Hum. Reprod. Advance Access originally published online on May 13, 2004
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Human Reproduction, Vol. 19, No. 7, 1580-1586,
July 2004
© 2004 European Society of Human Reproduction and Embryology
Four zona pellucida glycoproteins are expressed in the human*
1 Reproductive Biology and Genetics Group, Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, 2 Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham B15 2TG, 3 Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, 4 School of Chemical Sciences, University of Birmingham, Birmingham B15 2TT and 5 Division of Biology, School of Environmental and Applied Sciences, University of Derby, Derby DE22 1GB, UK
The first two authors contributed equally to this work.
6 To whom correspondence should be addressed. e-mail: c.l.barratt{at}bham.ac.uk
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Abstract |
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BACKGROUND: The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. METHODS: cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). RESULTS: All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. CONCLUSIONS: Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model
Key words: human/mouse ZPB/oocyte/proteomics/zona pellucida
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Introduction |
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Fertilization is a complex process which, to be successful, requires several steps. Critical early events include the binding of sperm to the zona pellucida (ZP) and subsequent induction of the acrosome reaction. The ZP is a matrix composed of glycoproteins which surrounds all mammalian oocytes and mediates several important roles such as binding sperm in a species-specific manner, inducing the acrosome reaction, preventing polyspermy and protecting the embryo prior to implantation. Experiments, primarily in the mouse, have led to the conclusion that the ZP is composed of three proteins (ZP1, ZP2 and ZP3). The murine ZP is the accepted model for ZP structure in higher vertebrates and is composed of repeating units of ZP2–ZP3 heterodimers arranged in filaments cross-linked by ZP1 dimers (Greve and Wassarman, 1985
). Experiments in the mouse, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)-purified zona proteins showed that ZP3 was the primary sperm ligand (Bleil and Wassarman, 1980a, 1983) and ZP2 was the secondary ligand binding to acrosome-reacted sperm and triggering events that are important for the prevention of polyspermy (Bleil and Wassarman, 1980b; Bleil et al., 1981). ZP1 is proposed to contribute to the structural integrity of the ZP matrix (Greve and Wassarman, 1985; Green, 1997; Wassarman, 1999).
Cloning of the ZP genes from a number of species has resulted in the introduction of an alternative nomenclature of ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3) based on gene size (Harris et al., 1994). However, it has now become apparent that human ZP1 and ZPB genes are, in fact, paralogues (Hughes and Barratt, 1999), and thus the human genome contains four ZP genes and not three. This is consistent with the identification of both ZP1 and ZPB genes in chicken (Bausek et al., 2000) and rat (accession number AF456325), and with recent molecular phylogenetic analysis of vertebrate ZP genes (Conner and Hughes, 2003; Spargo and Hope, 2003). These recent findings show a greater complexity in the number of zona genes across vertebrates than previously anticipated (Conner and Hughes, 2003; Lefièvre et al., 2003). In this context it is surprising that the presence of the mouse ZPB gene has not been reported.
Current models for the structure of mammalian zona pellucida are based upon the existence of three ZP proteins (Wassarman, 1999). If all four ZP genes are transcribed and translated in the human oocyte, a re-evaluation would be required, both of the structure of the ZP and, potentially, of the mechanisms of sperm–zona interaction. Thus, the aim of the present study was to investigate the ZP gene expression in the human oocyte and the protein composition of the human ZP.
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Materials and methods |
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mRNA purification and preparation of amplified oocyte cDNA in the human
Amplified cDNA preparations were prepared from human oocytes (four oocytes per sample). The oocytes were lysed in 3 µl of ice-cold lysis buffer (0.8% Igepal, Sigma, UK; 1 IU/µl RNase inhibitor, Gibco BRL, UK; and 5 mmol/l dithiothreitol, Gibco BRL, UK) and the released mRNA molecules bound to oligo-(dT)-linked magnetic beads (Dynabeads; Dynal UK). Synthesis of cDNA on the mRNA bound to the beads, and PCR amplification of the cDNA, were carried out using the SMARTTM cDNA library construction kit (Clontech, UK). Further details of the methods are given in Holding et al. (2000
).
PCR amplification of specific gene sequences in amplified cDNA preparations
Expression of four zona pellucida genes (ZP1, ZP2, ZP3 and ZPB), in the cDNA preparations derived from human oocytes was detected by PCR with the primers given in Table I. Primers for detection of the ubiquitously expressed genes 
-actin and hypoxanthine phosphoribosyl transferase (HPRT) as control genes are also given in Table I. PCR amplification was carried out in total volume of 25 µl of reaction mixture, containing 1 µl of cDNA (concentrations were adjusted by ethidium bromide staining of diluted aliquots compared with standard DNA), 200 µmol/l each of dNTP (Pharmacia), 1 µg each of primers, 1xPCR buffer (Perkin Elmer), using 1.25 IU of AmpliTaq DNA polymerase (Perkin Elmer). With the exception of ZP3, the parameters for the PCR for the zona pellucida genes were one cycle of 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 57°C for 1 min and 72°C for 1 min. For the detection of expression of the ZP3 gene, the PCR cycling parameters were one cycle of 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min. PCR cycles for the detection of the housekeeping genes were as follows: -actin, 35 cycles of 95°C for 1 min, 62°C for 1 min and 72°C for 1 min and HPRT, 35 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min. For the hemi-nested PCR for the expression of HPRT, 1 µl of the first round product was then transferred to 24 µl of reaction mixture for second round amplification, using the same conditions except for the substitution of the appropriate second round primers (Table I).
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Gel electrophoresis
Ten microlitres of PCR product were mixed with 2 µl of loading buffer and electrophoresed for 90 min at 120 V on a 1 or 2% agarose gel and visualized under UV light.
Isolation of human ZP
Metaphase II human oocytes which failed to fertilize after ICSI were donated by patients attending the Assisted Conception Unit, Birmingham Women’s Hospital (HFEA centre number 0119). All patients provided informed consent and the research was approved by the local research ethics committee (LREC 0554). Human ZP were mechanically isolated from oocytes. Briefly, several slits were made in the ZP using 10 µm subzonal insemination (SUZI) micropipettes (Conception Technologies, USA). The oocytes were then flushed out from one of the slits by rapidly introducing medium into the perivitelline space from behind the oocyte. The ZP were then washed five times with 50 mmol/l NH4HCO3, pH 7.2.
Solubilization of human ZP
Five isolated human ZP were solubilized in 300 µl of 50 mmol/l NH4HCO3, pH 7.2, for 1 h at 70°C. The sample was subsequently concentrated on a 10 kDa cut-off filter, transferred to a siliconized enzyme-linked immunosorbent assay plate and air-dried at 56°C. The dry spot was resuspended in trypsin solution (trypsin solution: 20 µg of trypsin was resuspended in 20 µl of 50 mmol/l acetic acid and then diluted to a final concentration of 12.5 ng/µl with 25 mmol/l NH4HCO3; sequencing grade modified trypsin, V5111, Promega UK Ltd) and incubated overnight at 37°C. The trypsin-digested human ZP were then resuspended in 6 µl of 1% formic acid and subjected to tandem mass spectrometry (MS/MS).
Mass spectrometry
Direct MS/MS analysis was used to identify the human ZP proteins. This allows direct identification of individual proteins from complex mixtures in very small samples. Lefièvre et al. (2003) previously identified the presence of tryptic peptides corresponding to the three known pig ZP proteins (ZP2, ZPB and ZP3) using only 10 isolated porcine ZP.
Tryptic peptides obtained from five isolated human ZP were subjected to MS/MS on a nanoESI Q-Tof mass spectrometer (Q-Tof Ultima GLOBAL; Micromass UK Ltd) following separation of peptides using capillary liquid chromatography (Waters Ltd, UK) with a 15 cm C18 PepMap column (75 µm i.d.x15 cm; Cat. No. 160396, Dionex Ltd, UK). Following MS/MS the raw data were processed using MassLynx 3.5 (Micromass). The resulting tryptic peptide de novo sequences data were then compared with non-identical protein sequence databases using MASCOT software (Matrix Science Ltd, UK). Finally, a BLAST search of the GenBank database was performed for each of the peptides obtained to confirm that the proteins identified by MASCOT was the unique match in the non-redundant protein database for a particular peptide sequence.
Mouse ZPB gene analysis
We adopted a bioinformatics approach to identify the mouse orthologue of the rat and human ZPB gene using available sequence data. A BLAST search of the non-redundant database with the rat ZPB sequence (NM_172330) was used to identify murine genomic sequence with significant sequence similarity.
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Results |
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ZP gene expression in human oocytes
To determine whether all four ZP genes were expressed, we amplified by PCR cDNA derived from four different samples of four human oocytes. PCR was performed with primers for human ZP1, ZP2, ZP3 and ZPB (Table I) on cDNA preparations from human oocytes. Figure 1 shows that, as expected, all four ZP genes are expressed in the human oocyte. This is the first demonstration that the ZP1 (Hughes and Barratt, 1999
) and ZPB genes (Harris et al., 1994) are expressed in the human oocyte. Previously only expression of ZP2 and ZP3 had been demonstrated by PCR and serial analysis of gene expression (SAGE) of human oocytes (Neilson et al., 2000).
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Human zona proteomics
In the present study we have used five human oocytes to investigate the number of distinct glycoproteins in the human ZP. The ZP were mechanically isolated from the oocytes, heat-solubilized, trypsin-digested and analysed using direct MS/MS. Results obtained from peptide sequencing and comparison with non-redundant protein sequence databases confirmed the presence of peptides from all four ZP proteins (ZP1, ZP2, ZP3 and ZPB) (Figure 2). Several tryptic peptide sequences were obtained for ZP2, ZP3 and ZPB (Figure 2), and one for ZP1 (505FTVATFALLDSGSQR519) (Figures 2 and 3). This latter tryptic peptide was compared with the human ZP1 peptide sequence identified by Hughes and Barratt (1999
). The quindecapeptide amino acid sequence is a perfect match, which indicates the presence of ZP1 in the human ZP.
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ZP1 is well recognized as being a low abundance protein (Bleil and Wassarman, 1980b
; Epifano et al., 1995; Green, 1997). Using the equivalent of several thousand human ZP, a recent MS/MS study on the mouse confirmed the low abundance of ZP1 in that species (Boja et al., 2003). Moreover, semiquantitative data in Figure 1 suggest that human ZP1 is less abundant than ZP2, ZP3 or ZPB. Due to the inherent low abundance of ZP1, combined with the paucity of human oocytes for research purposes, we have been unable to obtain further peptides for ZP1. Additional experiments have been performed using a variety of preparation methods including enzymatic deglycosylation of the ZP prior to the trypsin digestion as described by Boja et al. (2003). Although ZP2, ZP3 and ZPB were identified on each occasion, no peptides for ZP1 were obtained.
Existence of mouse ZPB
The current model of the structure of mouse ZP incorporates three glycoproteins, consistent with the detection, to date, of only three mouse ZP genes (ZP1, ZP2 and ZP3). Having established that a fourth ZP gene is present in the human genome (Hughes and Barratt, 1999) and that this gene is expressed (Figure 1) and protein present (Figure 2) in the human ZP, we undertook analysis of the mouse genome to determine whether a ZPB gene is present.
Using a bioinformatics approach, a putative mouse ZPB cDNA sequence was generated by comparison with rat ZPB sequence, coupled with manual identification of splice sites in the genomic sequence. This mapped to chromosome 13, a region of conserved synteny with human chromosome 1 (1q43) where human ZPB is found and rat chromosome 17 (17q12.1) where rat ZPB is found. However, this hypothetical sequence did not encode a continuous open reading frame. Alignment with the rat ZPB cDNA sequence revealed a number of insertions and deletions which resulted in shifts in the open reading frame and the generation of premature termination codons (Figure 4). To confirm the accuracy of the mouse genomic sequence, individual exons of the putative mouse ZPB gene were used to search the trace sequence database (http://www.ncbi.nlm.nih.gov/blast/tracemb). For each exon screened, the sequence was confirmed, with multiple independent trace sequences of both DNA strands (data not shown).
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Discussion |
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This paper presents the first evidence for the existence of four glycoproteins in the human ZP. The exact match of the quindecapeptide with the deduced ZP1 coding sequence (Hughes and Barratt, 1999
) in addition to the presence of mRNA transcript for ZP1 in four independent experiments strongly supports the premise that four ZP glycoproteins ZP1, ZP2, ZP3 and ZPB are expressed and present in the human. This study also represents the first protein analysis of the human ZP relying on protein sequencing rather than antibody-based approaches.
The difficulty in obtaining a definitive protein identification of ZP1 is primarily due to the low abundance of ZP1 in the mammalian ZP. For example, in the mouse, levels of ZP1 mRNA were four times lower compared to ZP2 and ZP3 (Epifano et al, 1995) and consequently only 56% of the ZP1 polypeptide chain was identified by direct MS/MS, compared to 96 and 100% for ZP2 and ZP3 respectively (Boja et al., 2003). However, despite its low abundance, experiments using ZP1 null mice demonstrated that ZP1 is an essential element of the ZP primarily required for the structural integrity of the zona matrix, although not involved directly in sperm binding (Rankin et al., 1999).
The demonstration of the existence of four human ZP proteins requires a re-interpretation of the numerous electrophoretic studies on the ZP. Although only three diffuse bands were observed on one-dimensional (1D) electrophoresis, the increased resolution provided by two-dimensional (2D) electrophoresis has previously suggested additional levels of complexity to the human ZP (Bercegeay et al., 1995; Moos et al., 1995). This is further complicated by the variable patterns of glycosylation observed. With hindsight, it is apparent that the ZP3-containing band on 1D electrophoresis most likely resolves into two proteins which correspond to ZP3 and ZPB [Yurewicz et al., 1987 (pig); Topper et al., 1997 (cow); Gupta et al., 1998 (human)].
The presence of the ZPB gene in a large number of species (cat, chick, cow, human, macaque, marmoset, pig, possum, rat and rabbit; see Lefièvre et al., 2003) raises the question: does the mouse have a ZPB orthologue? Our bioinformatic analysis demonstrates that, like humans and rats, the mouse has four ZP genes. However, comparative sequence analysis reveals that the mouse ZPB gene has acquired a number of changes making it unlikely that functional ZPB protein will be expressed (Figure 4). This in silico evidence is supported by recent data using MS analysis which failed to identify ZPB (Boja et al., 2003). Numerous peptides from ZP1, ZP2 and ZP3 were identified but the authors did not find any unassigned peptides which could correspond to ZPB.
Knockout experiments, in which murine ZP proteins were replaced by human equivalents (Rankin et al., 1998, 2003), showed that, although mouse sperm continued to bind to mouse oocytes engineered to express human ZP2 and ZP3 proteins, human sperm did not bind. One explanation for this result is that mouse oocytes express mouse ZP O-glycans on both mouse and human ZP3 (Dell et al., 2003) and that it is these O-glycans that are key to sperm binding (Wassarman, 1999). Another complementary explanation, derived from our results, is that human sperm have evolved to interact with a ZP composed of four, not three, proteins, with ZPB as well as ZP3 being required for sperm zona binding. This explanation is supported by data from other species, e.g. rabbit, bovine, porcine and macaque, which show that ZPB has sperm-binding properties (Prasad et al., 1996; Topper et al., 1997; Yurewicz et al., 1998; Govind et al., 2000). Thus, although the mouse ZP may support the hypothesis that the mammalian ZP has three proteins of which ZP3 is the primary sperm receptor, it may fall short as a hypothesis for sperm–zona interaction in other species. Consequently it appears that the model species for the study of mammalian fertilization may only serve as a good model for itself.
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Acknowledgements |
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We would like to express sincere thanks to Drs Bayard Storey and Stephen Publicover for critical reading of this manuscript. The authors also wish to thank the patients who participated in this investigation. This study was supported by Fonds québécois de la recherche sur la nature et les technologies (L.L.), Medical Research Council ROPA grant (C.L.R.B.), The Lord Dowding Fund for Humane Research (C.L.R.B. and I.A.B.), the Birmingham Women’s Hospital (C.L.R.B.), Biotechnology and Biological Sciences Research Council (M.M., A.S. and the Functional Genomics Laboratory of the University of Birmingham).
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FOOTNOTES |
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* Part of this work has been presented at ESHRE 2003 held in Madrid [L.Lefièvre et al. (2003), Hum. Reprod., 18 (Suppl. 1), 0–266: 91].
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Submitted on August 6, 2003; accepted on April 14, 2004.
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S. Assou, T. Anahory, V. Pantesco, T. Le Carrour, F. Pellestor, B. Klein, L. Reyftmann, H. Dechaud, J. De Vos, and S. Hamamah The human cumulus-oocyte complex gene-expression profile Hum. Reprod., July 1, 2006; 21(7): 1705 - 1719. [Abstract] [Full Text] [PDF]
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G. F. Clark and A. Dell Molecular Models for Murine Sperm-Egg Binding J. Biol. Chem., May 19, 2006; 281(20): 13853 - 13856. [Abstract] [Full Text] [PDF]
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P. Caballero-Campo, M. Chirinos, X. J. Fan, M. E. Gonzalez-Gonzalez, M. Galicia-Chavarria, F. Larrea, and G. L. Gerton Biological Effects of Recombinant Human Zona Pellucida Proteins on Sperm Function Biol Reprod, April 1, 2006; 74(4): 760 - 768. [Abstract] [Full Text] [PDF]
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R. K. Naz, S. K. Gupta, J. C. Gupta, H. K. Vyas, and a. G.P. Talwar Recent advances in contraceptive vaccine development: a mini-review Hum. Reprod., December 1, 2005; 20(12): 3271 - 3283. [Abstract] [Full Text] [PDF]
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T. Ebner, M. Moser, and G. Tews Possible applications of a non-contact 1.48 {micro}m wavelength diode laser in assisted reproduction technologies Hum. Reprod. Update, July 1, 2005; 11(4): 425 - 435. [Abstract] [Full Text] [PDF]
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Y. Shen, T. Stalf, C. Mehnert, U. Eichenlaub-Ritter, and H.-R. Tinneberg High magnitude of light retardation by the zona pellucida is associated with conception cycles Hum. Reprod., June 1, 2005; 20(6): 1596 - 1606. [Abstract] [Full Text] [PDF]
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M. Mannikko, R.-M. Tormala, T. Tuuri, A. Haltia, H. Martikainen, L. Ala-Kokko, J.S. Tapanainen, and J.T. Lakkakorpi Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF Hum. Reprod., June 1, 2005; 20(6): 1578 - 1585. [Abstract] [Full Text] [PDF]
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 S. Chakravarty, K. Suraj, and S. K. Gupta Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C Mol. Hum. Reprod., May 1, 2005; 11(5): 365 - 372. [Abstract] [Full Text] [PDF]
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S.J. Conner, L. Lefievre, D.C. Hughes, and C.L.R. Barratt Cracking the egg: increased complexity in the zona pellucida Hum. Reprod., May 1, 2005; 20(5): 1148 - 1152. [Abstract] [Full Text] [PDF]
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T. Hoodbhoy, S. Joshi, E. S. Boja, S. A. Williams, P. Stanley, and J. Dean Human Sperm Do Not Bind to Rat Zonae Pellucidae Despite the Presence of Four Homologous Glycoproteins J. Biol. Chem., April 1, 2005; 280(13): 12721 - 12731. [Abstract] [Full Text] [PDF]
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