Hum. Reprod. Advance Access originally published online on March 10, 2005
Human Reproduction 2005 20(5):1148-1152; doi:10.1093/humrep/deh835
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Cracking the egg: increased complexity in the zona pellucida
1 Reproductive Biology and Genetics Group, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, 2 Assisted Conception Unit, Birmingham Women's Hospital, Metchley Park Road, Edgbaston, Birmingham B15 2TG and 3 School of Biomedical and Natural Sciences, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
4 To whom correspondence should be addressed. Email: s.j.conner{at}bham.ac.uk
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Abstract |
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Top
Abstract
IntroductionA functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.
Key words: fertilization/human/zona pellucida/ZP1/ZPB
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Introduction |
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Although it is nearly 200 years since Prevost and Dumas (1824)
identified sperm as the fertilizing agents in semen, our understanding of the molecular mechanisms involved in fertilization is limited. The union between the sperm and the oocyte is arguably the most important interaction in biology. The initial contact for fertilization is between the sperm and the zona pellucida (ZP), the extracellular matrix that surrounds all vertebrate oocytes. The ZP is composed of a small number of glycoproteins; in addition to mediating the interaction with sperm, it is involved in the prevention of polyspermy and the protection of the developing embryo prior to implantation. The receptors responsible for the interaction of the male and female gametes have yet to be clearly identified. Evidence suggests that the composition of the ZP is more complicated than previously understood. As a consequence, it is time to consider the implications of a revised model for the structure of the ZP. |
There are four ZP genes in the human genome |
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The zona pellucida is typically described as containing three glycoproteins, ZP1, ZP2 and ZP3, also referred to as ZPB, ZPA and ZPC respectively. The increased availability of sequence data as the result of genome sequencing projects has led to a better understanding of the genetics of the ZP. The first realization of this was the finding that the human gene designated as ZPB was not the true orthologue of the mouse ZP1 gene (Hughes and Barratt, 1999
). (Orthologues are genes in different species that last shared a common ancestor at the time of species divergence Orthologues generally retain similar functions). The human genome was shown to contain an additional ZP gene that was the real homologue of the mouse ZP1 gene. This human gene was named ZP1. The chromosomal location of the human ZP1 gene (chromosome 11) is in a synteny group with the mouse ZP1 gene (chromosome 19). (Synteny group: portions of chromosomes within which the same genes are present in the same order in both species being compared.) Thus the human genome contains four ZP genes: ZP1, ZP2, ZP3 and ZPB, and not three as anticipated by the murine model. |
Time for a revised and simplified nomenclature? |
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The problem of confusing nomenclature regarding the ZP genes has been with us for some time. The ZP proteins were initially named ZP1, ZP2 and ZP3 for their sizes when separated by one-dimensional electrophoresis (Bleil and Wassarman, 1980
). An alphabetical system was later proposed, based on the length of the predicted protein, where ZPA=ZP2, ZPB=ZP1, and ZPC=ZP3 (Harris et al., 1994). Neither nomenclature was universally adopted, resulting in a dual naming system. There has also been misannotation, and, for some species, particularly in fish, completely new name derivation. The presence of genes for both ZP1 and ZPB in a number of species suggests that a revision of the gene nomenclature is needed. The greatest confusion in the literature regarding the zona pellucida is a failure to distinguish between ZP1 and ZPB, often as a result of a failure to recognize that these are two distinct genes. This lack of thoroughness over the genetics of the ZP genes was a major factor in the late discovery of the fourth human ZP gene. The subject of a simplified naming system has been raised by ourselves and others in two recent publications (Conner and Hughes, 2003; Spargo and Hope, 2003). Additionally the nomenclature committee at the National Center for Biotechnology Information (NCBI) have re-categorized the ZP genes. Under the new NCBI system, ZPB has been renamed ZP4 and thus the four ZP genes are termed ZP1, ZP2, ZP3 and ZP4. This proposal by the NCBI committee is uncomplicated and allows for the possible incorporation of new gene family members and thus we believe it should be supported by the reproductive biology community.
Figure 1 shows the major features of the four ZP genes. ZP1 and ZP4 are in fact paralogues. [Paralogues are genes (in the same or different species) that last shared a common ancestor prior to a gene duplication. Paralogues usually evolve different or more specialized functions], and can be seen to be structurally more closely related than the other ZP proteins, both having a trefoil domain. ZP1 and ZP4 have evolved from a common ancestor as a result of gene duplication (Conner and Hughes, 2003; Spargo and Hope, 2003).
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Do all species have four ZP genes? |
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Further support for the existence of distinct ZP1 and ZP4 genes came from the chicken, where both genes were subsequently identified (Bausek et al., 2000
). The rat and chimpanzee have recently been shown to have both ZP1 and ZP4 genes. These three species all have four ZP genes. Thus humans are not unique and this phenomenon exists in species at both extremes of the vertebrate phylum. Interestingly fish appear to have diverged from the vertebrate line sometime prior to the probable duplication that lead to the divergence of ZP1 and ZP4. Thus fish do not have ZP1 and ZP4 proteins but an ancestral protein (ZPX) that shows equal homology to both (Conner and Hughes, 2003). A phylogenetic tree of ZP sequences is shown in Figure 2. The grouping of the four classes of ZP protein can be clearly seen. To avoid confusion, fish ZP sequences have been omitted as there have been both genome and gene duplications giving rise to multiple gene copies of both ZPX and ZP3. Also missing are Xenopus ZPAX and ZPD sequences which fall outside the four main classes of ZP protein. Neither of these gene types appears to be present in higher vertebrates.
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Table I illustrates our current understanding of the occurrence of the four ZP genes across higher vertebrates. It should be remembered that the majority of these sequences resulted from projects looking for the specific gene sequences and thus the data show an ascertainment bias. Since most researchers have failed to recognize the existence of both ZP1 and ZP4 gene types, very few studies have looked for both. Many of the gaps may be filled as sequencing data continue to become available for more species.
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The existence of both ZP1 and ZP4 genes in chickens, rats, chimpanzees and humans implies that the gene duplication that permitted the divergence of the two genes occurred early in the vertebrate lineage (but subsequent to the divergence of the fish). Their persistence across the higher vertebrates indicates that both genes have been retained and thus have functional importance. This is particularly significant given that a number of proteins involved in reproduction, including ZP2 and ZP3, have been shown to be under high selective pressure, representing some of the most rapidly diverging genes (Swanson and Vacquier, 2002
). Thus one might reasonably predict that all higher vertebrates will have genes for both ZP1 and ZP4 and their preservation implies that they have functional importance. |
All four ZP genes are expressed in the human |
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While the number of ZP genes present in a given species is of interest in terms of evolution, the key question is whether this has functional importance. It is crucial to verify how many of the genes are expressed and therefore determine how many and which proteins constitute the zona pellucida. We have recently shown the presence of mRNA transcripts for all four ZP genes in human oocytes by PCR. In addition we have analysed by tandem mass spectrometry the composition of the human zona pellucida and identified the existence of all four ZP glycoproteins (ZP1, ZP2, ZP3 and ZP4) (Lefièvre et al., 2004
). |
Why does it matter if there is a fourth protein in the human ZP? |
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There is a paucity of direct functional data regarding the role of the four human ZP proteins. Proposed functions for ZP1, ZP2 and ZP3 again come from mouse studies. Mouse ZP1 is thought to contribute to the structural integrity of the ZP matrix acting as a linker molecule between ZP filaments (Greve and Wassarman, 1985
; Wassarman, 1988). In a number of studies ZP2 has been found to be involved in the secondary binding for acrosome-reacted sperm (Bleil et al., 1988; Mortillo and Wassarman, 1991; Tsubamoto et al., 1999). ZP3 is accepted to be the primary sperm receptor in the mouse responsible for binding to intact capacitated sperm and induction of the acrosome reaction (Bleil and Wassarman, 1983).
Mass spectrometry is a semi-quantitative technique and suggests that ZP4 levels in the human are equivalent to those of ZP3 and ZP2, with ZP1 being a rather minor component (Lefièvre et al., 2004). Recent experiments have shown that humanised mouse zonae expressing human ZP2 and ZP3 can bind mouse sperm but are unable to bind human sperm (Rankin et al., 2003). It is possible that this failure to bind is due to a requirement for species-specific glycosylation. Alternatively this result may reflect human sperm having evolved to bind to a zona pellucida consisting of four ZP proteins rather than three. Further to this is the possibility that ZP4 itself is required for direct interaction as part of the sperm receptor on the zona pellucida. There are data from a number of mammals (macaque, cow, and rabbit) supporting the hypothesis that ZP4 has sperm-binding activity (Prasad et al., 1996; Topper et al., 1997; Govind et al., 2000). In the pig, for example, the primary sperm receptor is a heterocomplex of ZP3 and ZP4 (Yurewicz et al., 1998). These data suggest that it is now necessary to establish which of the ZP proteins constitute the sperm receptor in humans.
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Is the mouse a good model for human fertilization? |
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Direct studies in the human are hampered by the small amount of biological material available for research. It is therefore necessary to have a good animal system as a model for human fertilization. The vast majority of studies in the last 25 years exploring the molecular mechanisms underlying fertilization have been done on the mouse. In light of the new evidence from humans it was important to establish whether the mouse too has a fourth ZP protein.
We were able to identify the murine ZP4 orthologue from available genomic sequences. Therefore like other vertebrates the mouse has a fourth ZP gene, ZP4. However, comparison of this gene sequence with the available rat ZP4 cDNA sequence revealed something unexpected. The murine ZP4 gene has acquired a number of deletions and encodes a truncated non-functional protein and appears to be a pseudogene (Lefièvre et al., 2004). The murine zona pellucida therefore ought to comprise only ZP1, ZP2 and ZP3. Recent mass spectrometry experiments on mouse zonae confirmed this composition (Boja et al., 2003).
The implications of this for the use of the mouse as a model species for human fertilization are considerable. While ZP proteins in both mouse and human continue to form the zona pellucida and presumably have similar functions, there will inevitably have been some divergence between proteins forming a three-protein matrix compared to those forming a four-protein matrix. Thus there are likely to be differences, perhaps small but potentially very significant, in the mechanisms of ZP formation and fertilization between the two species. It has recently been reported that, like humans, the rat expresses ZP1, ZP2, ZP3 and ZP4. The rat may therefore represent a better animal model for human fertilization than the mouse. However, there have been relatively few studies of fertilization in rat; for example, rat IVF is still poorly developed. Considerable effort will need to be put into characterization of the fertilization process of the rat to assess its usefulness as a model system for the human.
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ZP defects and female factor infertility: clinical significance |
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Currently there is no established pathology for defects in the ZP genes in humans. However, a recent preliminary study on patients with suspected female-factor infertility found a 2-fold increase in sequence variations in the ZP1 and ZP3 genes of the infertile women. Also two single base substitutions in the sequence of the ZP3 gene were found to exist with increased frequency compared to the fertile control group (Törmälä et al., 2004
). One of these mapped to a conserved motif in the upstream regulatory region of the gene, suggesting that changes in the expression levels of the ZP genes may lead to altered matrix formation with consequences for fertility. More well known are reports of zona thickness being predictive of a successful treatment outcome for assisted reproduction treatment (for example see Primi et al., 2004; Shiloh et al., 2004), although no molecular defects in such patients have been reported. A functional zona pellucida is critical not just for the early events of fertilization but also the latter stages of embryo development. The increased complexity of the human ZP now demands that we reconsider our reliance on the mouse model. Rapid developments in genomic and proteomic technologies now allow us to examine for the first time potential defects in zona genes and proteins associated with defined pathology.
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Acknowledgements |
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Work in the authors' laboratory is sponsored by the MRC, The Wellcome Trust, NHS and Fonds de recherche en santé du Québec. The authors also acknowledge Professor Christopher De Jonge for critical comments on the paper.
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Submitted on December 22, 2004; resubmitted on January 27, 2005; accepted on February 9, 2005.
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