BACKGROUND
molecules that are directly involved in reproduction are often subject to rapid evolutionary change  <cit> .
zonadhesin  is one such molecule that has undergone domain expansion  <cit>  and positive selection  <cit>  in mammals.
zan is a multi-domain sperm protein that is implicated in the species-specific binding of egg and sperm.
porcine  zan was first described by hardy et al.
<cit>  as a protein expressed by developing sperm that would bind to the zona pellucida of the egg.
since its initial discovery, zonadhesin has been identified in several other mammals, including mouse, human  <cit>  and rabbit  <cit> .
recent data suggest the processed zonadhesin localizes to the acrosomal matrix and binds the zona pellucida during the acrosome reaction  <cit> .
the discrete domains of mosaic proteins are known to be important in the evolution of new genes.
domain subunits can be rearranged, duplicated or deleted to produce a variety of proteins with different functions  <cit> .
zonadhesin structure is unique in its combination of protein domains.
all mammalian zonadhesin genes are predicted to encode: a signal peptide, a multiple meprin a <dig> antigen receptor tyrosine phosphatase mu  domain, multiple trypsin-like inhibitor  domains, multiple von willebrand d  cell adhesion domains, multiple hepta-peptide repeats that form the mucin domain, multiple epidermal growth factor  domains, a single transmembrane domain and short intracellular domain at the carboxyl terminus.
the domain order is the same for all mammals studied, with the main difference being the number of mam and vwd domains.
these individual domains each have a particular function and are found in many other mosaic proteins.
the extracellular vwd domain occurs in a family of immediate-early genes that are growth regulators and is thought to have an adhesive function.
this modular domain is found in a variety of mosaic proteins including von willebrand factor  <cit> , apolipoprotein b, vitellogenins, microsomal triglyceride transfer protein   <cit> , and mucins  <cit> .
biochemical studies of pig zonadhesin have shown that the zan precursor is processed and the mam domains are removed leaving the vwd domain to interact with the zona pellucida  <cit> .
while the role of the vwd in sperm-egg binding has been addressed, the role of the mucin-repeats and the mam domains is still unknown.
the mucin or muc domain is the primary functional domain of mucin proteins.
mucins are a diverse group of heavily glycosylated proteins that are the major component of mucus.
mucins function to lubricate surfaces and are the first line of defence against pathogens  <cit> .
most of the secreted mucins contain a domain with sequence similarity to vwd and a domain composed of a variable number of tandem repeats that code for serine-, threonine- and proline-rich repeat peptides that are potential glycosylation sites  <cit> .
two mucin-like genes that are similar to zonadhesin include alpha-tectorin, which is involved in non-syndromical autosomal dominant hearing impairment  <cit> , and fc fragment of igg binding protein .
by virtue of sequence identity, the closest relative to mammalian zonadhesin is fcgbp and this similarity is mostly seen in the til and vwd domains.
fcgbp is expressed in the mucosa of the small and large intestines, epithelial colon cells and the placenta and is thought to play a role in the mucosal immune system through the promotion of multivalent igg and the trapping of antigen igg complexes in the mucosa  <cit> .
the  <dig> amino acid mam domain distinguishes zonadhesin from other vwd and mucin repeat containing genes such as fcgbp.
mam domains have adhesive function and are found in several proteins including protein-tyrosine phosphatases, neuropilins and meprins.
meprins are metalloendopeptidases that have been found in the intestinal brush border and renal membranes of mammals  <cit> .
while their role in the zonadhesin protein is unknown, mam domains are important for multimerization  <cit>  and have conserved cysteine residues that are responsible for covalent interactions  <cit> .
when this study began, zonadhesin expression had only been described in the testes.
only zonadhesin was thought to encode mam domains, mucin repeats, and vwd domains and no non-mammalian zonadhesin orthologs had been reported.
for these reasons we were interested in atlantic salmon  zonadhesin-like ests from a gut-derived library that encoded the mam, mucin and vwd domains.
here we describe the cdna, genomic structure, and expression patterns of this atlantic salmon zonadhesin-like gene.
we also use comparative genomic and expression analyses to uncover additional zonadhesin-like genes, as well as orthologs of zonadhesin, in zebrafish  and puffer fish .
RESULTS
characterization of a zonadhesin-like gene in s. salar
a gene similar to mammalian zonadhesin was identified during an expressed sequence tag  analysis of atlantic salmon.
we assembled this zonadhesin-like gene  from five overlapping ests  <cit> ; the largest of which  was sequenced by primer walking from both directions and assembled to give a  <dig> bp sequence.
pcr primers were designed to the 3'-end of the est and used to amplify a probe for hybridization to atlantic salmon genomic dna and bacterial artificial chromosome  library filters .
probing of  <dig>  bac clones on five atlantic salmon genome bac library filters  <cit>  resulted in one positive bac.
this bac  was subcloned, sequenced, and assembled into a  <dig>  base pair contiguous sequence .
the assembly had more than  <dig> high quality sequence reads and  <dig> fold sequence coverage in most regions.
one gap of  <dig> bp was filled by pcr followed by sequencing from both directions.
an in silico restriction digest matched experimental digests.
the zonadhesin-like gene was the only gene found on this bac.
all other open reading frames were associated with line, sine and transposon related repetitive elements.
comparison of 722p <dig> against itself using dotter and pipmaker  <cit>  did not reveal any recent domain expansions or duplications.
the simple modular architecture research tool   <cit>  was used to identify conserved domains of the predicted protein .
the smart algorithm was used to detect three vwd domains , a vwc domain at , and two mam domains .
this tool also located three low complexity regions that corresponded to the mucin domains predicted by the computer program netoglyc  <dig>   <cit> .
these mucin domains occurred at positions 1091– <dig> and 1115– <dig> with a smaller low complexity region located between nucleotides  <dig> and  <dig>  these results were corroborated using the interpro domain prediction computer program  <cit> .
the smart and interpro domain prediction tools, in agreement with kyte-doolittle hydropathy data, did not find any transmembrane domains in the salmon zlg.
in contrast, the smart tool detected a transmembrane domain at the expected location for zonadhesins from other species.
using two distinct probes , only one copy of zlg was found in atlantic salmon by southern blot analysis .
however, two bands occurred when the same probes were used with rainbow trout genomic dna .
probe  <dig>  which contained sequence homologous to the 3'-utr of zlg , would be expected to be specific for this gene; however, probe  <dig> spanned vwd <dig> and could be expected to hybridize with related zonadhesin-like genes.
both probes gave similar results and only probe  <dig> data is shown in figure 1b.
southern blot analysis did not reveal any other genes in atlantic salmon other than zlg.
this suggests that if another zonadhesin-like gene does exist in atlantic salmon it is likely quite divergent from zlg.
the 5'-end of the zlg cdna coding sequence was obtained by pcr using primers designed to the predicted translation start site on the bac sequence.
the final assembly of the cdna was  <dig>  bp .
the total length of the mrna found by northern blot was just over  <dig> kb .
the additional length of the mrna is likely comprised of the 5'-utr of zlg.
the cdna aligned completely to the bac and the  <dig> consecutive exons of the zonadhesin-like gene utilized canonical splice sites.
the cdna has a predicted orf of  <dig>  bases that encodes a  <dig>  amino acid protein.
the predicted protein starts with a methionine and has a putative signal peptide of  <dig> amino acids.
a poly  signal of aataaa was identified by genscan  <cit>  at  <dig> bp from the start codon of the cdna,  <dig> bp downstream of the stop codon.
salmo salar zlg expression
semi-quantitative reverse transcription pcr was used to identify tissues expressing the zlg mrna .
liver, brain, kidney, spleen, foregut, midgut, hindgut and gonads were taken from one male and one female atlantic salmon.
male and female salmon showed expression in the liver, midgut and hindgut.
however, expression in the spleen only occurred in the male and expression in the foregut only occurred in the female.
a weak band was present in the ovarian sample and expression was not observed in the testes.
this gene does not appear to be expressed in either male or female brain or kidney.
northern blot results were similar to the rt-pcr results and showed a single band in male midgut and hindgut, and female midgut and foregut .
a weak band was seen in the northern blot analysis of liver from both male and female fish .
unlike the rt-pcr results, northern blot analysis did not detect a transcript in the male spleen and female hindgut.
this discrepancy could be due to the higher sensitivity of the pcr experiment.
the zlg expression pattern differed from the testes-specific expression known for mammalian zonadhesin.
to clarify the relationship between zan and zan-like genes, we looked for related genes in the genomes of other fish.
genomic analysis of the zebrafish zan locus
an initial inspection of the zv <dig>  whole genome shotgun  assembly of the d. rerio genome suggested there are two copies of zonadhesin that exist at two distinct loci.
one copy is found at linkage group  <dig>  and consists entirely of whole genome shotgun reads.
the second copy is found at linkage group  <dig>  and resides within a completely sequenced bac from the chori- <dig> library .
the zv <dig> assembly contained only one copy of zonadhesin that also assembles into linkage group  <dig>  it is important to note that the zv <dig> assembly consisted entirely of whole genome shotgun reads; the zv <dig> assembly incorporated finished bac sequence into the zv <dig> assembly; and that both assemblies are considered 'pre-assemblies' that need to be analyzed with caution.
the location of zonadhesin at linkage group  <dig> is supported by the fact that other genes with significant similarity to human chromosome 7q <dig> map to the same region of the d.rerio genome.
these genes include acetylcholine esterase, serpine, ap1s <dig>  unnamed product flj <dig>  unnamed protein product flj <dig> and mucin .
the regions surrounding the zan locus at both linkage groups were inspected for possible segmental duplications.
bac clones  that aligned to a  <dig> kb region surrounding the zan locus at linkage group  <dig>  or a  <dig> mb region at linkage group  <dig>  were obtained and aligned to both loci using the blastz algorithm through the multipipmaker web server.
our analysis showed that the zonadhesin-containing bac bx <dig> integrated completely into linkage group  <dig> and was ≅ 97% identical overall, not including indels.
seven additional bacs from the dkey library aligned to the scaffold and produced an acceptable tile through the entire zan locus .
in contrast, only a portion of bac bx <dig> and a portion of bx <dig>  aligned to the zan locus at linkage group  <dig>  this is suggestive of an assembly artifact that resulted in the assignment of a second zonadhesin gene to linkage group  <dig>
the differences between individual bacs, and between bacs and linkage group  <dig>  can be as high as 3%.
this amount of polymorphism is higher than the  <dig> % polymorphism rate expected from the whole genome shotgun sequence, which came from approximately  <dig>   <dig> day old embryos  <cit> .
despite this high rate of polymorphism, the existence of one zonadhesin locus at linkage group  <dig> is supported by the large tile of overlapping clones at linkage group  <dig>  and the rapidly evolving nature of zonadhesin genes  <cit> .
prediction of the zebrafish zan transcript and domain structure
we further analysed the zonadhesin gene found on the completely sequenced bac .
the  <dig>  amino acid translation product of the putative zan gene lacked a signal peptide, but contained the domain structure of: two mam domains; a mucin domain; nine vwd domains; and a transmembrane region.
this domain organization is typical of zonadhesin .
the entire zonadhesin gene was also contained within  <dig> unordered pieces from the clone dkey-3k <dig> .
from this clone we obtained a third putative zan transcript that was most similar to the complete zan sequence from bac bx <dig>  the predicted cdna from cr <dig>  differs from the cdna of bx <dig> by  <dig> %.
the putative zan protein encoded by bac cr <dig>  has a domain structure the same as the one encoded by the putative zan transcript of bac bx <dig>
zebrafish zonadhesin has expanded its vwd domains through recent tandem duplication.
the vwd domains  <dig>   <dig> and a fragment of  <dig> are encoded in  <dig> exons .
these exons have identical sizes to the exons  encoding the first four vwd domains.
furthermore, each group of  <dig> exons are symmetrical and flanked by phase '1' introns, which is evidence for recent domain expansion.
pair-wise alignment of bac bx <dig> against itself revealed these exons are found in three ≈  <dig> kb blocks that are 85–87% identical.
the zan locus at linkage group  <dig> only contains two of these ≈  <dig> kb repeats.
it is possible the third repeat was collapsed in the whole genome shotgun assembly process or the presence of two repetitive blocks is a true population variant.
danio rerio zonadhesin expression
to compare the expression pattern and verify the transcript size of the predicted zebrafish zan we extracted gut and testes rna and performed rt-pcr and northern blot analysis.
three different pcr primer sets were designed against three regions of the predicted zan cdna.
the first primer set was designed to amplify the exons encoding the mam domains through to the first vwd domain.
this primer set produced a doublet in both male and female  .
the female bands were approximately  <dig> bp larger than the male bands.
the differences between individuals may be due to a variation in mucin domain length since mucin domains have been shown to be variable in other genes  <cit> .
the second primer set flanked the ninth vwd domain and the third stretched from the epidermal growth factor domain to the cytoplasmic domain.
each primer set identified a zonadhesin transcript in the gut, but not in the testes .
these results were corroborated by northern blot analysis that found a single transcript of ≅  <dig> kb in the gut but not in the testes .
this ≅  <dig> kb transcript correlates to the mrna length predicted from the genomic sequence and supports the existence of a single zonadhesin gene in zebrafish.
genomic prediction t. rubripes zan cdna transcript and domain structure
analysis of the puffer fish genome assembly release  <dig>  revealed a putative zonadhesin gene in the same contig  as the ache gene.
this syntenic relationship was also found at the mammalian and zebrafish zan loci .
however, scaffold  <dig> has been split in the current fugu genome project release  leaving zonadhesin in a gap-free region of scaffold  <dig>  without ache.
puffer fish zonadhesin was predicted to contain  <dig> exons that coded for a protein of  <dig>  amino acids.
the predicted zonadhesin protein contained two mam domains at the n-terminus, a mucin domain, five vwd domains, an egf domain, a transmembrane domain and a short cytoplasmic domain .
this protein has the same domains in the same order as human zonadhesin.
no signal peptide was identified in the puffer fish zonadhesin.
however, this sequence may be incomplete since it was found at the end of the scaffold sequence.
expression of t. rubripes zonadhesin
evidence from the genbank database suggests that puffer fish zonadhesin is also expressed in the gut.
sequences can be found that have been isolated from gut-specific libraries , but there have been no zonadhesin sequences found with testes-specific expression.
structural similarity of d. rerio and t. rubripes to mammalian zan genes
although fish and mammals have not shared a common ancestor for an estimated  <dig> million years  <cit> , the domain structure of zonadhesin has been highly conserved .
however, domain numbers between species are variable and this variability appears to have been influenced by tandem duplication.
tandem duplications have occurred in both mammalian and fish species and are most prevalent in the vwd domain region .
it is this region which, at least in mammals, has been shown to be important for zona pellucida binding  <cit> .
while multiple vwd domains are found in all characterized zonadhesins, recent expansion of this domain is seen in the mouse and in the zebrafish.
repeated tandem duplication in the mouse zonadhesin gene resulted in  <dig> copies of a two-exon segment encoding a partial vwd <dig> domain that increased the length of the protein by over  <dig> amino acids  <cit> .
in zebrafish, the double duplication of two domains homologous to the puffer fish vwd <dig> and vwd <dig>  as well as a portion of the vwd <dig> domain , resulted in an additional  <dig> exons that encoded  <dig> additional full vwd domains and two partial vwd domains in the zebrafish zan .
the ancestral zonadhesin likely looked similar to the puffer fish zan as it is very similar in length and domain structure to most mammalian zonadhesins .
the puffer fish gene also has slightly higher identity with the human zonadhesin gene at 52%  compared with the 50% identity between zebrafish and human whereas the zebrafish and puffer fish genes are 62% identical.
the five vwd domains of the puffer fish also cluster with the four in human .
the last four vwd domains of puffer fish zan seem to be homologous to the last four human domains; although, the first vwd domain of puffer fish also has high similarity to the second domain in human.
this may be the result of an ancient duplication of the first two vwd domains in puffer fish and subsequent loss of the new first domain.
this inheritance is similar to that of the mam domains.
puffer fish zonadhesin has two mam domains, a structure matching the rabbit and pig proteins  <cit> .
neighbor-joining tree analysis of individual domains reveals that the mam <dig> domain of puffer fish is most similar to the mam <dig> and mam <dig> domains of human, while the mam <dig> domain of puffer fish groups with mam <dig> of human .
this pattern of similarity could be explained by a duplication of both mam domains in the human lineage and subsequent loss of the fourth domain.
this phylogeny, in combination with the conserved domain order and synteny between fish and mammal zonadhesin loci, supports the orthologous relationship of these genes.
genomic and phylogenetic analysis of genes with a domain content similar to zonadhesin
until this study, zonadhesin was generally thought to be unique in its domain content as no other genes were reported to contain mam, mucin, til and vwd domains.
however, the characterization of the salmon zlg revealed all of these domains, but in a different order .
the expression of zlg is similar to zebrafish zonadhesin which, in addition to domain content, established a possible evolutionary link between these genes.
examination of genbank sequences also revealed that zonadhesin-like genes have been found in gut tissues of other species; however, the automated annotation is based on the similarity of the til and vwd domains of fcgbp.
for example, there are three human colon ests  that are annotated as similar to zonadhesin; however, these genes align perfectly to the fcgbp gene at chromosome 19q <dig> .
similarly, the only mouse colon est that is annotated as similar to zonadhesin aligns to the fcgbp gene in the orthologous region at mouse chromosome  <dig>
we looked for zonadhesin-like genes in puffer fish, zebrafish and chicken  genome projects.
this search uncovered several regions with vwd-containing proteins without any detectable mam domains, some of these possibly related to fcgbp proteins.
one interesting exception was a zonadhesin-like gene found on zebrafish chromosome  <dig>  this putative zebrafish zlg encodes a  <dig>  amino acid protein containing three mam domains, the first of which is flanked by short  mucin-like low complexity regions.
the mam domains are followed by two vwd domains.
this gene structure is reminiscent of s. salar zlg and shows that a zonadhesin ortholog and a zan-like gene exist together in the zebrafish genome .
the search for zonadhesin-like genes in chicken  did not reveal an obvious zonadhesin ortholog but rather a prediction of a fcgbp-like protein residing on chromosome  <dig> .
we analysed the corresponding region of the g. gallus genome and utilized additional est evidence and in silico predictions to obtain a putative transcript that encodes a  <dig>  amino acid gene product.
this gene product contains three mam domains in addition to vwd and mucin-type o-glycosylation sites after each of the mam domains.
overall, this mam-mucin-til-vwd series of domains is reminiscent of all zonadhesins and is evidence for a common evolutionary origin of zonadhesin, the zonadhesin-like genes and the fc fragment of igg binding protein.
we extracted the mam and vwd domains of representative zonadhesin and zonadhesin-like genes and performed a phylogenetic analysis .
in addition to the clustering of fish and mammalian zonadhesin, both phylogenetic trees suggest an evolutionary relationship among the zonadhesin-like genes.
in particular, the zebrafish zlg mam <dig> and mam <dig> domains grouped with the salmon zlg mams as well as two of the chicken fcgbp-like mam domains .
the grouping of the mam domains of these proteins indicates that the zonadhesin-like genes represent a novel gene family that is distinct from zonadhesin.
although the phylogeny is more complex with many nodes not well supported, the evolutionary relationship between the fish zlgs and the chicken fcgbp-like gene was also observed for the vwd domain .
for example, the vwd <dig> domains of the fish zlgs clustered with the vwd <dig> and vwd <dig> domains of the chicken fcgbp-like gene.
a second clade consisting of the vwd <dig> domains of salmon and zebrafish zlg, and the vwd <dig> and vwd <dig> domain of the chicken fcgbp-like gene also formed.
the clade containing puffer fish zan vwd <dig>  human zan vwd <dig> and salmon zlg vwd <dig> also suggests that zonadhesin is closely related to the zonadhesin-like genes.
the human fcgbp vwd domains all formed a single clade except for vwd <dig> which grouped with the chicken fcgbp-like vwd <dig> and  <dig> .
overall, the phylogenies of the vwd and mam domains combined with the expression patterns of: fish zonadhesins, fish and chicken zonadhesin-like genes, fcgbp, mucin, and several mam containing proteins, suggest that these mosaic genes share a common ancestor.
zonadhesin in the context of other sperm-egg interacting proteins
many mammalian zona pellucida adhesion molecule candidates appear to have evolved from different physiological processes.
well known examples of sperm proteins with enzymatic function that have been 'hijacked' into playing non-enzymatic roles in sperm-egg interactions include: b4galt1/galtase , spam1/ph- <dig> , hk1/zrk/p <dig> , and arsa/slip <dig> .
evidence for immune-system hijacking events comes from the discovery of several complement system proteins in human spermatozoa, seminal plasma and follicular fluid .
the partial activation of the complement system  in acrosome-reacted spermatozoa suggests how components of a conserved immune system pathway could play a new role in sperm-egg recognition  <cit> .
although the function of the fish zonadhesin and zonadhesin-like genes is not known, their expression in the gut and absence of expression in the testes, combined with their homology to gut-expressed genes of the mucosal immune system , suggest that zonadhesin was also 'hijacked' by the mammalian reproductive system.
CONCLUSIONS
we identified zonadhesin genes in zebrafish and puffer fish that are similar in domain order and content to all known mammalian orthologs.
unlike all mammalian zonadhesin genes studied to date, zebrafish zan was expressed in the gut but not in the testes.
in addition to these orthologs, we characterized zonadhesin-like genes  in atlantic salmon, zebrafish and chicken.
while the atlantic salmon zlg contained the same domains found in zonadhesin, the order of these domains was altered and the expression was found predominantly in the gut and not in the testes.
overall, this suggests that zonadhesin's reproductive role evolved later in the mammalian lineage.

 molecules that are directly involved in reproduction are often subject to rapid evolutionary change. zonadhesin is one such molecule that has undergone domain expansion and positive selection in mammals. zan is a multi-domain sperm protein that is implicated in the species-specific binding of egg and sperm. porcine zan was first described by hardy et al. as a protein expressed by developing sperm that would bind to the zona pellucida of the egg. since its initial discovery, zonadhesin has been identified in several other mammals, including mouse, human and rabbit. recent data suggest the processed zonadhesin localizes to the acrosomal matrix and binds the zona pellucida during the acrosome reaction. the discrete domains of mosaic proteins are known to be important in the evolution of new genes. domain subunits can be rearranged, duplicated or deleted to produce a variety of proteins with different functions. zonadhesin structure is unique in its combination of protein domains. all mammalian zonadhesin genes are predicted to encode: a signal peptide, a multiple meprin a antigen receptor tyrosine phosphatase mu domain, multiple trypsin-like inhibitor domains, multiple von willebrand d cell adhesion domains, multiple hepta-peptide repeats that form the mucin domain, multiple epidermal growth factor domains, a single transmembrane domain and short intracellular domain at the carboxyl terminus. the domain order is the same for all mammals studied, with the main difference being the number of mam and vwd domains. these individual domains each have a particular function and are found in many other mosaic proteins. the extracellular vwd domain occurs in a family of immediate-early genes that are growth regulators and is thought to have an adhesive function. this modular domain is found in a variety of mosaic proteins including von willebrand factor, apolipoprotein b, vitellogenins, microsomal triglyceride transfer protein, and mucins. biochemical studies of pig zonadhesin have shown that the zan precursor is processed and the mam domains are removed leaving the vwd domain to interact with the zona pellucida. while the role of the vwd in sperm-egg binding has been addressed, the role of the mucin-repeats and the mam domains is still unknown. the mucin or muc domain is the primary functional domain of mucin proteins. mucins are a diverse group of heavily glycosylated proteins that are the major component of mucus. mucins function to lubricate surfaces and are the first line of defence against pathogens. most of the secreted mucins contain a domain with sequence similarity to vwd and a domain composed of a variable number of tandem repeats that code for serine-, threonine- and proline-rich repeat peptides that are potential glycosylation sites. two mucin-like genes that are similar to zonadhesin include alpha-tectorin, which is involved in non-syndromical autosomal dominant hearing impairment, and fc fragment of igg binding protein. by virtue of sequence identity, the closest relative to mammalian zonadhesin is fcgbp and this similarity is mostly seen in the til and vwd domains. fcgbp is expressed in the mucosa of the small and large intestines, epithelial colon cells and the placenta and is thought to play a role in the mucosal immune system through the promotion of multivalent igg and the trapping of antigen igg complexes in the mucosa. the amino acid mam domain distinguishes zonadhesin from other vwd and mucin repeat containing genes such as fcgbp. mam domains have adhesive function and are found in several proteins including protein-tyrosine phosphatases, neuropilins and meprins. meprins are metalloendopeptidases that have been found in the intestinal brush border and renal membranes of mammals. while their role in the zonadhesin protein is unknown, mam domains are important for multimerization and have conserved cysteine residues that are responsible for covalent interactions. when this study began, zonadhesin expression had only been described in the testes. only zonadhesin was thought to encode mam domains, mucin repeats, and vwd domains and no non-mammalian zonadhesin orthologs had been reported. for these reasons we were interested in atlantic salmon zonadhesin-like ests from a gut-derived library that encoded the mam, mucin and vwd domains. here we describe the cdna, genomic structure, and expression patterns of this atlantic salmon zonadhesin-like gene. we also use comparative genomic and expression analyses to uncover additional zonadhesin-like genes, as well as orthologs of zonadhesin, in zebrafish and puffer fish.  characterization of a zonadhesin-like gene in s. salar a gene similar to mammalian zonadhesin was identified during an expressed sequence tag analysis of atlantic salmon. we assembled this zonadhesin-like gene from five overlapping ests; the largest of which was sequenced by primer walking from both directions and assembled to give a bp sequence. pcr primers were designed to the 3'-end of the est and used to amplify a probe for hybridization to atlantic salmon genomic dna and bacterial artificial chromosome library filters. probing of bac clones on five atlantic salmon genome bac library filters resulted in one positive bac. this bac was subcloned, sequenced, and assembled into a base pair contiguous sequence. the assembly had more than high quality sequence reads and fold sequence coverage in most regions. one gap of bp was filled by pcr followed by sequencing from both directions. an in silico restriction digest matched experimental digests. the zonadhesin-like gene was the only gene found on this bac. all other open reading frames were associated with line, sine and transposon related repetitive elements. comparison of 722p against itself using dotter and pipmaker did not reveal any recent domain expansions or duplications. the simple modular architecture research tool was used to identify conserved domains of the predicted protein. the smart algorithm was used to detect three vwd domains, a vwc domain at, and two mam domains. this tool also located three low complexity regions that corresponded to the mucin domains predicted by the computer program netoglyc. these mucin domains occurred at positions 1091– and 1115– with a smaller low complexity region located between nucleotides and these results were corroborated using the interpro domain prediction computer program. the smart and interpro domain prediction tools, in agreement with kyte-doolittle hydropathy data, did not find any transmembrane domains in the salmon zlg. in contrast, the smart tool detected a transmembrane domain at the expected location for zonadhesins from other species. using two distinct probes, only one copy of zlg was found in atlantic salmon by southern blot analysis. however, two bands occurred when the same probes were used with rainbow trout genomic dna. probe which contained sequence homologous to the 3'-utr of zlg, would be expected to be specific for this gene; however, probe spanned vwd and could be expected to hybridize with related zonadhesin-like genes. both probes gave similar results and only probe data is shown in figure 1b. southern blot analysis did not reveal any other genes in atlantic salmon other than zlg. this suggests that if another zonadhesin-like gene does exist in atlantic salmon it is likely quite divergent from zlg. the 5'-end of the zlg cdna coding sequence was obtained by pcr using primers designed to the predicted translation start site on the bac sequence. the final assembly of the cdna was bp. the total length of the mrna found by northern blot was just over kb. the additional length of the mrna is likely comprised of the 5'-utr of zlg. the cdna aligned completely to the bac and the consecutive exons of the zonadhesin-like gene utilized canonical splice sites. the cdna has a predicted orf of bases that encodes a amino acid protein. the predicted protein starts with a methionine and has a putative signal peptide of amino acids. a poly signal of aataaa was identified by genscan at bp from the start codon of the cdna, bp downstream of the stop codon. salmo salar zlg expression semi-quantitative reverse transcription pcr was used to identify tissues expressing the zlg mrna. liver, brain, kidney, spleen, foregut, midgut, hindgut and gonads were taken from one male and one female atlantic salmon. male and female salmon showed expression in the liver, midgut and hindgut. however, expression in the spleen only occurred in the male and expression in the foregut only occurred in the female. a weak band was present in the ovarian sample and expression was not observed in the testes. this gene does not appear to be expressed in either male or female brain or kidney. northern blot results were similar to the rt-pcr results and showed a single band in male midgut and hindgut, and female midgut and foregut. a weak band was seen in the northern blot analysis of liver from both male and female fish. unlike the rt-pcr results, northern blot analysis did not detect a transcript in the male spleen and female hindgut. this discrepancy could be due to the higher sensitivity of the pcr experiment. the zlg expression pattern differed from the testes-specific expression known for mammalian zonadhesin. to clarify the relationship between zan and zan-like genes, we looked for related genes in the genomes of other fish. genomic analysis of the zebrafish zan locus an initial inspection of the zv whole genome shotgun assembly of the d. rerio genome suggested there are two copies of zonadhesin that exist at two distinct loci. one copy is found at linkage group and consists entirely of whole genome shotgun reads. the second copy is found at linkage group and resides within a completely sequenced bac from the chori- library. the zv assembly contained only one copy of zonadhesin that also assembles into linkage group it is important to note that the zv assembly consisted entirely of whole genome shotgun reads; the zv assembly incorporated finished bac sequence into the zv assembly; and that both assemblies are considered'pre-assemblies' that need to be analyzed with caution. the location of zonadhesin at linkage group is supported by the fact that other genes with significant similarity to human chromosome 7q map to the same region of the d.rerio genome. these genes include acetylcholine esterase, serpine, ap1s unnamed product flj unnamed protein product flj and mucin. the regions surrounding the zan locus at both linkage groups were inspected for possible segmental duplications. bac clones that aligned to a kb region surrounding the zan locus at linkage group or a mb region at linkage group were obtained and aligned to both loci using the blastz algorithm through the multipipmaker web server. our analysis showed that the zonadhesin-containing bac bx integrated completely into linkage group and was ≅ 97% identical overall, not including indels. seven additional bacs from the dkey library aligned to the scaffold and produced an acceptable tile through the entire zan locus. in contrast, only a portion of bac bx and a portion of bx aligned to the zan locus at linkage group this is suggestive of an assembly artifact that resulted in the assignment of a second zonadhesin gene to linkage group the differences between individual bacs, and between bacs and linkage group can be as high as 3%. this amount of polymorphism is higher than the % polymorphism rate expected from the whole genome shotgun sequence, which came from approximately day old embryos. despite this high rate of polymorphism, the existence of one zonadhesin locus at linkage group is supported by the large tile of overlapping clones at linkage group and the rapidly evolving nature of zonadhesin genes. prediction of the zebrafish zan transcript and domain structure we further analysed the zonadhesin gene found on the completely sequenced bac. the amino acid translation product of the putative zan gene lacked a signal peptide, but contained the domain structure of: two mam domains; a mucin domain; nine vwd domains; and a transmembrane region. this domain organization is typical of zonadhesin. the entire zonadhesin gene was also contained within unordered pieces from the clone dkey-3k. from this clone we obtained a third putative zan transcript that was most similar to the complete zan sequence from bac bx the predicted cdna from cr differs from the cdna of bx by %. the putative zan protein encoded by bac cr has a domain structure the same as the one encoded by the putative zan transcript of bac bx zebrafish zonadhesin has expanded its vwd domains through recent tandem duplication. the vwd domains and a fragment of are encoded in exons. these exons have identical sizes to the exons encoding the first four vwd domains. furthermore, each group of exons are symmetrical and flanked by phase'1' introns, which is evidence for recent domain expansion. pair-wise alignment of bac bx against itself revealed these exons are found in three ≈ kb blocks that are 85–87% identical. the zan locus at linkage group only contains two of these ≈ kb repeats. it is possible the third repeat was collapsed in the whole genome shotgun assembly process or the presence of two repetitive blocks is a true population variant. danio rerio zonadhesin expression to compare the expression pattern and verify the transcript size of the predicted zebrafish zan we extracted gut and testes rna and performed rt-pcr and northern blot analysis. three different pcr primer sets were designed against three regions of the predicted zan cdna. the first primer set was designed to amplify the exons encoding the mam domains through to the first vwd domain. this primer set produced a doublet in both male and female. the female bands were approximately bp larger than the male bands. the differences between individuals may be due to a variation in mucin domain length since mucin domains have been shown to be variable in other genes. the second primer set flanked the ninth vwd domain and the third stretched from the epidermal growth factor domain to the cytoplasmic domain. each primer set identified a zonadhesin transcript in the gut, but not in the testes. these results were corroborated by northern blot analysis that found a single transcript of ≅ kb in the gut but not in the testes. this ≅ kb transcript correlates to the mrna length predicted from the genomic sequence and supports the existence of a single zonadhesin gene in zebrafish. genomic prediction t. rubripes zan cdna transcript and domain structure analysis of the puffer fish genome assembly release revealed a putative zonadhesin gene in the same contig as the ache gene. this syntenic relationship was also found at the mammalian and zebrafish zan loci. however, scaffold has been split in the current fugu genome project release leaving zonadhesin in a gap-free region of scaffold without ache. puffer fish zonadhesin was predicted to contain exons that coded for a protein of amino acids. the predicted zonadhesin protein contained two mam domains at the n-terminus, a mucin domain, five vwd domains, an egf domain, a transmembrane domain and a short cytoplasmic domain. this protein has the same domains in the same order as human zonadhesin. no signal peptide was identified in the puffer fish zonadhesin. however, this sequence may be incomplete since it was found at the end of the scaffold sequence. expression of t. rubripes zonadhesin evidence from the genbank database suggests that puffer fish zonadhesin is also expressed in the gut. sequences can be found that have been isolated from gut-specific libraries, but there have been no zonadhesin sequences found with testes-specific expression. structural similarity of d. rerio and t. rubripes to mammalian zan genes although fish and mammals have not shared a common ancestor for an estimated million years, the domain structure of zonadhesin has been highly conserved. however, domain numbers between species are variable and this variability appears to have been influenced by tandem duplication. tandem duplications have occurred in both mammalian and fish species and are most prevalent in the vwd domain region. it is this region which, at least in mammals, has been shown to be important for zona pellucida binding. while multiple vwd domains are found in all characterized zonadhesins, recent expansion of this domain is seen in the mouse and in the zebrafish. repeated tandem duplication in the mouse zonadhesin gene resulted in copies of a two-exon segment encoding a partial vwd domain that increased the length of the protein by over amino acids. in zebrafish, the double duplication of two domains homologous to the puffer fish vwd and vwd as well as a portion of the vwd domain, resulted in an additional exons that encoded additional full vwd domains and two partial vwd domains in the zebrafish zan. the ancestral zonadhesin likely looked similar to the puffer fish zan as it is very similar in length and domain structure to most mammalian zonadhesins. the puffer fish gene also has slightly higher identity with the human zonadhesin gene at 52% compared with the 50% identity between zebrafish and human whereas the zebrafish and puffer fish genes are 62% identical. the five vwd domains of the puffer fish also cluster with the four in human. the last four vwd domains of puffer fish zan seem to be homologous to the last four human domains; although, the first vwd domain of puffer fish also has high similarity to the second domain in human. this may be the result of an ancient duplication of the first two vwd domains in puffer fish and subsequent loss of the new first domain. this inheritance is similar to that of the mam domains. puffer fish zonadhesin has two mam domains, a structure matching the rabbit and pig proteins. neighbor-joining tree analysis of individual domains reveals that the mam domain of puffer fish is most similar to the mam and mam domains of human, while the mam domain of puffer fish groups with mam of human. this pattern of similarity could be explained by a duplication of both mam domains in the human lineage and subsequent loss of the fourth domain. this phylogeny, in combination with the conserved domain order and synteny between fish and mammal zonadhesin loci, supports the orthologous relationship of these genes. genomic and phylogenetic analysis of genes with a domain content similar to zonadhesin until this study, zonadhesin was generally thought to be unique in its domain content as no other genes were reported to contain mam, mucin, til and vwd domains. however, the characterization of the salmon zlg revealed all of these domains, but in a different order. the expression of zlg is similar to zebrafish zonadhesin which, in addition to domain content, established a possible evolutionary link between these genes. examination of genbank sequences also revealed that zonadhesin-like genes have been found in gut tissues of other species; however, the automated annotation is based on the similarity of the til and vwd domains of fcgbp. for example, there are three human colon ests that are annotated as similar to zonadhesin; however, these genes align perfectly to the fcgbp gene at chromosome 19q. similarly, the only mouse colon est that is annotated as similar to zonadhesin aligns to the fcgbp gene in the orthologous region at mouse chromosome we looked for zonadhesin-like genes in puffer fish, zebrafish and chicken genome projects. this search uncovered several regions with vwd-containing proteins without any detectable mam domains, some of these possibly related to fcgbp proteins. one interesting exception was a zonadhesin-like gene found on zebrafish chromosome this putative zebrafish zlg encodes a amino acid protein containing three mam domains, the first of which is flanked by short mucin-like low complexity regions. the mam domains are followed by two vwd domains. this gene structure is reminiscent of s. salar zlg and shows that a zonadhesin ortholog and a zan-like gene exist together in the zebrafish genome. the search for zonadhesin-like genes in chicken did not reveal an obvious zonadhesin ortholog but rather a prediction of a fcgbp-like protein residing on chromosome. we analysed the corresponding region of the g. gallus genome and utilized additional est evidence and in silico predictions to obtain a putative transcript that encodes a amino acid gene product. this gene product contains three mam domains in addition to vwd and mucin-type o-glycosylation sites after each of the mam domains. overall, this mam-mucin-til-vwd series of domains is reminiscent of all zonadhesins and is evidence for a common evolutionary origin of zonadhesin, the zonadhesin-like genes and the fc fragment of igg binding protein. we extracted the mam and vwd domains of representative zonadhesin and zonadhesin-like genes and performed a phylogenetic analysis. in addition to the clustering of fish and mammalian zonadhesin, both phylogenetic trees suggest an evolutionary relationship among the zonadhesin-like genes. in particular, the zebrafish zlg mam and mam domains grouped with the salmon zlg mams as well as two of the chicken fcgbp-like mam domains. the grouping of the mam domains of these proteins indicates that the zonadhesin-like genes represent a novel gene family that is distinct from zonadhesin. although the phylogeny is more complex with many nodes not well supported, the evolutionary relationship between the fish zlgs and the chicken fcgbp-like gene was also observed for the vwd domain. for example, the vwd domains of the fish zlgs clustered with the vwd and vwd domains of the chicken fcgbp-like gene. a second clade consisting of the vwd domains of salmon and zebrafish zlg, and the vwd and vwd domain of the chicken fcgbp-like gene also formed. the clade containing puffer fish zan vwd human zan vwd and salmon zlg vwd also suggests that zonadhesin is closely related to the zonadhesin-like genes. the human fcgbp vwd domains all formed a single clade except for vwd which grouped with the chicken fcgbp-like vwd and. overall, the phylogenies of the vwd and mam domains combined with the expression patterns of: fish zonadhesins, fish and chicken zonadhesin-like genes, fcgbp, mucin, and several mam containing proteins, suggest that these mosaic genes share a common ancestor. zonadhesin in the context of other sperm-egg interacting proteins many mammalian zona pellucida adhesion molecule candidates appear to have evolved from different physiological processes. well known examples of sperm proteins with enzymatic function that have been'hijacked' into playing non-enzymatic roles in sperm-egg interactions include: b4galt1/galtase, spam1/ph-, hk1/zrk/p, and arsa/slip. evidence for immune-system hijacking events comes from the discovery of several complement system proteins in human spermatozoa, seminal plasma and follicular fluid. the partial activation of the complement system in acrosome-reacted spermatozoa suggests how components of a conserved immune system pathway could play a new role in sperm-egg recognition. although the function of the fish zonadhesin and zonadhesin-like genes is not known, their expression in the gut and absence of expression in the testes, combined with their homology to gut-expressed genes of the mucosal immune system, suggest that zonadhesin was also'hijacked' by the mammalian reproductive system.  we identified zonadhesin genes in zebrafish and puffer fish that are similar in domain order and content to all known mammalian orthologs. unlike all mammalian zonadhesin genes studied to date, zebrafish zan was expressed in the gut but not in the testes. in addition to these orthologs, we characterized zonadhesin-like genes in atlantic salmon, zebrafish and chicken. while the atlantic salmon zlg contained the same domains found in zonadhesin, the order of these domains was altered and the expression was found predominantly in the gut and not in the testes. overall, this suggests that zonadhesin's reproductive role evolved later in the mammalian lineage. 
we characterized likely orthologs of zonadhesin in both t. rubripes and d. rerio and uncovered zonadhesin-like genes in s. salar and d. rerio. the expression patterns of fish zonadhesin and zonadhesin-like genes suggest that the reproductive role of zonadhesin evolved later in the mammalian lineage. analysis of puffer fish and zebrafish sequence data identified zonadhesin genes that share the same domain order, content, and a conserved syntenic relationship with mammalian zonadhesin. the genomic structure and testes-specific expression of zonadhesin is known for many mammalian species. zlg is similar in adhesion domain content to mammalian zonadhesin; however, the domain order is altered. here we investigate the genomic structure and expression of zonadhesin-like genes in three species of fish. the cdna and corresponding genomic locus of a zonadhesin-like gene in atlantic salmon were sequenced. unlike mammalian zonadhesin, d. rerio zan and s. salar zlg were expressed in the gut and not in the testes. all zonadhesin genes characterized to date consist of meprin a antigen receptor tyrosine phosphatase mu domains, mucin tandem repeats, and von willebrand adhesion domains. each of these genes contains mam, mucin, and vwd domains. a zonadhesin-like gene in d. rerio was also identified. while these domains are associated with several proteins that show prominent gut expression, their combination is unique to zonadhesin and zonadhesin-like genes in vertebrates.
 the mosaic sperm protein zonadhesin has been characterized in mammals and is implicated in species-specific egg-sperm binding interactions. the genomic structure and testes-specific expression of zonadhesin is known for many mammalian species. all zonadhesin genes characterized to date consist of meprin a antigen receptor tyrosine phosphatase mu domains, mucin tandem repeats, and von willebrand adhesion domains. here we investigate the genomic structure and expression of zonadhesin-like genes in three species of fish.  the cdna and corresponding genomic locus of a zonadhesin-like gene in atlantic salmon were sequenced. zlg is similar in adhesion domain content to mammalian zonadhesin; however, the domain order is altered. analysis of puffer fish and zebrafish sequence data identified zonadhesin genes that share the same domain order, content, and a conserved syntenic relationship with mammalian zonadhesin. a zonadhesin-like gene in d. rerio was also identified. unlike mammalian zonadhesin, d. rerio zan and s. salar zlg were expressed in the gut and not in the testes.  we characterized likely orthologs of zonadhesin in both t. rubripes and d. rerio and uncovered zonadhesin-like genes in s. salar and d. rerio. each of these genes contains mam, mucin, and vwd domains. while these domains are associated with several proteins that show prominent gut expression, their combination is unique to zonadhesin and zonadhesin-like genes in vertebrates. the expression patterns of fish zonadhesin and zonadhesin-like genes suggest that the reproductive role of zonadhesin evolved later in the mammalian lineage. 