1 Reproductive Biology and Genetics Group, Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, 2 Assisted Conception Unit, Birmingham Womens 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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Key words: human/mouse ZPB/oocyte/proteomics/zona pellucida
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Introduction |
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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 spermzona 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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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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Isolation of human ZP
Metaphase II human oocytes which failed to fertilize after ICSI were donated by patients attending the Assisted Conception Unit, Birmingham Womens 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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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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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 spermzona 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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FOOTNOTES |
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References |
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Bercegeay S, Jean M, Lucas H and Barriere P (1995) Composition of human zona pellucida as revealed by SDSPAGE after silver staining. Mol Reprod Dev 41,355359.[ISI][Medline]
Bleil JD and Wassarman PM (1980a) Mammalian spermegg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20,873882.[ISI][Medline]
Bleil JD and Wassarman PM (1980b) Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocytes zona pellucida. Dev Biol 76,185202.[ISI][Medline]
Bleil JD and Wassarman PM (1983) Spermegg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol 95,317324.[ISI][Medline]
Bleil JD, Beall CF and Wassarman PM (1981) Mammalian spermegg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Dev Biol 86,189197.[ISI][Medline]
Boja ES, Hoodbhoy T, Fales HM and Dean J (2003) Structural characterization of native mouse zona pellucida proteins using mass spectrometry. J Biol Chem 278,3418934202.
Conner SJ and Hughes DC (2003) Analysis of fish ZP1/ZPB homologous genes-evidence for both genome duplication and species-specific amplification models of evolution. Reproduction 126,347352.
Dell A, Chalabi S, Easton RL, Haslam SM, Sutton-Smith M, Patankar MS, Lattanzio F, Panico M, Morris HR and Clark GF (2003) Murine and human zona pellucida 3 derived from mouse eggs express identical O-glycan. Proc Natl Acad Sci USA 100,1563115636.
Epifano O, Liang L, Familari M, Moos MC and Dean J (1995) Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121,19471956.
Gibbs RA, Nguyen P-N, McBride LJ, Koepf SM and Caskey CT (1989) Identification of mutations leading to LeschNyhan syndrome by automated direct DNA sequencing of in-vitro amplified cDNA. Proc Natl Acad Sci USA 86,19191923.[Abstract]
Govind CK, Hasegawa A, Koyama K and Gupta SK (2000) Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of spermegg binding. Biol Reprod 62,6775.
Green DP (1997) Three-dimensional structure of the zona pellucida. Rev Reprod 2,147156.
Greve JM and Wassarman PM (1985) Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. J Mol Biol 181,253264.[ISI][Medline]
Gupta SK, Yurewicz EC, Sacco AG, Kaul R, Jethanandani P and Govind CK (1998) Human zona pellucida glycoproteins: characterization using antibodies against recombinant non-human primate ZP1, ZP2 and ZP3. Mol Hum Reprod 4,10581064.[Abstract]
Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz EC and Sacco AG (1994) Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Seq 4,361393.[ISI][Medline]
Higgins D., Thompson J, Gibson T, Thompson JD, Higgins DG and Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22,46734680.[Abstract]
Holding C, Bolton V and Monk M (2000) Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos. Mol Hum Reprod 6,801809.
Hughes DC and Barratt CLR (1999) Identification of the true human orthologue of the mouse Zp1 gene: evidence for greater complexity in the mammalian zona pellucida? Biochim Biophys Acta 1447,303306.[Medline]
Lefièvre L, Barratt CLR, Harper CV, Conner SJ, Flesch FM, Deeks E, Moseley FLC, Pixton KL, Brewis IA and Publicover SJ (2003) Physiological and proteomic approaches to study pre fertilisation events in the human. Reprod BioMed Online 7,419427.[Medline]
Moos J, Faundes D, Kopf GS and Schultz RM (1995) Composition of the human zona pellucida and modifications following fertilization. Mol Hum Reprod 10,24672471.
Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF, Stanton JA and Green DP (2000) Molecular phenotype of the human oocyte by PCRSAGE. Genomics 63,1324.[CrossRef][ISI][Medline]
Ponte P, Ng SY, Engel J, Gunning P and Kedes L (1984) Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucl Acid Res 12,16871696.[Abstract]
Prasad SV, Wilkins B, Skinner SM and Dunbar BS (1996) Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system. Mol Reprod Dev 43,519529.[CrossRef][ISI][Medline]
Rankin TL, Tong ZB, Castle PE, Lee E, Gore-Langton R, Nelson LM and Dean J (1998) Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding. Development 125,24152424.
Rankin T, Talbot P, Lee E and Dean J (1999) Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss. Development 126,38473855.
Rankin TL, Coleman JS, Epifano O, Hoodbhoy T, Turner SG, Castle PE, Lee E, Gore-Langton R and Dean J (2003) Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs. Dev Cell 5,3343.[ISI][Medline]
Spargo SC and Hope RM (2003) Evolution and nomenclature of the zona pellucida gene family. Biol Reprod 68,358362.
Topper EK, Kruijt L, Calvete J, Mann K, Topfer-Petersen E and Woelders H (1997) Identification of bovine zona pellucida glycoproteins. Mol Reprod Dev 46,344350.[CrossRef][ISI][Medline]
Wassarman PM (1999) Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96,175183.[ISI][Medline]
Yurewicz EC, Sacco AG and Subramanian MG (1987) Structural characterization of the Mr = 55,000 antigen (ZP3) of porcine oocyte zona pellucida. Purification and characterization of alpha- and beta-glycoproteins following digestion of lactosaminoglycan with endo-beta-galactosidase. J Biol Chem 262,564571.
Yurewicz EC, Sacco AG, Gupta SK, Xu N and Gage DA (1998) Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles. J Biol Chem 273,74887494.
Submitted on August 6, 2003; accepted on April 14, 2004.