BACKGROUND
since the discovery of sry  <cit> , numerous genes encoding proteins containing hmg  domain have been identified throughout the animal kingdom  <cit> .
genes with their hmg domains similar to that of sry  by at least 50% were named as sox  genes  <cit> .
at present, sox genes encoding transcription factors, count about  <dig> members in vertebrates  <cit> .
based on differing levels of structural, organizational similarities and other features, sox genes can be subdivided into  <dig> groups, named from a to j  <cit> .
members of the same group are similar to each other within and outside the hmg-box.
however, members from different groups share lower degree of identity in the hmg-box, and no significant identity outside this domain.
many of the sox proteins, like sry, sox <dig>  sox <dig>  sox <dig> and sox <dig>  are encoded by a single exon  <cit> .
in contrast, others, such as sox <dig>  sox <dig>  sox <dig> and sox <dig>  consist of multiple exons  <cit> .
in addition, the sox genes are scattered on different chromosomes and are not clustered  <cit> .
apart from the sry and sox <dig> located on the y- and x-chromosome respectively, other sox genes are autosomal  <cit> .
sox <dig> , the only member of group h, has been characterized merely in a few species so far.
it was firstly isolated from mouse  and human .
in these species, two transcripts of sox <dig> mrna were found, one of which encodes a truncated protein lacking partial c-terminal region due to a frame shift  <cit> .
sox <dig> was considered to be involved in mammalian spermatogonial differentiation and spermatogenesis  <cit> .
as no sequences of sox <dig> were available from any other groups of animals, including the sequenced fish genomes at that time, some scholars even speculated that sox <dig> was specific to mammals  <cit> .
in zebrafish , a sox <dig> gene has been reported  <cit> , however, it was proved to be sox <dig> by genbank blast and phylogenetic analysis.
interestingly, in our present study, sox <dig> was isolated from the nile tilapia  accidentally when cloning sox9b.
subsequently, by blast search and data mining, sox <dig> was also found to exist in genome sequences of the chicken , green anole lizard , western clawed frog , sea squirts , lancelets , acorn worm , sea anemone , and ests  of the channel catfish , guppy , fathead minnow , little skate , dogfish , the california mussel  and snail .
thus we report here that sox <dig> exists widely throughout the animal kingdom rather than specific to mammals as reported previously.
more importantly, we isolated four alternatively spliced isoforms of sox <dig>  and studied their temporal and spatial expression patterns during normal and sex reversed gonadal development in tilapia.
our data suggest that sox <dig> may play key roles in gonadal development, and the roles may be different at different stages and sexes.
methods
animals
the nile tilapias for this study were reared in large tanks with a circulating aerated freshwater system.
fish were maintained at ambient temperature  under natural photoperiod.
all genetic females  and males  were obtained by artificial fertilization of eggs from normal females  with sperms from either sex reversed males  or super males , respectively  <cit> .
then, artificially fertilized eggs were cultured in recirculating water at 26°c to obtain fry.
all animal experiments conformed to the guide for care and use of laboratory animals and were approved by the institutional committee of laboratory animal experimentation at southwest university, china.
drug treatment
xx males and xy females were generated by treating fry with estrogen receptor antagonist tamoxifen   and 17β-estradiol  , respectively.
fish feed were sprayed with 95% ethanol containing tam,  <dig> μg/g; e <dig>   <dig> μg/g.
control fish were fed with 95% ethanol sprayed feed.
drug treatment was applied to the fry from  <dig> to  <dig> days after hatching , the critical period of the nile tilapia sex differentiation  <cit> .
e <dig>  and tam  treatment resulted in complete sex reversal in tilapia, respectively  <cit> .
cloning of tilapia sox <dig> cdna and genomic dna
the smart race cdna of the gonads, which was used to perform both 5' and 3'-rapid implication of cdna ends  to obtain full-length cdna of tilapia sox <dig>  were synthesized according to the manufacturer's instruction .
the sox <dig> 5' cdna end was amplified accidentally with primers for sox9b , and confirmed by blastx http://blast.ncbi.nlm.nih.gov/.
subsequently, 3' race primers were designed to amplify 3' cdna end for tilapia sox <dig>  finally, to verify the full-length sequences of sox <dig> cdna, end-to-end pcr was performed and four mrna isoforms were identified by using 5'- and 3'-utr primers .
in order to determine the gene structure of the tilapia sox <dig>  the genomic dna sequence of this gene was amplified.
genomic dna was prepared from mature testis and ovary.
a  <dig> bp sox <dig> gene fragment with complete cds  and four introns,  <dig>   <dig>   <dig> and  <dig> bp in length, was obtained by genomic pcr using end primers t30utr-f and t30utr-r. all the primers and sequences used in the present study were listed in table  <dig>  the pcr products were resolved on a  <dig> % agarose gel and the target dna fragments were purified using qiaquick gel extraction kit .
all the fragments obtained were cloned into pgem-t vector  and bi-directional sequencing was performed by the dideoxy chain termination method using an abi prism  <dig> dna genetic analyzer .
data mining and sequence analyses
the following genome databases were searched to explore the presence of sox30-like sequences in other vertebrate and invertebrate species, including the ucsc genome bioinformatics at: http://genome.ucsc.edu/, the ncbi databases at http://blast.ncbi.nlm.nih.gov/, the silk db databases at http://silkworm.genomics.org.cn/, the jgi databases at http://genome.jgi-psf.org/nemve1/nemve <dig> home.html, the ensembl databases http://www.ensembl.org/biomart/martview/ and the gene db databases at http://www.genedb.org/genedb/smansoni/.
sox30-like sequences were identified from seven species by blast search against genome sequences and further confirmed in est databases, i.e., chicken , anole lizard , frog , sea squirts , lancelets , acorn worm  and sea anemones , but failed to identify it from other  <dig> animal species with genome sequence information.
partial or complete sox30-like sequences were also identified by blast search against the ncbi est databases from channel catfish , guppy , fathead minnow , little skate , dogfish , the california mussel  and snail .
the orientation and chromosomal position of sox <dig> and its adjacent genes thg1l  and adam <dig>  were determined manually from the gene orientations listed in biomart from the ensembl database.
synteny analyses of sox <dig>  thg1l and adam <dig> in human, mouse, chicken, anole lizard and frog were performed by comparing the cdna sequences with genome sequence using blast search from the ucsc genome bioinformatics or by search in biomart from the ensembl database.
the multiple alignment software dnastar and clustalx were employed to analyze the nucleotide sequences and their deduced amino acid  sequences.
based on the alignment results, a phylogenetic tree of sox <dig> proteins was constructed using the fruitfly  soxf as an outgroup.
the sox <dig> sequences isolated from chicken, anole lizard, frog, channel catfish, guppy, fathead minnow, little skate, dogfish, sea squirts, lancelets, acorn worm, the california mussel, snail and sea anemones were also included in the phylogenetic analysis.
the tree was constructed using the neighbor-joining method.
the scores represent bootstrap values of  <dig> trials, indicating the credibility of each branch.
all the aa sequences used in the phylogenetic analysis were obtained from genbank, ucsc and jgi genome bioinformatics, except that from tilapia.
the genbank accession numbers and scaffold numbers of these sequences are listed in table  <dig>
soxf
note.
* partial sequences identified from databases and first named in this report.
analysis of sox <dig> expression by rt-pcr and semiquantitative rt-pcr
total rnas  were isolated from various tissues of adults   and the gonads of monosex .
thereafter, total rnas were treated with dnase i to eliminate the genomic dna contamination.
then first strand cdnas were synthesized and rt-pcr was carried out to check the expression levels of tilapia sox <dig> according to methods described previously  <cit> .
the templates for positive and negative controls were set with sox <dig> plasmid dna and deionized water, respectively.
a 342-bp fragmentofβ-actin was amplified as internal control to test the quality of the cdnas used in the pcr.
the pcr products were subjected to agarose gel  electrophoresis.
to examine the influence of drug treatment on sox <dig> gene expression, gonads of  <dig> individuals were collected from each control group  and drug treated group  when the fish were adult at 240dah.
semi-quantitative rt-pcr  <cit>  was performed to measure the mrna levels using the primers  described in the previous section.
a series of pcrs with different cycle numbers  were performed to determine the linear phase of the amplification using  <dig> to  <dig> diluted cdna template.
based on these pilot experiments,  <dig> cycles for β-actin and  <dig> cycles for the target gene were chosen and applied to the subsequent semi-quantitative rt-pcr analyses.
band intensities resulting from the pcr amplification were analyzed using the image analysis software quantity one .
sox <dig> alternatively spliced mrna levels were expressed relative to that of β-actin in each sample.
in our study, β-actin expression was found to be unaffected by the drug treatment  <cit> .
results obtained were expressed as mean values ± s.e.m.
from five individual fish.
data analyses were performed using one-way anova and the least significant difference on the graphpad prism  <dig> software .
in situ hybridization
tilapia gonads from  <dig>   <dig> and 210dah were dissected and fixed in 4% paraformaldehyde  in  <dig> × pbs  at 4°c overnight.
after fixation, gonads were embedded in paraffin.
cross-sections of  <dig> μm were cut with a sliding microtome.
probes of both sense and antisense digoxigenin -labeled rna strands were transcribed in vitro from a linearized tilapia sox <dig> plasmid, using the rna labeling kit .
in situ hybridization was carried out as follows: sections were deparaffinized, hydrated and treated with proteinase k  and then hybridized with the sense or antisense dig-labeled rna probe at 60°c for 16- <dig> h. the hybridization signals were then detected using alkaline phosphatase-conjugated anti-dig antibody  and nbt as the chromogen  <cit> .
RESULTS
molecular cloning of tilapia sox30
the sox <dig> genomic dna and four alternatively spliced cdnas were cloned from the nile tilapia.
the genomic dna is  <dig> bp long , containing  <dig> exons,  <dig>   <dig>   <dig>   <dig> and  <dig> bp, respectively, and four introns .
all introns share the typical intron characteristics .
the isolated full-length sox <dig> cdna  is  <dig> bp , containing an open reading frame  of  <dig> bp encoding a putative protein of  <dig> aa, a  <dig> bp 5' untranslated region , and a  <dig> bp 3' utr.
the other three alternatively spliced isoforms  amplified by the same pair of primers were  <dig>   <dig> and  <dig> bp shorter than the orf of isoform-i due to exon skipping.
the four isoforms share the same 5'-utr, while only isoform-i and -iii share the same 3'-utr.
isoform-ii and -iv have 3'-utrs  <dig> bp longer than those of isoform-i and -iii due to the reading frame shift and introduction of a stop codon which is  <dig> bp upstream of the stop codon found in isoform-i and -iii transcripts.
further analysis suggested that these isoforms  contain  <dig>   <dig>   <dig> and  <dig> exons, respectively.
compared with the longest isoform , part of exon  <dig>  exon  <dig>  exon  <dig> and exon  <dig> was spliced off in isoform-ii , -iii , and -iv , respectively .
sequence, phylogeney and synteny analyses
multiple alignment of the aa sequences of sox <dig> s from various species revealed that sox <dig> was poorly conserved except in the hmg-box, the characteristic dna binding domain .
the hmg-boxes of tilapia and guppy sox <dig> shared the highest similarity , whereas the whole peptides of both shared rather low similarity .
compared with sox <dig> from mammals, tilapia sox <dig> is shorter in both n- and c-terminal regions .
as was shown in figure  <dig>  sox <dig> s isolated from tilapia and other species were clustered into the same clade with the mammalian sox <dig> s, indicating that they are genuine sox <dig> s. synteny analyses showed that sox <dig> and its adjacent gene thg1l and adam <dig> were located on the same chromosome/scaffold in the same gene order in human, mouse, chicken, anole lizard and frog .
tissue expression of sox <dig> gene
tissue distribution analysis using rt-pcr revealed that only three of the four isoforms of tilapia sox <dig>  were expressed in adult.
they were expressed exclusively in gonads, with isoform-i higher in the testis; isoform-iv higher in the ovary; while isoform-ii only in the testis, not in the ovary .
ontogeny of sox <dig> expression in tilapia gonads
sox <dig> expression was detected in gonads of tilapia during sex determination, differentiation and later development by rt-pcr.
isoform-i mrna was detected consistently in the gonads at  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> dah with a very interesting expression profile: ovary only at  <dig> and  <dig> dah; both ovary and testis from  <dig> dah onwards, higher in testis than in ovary from  <dig> dah to adult .
isoform-ii showed a male-specific expression from  <dig> dah to adult.
isoform-iii was detected exclusively in male gonad at  <dig> dah only, and no expression was detected during any other developmental stages.
isoform-iv started to express in both testis and ovary from  <dig> dah onwards, with higher level in the ovary from  <dig> dah to adult.
expression of sox <dig> in sex-reversed gonads
consistent with tissue distribution results, three isoforms  were detected in adult gonads by semi-quantitative rt-pcr.
isoform-i and -iv mrna were detected in gonads of all four groups, i.e.
xx  and xy  control fish and sex reversed xx  fish and sex reversed xy  fish.
isoform-i was significantly down-regulated in sex reversed xy  gonads, but up-regulated in sex reversed xx  gonads, being consistent with the result of tissue distribution and ontogeny analyses that isoform-i was expressed higher in the testis; isoform-iv was up-regulated in sex reversed xy  gonads, but down-regulated in sex reversed xx  gonads, which was consistent with that isoform-iv was expressed higher in the ovary.
isoform-ii mrna was only detected in normal xy  and sex reversed xx  gonads but not in normal xx  and sex reversed xy  gonads, consistent with that isoform-ii showed a male gonad-specific expression in tissue distribution and ontogeny analyses.
these data indicated that the expressions of sox <dig> are phenotypically related rather than genotypically related .
expression of sox <dig> by in situ hybridization in tilapia gonads
to ascertain which population of cells in the developing gonads expresses sox <dig>  ish was performed using gonads at  <dig>   <dig>   <dig> and  <dig> dah.
specific signals were observed in the male  germ cells at  <dig> dah  and the sperms of the testis at  <dig> and  <dig> dah .
in contrast, specific signals were detected in the female  somatic cells at  <dig> dah , interstitial cells of the ovary at  <dig> dah , and theca and granulosa cells of the ovary at  <dig> dah , but no signal was detected in both sexes at  <dig> dah .
taking the ontogeny results into consideration, we conclude that isoform-iii is restricted to the germ cells in male gonads and isoform-i is expressed in the somatic cells of the female gonads at  <dig> dah.
however, due to the overlapping probes , we failed to distinguish the signals detected at  <dig> and  <dig> dah gonads.
at least three and two isoforms could produce the signals detected in the testis and the ovary, respectively at these stages.
discussion
bioinformatics and phylogeney analyses of sox30
in the present study, a novel sry-related gene was isolated from the nile tilapia accidentally when cloning sox9b.
it was characterized as sox <dig> by subsequent blast against genbank for its relatively high identity with its mammalian orthologs, which was further confirmed by phylogenetic and syntenic analyses.
to date, sox <dig> has been reported only in mammals  <cit> , but not in any non-mammalian vertebrates  and invertebrates.
our data, for the first time, provided the solid proofs for the existence of sox <dig> in a teleost fish, the nile tilapia.
subsequently, genome databases of fishes, including zebrafish, medaka , stickleback , takifugu  and tetraodon  were searched for possible sox30-like genes.
however, it seems that this gene does not exist in any database of these teleosts.
est data bases were also searched afterward, and complete or partial sox <dig> sequences were successfully isolated from the channel catfish, guppy, fathead minnow, little skate and dogfish.
further screening of the genome sequences of other animals showed that sox <dig> was also present in other chordates, including chicken, anole lizard, frog, sea squirts, lancelets and acorn worm , and non-chordate invertebrates, including the california mussel, snail and sea anemone , while not found in the nematode , fruitfly and sea lamprey.
a recent report stated the existence of sox30-like sequence in sea urchin   <cit> , but we failed to find in the genome and est databases by blast search.
these findings suggested that sox <dig> is not specific to mammals, but also exists in other vertebrates and invertebrates.
then, why we failed to find sox30-like sequences in all sequenced teleost genomes?
there are two possible explanations.
one is that the genome sequences from these species have not covered all the genome yet; and the other is that sox <dig> was secondarily lost in these species during evolution.
however, the chance for the latter possibility is quite small because of the following two reasons, 1) cyprinids and ictalurids, such as fathead minnow and channel catfish, are relatively primitive teleosts while cichlids and poeciliids, such as tilapia and guppy, are relatively advanced teleosts.
it is unlikely that sox <dig> was lost in all the other teleosts except the primitive and advanced species; 2) both fathead minnow and zebrafish belong to the same family, cyprinidae.
it is difficult to believe that sox <dig> was secondarily lost in the zebrafish but not in another closely related species, fathead minnow.
therefore, we tend to accept the former explanation.
to confirm this, further cloning sox <dig> from other teleosts is required.
sox <dig> was found not only in chordates such as human, mouse, chicken, frog, tilapia, channel catfish, guppy, fathead minnow, little skate, dogfish, sea squirts, lancelets and acorn worm, but also in non-chordate invertebrate species, such as the california mussel, snail and sea anemone, indicating that it had already appeared before or with the emergence of coelenterates.
then, it is of interests to know from which ancestor gene sox <dig> was derived.
group h, which only consists of sox <dig>  shared the highest similarity to group f in  <dig> sox groups , suggesting that they may share a common ancestor.
however, the exact timing for the divergence of the two groups is still unclear.
its presence in the relatively primitive invertebrate species, sea anemones  and its position  in the phylogenetic tree  suggested that sox <dig> seemed to be one of the oldest members in the sox family, which is consistent with the previous conclusion  <cit> .
sequence and gene structure analyses of sox30
compared with its counterparts from mammals, tilapia sox <dig> genomic sequence was smaller in size due to compression of intron sequences.
similar phenomenon has been found for sox <dig> in sea squirts, lancelets and acorn worm.
alignment of aa sequences indicated that sox <dig> s was poorly conserved, even in the highly conserved hmg-box among both distant and closely related species, suggesting high diversity and rapid evolution of these proteins.
this also explains why sox <dig> has not been successfully isolated from non-mammalian animals until the accident cloning of tilapia sox <dig> in this study.
gene structure analysis showed that tilapia sox <dig>  like some other sox group members, such as sox <dig>  sox <dig> and sox <dig>  <cit> , was also characterized by the splitting of the hmg-box by an intron.
the same is true to sox <dig> of acorn worm, lancelets, sea squirts, frog, chicken, mouse and human, and the location of the intron is conserved among these species.
these findings indicated that this intron had already been fixed before or with the emergence of chordates.
alternative splicing  is emerging as one of the most important mechanisms to control vertebrate gene expression.
existing data indicate that as much as 76% of genes generate alternatively spliced products  <cit> .
as has been associated with a regulatory system in tissue- or stage-specific splicing mechanisms by which expression may be regulated.
this regulation may be achieved by the introduction of premature stop codons that function as an on-off gene expression switch  <cit> .
previous study showed that two different mrna isoforms of sox <dig>  sox <dig> have been isolated in mouse and two different mrna isoforms of sox <dig> in frog because of as  <cit> .
in mice, the sox <dig> isoform is expressed at a low level in the testis throughout postnatal development, while the t-sox <dig> isoform is expressed abundantly in the testis, predominantly in postmeiotic germ cells  <cit> .
based on these results, they suggested that a switch from sox <dig> to the t-sox <dig> isoform may alter the function of sox <dig> at the meiotic and postmeiotic phases during spermatogenesis in mice  <cit> .
our data demonstrated that tilapia sox <dig> has four alternatively spliced isoforms.
early termination of the stop codon was found in the isoform-ii and -iv transcripts of tilapia sox <dig>  suggesting that the mechanism mentioned above may be also responsible for regulation of sox <dig> gene expression in tilapia.
besides, similar to mouse sox <dig>  four isoforms of sox <dig> were also expressed in a non-parallel manner during gonadal development of tilapia.
thus, there may be some switches from one to the other isoform to alter the function of sox <dig> during gonadal development.
it is worthwhile to note that the hmg-box region was deleted partially in isoform-iii and completely in isoform-iv, respectively, which resulted in truncated proteins lacking most parts or all of the hmg-box domain and the nuclear localization signal.
it is interesting to know whether these protein isoforms are ever made, but now there is no antibody available because sox <dig> is poorly conserved among species of different classes of animals, and therefore, the available antibody against mammalian sox <dig> is not suitable for usage in fish.
there are reports showing that alternatively spliced isoforms of sox <dig> and sox <dig>  without functional dna binding domain or c-terminus, very similar to tilapia sox <dig> isoforms, were translated into proteins in mammals  <cit> .
based on those findings, we speculated that all isoforms of sox <dig> in tilapia may also be translated into the protein products.
as sox <dig> gene, like sry and some other sox genes, uses the single hmg-box for dna-binding, it would be very interesting to know what these truncated isoforms do and how they function.
whether this would result in different capacity of transcription activation, e.g.
complete non-functional factors or dominant negative mutants of the wild type sox <dig>  is still an open question.
the expression pattern and functional relevance of sox <dig> in tilapia
previous reports in mammals showed that human and mouse sox <dig> are exclusively expressed in normal adult testis and specifically in germ cells  <cit> .
this expression pattern suggests that sox <dig> may be involved in mammalian spermatogonial differentiation and spermatogenesis  <cit>  however, it is still unclear in which cell type sox <dig> is expressed in the mammalian ovary even though there was report indicating its possible expression in mouse oocytes  <cit> .
in the present study, sox <dig> started to express in gonads from  <dig> dah, earlier than the morphological gonadal differentiation period  in tilapia and showed a gonad specific expression pattern at least in adults.
of course, expression of sox <dig> in extra-gonadal tissues in other stages of development cannot be completely excluded.
meanwhile, expression of each alternatively spliced isoform showed a clear sexual dimorphism in gonads.
however, none of the four isoforms of sox <dig> were detected at  <dig> dah, the critical period of tilapia molecular sex determination, which excluded its role as the sex determining gene in tilapia.
in sex reversed adult gonads, three types of sox <dig> also exhibited a phenotypic sex-related expression pattern.
sox <dig> expression was restricted to the germ cells at  <dig> dah and later to sperms in male gonads, indicating its possible involvement in spermatogonial differentiation and spermatogenesis in male fish, like in human and mouse.
in female, it was expressed in the somatic cells in female gonads at  <dig>   <dig> and  <dig> dah, respectively.
sox <dig> is a strong regulator of the sf- <dig>  <cit> , the most important steroidogenic factor found in all vertebrates, including fish  <cit> .
our in situ hybridization data demonstrated clearly that sox <dig> is expressed in the somatic cells, especially steroidogenic cells of the ovary, colocalized with sf- <dig> in tilapia  <cit> .
taken together, we speculate that sox <dig> may be an important regulator for somatic differentiation and steroidogenesis in female fish as well.
both isoform i and iv are expressed in the ovary after  <dig> dah.
although isoform iv is expressed much higher than isoform i it lacks the dna binding hmg-box, and therefore, it is unlikely that this isoform regulates sf- <dig> expression by direct binding to the promoter.
however, the possibility that isoform iv might function as a dominant negative mutant of the wild type molecule  can not be excluded because it still has the other functional domains, such as transactivation domain.
more works need to be done to unravel this.
in addition to the gonad specific expression of sox <dig> in human, mouse and tilapia, all sox <dig> est sequences were also derived from gonads  demonstrating that sox <dig> may also be a gonad specific gene in these species.
these data further supported that sox <dig> plays a key role in gonadal differentiation and development, which might be conserved among species in the animal kingdom.
moreover, four alternatively spliced sox <dig> isoforms exhibited different temporal and spatial expression patterns in tilapia gonads.
alternative splicing of sox <dig> mrnas and different temporal expression pattern of the spliced isoforms had also been reported in human and mouse  <cit> .
therefore, we speculate that sox <dig> may be involved in gonadal differentiation and development in different sexes, at different stages and in different cell types of gonads in the animal kingdom by as.
further study on sox <dig> in other species is required to confirm this.
the low similarity between tilapia sox <dig> and its mammalian counterpart raises a question of whether tilapia sox <dig> is a genuine sox <dig> or just a new sox member of the group h. to answer this, we analyzed the synteny of sox <dig> and its adjacent genes in human, mouse, chicken, anole lizard and frog .
sox <dig> and its adjacent gene thg1l and adam <dig> were found to be located on the chromosome  <dig>   <dig>   <dig> and scaffold_ <dig> and _ <dig> in human, mouse, chicken, anole lizard and frog, respectively.
the conservation in synteny between non-mammalian species and mammals indirectly support that our isolated sox <dig> s are genuine orthologs of mammalian sox <dig>
CONCLUSIONS
for the first time, sox <dig> was found to exist in non-mammalian vertebrates and invertebrates by cloning, genome and est database analyses.
the probable gonad specific expression in all species analyzed and the temporal and spatial expression of the alternatively spliced isoforms indicated that sox <dig> may play a key role in gonadal differentiation and development, and the function of sox <dig> may be regulated by as and expression in different gender, different stages and different cell types in gonads in a wide variety of animals.
authors' contributions
fh carried out most of experiments, data mining and drafted the manuscript.
zjw co-carried out most of experiments and co-draft the manuscript.
frw participated in ish.
zhl participated in some of expression study.
bfh participated in gene cloning.
dsw designed and supervised the experiment, analyzed data and critically edited the manuscript.
all authors read and approved the final manuscript.

 since the discovery of sry, numerous genes encoding proteins containing hmg domain have been identified throughout the animal kingdom. genes with their hmg domains similar to that of sry by at least 50% were named as sox genes. at present, sox genes encoding transcription factors, count about members in vertebrates. based on differing levels of structural, organizational similarities and other features, sox genes can be subdivided into groups, named from a to j. members of the same group are similar to each other within and outside the hmg-box. however, members from different groups share lower degree of identity in the hmg-box, and no significant identity outside this domain. many of the sox proteins, like sry, sox sox sox and sox are encoded by a single exon. in contrast, others, such as sox sox sox and sox consist of multiple exons. in addition, the sox genes are scattered on different chromosomes and are not clustered. apart from the sry and sox located on the y- and x-chromosome respectively, other sox genes are autosomal. sox, the only member of group h, has been characterized merely in a few species so far. it was firstly isolated from mouse and human. in these species, two transcripts of sox mrna were found, one of which encodes a truncated protein lacking partial c-terminal region due to a frame shift. sox was considered to be involved in mammalian spermatogonial differentiation and spermatogenesis. as no sequences of sox were available from any other groups of animals, including the sequenced fish genomes at that time, some scholars even speculated that sox was specific to mammals. in zebrafish, a sox gene has been reported, however, it was proved to be sox by genbank blast and phylogenetic analysis. interestingly, in our present study, sox was isolated from the nile tilapia accidentally when cloning sox9b. subsequently, by blast search and data mining, sox was also found to exist in genome sequences of the chicken, green anole lizard, western clawed frog, sea squirts, lancelets, acorn worm, sea anemone, and ests of the channel catfish, guppy, fathead minnow, little skate, dogfish, the california mussel and snail. thus we report here that sox exists widely throughout the animal kingdom rather than specific to mammals as reported previously. more importantly, we isolated four alternatively spliced isoforms of sox and studied their temporal and spatial expression patterns during normal and sex reversed gonadal development in tilapia. our data suggest that sox may play key roles in gonadal development, and the roles may be different at different stages and sexes. methods animals the nile tilapias for this study were reared in large tanks with a circulating aerated freshwater system. fish were maintained at ambient temperature under natural photoperiod. all genetic females and males were obtained by artificial fertilization of eggs from normal females with sperms from either sex reversed males or super males, respectively. then, artificially fertilized eggs were cultured in recirculating water at 26°c to obtain fry. all animal experiments conformed to the guide for care and use of laboratory animals and were approved by the institutional committee of laboratory animal experimentation at southwest university, china. drug treatment xx males and xy females were generated by treating fry with estrogen receptor antagonist tamoxifen and 17β-estradiol, respectively. fish feed were sprayed with 95% ethanol containing tam, μg/g; e μg/g. control fish were fed with 95% ethanol sprayed feed. drug treatment was applied to the fry from to days after hatching, the critical period of the nile tilapia sex differentiation. e and tam treatment resulted in complete sex reversal in tilapia, respectively. cloning of tilapia sox cdna and genomic dna the smart race cdna of the gonads, which was used to perform both 5' and 3'-rapid implication of cdna ends to obtain full-length cdna of tilapia sox were synthesized according to the manufacturer's instruction. the sox 5' cdna end was amplified accidentally with primers for sox9b, and confirmed by blastx http://blast.ncbi.nlm.nih.gov/. subsequently, 3' race primers were designed to amplify 3' cdna end for tilapia sox finally, to verify the full-length sequences of sox cdna, end-to-end pcr was performed and four mrna isoforms were identified by using 5'- and 3'-utr primers. in order to determine the gene structure of the tilapia sox the genomic dna sequence of this gene was amplified. genomic dna was prepared from mature testis and ovary. a bp sox gene fragment with complete cds and four introns, and bp in length, was obtained by genomic pcr using end primers t30utr-f and t30utr-r. all the primers and sequences used in the present study were listed in table the pcr products were resolved on a % agarose gel and the target dna fragments were purified using qiaquick gel extraction kit. all the fragments obtained were cloned into pgem-t vector and bi-directional sequencing was performed by the dideoxy chain termination method using an abi prism dna genetic analyzer. data mining and sequence analyses the following genome databases were searched to explore the presence of sox30-like sequences in other vertebrate and invertebrate species, including the ucsc genome bioinformatics at: http://genome.ucsc.edu/, the ncbi databases at http://blast.ncbi.nlm.nih.gov/, the silk db databases at http://silkworm.genomics.org.cn/, the jgi databases at http://genome.jgi-psf.org/nemve1/nemve home.html, the ensembl databases http://www.ensembl.org/biomart/martview/ and the gene db databases at http://www.genedb.org/genedb/smansoni/. sox30-like sequences were identified from seven species by blast search against genome sequences and further confirmed in est databases, i.e., chicken, anole lizard, frog, sea squirts, lancelets, acorn worm and sea anemones, but failed to identify it from other animal species with genome sequence information. partial or complete sox30-like sequences were also identified by blast search against the ncbi est databases from channel catfish, guppy, fathead minnow, little skate, dogfish, the california mussel and snail. the orientation and chromosomal position of sox and its adjacent genes thg1l and adam were determined manually from the gene orientations listed in biomart from the ensembl database. synteny analyses of sox thg1l and adam in human, mouse, chicken, anole lizard and frog were performed by comparing the cdna sequences with genome sequence using blast search from the ucsc genome bioinformatics or by search in biomart from the ensembl database. the multiple alignment software dnastar and clustalx were employed to analyze the nucleotide sequences and their deduced amino acid sequences. based on the alignment results, a phylogenetic tree of sox proteins was constructed using the fruitfly soxf as an outgroup. the sox sequences isolated from chicken, anole lizard, frog, channel catfish, guppy, fathead minnow, little skate, dogfish, sea squirts, lancelets, acorn worm, the california mussel, snail and sea anemones were also included in the phylogenetic analysis. the tree was constructed using the neighbor-joining method. the scores represent bootstrap values of trials, indicating the credibility of each branch. all the aa sequences used in the phylogenetic analysis were obtained from genbank, ucsc and jgi genome bioinformatics, except that from tilapia. the genbank accession numbers and scaffold numbers of these sequences are listed in table soxf note. * partial sequences identified from databases and first named in this report. analysis of sox expression by rt-pcr and semiquantitative rt-pcr total rnas were isolated from various tissues of adults and the gonads of monosex. thereafter, total rnas were treated with dnase i to eliminate the genomic dna contamination. then first strand cdnas were synthesized and rt-pcr was carried out to check the expression levels of tilapia sox according to methods described previously. the templates for positive and negative controls were set with sox plasmid dna and deionized water, respectively. a 342-bp fragmentofβ-actin was amplified as internal control to test the quality of the cdnas used in the pcr. the pcr products were subjected to agarose gel electrophoresis. to examine the influence of drug treatment on sox gene expression, gonads of individuals were collected from each control group and drug treated group when the fish were adult at 240dah. semi-quantitative rt-pcr was performed to measure the mrna levels using the primers described in the previous section. a series of pcrs with different cycle numbers were performed to determine the linear phase of the amplification using to diluted cdna template. based on these pilot experiments, cycles for β-actin and cycles for the target gene were chosen and applied to the subsequent semi-quantitative rt-pcr analyses. band intensities resulting from the pcr amplification were analyzed using the image analysis software quantity one. sox alternatively spliced mrna levels were expressed relative to that of β-actin in each sample. in our study, β-actin expression was found to be unaffected by the drug treatment. results obtained were expressed as mean values ± s.e.m. from five individual fish. data analyses were performed using one-way anova and the least significant difference on the graphpad prism software. in situ hybridization tilapia gonads from and 210dah were dissected and fixed in 4% paraformaldehyde in × pbs at 4°c overnight. after fixation, gonads were embedded in paraffin. cross-sections of μm were cut with a sliding microtome. probes of both sense and antisense digoxigenin -labeled rna strands were transcribed in vitro from a linearized tilapia sox plasmid, using the rna labeling kit. in situ hybridization was carried out as follows: sections were deparaffinized, hydrated and treated with proteinase k and then hybridized with the sense or antisense dig-labeled rna probe at 60°c for 16- h. the hybridization signals were then detected using alkaline phosphatase-conjugated anti-dig antibody and nbt as the chromogen.  molecular cloning of tilapia sox30 the sox genomic dna and four alternatively spliced cdnas were cloned from the nile tilapia. the genomic dna is bp long, containing exons, and bp, respectively, and four introns. all introns share the typical intron characteristics. the isolated full-length sox cdna is bp, containing an open reading frame of bp encoding a putative protein of aa, a bp 5' untranslated region, and a bp 3' utr. the other three alternatively spliced isoforms amplified by the same pair of primers were and bp shorter than the orf of isoform-i due to exon skipping. the four isoforms share the same 5'-utr, while only isoform-i and -iii share the same 3'-utr. isoform-ii and -iv have 3'-utrs bp longer than those of isoform-i and -iii due to the reading frame shift and introduction of a stop codon which is bp upstream of the stop codon found in isoform-i and -iii transcripts. further analysis suggested that these isoforms contain and exons, respectively. compared with the longest isoform, part of exon exon exon and exon was spliced off in isoform-ii, -iii, and -iv, respectively. sequence, phylogeney and synteny analyses multiple alignment of the aa sequences of sox s from various species revealed that sox was poorly conserved except in the hmg-box, the characteristic dna binding domain. the hmg-boxes of tilapia and guppy sox shared the highest similarity, whereas the whole peptides of both shared rather low similarity. compared with sox from mammals, tilapia sox is shorter in both n- and c-terminal regions. as was shown in figure sox s isolated from tilapia and other species were clustered into the same clade with the mammalian sox s, indicating that they are genuine sox s. synteny analyses showed that sox and its adjacent gene thg1l and adam were located on the same chromosome/scaffold in the same gene order in human, mouse, chicken, anole lizard and frog. tissue expression of sox gene tissue distribution analysis using rt-pcr revealed that only three of the four isoforms of tilapia sox were expressed in adult. they were expressed exclusively in gonads, with isoform-i higher in the testis; isoform-iv higher in the ovary; while isoform-ii only in the testis, not in the ovary. ontogeny of sox expression in tilapia gonads sox expression was detected in gonads of tilapia during sex determination, differentiation and later development by rt-pcr. isoform-i mrna was detected consistently in the gonads at and dah with a very interesting expression profile: ovary only at and dah; both ovary and testis from dah onwards, higher in testis than in ovary from dah to adult. isoform-ii showed a male-specific expression from dah to adult. isoform-iii was detected exclusively in male gonad at dah only, and no expression was detected during any other developmental stages. isoform-iv started to express in both testis and ovary from dah onwards, with higher level in the ovary from dah to adult. expression of sox in sex-reversed gonads consistent with tissue distribution results, three isoforms were detected in adult gonads by semi-quantitative rt-pcr. isoform-i and -iv mrna were detected in gonads of all four groups, i.e. xx and xy control fish and sex reversed xx fish and sex reversed xy fish. isoform-i was significantly down-regulated in sex reversed xy gonads, but up-regulated in sex reversed xx gonads, being consistent with the result of tissue distribution and ontogeny analyses that isoform-i was expressed higher in the testis; isoform-iv was up-regulated in sex reversed xy gonads, but down-regulated in sex reversed xx gonads, which was consistent with that isoform-iv was expressed higher in the ovary. isoform-ii mrna was only detected in normal xy and sex reversed xx gonads but not in normal xx and sex reversed xy gonads, consistent with that isoform-ii showed a male gonad-specific expression in tissue distribution and ontogeny analyses. these data indicated that the expressions of sox are phenotypically related rather than genotypically related. expression of sox by in situ hybridization in tilapia gonads to ascertain which population of cells in the developing gonads expresses sox ish was performed using gonads at and dah. specific signals were observed in the male germ cells at dah and the sperms of the testis at and dah. in contrast, specific signals were detected in the female somatic cells at dah, interstitial cells of the ovary at dah, and theca and granulosa cells of the ovary at dah, but no signal was detected in both sexes at dah. taking the ontogeny results into consideration, we conclude that isoform-iii is restricted to the germ cells in male gonads and isoform-i is expressed in the somatic cells of the female gonads at dah. however, due to the overlapping probes, we failed to distinguish the signals detected at and dah gonads. at least three and two isoforms could produce the signals detected in the testis and the ovary, respectively at these stages. discussion bioinformatics and phylogeney analyses of sox30 in the present study, a novel sry-related gene was isolated from the nile tilapia accidentally when cloning sox9b. it was characterized as sox by subsequent blast against genbank for its relatively high identity with its mammalian orthologs, which was further confirmed by phylogenetic and syntenic analyses. to date, sox has been reported only in mammals, but not in any non-mammalian vertebrates and invertebrates. our data, for the first time, provided the solid proofs for the existence of sox in a teleost fish, the nile tilapia. subsequently, genome databases of fishes, including zebrafish, medaka, stickleback, takifugu and tetraodon were searched for possible sox30-like genes. however, it seems that this gene does not exist in any database of these teleosts. est data bases were also searched afterward, and complete or partial sox sequences were successfully isolated from the channel catfish, guppy, fathead minnow, little skate and dogfish. further screening of the genome sequences of other animals showed that sox was also present in other chordates, including chicken, anole lizard, frog, sea squirts, lancelets and acorn worm, and non-chordate invertebrates, including the california mussel, snail and sea anemone, while not found in the nematode, fruitfly and sea lamprey. a recent report stated the existence of sox30-like sequence in sea urchin, but we failed to find in the genome and est databases by blast search. these findings suggested that sox is not specific to mammals, but also exists in other vertebrates and invertebrates. then, why we failed to find sox30-like sequences in all sequenced teleost genomes? there are two possible explanations. one is that the genome sequences from these species have not covered all the genome yet; and the other is that sox was secondarily lost in these species during evolution. however, the chance for the latter possibility is quite small because of the following two reasons, 1) cyprinids and ictalurids, such as fathead minnow and channel catfish, are relatively primitive teleosts while cichlids and poeciliids, such as tilapia and guppy, are relatively advanced teleosts. it is unlikely that sox was lost in all the other teleosts except the primitive and advanced species; 2) both fathead minnow and zebrafish belong to the same family, cyprinidae. it is difficult to believe that sox was secondarily lost in the zebrafish but not in another closely related species, fathead minnow. therefore, we tend to accept the former explanation. to confirm this, further cloning sox from other teleosts is required. sox was found not only in chordates such as human, mouse, chicken, frog, tilapia, channel catfish, guppy, fathead minnow, little skate, dogfish, sea squirts, lancelets and acorn worm, but also in non-chordate invertebrate species, such as the california mussel, snail and sea anemone, indicating that it had already appeared before or with the emergence of coelenterates. then, it is of interests to know from which ancestor gene sox was derived. group h, which only consists of sox shared the highest similarity to group f in sox groups, suggesting that they may share a common ancestor. however, the exact timing for the divergence of the two groups is still unclear. its presence in the relatively primitive invertebrate species, sea anemones and its position in the phylogenetic tree suggested that sox seemed to be one of the oldest members in the sox family, which is consistent with the previous conclusion. sequence and gene structure analyses of sox30 compared with its counterparts from mammals, tilapia sox genomic sequence was smaller in size due to compression of intron sequences. similar phenomenon has been found for sox in sea squirts, lancelets and acorn worm. alignment of aa sequences indicated that sox s was poorly conserved, even in the highly conserved hmg-box among both distant and closely related species, suggesting high diversity and rapid evolution of these proteins. this also explains why sox has not been successfully isolated from non-mammalian animals until the accident cloning of tilapia sox in this study. gene structure analysis showed that tilapia sox like some other sox group members, such as sox sox and sox, was also characterized by the splitting of the hmg-box by an intron. the same is true to sox of acorn worm, lancelets, sea squirts, frog, chicken, mouse and human, and the location of the intron is conserved among these species. these findings indicated that this intron had already been fixed before or with the emergence of chordates. alternative splicing is emerging as one of the most important mechanisms to control vertebrate gene expression. existing data indicate that as much as 76% of genes generate alternatively spliced products. as has been associated with a regulatory system in tissue- or stage-specific splicing mechanisms by which expression may be regulated. this regulation may be achieved by the introduction of premature stop codons that function as an on-off gene expression switch. previous study showed that two different mrna isoforms of sox sox have been isolated in mouse and two different mrna isoforms of sox in frog because of as. in mice, the sox isoform is expressed at a low level in the testis throughout postnatal development, while the t-sox isoform is expressed abundantly in the testis, predominantly in postmeiotic germ cells. based on these results, they suggested that a switch from sox to the t-sox isoform may alter the function of sox at the meiotic and postmeiotic phases during spermatogenesis in mice. our data demonstrated that tilapia sox has four alternatively spliced isoforms. early termination of the stop codon was found in the isoform-ii and -iv transcripts of tilapia sox suggesting that the mechanism mentioned above may be also responsible for regulation of sox gene expression in tilapia. besides, similar to mouse sox four isoforms of sox were also expressed in a non-parallel manner during gonadal development of tilapia. thus, there may be some switches from one to the other isoform to alter the function of sox during gonadal development. it is worthwhile to note that the hmg-box region was deleted partially in isoform-iii and completely in isoform-iv, respectively, which resulted in truncated proteins lacking most parts or all of the hmg-box domain and the nuclear localization signal. it is interesting to know whether these protein isoforms are ever made, but now there is no antibody available because sox is poorly conserved among species of different classes of animals, and therefore, the available antibody against mammalian sox is not suitable for usage in fish. there are reports showing that alternatively spliced isoforms of sox and sox without functional dna binding domain or c-terminus, very similar to tilapia sox isoforms, were translated into proteins in mammals. based on those findings, we speculated that all isoforms of sox in tilapia may also be translated into the protein products. as sox gene, like sry and some other sox genes, uses the single hmg-box for dna-binding, it would be very interesting to know what these truncated isoforms do and how they function. whether this would result in different capacity of transcription activation, e.g. complete non-functional factors or dominant negative mutants of the wild type sox is still an open question. the expression pattern and functional relevance of sox in tilapia previous reports in mammals showed that human and mouse sox are exclusively expressed in normal adult testis and specifically in germ cells. this expression pattern suggests that sox may be involved in mammalian spermatogonial differentiation and spermatogenesis however, it is still unclear in which cell type sox is expressed in the mammalian ovary even though there was report indicating its possible expression in mouse oocytes. in the present study, sox started to express in gonads from dah, earlier than the morphological gonadal differentiation period in tilapia and showed a gonad specific expression pattern at least in adults. of course, expression of sox in extra-gonadal tissues in other stages of development cannot be completely excluded. meanwhile, expression of each alternatively spliced isoform showed a clear sexual dimorphism in gonads. however, none of the four isoforms of sox were detected at dah, the critical period of tilapia molecular sex determination, which excluded its role as the sex determining gene in tilapia. in sex reversed adult gonads, three types of sox also exhibited a phenotypic sex-related expression pattern. sox expression was restricted to the germ cells at dah and later to sperms in male gonads, indicating its possible involvement in spermatogonial differentiation and spermatogenesis in male fish, like in human and mouse. in female, it was expressed in the somatic cells in female gonads at and dah, respectively. sox is a strong regulator of the sf-, the most important steroidogenic factor found in all vertebrates, including fish. our in situ hybridization data demonstrated clearly that sox is expressed in the somatic cells, especially steroidogenic cells of the ovary, colocalized with sf- in tilapia. taken together, we speculate that sox may be an important regulator for somatic differentiation and steroidogenesis in female fish as well. both isoform i and iv are expressed in the ovary after dah. although isoform iv is expressed much higher than isoform i it lacks the dna binding hmg-box, and therefore, it is unlikely that this isoform regulates sf- expression by direct binding to the promoter. however, the possibility that isoform iv might function as a dominant negative mutant of the wild type molecule can not be excluded because it still has the other functional domains, such as transactivation domain. more works need to be done to unravel this. in addition to the gonad specific expression of sox in human, mouse and tilapia, all sox est sequences were also derived from gonads demonstrating that sox may also be a gonad specific gene in these species. these data further supported that sox plays a key role in gonadal differentiation and development, which might be conserved among species in the animal kingdom. moreover, four alternatively spliced sox isoforms exhibited different temporal and spatial expression patterns in tilapia gonads. alternative splicing of sox mrnas and different temporal expression pattern of the spliced isoforms had also been reported in human and mouse. therefore, we speculate that sox may be involved in gonadal differentiation and development in different sexes, at different stages and in different cell types of gonads in the animal kingdom by as. further study on sox in other species is required to confirm this. the low similarity between tilapia sox and its mammalian counterpart raises a question of whether tilapia sox is a genuine sox or just a new sox member of the group h. to answer this, we analyzed the synteny of sox and its adjacent genes in human, mouse, chicken, anole lizard and frog. sox and its adjacent gene thg1l and adam were found to be located on the chromosome and scaffold_ and _ in human, mouse, chicken, anole lizard and frog, respectively. the conservation in synteny between non-mammalian species and mammals indirectly support that our isolated sox s are genuine orthologs of mammalian sox  for the first time, sox was found to exist in non-mammalian vertebrates and invertebrates by cloning, genome and est database analyses. the probable gonad specific expression in all species analyzed and the temporal and spatial expression of the alternatively spliced isoforms indicated that sox may play a key role in gonadal differentiation and development, and the function of sox may be regulated by as and expression in different gender, different stages and different cell types in gonads in a wide variety of animals. authors' contributions fh carried out most of experiments, data mining and drafted the manuscript. zjw co-carried out most of experiments and co-draft the manuscript. frw participated in ish. zhl participated in some of expression study. bfh participated in gene cloning. dsw designed and supervised the experiment, analyzed data and critically edited the manuscript. all authors read and approved the final manuscript. 
our data suggest that expressions of sox isoforms are related to stage and phenotypic-sex, observed in the germ cells of male gonad and in somatic cells of the female gonad. their temporal and spatial expression patterns during normal and sex reversed gonadal development were investigated by rt-pcr and in situ hybridization. expression analyses revealed that sox may be involved in female and male gonadal development at different stages by alternative splicing. members of the sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. screening of available genome and est databases and phylogenetic analysis showed that sox also exists in non-mammalian vertebrates and invertebrates, which was further supported by synteny analyses. sox cdna was cloned from the nile tilapia by rt-pcr and race. sox is not a gene only existed in mammals, but exists widely throughout the animal kingdom as supported by our bioinformatic, phylogenetic and syntenic analyses. tissue expression in human, mouse and tilapia suggested that sox was probably a gonad-specific gene, which was also supported by the fact that sox est sequences were obtained from gonads of the animal species.
 members of the sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. among these members, sox had been considered to exist only in mammals since its discovery, and its exact function remains unclear.  sox cdna was cloned from the nile tilapia by rt-pcr and race. screening of available genome and est databases and phylogenetic analysis showed that sox also exists in non-mammalian vertebrates and invertebrates, which was further supported by synteny analyses. tissue expression in human, mouse and tilapia suggested that sox was probably a gonad-specific gene, which was also supported by the fact that sox est sequences were obtained from gonads of the animal species. in addition, four alternatively spliced isoforms were isolated from tilapia gonad. their temporal and spatial expression patterns during normal and sex reversed gonadal development were investigated by rt-pcr and in situ hybridization. our data suggest that expressions of sox isoforms are related to stage and phenotypic-sex, observed in the germ cells of male gonad and in somatic cells of the female gonad.  sox is not a gene only existed in mammals, but exists widely throughout the animal kingdom as supported by our bioinformatic, phylogenetic and syntenic analyses. it is very likely that sox is expressed exclusively in gonads. expression analyses revealed that sox may be involved in female and male gonadal development at different stages by alternative splicing. 