes were exposed to the complete set of manipulations normally experienced during cryopreservation and appropriate control groups. A small but significant decrease in the rate of fertilization (82.8%) was apparent in oocytes exposed to the manipulations before and after freezing compared with controls (92.2%). The freeze-thaw-induced changes in the zona pellucida therefore occur primarily during the complete freeze-thaw cycle itself and not the manipulations before and after freezing and are responsible for the decreased rate of fertilization observed in frozen-thawed oocytes.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.