MOLECULAR REPRODUCTION AND DEVELOPMENT 70:133–145 (2005)

Isolation and Mapping the Chicken Zona PellucidaGenes: An Insight Into the Evolution of OrthologousGenes in Different SpeciesJACQUELINE SMITH,1* IAN R. PATON,1 DAVID C. HUGHES,2 AND DAVID W. BURT1

1Division of Genomics and Bioinformatics, Roslin Institute, Roslin (Edinburgh), Midlothian, United Kingdom2Biological Sciences Research Group, Division of Biological, Pharmaceutical and Forensic Sciences, School of Education,Health, and Sciences, University of Derby, Derby, United Kingdom

ABSTRACT The avian oocyte is surroundedby a specialized extracellular glycoproteinaceous ma-trix, the perivitelline membrane, which is equivalent tothe zona pellucida (ZP) in mammals and the chorionin teleosts. A number of related ZP genes encode theproteins that make up this matrix. These proteins playan important role in the sperm/egg interaction andmay be involved in speciation. The human genome isknown to contain ZP1, ZP2, ZP3, and ZPB genes,while a ZPAX gene has also been identified in Xenopus.The rapid evolution of these genes has confused thenomenclature and thus orthologous relationshipsacross species. In order to clarify these homologies,we have identified ZP1, ZP2, ZPC, ZPB, and ZPAXgenes in the chicken and mapped them to chromo-somes 5, 14, 10, 6, and 3, respectively, establishingconserved synteny with human and mouse. The aminoacid sequences of these genes were compared to theorthologous genes in human, mouse, and Xenopus,and have given us an insight into the evolution of thesegenes in a variety of different species. The presence ofthe ZPAX gene in the chicken has highlighted a patternof probable gene loss by deletion in mouse and geneinactivation by deletion, and base substitution inhuman. Mol. Reprod. Dev. 70: 133–145, 2005.� 2005 Wiley-Liss, Inc.

Key Words: chicken; zona pellucida; evolution;gene loss; gene duplication; orthology; parology

INTRODUCTION

The zona pellucida (ZP) matrix that surroundsvertebrate oocytes is involved in sperm binding and alsogives support to the oocyte as it travels down the oviduct.The equivalent structure in avian species is theperivitelline membrane. The ZP is traditionally believedto be composed of three major glycoproteins encoded byZP1, ZP2, and ZP3. The initial nomenclature was basedon the mobility of the mouse ZP proteins on SDS–PAGE,and with the cloning of the ZP1, ZP2, and ZP3 genesfrom mouse and other species (reviewed in McLeskeyet al., 1998), the presence of a common motif, the ZPdomain was noted (Bork and Sander, 1992). In anattempt to clarify the nomenclature, Harris et al. (1994)

proposed an alternative nomenclature based on genetranscript size, ZPA (ZP2), ZPB (ZP1), and ZPC (ZP3),although this has been inconsistently applied in theliterature. Further clarification came with the realiza-tion that ZP1 in the mouse and ZPB in other mammalssuch as man, were not orthologous but paralogous, andthat the human genome included genes for both ZPB andZP1 (Hughes and Barratt, 1999). This classification intofour gene classes was strengthened by the isolation ofthe chickenZP1 gene (Bausek et al., 2000) and by recentlarge-scale comparisons of vertebrate ZP genes (Connerand Hughes, 2003; Spargo and Hope, 2003). It has beenconfirmed recently that all four ZP genes are expressedin oocytes, and that the human ZP does indeed containfour ZP proteins (Lefievre et al., 2004). Spargo and Hope(2003) have also attempted to clarify the nomenclatureof the vertebrate ZP genes, and so the nomenclatureproposed is summarized in Table 1 and will be usedthroughout. It has been demonstrated that the mam-malian ZP2 and ZP3 genes are rapidly evolving, underpositive Darwinian selection (Swanson and Vacquier,2002). In addition, the combination of genome duplica-tion coupled with species-specific gene amplificationand/or attrition of theZP genes in euteleost fish (Connerand Hughes, 2003) have all served to complicate thedetermination of the relationships within the vertebrateZP genes. However, the identification and mappingof the chicken genes has allowed us to confirm thehomologies presented in the earlier review (Spargo andHope, 2003).

In the mouse, the ZPC protein is involved in inductionof the sperm acrosome reaction, while ZPA mediates thesperm/egg binding interaction (McLeskey et al., 1998),although from other mammals there is evidence that the

� 2005 WILEY-LISS, INC.

Sequence submissions: Sequences were submitted to EMBL underaccession numbers: BN000517 (ZPA gene); AJ698915 (ZPX1 gene);and AJ703825-AJ703886 (sequence sampling data from ZP BACs)

Grant sponsor: Biotechnology and Biological Science Research Council(BBSRC).

*Correspondence to: Dr. Jacqueline Smith, Division of Genomics andBioinformatics, Roslin Institute (Edinburgh), Roslin, MidlothianEH25 9PS, United Kingdom. E-mail: Jacqueline.Smith@bbsrc.ac.uk

Received 28 August 2004; Accepted 12 August 2004Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/mrd.20197

TABLE 1. Details of Known Zona Pellucida (ZP) Genes

Species name Common name Chromosome Accession no. Other accessions SpeciesCurrent

name Old names

Bos taurus Cow AB042652 NM_173975 Bta ZPB2 ZPB/ZP4Callithrix jacchus White-tufted-ear

marmosetY10767 Cja ZPA ZP2

Callithrix jacchus White-tufted-earmarmoset

Y10822 Cja ZPB2 ZP1

Canis familiaris Dog U05779 D45069 Cfa ZPA ZPA/ZP2Canis familiaris Dog U05780 D45070 Cfa ZPC ZPC/ZP3Carassius auratus Goldfish Z48974 Cau ZPC ZP3Coturnix japonica Japanese quail AB061520 Cjap ZPB1 ZP1Cyprinus carpio Common carp L41637 L41639 Cca ZPCa ZP3ACyprinus carpio Common carp L41638 Cca ZPCb ZP3BCyprinus carpio Common carp Z72491 Cca ZPBa ZP2bCyprinus carpio Common carp Z72492 Cca ZPBb ZP2Cyprinus carpio Common carp Z72493 Cca ZPBc ZP2cDanio rerio Zebrafish AF095457 Dre ZPC ZP3Danio rerio Zebrafish AF331968 Dre ZPB ZP2Felis catus Cat U05776 D45067 Fca ZPA ZPAFelis catus Cat U05777 Fca ZPB2 ZPBFelis catus Cat U05778 D45068 Fca ZPC ZPC/ZP3Gallus gallus Chicken 6q12-q14 AB025428 Gga ZPB2 ZPBGallus gallus Chicken 10 AB031033 D89097 Gga ZPC ZP3/ZPCGallus gallus Chicken 11 AB114441 Gga ZPX2 ZPDGallus gallus Chicken 5p11-q11 AJ289697 Gga ZPB1 ZP1Gallus gallus Chicken 3q33-q35 AJ698915 Gga ZPX1 ZPAXGallus gallus Chicken 14 BN000517 Gga ZPA ZP2Homo sapiens Human 16p12 M90366 16p12 Hsa ZPA ZPA/ZP2Homo sapiens Human 7q11.23 NM_007155 7q11.23, A18567 Hsa ZPC ZP3/ZPCHomo sapiens Human 1q42.11 NM_021186 1q42.11-q42.3,

U05781Hsa ZPB2 ZP4/ZPB

Homo sapiens Human 11q12.2 XM_172861 Hsa ZPB1 ZP1Homo sapiens Human 2p24 Hsa ZPX1 ZPAXpMacaca fascicularis Crab-eating macaque AY222647 AY222648 Mfa ZPB2 ZPBMacaca radiata Macaque X82639 Mra ZPC ZP3Macaca radiata Macaque Y10381 Y10382,Y10383 Mra ZPB2 ZP1Macaca radiata Macaque Y10690 Mra ZPA ZP2Mesocricetus auratus Golden hamster M63629 Mau ZPC ZP3Microtus brandti Brandt’s vole AF304487 Mbr ZPC ZPCMus musculus House mouse 19 (2 cM) NM_009580 19 (2 cM) Mmu ZPB1 ZP1Mus musculus House mouse 7 (56 cM) NM_011775 7 (56 cM) Mmu ZPA ZP2Mus musculus House mouse 5 (77 cM) X14376 NM_011776 5 (77 cM) Mmu ZPC ZP3Mus musculus House mouse 13 XM_138427 Mmu ZPB2 ZPBNotomys alexis Hopping mouse AY078054 Nal ZPC ZPCOncorhynchus mykiss Rainbow trout AF231706 Omy ZPBa VEP alphaOncorhynchus mykiss Rainbow trout AF231707 Omy ZPBb VEP betaOncorhynchus mykiss Rainbow trout AF231708 Omy ZPC VEP gammaOryctolagus cuniculus Rabbit L12167 Ocu ZPA 75 kDaOryctolagus cuniculus Rabbit M58160 Ocu ZPB2 ZPOryzias latipes Japanese medaka AF128807 Ola ZPX1 ZPAOryzias latipes Japanese medaka AF128808 Ola ZPBa ZPBOryzias latipes Japanese medaka AF128809 Ola ZPCe ZPC protein 1Oryzias latipes Japanese medaka AF128811 Ola ZPCd ZPC protein 3Oryzias latipes Japanese medaka AF128812 Ola ZPCb ZPC protein 4Oryzias latipes Japanese medaka AF128813 Ola ZPCa ZPC protein 5Oryzias latipes Japanese medaka D38630 Ola ZPCc L-SFOryzias latipes Japanese medaka D89609 Ola ZPBb choriogenin HPapio cynocephalus Yellow baboon AY222646 Pcy ZPB2 ZPBPseudomys australis Plains rat AY078055 Pau ZPC ZPCPseudopleuronectes

americanusWinter flounder U03674 Pam ZPB ZP

Rattus norvegicus Rat AB000928 Rno ZPB1 ZP1Rattus norvegicus Rat AB000929 NM_031150 Rno ZPA ZP2Rattus norvegicus Rat AF456325 NM_172330 Rno ZPB2 ZP4/ZPBRattus norvegicus Rat D78482 Y10823 Rno ZPC ZP3Salmo salar Salmon AJ000665 SSa ZPB ZPBSus scrofa Pig L22169 D45065 Ssc ZPC ZP3-beta/ZP3Sus scrofa Pig S74651 L22170, D45064 Ssc ZPA ZP1/ZP2Trichosurus vulpecula Brush-tailed possum AF079524 Tvu ZPC ZP3Trichosurus vulpecula Brush-tailed possum AF263013 Tvu ZPB2 ZPBTrichosurus vulpecula Brush-tailed possum AF263014 Tvu ZPA ZPAXenopus laevis African clawed frog AF038151 Xla ZPA 69 kDa/ZP2Xenopus laevis African clawed frog AF225906 Xla ZPX1 ZPAXXenopus laevis African clawed frog U44949 Xla ZPX2 ZPAXenopus laevis African clawed frog U44950 Xla ZPB 37 kDa/ZPBXenopus laevis African clawed frog U44952 Xla ZPC ZPC/ZP3

134 J. SMITH ET AL.

primary sperm receptor is a complex of both ZPC andZPB2 (Yurewicz et al., 1998). The egg envelope ofXenopus is made up of several glycoproteins, one ofwhich is ZPX1, a ZP2-like protein, the gene for which hasrecently been identified (Lindsay et al., 2001) and ZPD(Lindsay et al., 2002) which, although contains a ZPdomain, shows greater similarity to nonegg-envelopeproteins in other species, for example, alpha tectorin anduromodulin. The sequence of the chicken ZPD gene hasalso recently been elucidated (GenBank: AB114441).Another geneZPB2, which is a member of the ZP1 (ZPB1)family, has been identified in various species includinghuman, mouse, chicken, Xenopus, and fish (Table 1).

In mammals, the ZP proteins are expressed in oocytes.However, chicken ZPB1 is synthesized in the liver underthe control of oestrogen and transported to the folliclevia the bloodstream (Bausek et al., 2000), and chickenZPC is synthesized and secreted in the granulosacells surrounding the oocyte (Waclawek et al., 1998;Takeuchi et al., 1999). ZPC is the major component of theperivitelline membrane in chicken (Takeuchi et al.,1999). In teleosts, theZP genes are expressed in both theovary and the liver; for example, ZPB1/ZPB2 relatedgenes are expressed in the ovary (Kanamori, 2000), theliver (Hyllner et al., 2001), or the ovary and the liver(Conner and Hughes, 2003).

The sequences of the chicken ZPB1, ZPB2, and ZPCgenes have previously been published. Here, we reportthe genetic and physical mapping of these genes andthe identification and subsequent mapping of novelchicken ZP genes—ZPA and ZPX1 (EMBL accessions:BN000517 and AJ698915). We compare these chickenprotein sequences to the ZP proteins from human,mouse, and Xenopus, and a broader combined analysisof sequence homology and conservation of synteny hasallowed us to determine an unambiguous relationshipbetween ZP family members in vertebrates and hasgiven us an insight into the evolution of this family ofgenes.

MATERIALS AND METHODS

Preparation of Probes for Hybridization

Chicken ZPB1. cDNA, as described in Bausek et al.(2000), was digested with NcoI to isolate the insert.Chicken ZPC. cDNA as described in Takeuchi et al.

(1999) was digested with EcoRI.Chicken ZPB2. An EcoRI digest of a ZPB2 cDNA

released the insert for use as a probe (Matsuda,unpublished). Restriction digestion of chicken ESTclones 603269036F1 and 603774195F1 provided probesfor ZPA and ZPX1, respectively. Each probe DNA(300 ng) was labeled with P32a-dCTP using the Prime-a-Gene kit (Promega, Madison, WI)

Isolation of BACs Containing ZP Genes

Chicken probes forZPB1,ZPA,ZPC,ZPB2, andZPX1were hybridized to the chicken BAC library (Crooijmanset al., 2000) overnight at 658C in 10% PEG8000; 7% SDS;1.5� SSC. Filters were then washed twice in 2� SSC;

0.1% SDS, and once in 0.5� SSC; 0.1% SDS at 658C,and exposed to autoradiographic film for 4 hr at roomtemperature.

Fluorescence In SituHybridization

Chicken metaphase chromosome spreads were pre-pared from chicken embryo fibroblasts after treatmentwith colcemid solution. Cells were swollen by treatmentwith hypotonic solution, fixed in methanol:acetic acidfixative (3:1), and dropped onto methanol-cleaned slidesand allowed to air dry. BAC probes were labeled withbiotin-16-d-UTP by nick translation (Rigby et al., 1977).Labeled probe (100 ng) precipitated with 6 mg salmonsperm DNA was used in each experiment. Probes werehybridized to chromosomes overnight at 378C in amoist chamber. Hybridization was detected by incuba-tion with avidin-Cy3. Chromosomes were stained withDAPI and images captured using a Nikon Microphot-SAmicroscope fitted with a cooled CCD camera (DigitalPixel, Brighton, UK) and analyzed with IPLab software(Scanalytics, Inc., Fairfax, VA).

Subcloning

BAC DNA (5 mg) was partially digested with therestriction enzyme Cvi JI (Cambio, Cambridge, UK)that cuts at the consensus PuG/CPy (Xia et al., 1987).Digestion was carried out at 378C for 1 hr using 0.01 Uenzyme. DNA fragments in the size range 1–3 kb wereexcised from an agarose gel and the DNA extractedusing a QiaexII DNA extraction kit (Qiagen GmbH,Hilden, Germany). DNA fragments were subcloned intoEcoRV-restricted pBluescriptSKþ and sequenced asdescribed.

DNA Sequencing and Analysis of BACs

Thirty-two subclones were shotgun-sequenced fromeach BAC. Sequencing reactions were carried out usinga Big Dye sequencing kit (Applied Biosystems, War-rington, UK) and separated on an ABI 377 automaticsequencer. Following sequencing, vector and poorquality sequences were removed. Contig assembly wascarried out using the Seqman program (DNAStar). Theremaining sequence data were searched against thenonredundant nucleotide and protein databases usingthe BLAST algorithm (Altschul et al., 1990).

Genetic Mapping

Polymorphisms were identified in PCR-amplifiedDNA as single strand conformational polymorphisms(SSCP). Markers were scored on the East Lansingreference population (Crittenden et al., 1993) and theirpositions recalculated on the consensus linkage map(Groenen et al., 2000).

Linkage Analysis

Linkage analysis was carried out using the MapManager program (Manly, 1993), and recombinationfractions were converted to centiMorgans by means ofthe Kosambi mapping function (Kosambi, 1944).

CHICKEN ZONA PELLUCIDA GENES 135

Phylogenetic Analysis

Sequences included in the phylogenetic analysis of theZP gene family are listed in Table 1. This table includesthe GenBank accession numbers of the gene sequences(mostly cDNAs and a few genomic sequences), thespecies names, and suggested names for the ZP genes(based on the recommendations of Spargo and Hope,2003). In addition, other accession numbers of identicalsequences are also given, if they exist in the currentdatabases. In this paper, we use the amino acid sequenceto infer phylogenetic relationships, since synonymousnucleotide differences were saturated for pairs ofsequences separated by more than 150 million years ofevolution. Amino acid sequence alignments of the ZPproteins were carried out using CLUSTAL X (Thompsonet al., 1997) and BioEdit (see www References) withdefault parameter settings. Since the entire ZP genefamily was aligned, many gaps were expected with sucha diverse set of sequences. Only the gap-free (or at leastfew gaps, where gaps are treated as ‘‘missing’’ aminoacids) segments were used in the calculation of gene-trees, since the alignments in the gap-rich regions werelikely to be unreliable. In total, 68 nonidentical se-quences with 292 amino acid sites were used in themaximum parsimony and bootstrap analyses (PAUP*version 4.0, Swofford, 2002) and quartet puzzling(TREE-PUZZLE version 5.1, Schmidt et al., 2002).TreeView (Page, 1996) was used to display phylogenetictrees created by PAUP* or TREE-PUZZLE. The wwwsources for all programs used in this paper are listed inthe references.

RESULTS

Isolation of ZP BACs

After hybridization of the various ZP probes to thechicken BAC library, the following positive clones wereidentified: ZPB1: 35-O18, 107-E21, and 110-M4. ZPA:122-G19. ZPC: 30-I3, 73-O10, and 108-C9. ZPB2: 25-F18, 25-P8, 44-H2, 81-O24, 86-J8, 86-K17, 87-K24, and121-O9. ZPX1: 13-L17, 36M11, 98J5, and 119H7. EightBACs were identified as positive upon hybridizationwith the ZPB2 probe. The library represents fivefoldcoverage of the genome, so it was a possibility that twodifferent genes were being highlighted. The eight cloneswere tested by PCR for amplification of ZPB2 sequence.Two sets of primers were used: one pair developed fromthe 50-end of the published chicken ZPB2 sequence(GenBank: AB025428) and one pair from the 30-end. These are: 50-F:TTTGATGGGTGTTGTAGGGC50-R:ACAAAGGAAGCATTCCCCTC; and 30-F:CTCTGG-GTTTTGCTGCTGTG 30-R:TGCAAGACAGGTCCCTC-TAAG.

Individual BAC colonies were diluted with 50 ml dH2Oand 5 ml used in 15 ml colony PCR reactions. All eightBACs were found to be positive by PCR. However, two ofthe BACs only amplified with the 30-primers: 25-F18 and87-K24 and two of the BACs only amplified with the 50-primer: 81-O24 and 86-J8. The other four BACs all gaveproduct with both sets of primers.

Physical and Genetic Mapping of ZP Genes

Each of the putative ZP BACs was physically mappedby FISH. The three BACs which were identified by theZPB1 probe were all found to hybridize to chickenchromosome 5p11-q11 (RBG; Flpter: 0.11). The BACidentified with the ZPA probe hybridized to one of themedium-sized microchromosomes (13–18). The threeZPC clones were localized to one of the large micro-chromosomes (chromosomes 9–12), while the eightZPB2 BACs hybridized to chromosome 6q12-14 (RBG;Flpter: 0.45). The fourZPX1 clones were found to map to3q33-35 (RBG; Flpter: 0.87) (Fig. 1). Primers weredeveloped from the published sequences of the chickenZPB1 (GenBank: AJ289697), ZPC (GenBank:AB031033), and ZPB2 (accession no. AB025428) genesand from the sequences of EST clone 603269036F1(GenBank: BU472281) (ZPA) and from sample sequencefrom BAC 13-L17 (EMBL: AJ703861) (ZPX1) and usedto develop SSCP markers. The genetic positions weredetermined and the genes confirmed as mapping tochromosome 5 (ZPB1), chromosome 14 (ZPA), chromo-some 10 (ZPC), chromosome 6 (ZPB2), and chromosome3 (ZPX1) (Table 2).

Sequencing of BACs

Shotgun sequence was obtained from BAC 107-E21(ZPB1), BAC 122-G19 (ZPA), BAC 73-O10 (ZPC), BAC87-K24 (ZPB2), and BAC 13-L17 (ZPX1) and ZPsequences identified, (GenBank: AY268032-AY68036).Shotgun sequencing of the chicken BACs confirmed thepresence of theZP genes and also identified the presenceof other genes. BAC 107-E21 was found to contain notonly ZPB1 but also the gene for the transmembraneglycoprotein CD5, pepsinogen A (PgA), and the ortholo-gue of the gene for the human nuclear membrane pro-tein NMP200. Human pepsinogen A maps to HSA11q13and NMP200 to HSA11q12.2, adding to the knownconserved synteny between chicken chromosome 5 andhuman chromosome 11. Shotgun sequence from theZPA-containing BAC 122-G19 identified the DNAH3homologue. This gene maps to chromosome 16p12 inhuman, the same region thatZPA is located. Sequencingof BAC 73-O10 confirmed the presence of the ZPC geneand also identified the cytochrome P-450 gene and thehomologue of the hypothetical human gene FLJ20452.Human cytochrome P-450 is known to map to HSA7q21and FLJ20452 to HSA15q33, thus confirming that thesynteny between chicken chromosome 10 and humanchromosome 15 is interrupted by a small region ofhuman chromosome 7, and showing that BAC 73-O10encompasses sequence covering a human chromosomalbreakpoint. Sequencing of a ZPX1 BAC (13-L17)identified the chicken homologue of human FLJ22116,which is known to encode a member of the SMC family ofgenes. This locus is found on human chromosome 2p in aregion where we have found a ZPX1 pseudogene to belocated (GenBank: XM_377732). Figure 2 shows theamino acid alignment of the Xenopus ZPX1 gene andthe predicted chicken sequence with the translated

136 J. SMITH ET AL.

sequence of the homologous region of human chromo-some 2. Sequence homology is still clearly visiblebetween the three species. However, the humansequence no longer contains a conserved ZP domainand many deletions are seen when compared with theamphibian and avian sequences. Sequencing of theZPB2-containing BAC 87-K24 did not highlight anyother genes than the ZP gene.

Elucidation of the Full-Length Sequence ofChicken ZPA and ZPX1 Genes

The ESTs that represent the chicken ZPA and ZPX1genes were used to search the chicken genome sequenceusing the Ensembl browser (Hubbard et al., 2002). Theprotein sequence was predicted by the Genomescanprogram (Yeh et al., 2001) and the full-length cDNA

Fig. 1. Fluorescence in situ hybridization of (A) BAC 107-E21(ZPB1), (B) BAC 122-G19 (ZPA), (C) BAC 73-O10 (ZPC), (D) BAC 81-O24 (ZPB2), and (E) BAC 13-L17 (ZPX1) to chicken metaphasechromosomes. Probes are labeled with biotin-16-dUTP by nick trans-lation and detected by the addition of avidin-Cy3. Chromosomes are

stained with DAPI. ZPB1 maps to chromosome 5p11-q11 (only onechromosome 5 seen in the spread), ZPA maps to chromosome 14, ZPCmaps to chromosome 10, ZPB2 maps to 6q12-14, and ZPX1 maps to3q33-35.

CHICKEN ZONA PELLUCIDA GENES 137

sequence by analysis with the GeneWise algorithm(Birney and Durbin, 2000). The ZPA sequence is onchromosome 14 between bases 39517218 and 39522799on chromosome UN in genome assembly (genomiccontig 164.44-accession number: AADN01078972.1),and the ZPX1 sequence lies on chromosome 3 betweenbases 98449306 and 98463626 (genomic contigs 27.188and 27.189-accession numbers: AADN01041695 andAADN01041696). The predicted sequences are in theEMBL database under accession nos. BN000517 andAJ698915, respectively.

Comparison of Chicken, Human,and Mouse Orthologues

The amino acid sequence from theZP genes of human,mouse, chicken, andXenopuswere all compared againsteach other and levels of identity amongst the genesdetermined (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) (Table 3).

Phylogenetic Analysis

Two methods were used to construct ZP gene-trees,maximum parsimony (Swofford, 2002) and maximumlikelihood estimation based on quartet puzzling(Schmidt et al., 2002). Both gave similar results, at leastfor the major branches of the phylogeny.

Maximum parsimony. A consensus tree calculatedby bootstrap analysis of 200 replicates based on max-imum parsimony is shown in Figure 3. Most of the majorbranching orders were well supported in over 70% ofreplicates. These represent the different ZP paralogues:ZPA, ZPB1, ZPB2, ZPBC, ZPX1, and ZPX2. Branchingorders of some groups were only supported by maximumparsimony bootstrap values of 56%–68%, but these werebetween orthologous genes rather than between para-logues, the major focus of this paper.

Maximum likelihood method using quartetpuzzling. TREE-PUZZLE maximum likelihood dis-tances were calculated using the JTT (Jones et al.,1992) model of amino acid substitution and assumeda uniform rate of heterogeneity between sites. Theaverage distance over all possible pairs of sequences was2.39. Quartet puzzling was used to choose the mostlikely tree. 25,000 puzzling steps and 864,501 quartetswere analyzed. Ninety-two percent of quartets werefully resolved into unique topologies and 5% were com-pletely unresolved. Figure 4 shows the percentagesupport or reliability value (Schmidt et al., 2002).

Both maximum parsimony and quartet puzzlingsupported similar tree topologies; however, TREE-PUZZLE was less able to resolve deep branches.

DISCUSSION

Genes for ZPB1, ZPA, ZPC, ZPB2, and ZPX1 havenow been isolated and both physically and geneticallymapped in the chicken. This has shown that each ZPgene is found on a different chromosome and is not foundclustered as is sometimes seen with members of othergene families. Mapping of these genes placed ZPB1on chromosome 5, ZPA on chromosome 14, ZPC on

TABLE

2.Mappin

gofth

eChickenZP

Genes

Gen

eL

ocu

sF

ISH

(RB

G)

EL

a(c

M)

Con

sen

sus

(cM

)F

pri

mer

Rp

rim

erT

emp

eratu

re(8

C)

Siz

e(b

p)

ZPB1

RO

S0273

5p

11-q

11

0.0

6G

TG

CC

AC

CT

GT

CA

CA

GC

CT

CT

GC

TG

CC

TT

CC

TG

TC

C60

�160

ZPA

RO

S0284

Ch

rom

osom

e14

49.1

65

AG

AC

CC

AA

CG

TC

AC

TT

CC

AC

CC

AC

GG

GA

TG

GA

AT

AC

AG

TC

55

�450

ZPC

RO

S0272

Ch

rom

osom

e10

97.8

24

AG

AA

CA

CC

CT

TC

GT

GT

CA

CC

CT

TG

GT

CC

CT

CC

AT

AG

CA

TC

60

257

ZPB2

RO

S0271

6q12-1

462.1

63

TT

TG

AT

GG

GT

GT

TG

TA

GG

GC

AC

AA

AG

GA

AG

CA

TT

CC

CC

TC

60

189

ZPX1

RO

S0295

3q33-3

5305.6

273

AC

CA

GC

AG

TT

CT

TC

CA

GC

TC

GT

TG

CT

CA

TT

TG

TG

CA

GG

AG

60

276

aE

ast

Lan

sin

gre

fere

nce

pop

ula

tion

.

138 J. SMITH ET AL.

Fig. 2. Boxshade alignment (http://www.ch.embnet.org/software/BOX_form.html) of the amino acidsequences from the Xenopus ZPX1 protein (GenBank: AF225906) and the predicted chicken sequence(EMBL: AJ698915) with the translated homologous region of human DNA (GenBank: XM_377732). Thezona pellucida (ZP) domain is underlined, where it is clearly seen that deletions have rendered this regioninactive in the human sequence.

chromosome 10, ZPB2 on chromosome 6, and ZPX1 onchromosome 3. HumanZPB1 is found on chromosome 11and mouse ZPB1 on chromosome 19 (Hughes andBarratt, 1999). This GGA5/HSA11/MMU19 region isknown to show conserved synteny amongst the threespecies (Schmid et al., 2000; http://www.ncbi.nlm.nih.gov/Homology/view.cgi?map¼ncbi_mgd&chr¼11&tax_id¼ 9606&mode¼text). Likewise, ZPA maps to chromo-some 14, which shows conserved synteny with HSA16p,the location of human ZPA. ZPC is located on chromo-some 7 in the human. This breaks up a large area ofconserved synteny with chicken chromosome 10 andhuman chromosome 15. Similarly, ZPB2 is found onhuman chromosome 1, disrupting a region of conserva-tion between chicken chromosome 6 and human chro-mosome 10. Conserved synteny is maintained by ZPX1mapping to chromosome 3 in a region that is alreadyshown to be homologous to human 2p (Fig. 5).

The amino acid sequences of the human ZP genes[ZPB1 (Hughes and Barratt, 1999), ZPA (Liang andDean, 1993), ZPC (Chamberlin and Dean, 1990), andZPB2 (Harris et al., 1994)], the mouse genes [ZPB1(Epifano et al., 1995), ZPA (Liang et al., 1990), ZPC(Kinloch and Wassarman, 1989), and ZPB2], thechicken genes ZPB1 (Bausek et al., 2000), ZPA, ZPC(Takeuchi et al., 1999), ZPB2 (Takeuchi et al., 2001),and ZPX1)] and the Xenopus genes [ZPA (Tian et al.,1999), ZPB (Kubo et al., 2000), ZPC (Kubo et al., 1997),and ZPX1 (Lindsay et al., 2001)] were all comparedagainst each other and levels of identity determinedamongst the genes in each species. Genes involved inreproduction have previously been shown to be highlydivergent compared to genes that are not involved inthis process (Vacquier, 1998; Swanson and Vacquier,2002). The rapid evolution of these genes is thought to beinvolved in the speciation process. The high level ofdivergence amongst the ZP genes is reflected in theamino acid alignments as shown in Table 3.

Using both phylogenetic and comparative map-ping methods we were able to show that all six majorbranches represent distinct ZP paralogues: ZPA, ZPB1,ZPB2, ZPBC, ZPX1, and ZPX2.

From conserved synteny and the levels of homologybetween species, it can be seen that human ZP1, mouseZp1, and chicken ZP1 are all orthologues. Human ZP2,mouse Zp2, and Xenopus ZPA are orthologous, as arehuman ZP3, mouse Zp3, chicken ZPC, and XenopusZPC. Human ZPB, mouse Zpb, chicken ZPB, andXenopus ZPB are also orthologous genes. ZPB1 andZPB2 are genes that are closely related to each other(Hughes and Barratt, 1999) and appear to only exist asdistinct genes in mammals and birds. Xenopus does notappear to contain a ZPB1 homologue, although it has aclosely-related ZPB2 gene. From analysis of vertebrateZPB1/ZPB2 genes it is likely that an ancestral ZPB1/ZPB2 gene underwent independent duplications in theteleost lineage and in the higher vertebrate lineage(Bausek et al., 2000; Conner and Hughes, 2003;Kanamori et al., 2003; Spargo and Hope, 2003). Ifindeed Xenopus does not have a ZPB1 gene, this would

TABLE

3.PercentId

entity

inth

eAmin

oAcid

Sequenceofth

eZP

Genes

HS

A-Z

PB

1H

SA

-ZP

AH

SA

-ZP

CH

SA

-ZP

B2

MM

U-Z

PB

1M

MU

-ZP

AM

MU

-ZP

CM

MU

-ZP

B2

GG

A-Z

PB

1G

GA

-ZP

AG

GA

-ZP

CG

GA

-ZP

B2

GG

A-Z

PX

1X

LA

-ZP

AX

LA

-ZP

B2

XL

A-Z

PC

XL

A-Z

PX

1

HS

A-Z

PB

1100%

HS

A-Z

PA

91/2

71

(33%

)100%

HS

A-Z

PC

70/3

01

(23%

)62/3

00

(20%

)100%

HS

A-Z

PB

2158/3

43

(46%

)121/3

04

(39%

)61/2

73(2

2%

)100%

MM

U-Z

PB

1388/6

04

(64%

)97/2

65

(36%

)71/3

08

(23%

)167/3

53

(47%

)100%

MM

U-Z

PA

99/2

88

(34%

)413/7

29

(56%

)47/2

32

(20%

)112/3

04

(36%

)95/2

70

(35%

)100%

MM

U-Z

PC

66/2

86

(23%

)48/1

36

(25%

)240/3

48

(68%

)61/2

87

(21%

)0

41/1

81

(22%

)100%

MM

U-Z

PB

283/2

89

(28%

)73/2

86

(25%

)0

131/2

78

(47%

)84/2

94

(28%

)69/2

89

(23%

)0

100%

GG

A-Z

PB

1187/3

38

(55%

)95/3

03

(31%

)65/2

94

(22%

)162/3

55

(45%

)190/3

71

(51%

)107/3

03

(35%

)68/2

90

(23%

)83/2

80

(29%

)100%

GG

A-Z

PA

85/2

87

(29%

)274/6

63

(41%

)0

91/2

80

(32%

)86/2

82

(30%

)242/6

57

(36%

)0

65/2

58

(25%

)76/2

79

(27%

)100%

GG

A-Z

PC

048/2

38

(20%

)178/3

24

(54%

)0

054/2

32

(23%

)178/3

79

(46%

)0

59/2

77

(21%

)0

100%

GG

A-Z

PB

2171/3

42

(50%

)124/3

24(3

8%

)57/2

53

(22%

)262/4

99

(52%

)170/3

42

(49%

)119/3

06

(38%

)62/2

60

(23%

)104/2

85

(36%

)22/7

1(3

0%

)93/2

73

(34%

)44/2

05

(21%

)100%

GG

A-Z

PX

10

00

00

00

00

00

0100%

XL

A-Z

PA

104/3

30

(31%

)252/6

28

(40%

)55/2

21

(24%

)125/3

76

(33%

)89/2

46

(36%

)229/6

23

(36%

)0

50/1

74

(28%

)94/2

81

(33%

)241/6

54

(36%

)55/2

38

(23%

)122/3

11

(39%

)0

100%

XL

A-Z

PB

2181/5

47

(33%

)104/3

09

(33%

)55/2

61

(21%

)202/4

61

(43%

)178/5

29

(33%

)107/3

26

(32%

)50/2

37

(21%

)82/2

84

(28%

)140/3

35

(41%

)90/2

85

(31%

)0

225/5

00

(45%

)0

118/3

10

(38%

)100%

XL

A-Z

PC

72/3

31

(21%

)57/2

56

(22%

)155/3

32

(46%

)62/2

92

(21%

)52/2

26

(23%

)48/2

40

(20%

)160/4

13

(38%

)0

62/2

88

(21%

)0

168/3

56

(47%

)46/2

10

(21%

)0

54/2

22

(24%

)49/2

24

(21%

)100%

XL

A-Z

PX

197/3

13

(30%

)135/4

97

(27%

)62/2

75

(22%

)99/3

13

(31%

)87/3

00

(29%

)109/3

06

(35%

)0

51/2

11

(24%

)87/2

82

(30%

)79/2

71

(29%

)42/1

50

(28%

)100/2

98

(33%

)372/8

30

(44%

)118/3

56

(33%

)87/2

74

(31%

)54/2

51

(21%

)100%

140 J. SMITH ET AL.

place this duplication after the divergence of amphi-bians and mammals (360Mya), but before the diver-gence of birds and mammals (300Mya). However, if thegene has just not been found yet, or if it has been deleted,it would indicate that the duplication of ZPB1 fromZPB2 occurred further back, in the common ancestor.The level of homology betweenXenopusZPB2 andZPB1

and ZPB2 genes in other species is similar to the levelof homology between ZPB1 and ZPB2 within species(28%–46%, see Table 3), indicating that the Xenopusgene is probably the ancestral gene and that the ZPB2/ZPB1 duplication most likely occurred after the diver-gence of amphibians and mammals. The ZP gene-treesupports an independent model of ZPB2 gene evolution

Fig. 3. Maximum parsimony (PAUP*) analysis of theZP gene family was carried out using the followingparameters: Tree lengths 4,856, consistency index¼ 0.54, homoplasy index¼ 0.46, retention index¼ 0.74,rescaled consistency index¼ 0.40. This figure shows the bootstrap 50% majority-rule consensus tree (200replicates) based on maximum parsimony of 5255 trees (using tree weights).

CHICKEN ZONA PELLUCIDA GENES 141

in fish from the ZPB2 genes in amphibians/birds/mammals (Conner and Hughes, 2003). It can be seenthat the fish ZP genes have independently duplicatedmany times, distinct from the major duplication thatgave rise to the ZPB1 and ZPB2 genes in birds andmammals. Evidence for the theory that ZPB1 and ZPB2arose from the duplication of a single gene is presentedin the mouse. Although these two genes are found to belocated on different chromosomes in chicken, human,and mouse, the loci around each of these genes in mouseare seen to reside on chromosome 19, indicating thatZPB1andZPB2were both once together in the ancestralgenome (Fig. 5).

It is interesting to note that the chicken ZPB1sequence contains a region of highly repetitive sequence(around exon 3) that also codes for a glutenin-likedomain (Bausek et al., 2000). None of the otherZP genesshow this feature and nor do the shorter human andmouse ZP1 genes. It is, however, found in the publishedquail sequence (GenBank: AB061520), thus suggestingthat this part of the gene sequence is unique to the avian

lineage, appearing after the divergence of birds andmammals (300Mya).

Novel ZP and ZP-related genes continue to be dis-covered. For instance, medaka and Fugu also contain ahost of other ZP genes that belong to the ZPC family(Kanamori et al., 2003). The presence of these variousZPC genes alludes to a duplication event havingoccurred in this gene in the ancient teleost lineage.The analysis presented here clearly shows that thegrouping of ZPC genes for Gallus gallus and Tricho-surus vulpecula, reported by Spargo and Hope (2002),are orthologues of the apparently distantly relatedeutherian ZPC genes.

These authors also noted that since the duplicationevent that gave rise to the ZPA gene occurred before thedivergence of fish and amphibians, that fish must have/or had a copy of the ZPA gene. Various models wereproposed to explain this observation, including (1)the fish ZPA gene has not been cloned yet, (2) the fishZPA gene has been lost, and (3) that the ZPX1 gene isactually a ZPA orthologue. The data presented in

Fig. 4. Support for internal branches of an unrooted quartet puzzling tree topology, based on maximumlikelihood analysis. Branch lengths were calculated using the JTT substitution model and a model ofuniform rate heterogeneity. Levels of support for each branch are shown in percentages.

142 J. SMITH ET AL.

Figure 3 appears to support either a gene loss model orone in which the fish ZPA gene has not yet been cloned.

The chicken ZPX1 gene, orthologous to XenopusZPAX, is a ZP2-like gene that, until now, had only been

found in amphibians and teleosts such as medakaand Fugu (http://fugu.hgmp.mrc.ac.uk scaffold nos.S006926 and S007855)—species that show no ZPAorthologues, either because the genes have not yet been

Fig. 5. Chicken/human/mouse comparative maps of the regionsaround the zona pellucida genes. Conserved synteny is seen around theZPB1, ZPA, and ZPX1 loci. ZPC is located within a small region ofconservation, which breaks up larger blocks of synteny in the human andmouse.ZPB2 is seen to be an insertion in the human chromosome, which

interruptsa largeregionofsyntenybetweenchickenandhuman.Humanmapping information was obtained from the Genecards website (http://bioinformatics. weizmann.ac.il/cards/) and mouse loci positions fromNCBIMapViewer(http://www.ncbi.nlm.nih.gov/mapview/)andfromtheEnsembl genome server (http://www.ensembl.org/Mus_musculus/).

CHICKEN ZONA PELLUCIDA GENES 143

identified in these species, or they do not exist. It isinteresting to find that the chicken exhibits all five ZPgenes: ZPB1, ZPB2, ZPA, ZPC, and ZPX1—a situationas yet, unseen in any other vertebrate. The ancestralchicken, therefore, provides us with a good model forstudying the evolution of this gene family. The presenceor absence of the ZPX1 gene in different species high-lights the mechanisms that are occurring. The ancientspecies such as fish, amphibians, and birds, all containat least one ZPAX gene. However, in the rapidly evolv-ing murine genome, this particular gene has been lost.The human genome also does not contain a functionalZPX1 gene. However, there are remnants of this genefound on chromosome 2 in a region that shares con-served synteny with chicken chromosome 3, where theavian orthologue is located. So, although ZPX1 has notbeen completely deleted in human, as appears to be thecase in mouse, the function has been lost throughevolutionary mutation. Similarly, a search for ortholo-gues of chicken ZPX2 [chromosome 11: Genomicsequence 496,557–499,484)] failed to detect any ortho-logues in either human or mouse genes, indicating geneloss in both species.

Overall, the phylogeny is consistent with the hypoth-esis of Spargo and Hope (2002), who suggested an initialduplication event giving rise to ZPC in one branch andZPA, ZPB, etc. in other branches. Another possibilitywould be ZPX2 as being derived from the most ancientduplication event. The identification of a chicken ZPX2orthologue clearly defines this group as another ZPparalogue.

ACKNOWLEDGMENTS

The authors thank Franz Wohlrab (University andBiocenter of Vienna, Austria) for supplying the ZPB1probe and Tsukasa Matsuda (Nagoya University,Japan) for the ZPC and ZPB2 probes. The authors alsothank Frazer Murray (Roslin Institute) for sequenc-ing the BAC clones. The chicken BACs (Wageningenlibrary) were obtained from the HGMP in Hinxton, UK.

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CHICKEN ZONA PELLUCIDA GENES 145