BACKGROUND
genetic differences between the sexes can broadly be separated into two groups: differences in the transcription level, where the abundance of a particular gene transcript differs between the sexes , and heterogametic sex chromosomes that are present in one sex and absent in the other.
these two mechanisms can occur together, and often species that lack differentiated sex chromosomes exhibit sex specific gene expression  <cit> .
many studies have shown that sex-biased differences in gene expression are present after sex determination and differentiation has taken place; sex bias in gene expression has been documented in multiple species including fruit flies   <cit> , the worm   <cit> , the mouse   <cit> , chicken   <cit> , the flour beetle   <cit>  and zebra fish   <cit>  .
most  studies have found male bias in gene expression, with more genes upregulated in mature males than mature females .
this is hypothesized to be due to strong sexual selection in males in the form of female choice, and/or sperm competition  <cit> .
patterns of sex-biased gene expression are extremely variable both within and between species, and there are marked differences in the proportion, number, and identity of genes that are differentially expressed depending on the tissue type and developmental stage examined.
for example 4% of the adult whole-body transcriptome exhibited sex-biased expression in mice  <cit>  to as high as 88% in drosophila  <cit> .
sex-biased expression also varies within different tissues of the same individual.
for example, yang et al.
<cit>  studied sex-biased expression in mus and found that brain tissue exhibits fewer sex-biased genes  than muscle , adipose  and liver  tissue.
other studies  report similar differences with the greatest percentage of sex-biased expression frequently being in the gonad transcriptome of sexually mature adults .
although sex-bias in expression seems to be a common phenomenon in many different species, most studies have used sexually mature specimens or individuals  that have already undergone differentiation of the gonads .
however, substantially less information is available on the level of sex-biased expression in un-differentiated embryos.
studies in the pre-implantation stage of embryogenesis in mouse found that roughly 3% of the transcriptome is differentially expressed between the sexes  <cit> .
in chicken, levels of sex-biased expression in embryos after differentiation have been compared with adults, and unsurprisingly found far less sex-biased expression in embryos  <cit> .
this suggests that the amount of sex-biased expression in the transcriptome changes throughout development through the juvenile and adult stages, and that sex-biased expression patterns may be present very early in development.
the extent to which biases in gene expression occur prior to gonad differentiation, however, have not been fully explored.
genetic differences between the sexes also occur due to the presence of heteromorphic sex chromosomes.
genetic sex determination  is most often thought of as being initiated by a switch in one sex that begins the gonad differentiation cascade .
however, despite the widespread importance of genetic sex determination across taxonomic groups, surprisingly little is known about the genes involved in the cascade in fishes .
unlike mammals , fish species with gsd appear to use a range of different loci, with dmy in medaka the only known example of a primary switch  <cit> .
karyotype and inheritance studies have shown there are a number of fish species that have gsd, but the loci responsible have yet to be determined.
sex can also be determined by a set of genes presumably on both the sex chromosomes and the autosomes that act in concert to determine sex, e.g.
many species of livebearers including the platyfish xiphophorus maculatus .
rainbow trout  are known to have gsd, but the mechanisms triggering the differentiation of testes and ovaries during development have not yet been revealed.
however, a number of studies have identified candidate sex determination genes that are differentially expressed as early as the onset of exogenous feeding, and presumably during the differentiation process  <cit> , but no studies have yet identified the sex determination gene on the y chromosome.
moreover, these studies evaluated candidate genes known to be important for sex determination in other species.
a whole-transcriptome approach may be better suited for determining as-yet unidentified sex determination genes, while also revealing the patterns of sex bias in gene expression at the earliest time points in development.
our aims here were twofold: 1) to evaluate differences in gene expression between the sexes in pre-hatch embryos of rainbow trout on a genome-wide level using a microarray analysis to: a) determine how much of the transcriptome appears to be differentially expressed between the sexes early on in development , and b) identify genes which are known to be involved in the sex differentiation cascade in model organisms, and 2) to characterize the expression profiles of a set of candidate sex genes that are known to be differentially expressed at the onset of exogenous feeding  in an attempt to ascertain the time point earlier in development when these genes are being turned on.
this study focuses on three development time points:  <dig> days post-fertilization ,  <dig> dpf  and  <dig> dpf .
this combination of approaches identifies sex bias in gene expression at some of the earliest time points in development on a genome-wide level, even before the onset of sex determination and phenotypic sex differentiation, while also evaluating the timing of genes known for their role in the sex determination cascade in other species.
RESULTS
samples used in this experiment were from a fourth generation backcross between two clonal lines of rainbow trout , as described in xu et al.
<cit> .
one line  possessed alleles for fast development, and the other line  possessed alleles for slower development at a major qtl for embryonic development rate.
here we use samples from this cross to investigate sex differences in the expression in the early stages of development of o. mykiss.
microarray data results of differential expression between the sexes
microarray data from the experiments have been submitted to the gene expression omnibus database http://www.ncbi.nlm.nih.gov/geo/ according to miame guidelines.
the series accession number is gse <dig>  controlling for the false discovery rate in multiple tests,  <dig> features were identified as differentially expressed between female and male embryos in at least one comparison.
more features were differentially expressed between the sexes in samples from osu/cw than osu/osu; these included  <dig> features from osu/cw in  <dig> dpf embryos compared to  <dig> from osu/osu,  <dig> features in  <dig> dpf embryos from osu/cw compared to  <dig> features from osu/osu, and  <dig> features in  <dig> dpf embryos from osu/cw compared to  <dig> from osu/osu.
to focus on features with large expression differences, there were  <dig>  features with a minimum of a 3-fold change expression difference between the sexes in at least one comparison.
the top  <dig> features with the greatest differences in expression at  <dig>   <dig> and  <dig> dpf are shown in tables  <dig>   <dig> and  <dig> .
of the features differentially expressed at  <dig> dpf,  <dig> features were higher expressed in males, and  <dig> were greater expressed in females.
the most common go terms associated with these features were cell cycle regulation , muscle contraction and development  and transport .
in the  <dig> dpf samples  <dig> features were higher expressed in males, and  <dig> were higher expressed in females.
the most common go terms were cell cycle regulation , protein metabolism  and lipid metabolism .
in the  <dig> dpf samples  <dig> features were upregulated in males and  <dig> features were upregulated in females.
the most common go terms were cell cycle regulation , protein synthesis  and transport .
only a limited number of differentially expressed features between the sexes were shared between different time points;  <dig> out of  <dig>  features in osu/cw samples and  <dig> out of  <dig>  in osu samples .
.
a small number of sex-biased features were shared between genotypes at the same time point with  <dig> out of  <dig>  at 15dpf ,  <dig> out of  <dig>  at  <dig> dpf  and  <dig> out of  <dig>  at  <dig> dpf .
these results indicate that very few differentially expressed genes are shared between genotypes, or between developmental time points, suggesting that expression patterns are rapidly changing during early stages of development, and that the individuals with the different qtl genotypes are developing at different rates  <cit> .
cluster analysis grouped the  <dig> features into  <dig> discrete clusters .
most of these clusters had only a few features, but clusters  <dig> and  <dig> had  <dig> and  <dig> features respectively.
cluster  <dig> grouped those features that were upregulated in osu/cw females at  <dig> dpf, and features within cluster  <dig> showing upregulation in osu/cw males at  <dig> dpf.
clustering analysis suggests that samples from the same developmental time point show more similar expression profiles than samples from different time points from the same genotype.
for example, osu/cw samples at  <dig> dpf are more similar to samples from to osu/osu at  <dig> dpf than osu/cw samples from either  <dig> or  <dig> dpf.
this trend is similar in samples from all three time points.
as mentioned, previous studies have documented a bias in the number of differentially expressed featured between the sexes .
we compared fold change values of differentially expressed features between the sexes to determine if there was bias in the directionality of differentially regulated genes between the sexes .
for each gene the fold change represents the magnitude and direction of differential expression between the sexes.
there appears to be little difference in the number of genes differentially expressed between the sexes at  <dig> dpf in both genotypes, with marginally more female expressed genes than male expressed genes in osu/cw  .
there is a similar difference in the opposite direction in samples from osu/osu where  <dig> features have a three-fold upregulation in females compared to  <dig> in males.
samples from later developmental time points seem to show higher differences in fold expression between the sexes.
at  <dig> dpf there are more features from both osu/cw and osu/osu that exhibit male bias expression with  <dig> features with a three-fold difference from osu/cw  and  <dig> features with a three-fold difference from male osu/osu samples .
very similar patterns were seen in samples from  <dig> dpf with males from both osu/cw and osu/osu showing a greater number of three-fold difference genes than females .
the majority of the  <dig> sex-biased genes did not show big differences in expression between the sexes, and showed ratios less than 2: <dig>
go enrichment analyses
go enrichment analyses determined that only  <dig> go terms identified from the  <dig> differentially expressed features between the sexes were upregulated compared to the whole 16k grasp chip.
these go functions were cellular macromolecule metabolic process, , macromolecule metabolic process  and cellular protein metabolic process .
when comparing the features identified from one sex to the total set of differentially expressed genes,  <dig> go terms were statistically different between the sexes .
of these  <dig> were differentially expressed in osu/cw samples  and  <dig> were differentially expressed in osu/osu samples .
for the osu/cw samples,  <dig> go terms were upregulated in males compared to  <dig> that were upregulated in females.
for the osu/osu samples,  <dig> go terms were upregulated in males compared to  <dig> go terms in females.
these differences suggest that in addition to more features being upregulated in males, more go terms  are also upregulated in males compared to females.
the ratio of go terms upregulated in males compared to females changed between the three developmental time points.
for example, within osu/cw samples there were  <dig> go terms upregulated in males compared to  <dig> go terms in females at  <dig> dpf,  <dig> go terms in males to  <dig> go terms in females at  <dig> dpf and finally one go term being upregulated in males at  <dig> dpf and none in females.
these patterns suggest that the greatest differences in the functional categories differentially regulated between the sexes were at  <dig> and  <dig> dpf.
numbers of functional go categories that were enriched from osu/osu were generally low but again the ratio of male to female upregulated genes varied between time points.
all six go terms differentially expressed at  <dig> dpf were upregulated in males, whereas the  <dig> go terms differentially expressed between the sexes at  <dig> dpf were upregulated in females at  <dig> dpf.
some enriched go categories were shared between different developmental time points and between different genotypes with some terms switching between upregulation in males and upregulation in females.
for example structural molecular activity was upregulated in males at  <dig> dpf in samples from osu/cw , but upregulated in females at  <dig> dpf in osu/cw .
however, some go terms that were shared between the two genotypes showed similar patterns of expression such as cellular biosynthesis process, which was upregulated in males in samples from osu/cw at  <dig> dpf  and the same result was found in samples from osu/osu at  <dig> dpf .
enrichment analysis on the  <dig> clusters shown in figure  <dig> failed to identify any go terms that were differentially expressed compared to both the complete  and sex-biased  datasets.
this suggests that patterns of similar expression within clusters are not due to shared functionality.
thirty-eight go terms were found to be significantly under expressed in  <dig> clusters  but this is almost certainly due to small cluster size rather than a cluster of genes with shared function.
qrt-pcr of candidate sex genes
we further examined whether genes with a potential role in sex determination were differentially expressed between male and female embryos.
these genes were identified using two approaches: 1) based on go term analyses of differentially expressed genes on the microarray ,  <dig> candidate sex determining genes were identified for further study.
of these only  <dig>  amplified with qrt-pcr methods.
2) of  <dig> candidate sex genes described in baron et al  <cit>  we investigated  <dig> genes .
all  <dig> genes amplified product of the expected size .
note that cyp19a1a  was the only candidate sex gene identified from both the microarray and was in baron et al's study.
both sets of genes were examined in the original three developmental time points, and a subset of six genes from baron et al.
<cit>   as well as zonadhesin and cyp19a1a identified from the microarray were investigated at three additional developmental times points .
there is a common pattern in six of the genes  which show a higher peak of expression in  <dig> dpf  samples than the other developmental time points.
at  <dig> dpf, the expression was higher in the males for ovol <dig>  wt <dig>  foxl2b and dmrt <dig> and higher in the females for zonadhesin and cyp19a1a.
looking at each time point only ovol <dig> shows a significant difference in expression between the sexes with higher expression in males than in females .
sex was significant when it was considered as an interaction term with dpf  and with qtl .
qtl was a significant factor in ovol <dig>  zonadhesin and cyp19a1a although in all cases it was only marginally significant .
the other  <dig> sex genes did not show a significant relationship with sex either as a main or interaction term except solt <dig> where the sex*dpf interaction was significant .
most of these genes showed very similar levels of expression across all three developmental time periods .
most of the candidate sex genes except for the ones discussed above show constant low levels of expression throughout all developmental time points and in both sexes  .
scatter plots shown in figure  <dig> plot the mean relative expression values of the male and female samples per gene averaged between the early developmental time points  and the late developmental time points .
of note, nr5a <dig> appears to be highly expressed in male samples from early developmental time points, whereas the other samples seem roughly similarly expressed between males and females.
ovol <dig> has a two fold increase in expression in males than in females in later stages in development.
cyp19a1a and nr0b <dig> show higher expression in females than in males at later stages of development.
candidate gene linkage mapping
where possible, candidate sex genes were mapped to determine if any map to the putative sex determining region on the rainbow trout sex chromosomes.
four of the candidate sex determining genes  have already been mapped in rainbow trout, and so were not mapped here; none of these map to the sex chromosome  <cit> .
of the remaining  candidate sex determining genes, three  failed to produce clean sequence product, despite primer redesign and optimization.
this suggests that these genes are duplicated in the rainbow trout.
of the eight remaining genes all had at least one polymorphic base within the sequence and were genotyped in the osu × cw doubled haploid mapping population  <cit> .
one of these genes  did not produce significant linkage with any other marker and remains in an unmapped region of the genome.
none of the remaining seven genes mapped to the sex linkage group  .
discussion
patterns of gene expression between the sexes
repeatedly, microarray experiments have found that a substantial portion of the transcriptome is differentially expressed between the sexes .
most of these studies have focused on model organisms such as drosophila, c. elegans and mus, with relatively few studies on non-model species that lack a complete genome sequence.
moreover, very few studies have included individuals that have not yet undergone sexual differentiation.
our study mined data from a microarray project to find a total of  <dig> genes that showed a significant difference in expression between the sexes before the completion of sexual differentiation.
in so doing, it is not only one of the few studies to evaluate sex bias in the transcriptome in a non-model species, but is also one of the first studies to examine expression patterns between the genotypic sexes in embryos before the completion of gonad differentiation.
most genes differentially expressed between the sexes in this study are involved in processes such as cell cycle function and general structure, and are not necessarily associated with sexual development.
this suggests that there are inherent differences in the transcriptome of male and female rainbow trout, and that these differences are present early in development.
other microarray studies in ovarian o. mykiss samples  also found a wide range of genes differentially expressed in the ovaries, many of which had no obvious sex function  <cit> .
these included transcripts that encode various elastases, cathepsins, proteases and immune function transcripts, suggesting that rainbow trout are exhibiting broad differences in transcriptome expression between the sexes throughout life.
an expression quantitative trait loci  study in an f <dig> cross between "dwarf" and "normal" lake whitefish  found strong evidence for more eqtls in females than in males.
however, the genes underlying these eqtls were found not to be differentially expressed between the sexes, strongly suggesting pleiotropic sex-linked effects in the transcriptome, at least within white muscle, the tissue used for microarray analysis  <cit> .
here we find a relatively low proportion of the transcriptome that exhibits sex bias in gene expression when compared to other studies, but it is important to consider the developmental stages of the samples.
in our study,  <dig> % of the features showed sex-biased gene expression  in at least one of the three developmental time points in one of the two studied qtl genotypes.
these numbers are low when compared to results in drosophila, where a much higher percentage of sex-biased expression in mature adults  with most studies reporting a number near 50%  <cit> .
studies from whole individual adult tribolium  <cit>  and adult zebra fish  <cit>  report around 20% and 38%, respectively.
similar numbers have been found in mice ; although note that like in drosophila, sex-biased expression is very variable depending on tissue type with up to 72% of genes from liver to 13% of genes from brain  <cit> .
taken together these studies suggest that sex-biased gene expression is commonplace, however it seems to be variable both within and among species and variable with regard to tissue type and developmental stage.
there are a number of potential reasons for why we see fewer genes showing sex-biased expression compared to prior studies.
firstly, most other studies have focused on developmental stages that have occurred after sex differentiation, so presumably the samples used in such studies were actively expressing genes linked to sex and gamete functions  <cit> .
in salmonids gonad differentiation tends to occur soon after the onset of hatching  <cit>  which is around our oldest time point of sampling.
so it is perhaps surprising that we did not find more sex-biased genes, and genes with an obvious sex function in samples from our oldest developmental point .
secondly, the absolute numbers of genes that are being expressed in embryos are less compared to the number of genes that are expressed in adults.
many of the sex and gamete function processes do not begin at such early stages of development and transcripts of such genes, presumably, would not be present .
thirdly, the microarray is not a complete survey of the transcriptome and so there will be sex genes and sex-biased genes that were not surveyed.
for example, none of the  <dig> candidate sex genes chosen for qrt-pcr study were on the microarray.
although these  <dig> genes did not show significant differences in expression between the sexes there are many other genes that have been shown to be differentially expressed at the onset of exogenous feeding in o. mykiss  <cit>  that were not studied here.
these reasons are not mutually exclusive, and all three undoubtedly help explain our results.
other studies have also found low levels of sex-bias expression in embryos, for example roughly  <dig> % of the chicken embryo gonad transcriptome is female biased and  <dig> % male biased  <cit> .
this number increases to  <dig> % and  <dig> % in the adult gonads of females and males, respectively.
although the proportions of the transcriptome that was differentially expressed in chicken is similar to this study in rainbow trout, it is important to note key differences in the study designs.
in our study, whole embryos were used from developmental time points prior to gross morphological differentiation of the gonads, while in  <cit>  gonad tissue was used from embryos after sex differentiation.
previous studies have examined sex-bias in gene expression to test hypotheses about the influence of sexual selection on gene expression.
as other authors have suggested, a male bias in gene expression could provide support for a higher rate of functional evolution in genes important for sex and reproduction in males, where selective pressure on males in the form of male-male competition for mates is high  <cit> .
we however, found roughly equal expression between the number of male biased and female biased genes, with only slightly higher number of male biased genes in samples from both lines at  <dig> dpf and slightly higher number of female biased genes in osu/osu at  <dig> dpf.
moreover, our enrichment analysis failed to identify any key functional processes with sex bias in gene expression and moreover within and among genes there was no consistent directionality to sex-biased in expression across time points .
all of the sex-biased genes and candidate sex determining genes showed spiky patterns of expression, suggesting rapid change in the transcriptome of the developing embryo.
many other studies using adult individuals report large differences in the number of sex-biased genes with, frequently, many more male-biased than female-biased genes  <cit> , supporting the idea that sex bias towards males could be a product of sexual selection.
however, studies in both drosophila  <cit>  and zebra fish  <cit>  have found more female-biased genes than male-biased genes.
these studies reinforce the idea that results can be very dependent on which transcriptomes are being investigated.
for example, studies in the gonads  <cit>  and whole body transcriptome  <cit>  of drosophila produce different results.
in a hypothetical situation where sexual selection arises through sperm competition, sex bias may be expected to occur only at the level of the gonad and potentially swamped by signals from other tissues in whole body analyses .
in contrast, sex bias in gene expression arising from selection in the form of male-male competition through alternative behaviors, ornamentation, or other 'phenotypes' outside of the gonads themselves could be found in the transcriptome of multiple tissues or at the level of the whole body.
moreover, the way in which animals used for transcriptome studies are maintained prior to tissue sampling can have a large effect.
for example,  <cit>  separated male and female zebra fish whereas  <cit>  kept both sexes together immediately prior to sampling and in so doing, may have modified the expression of sex related genes.
in other words, it is clear that the way individuals are reared prior to sampling can have an effect on the results of the experiment.
this potentially could lead to misleading interpretations of the effects of sexual selection on sex-bias expression, as behavioral and physiological interactions among members of the opposite sex can elicit changes in gene expression.
finally, the degree to which gene expression early in life is genetically correlated to gene expression late in life when sexual selection is prominent also remain as questions to be explored in future studies of model and non-model organisms where sexual selection is prevalent.
candidate sex determination genes
studies on post hatch rainbow trout indicate that genes known to be involved in the sex determination process are differentially expressed between the sexes  <cit> .
however, we are still unclear as to when expression of these genes is initiated as the majority of the candidate sex determination genes were differentially expressed at the earliest point of sampling .
the o. mykiss embryos in our study were reared at 11°c and hatched at  <dig> dpf , and therefore, our sampling procedure should span the point of sex determination.
it has been found that differences in expression between the sexes in these genes tend to spike more at specific time points, rather than show consistent differences in multiple development points  <cit> .
our results show a similar trend in that out of  <dig> candidate sex genes only ovol <dig> showed a statistically significant difference in expression between male and female samples across all developmental time points sampled, although many genes showed differences in expression at specific time points.
in the fruit fly  ovol <dig> is required for female germ line determination and differentiation  <cit> .
ovol <dig> is also expressed in sheep  ovaries immediately prior to gonad differentiation  <cit> .
here we find upregulation of ovol <dig> in males at 15dpf and females at  <dig> dpf.
of the other genes that showed a significant difference between the sexes, nr5a <dig> expression has been shown to be directly affected by sox <dig> in mammals and wt <dig> in reptiles and is turned on after male determination  <cit> .
with so few candidate genes showing differences between the sexes, it is important to give pause as to why this might be.
results from studies in mouse suggest that the window of differential expression of sex determining genes is small .
perhaps we have missed the specific time point when these genes are upregulated?
increasing sampling effort just before the onset of hatch to the point of first exogenous feeding, and using a whole-transcriptome approach, could prove fruitful.
mapping of candidate sex differentiation genes
the fact that none of the candidate sex genes mapped to the sex chromosome in this study further suggests that a master control gene for the determination of sex in o. mykiss remains elusive.
linkage mapping of other candidate sex determining genes in other populations of o. mykiss also found no genes mapping to the sex chromosome.
a total of  <dig> candidate sex genes have been mapped prior to this study, including  <dig> different transcripts of wt <dig>  <cit> , three sox <dig> loci  <cit>  and  <dig> sox <dig> genes, amh, dmrt <dig> and nrob <dig>  <cit> .
the addition of fst, ovo <dig>  zonadhesin, igf <dig>  foxl2a, cyp19a1a and wt <dig> brings the total number of loci to  <dig>  the sex chromosomes of fishes  are at the early stages of differentiation  and this in part helps explain why only one sex determining gene is known in any fish .
in order to identify the master sex gene in rainbow trout a concentrated sequencing effort of the sex chromosome is needed.
CONCLUSIONS
here we found evidence that a proportion of the transcriptome is differentially expressed between the sexes at early stages of development in rainbow trout, prior to the morphological differentiation of the gonads.
most of the features were not connected with obvious sex function suggesting that there is sexual dimorphism in gene expression even prior to sex differentiation, and the processes that are differentially regulated are varied and complex.
the early age of our sampling precluded us from looking at the transcriptome of the gonads; however the fact that so many genes are differentially expressed so early in development is a novel and interesting find.
in addition, we found evidence that several candidate sex determining genes showed a significant difference in expression between males and females.
in all cases, this difference was seen at one or at most two time points only, suggesting that the transcriptome of o. mykiss embryos goes through rapid changes early in development.
it is possible that our sampling scheme has missed the key window of sexual differentiation in o. mykiss, to that end a more thorough sampling effort to include embryos after  <dig> dpf and before  with  <dig> dpf as well as earlier points in development could further our understanding of embryonic development in o. mykiss.

 genetic differences between the sexes can broadly be separated into two groups: differences in the transcription level, where the abundance of a particular gene transcript differs between the sexes, and heterogametic sex chromosomes that are present in one sex and absent in the other. these two mechanisms can occur together, and often species that lack differentiated sex chromosomes exhibit sex specific gene expression. many studies have shown that sex-biased differences in gene expression are present after sex determination and differentiation has taken place; sex bias in gene expression has been documented in multiple species including fruit flies, the worm, the mouse, chicken, the flour beetle and zebra fish. most studies have found male bias in gene expression, with more genes upregulated in mature males than mature females. this is hypothesized to be due to strong sexual selection in males in the form of female choice, and/or sperm competition. patterns of sex-biased gene expression are extremely variable both within and between species, and there are marked differences in the proportion, number, and identity of genes that are differentially expressed depending on the tissue type and developmental stage examined. for example 4% of the adult whole-body transcriptome exhibited sex-biased expression in mice to as high as 88% in drosophila. sex-biased expression also varies within different tissues of the same individual. for example, yang et al. studied sex-biased expression in mus and found that brain tissue exhibits fewer sex-biased genes than muscle, adipose and liver tissue. other studies report similar differences with the greatest percentage of sex-biased expression frequently being in the gonad transcriptome of sexually mature adults. although sex-bias in expression seems to be a common phenomenon in many different species, most studies have used sexually mature specimens or individuals that have already undergone differentiation of the gonads. however, substantially less information is available on the level of sex-biased expression in un-differentiated embryos. studies in the pre-implantation stage of embryogenesis in mouse found that roughly 3% of the transcriptome is differentially expressed between the sexes. in chicken, levels of sex-biased expression in embryos after differentiation have been compared with adults, and unsurprisingly found far less sex-biased expression in embryos. this suggests that the amount of sex-biased expression in the transcriptome changes throughout development through the juvenile and adult stages, and that sex-biased expression patterns may be present very early in development. the extent to which biases in gene expression occur prior to gonad differentiation, however, have not been fully explored. genetic differences between the sexes also occur due to the presence of heteromorphic sex chromosomes. genetic sex determination is most often thought of as being initiated by a switch in one sex that begins the gonad differentiation cascade. however, despite the widespread importance of genetic sex determination across taxonomic groups, surprisingly little is known about the genes involved in the cascade in fishes. unlike mammals, fish species with gsd appear to use a range of different loci, with dmy in medaka the only known example of a primary switch. karyotype and inheritance studies have shown there are a number of fish species that have gsd, but the loci responsible have yet to be determined. sex can also be determined by a set of genes presumably on both the sex chromosomes and the autosomes that act in concert to determine sex, e.g. many species of livebearers including the platyfish xiphophorus maculatus. rainbow trout are known to have gsd, but the mechanisms triggering the differentiation of testes and ovaries during development have not yet been revealed. however, a number of studies have identified candidate sex determination genes that are differentially expressed as early as the onset of exogenous feeding, and presumably during the differentiation process, but no studies have yet identified the sex determination gene on the y chromosome. moreover, these studies evaluated candidate genes known to be important for sex determination in other species. a whole-transcriptome approach may be better suited for determining as-yet unidentified sex determination genes, while also revealing the patterns of sex bias in gene expression at the earliest time points in development. our aims here were twofold: 1) to evaluate differences in gene expression between the sexes in pre-hatch embryos of rainbow trout on a genome-wide level using a microarray analysis to: a) determine how much of the transcriptome appears to be differentially expressed between the sexes early on in development, and b) identify genes which are known to be involved in the sex differentiation cascade in model organisms, and 2) to characterize the expression profiles of a set of candidate sex genes that are known to be differentially expressed at the onset of exogenous feeding in an attempt to ascertain the time point earlier in development when these genes are being turned on. this study focuses on three development time points: days post-fertilization, dpf and dpf. this combination of approaches identifies sex bias in gene expression at some of the earliest time points in development on a genome-wide level, even before the onset of sex determination and phenotypic sex differentiation, while also evaluating the timing of genes known for their role in the sex determination cascade in other species.  samples used in this experiment were from a fourth generation backcross between two clonal lines of rainbow trout, as described in xu et al.. one line possessed alleles for fast development, and the other line possessed alleles for slower development at a major qtl for embryonic development rate. here we use samples from this cross to investigate sex differences in the expression in the early stages of development of o. mykiss. microarray data results of differential expression between the sexes microarray data from the experiments have been submitted to the gene expression omnibus database http://www.ncbi.nlm.nih.gov/geo/ according to miame guidelines. the series accession number is gse controlling for the false discovery rate in multiple tests, features were identified as differentially expressed between female and male embryos in at least one comparison. more features were differentially expressed between the sexes in samples from osu/cw than osu/osu; these included features from osu/cw in dpf embryos compared to from osu/osu, features in dpf embryos from osu/cw compared to features from osu/osu, and features in dpf embryos from osu/cw compared to from osu/osu. to focus on features with large expression differences, there were features with a minimum of a 3-fold change expression difference between the sexes in at least one comparison. the top features with the greatest differences in expression at and dpf are shown in tables and. of the features differentially expressed at dpf, features were higher expressed in males, and were greater expressed in females. the most common go terms associated with these features were cell cycle regulation, muscle contraction and development and transport. in the dpf samples features were higher expressed in males, and were higher expressed in females. the most common go terms were cell cycle regulation, protein metabolism and lipid metabolism. in the dpf samples features were upregulated in males and features were upregulated in females. the most common go terms were cell cycle regulation, protein synthesis and transport. only a limited number of differentially expressed features between the sexes were shared between different time points; out of features in osu/cw samples and out of in osu samples.. a small number of sex-biased features were shared between genotypes at the same time point with out of at 15dpf, out of at dpf and out of at dpf. these results indicate that very few differentially expressed genes are shared between genotypes, or between developmental time points, suggesting that expression patterns are rapidly changing during early stages of development, and that the individuals with the different qtl genotypes are developing at different rates. cluster analysis grouped the features into discrete clusters. most of these clusters had only a few features, but clusters and had and features respectively. cluster grouped those features that were upregulated in osu/cw females at dpf, and features within cluster showing upregulation in osu/cw males at dpf. clustering analysis suggests that samples from the same developmental time point show more similar expression profiles than samples from different time points from the same genotype. for example, osu/cw samples at dpf are more similar to samples from to osu/osu at dpf than osu/cw samples from either or dpf. this trend is similar in samples from all three time points. as mentioned, previous studies have documented a bias in the number of differentially expressed featured between the sexes. we compared fold change values of differentially expressed features between the sexes to determine if there was bias in the directionality of differentially regulated genes between the sexes. for each gene the fold change represents the magnitude and direction of differential expression between the sexes. there appears to be little difference in the number of genes differentially expressed between the sexes at dpf in both genotypes, with marginally more female expressed genes than male expressed genes in osu/cw. there is a similar difference in the opposite direction in samples from osu/osu where features have a three-fold upregulation in females compared to in males. samples from later developmental time points seem to show higher differences in fold expression between the sexes. at dpf there are more features from both osu/cw and osu/osu that exhibit male bias expression with features with a three-fold difference from osu/cw and features with a three-fold difference from male osu/osu samples. very similar patterns were seen in samples from dpf with males from both osu/cw and osu/osu showing a greater number of three-fold difference genes than females. the majority of the sex-biased genes did not show big differences in expression between the sexes, and showed ratios less than 2: go enrichment analyses go enrichment analyses determined that only go terms identified from the differentially expressed features between the sexes were upregulated compared to the whole 16k grasp chip. these go functions were cellular macromolecule metabolic process,, macromolecule metabolic process and cellular protein metabolic process. when comparing the features identified from one sex to the total set of differentially expressed genes, go terms were statistically different between the sexes. of these were differentially expressed in osu/cw samples and were differentially expressed in osu/osu samples. for the osu/cw samples, go terms were upregulated in males compared to that were upregulated in females. for the osu/osu samples, go terms were upregulated in males compared to go terms in females. these differences suggest that in addition to more features being upregulated in males, more go terms are also upregulated in males compared to females. the ratio of go terms upregulated in males compared to females changed between the three developmental time points. for example, within osu/cw samples there were go terms upregulated in males compared to go terms in females at dpf, go terms in males to go terms in females at dpf and finally one go term being upregulated in males at dpf and none in females. these patterns suggest that the greatest differences in the functional categories differentially regulated between the sexes were at and dpf. numbers of functional go categories that were enriched from osu/osu were generally low but again the ratio of male to female upregulated genes varied between time points. all six go terms differentially expressed at dpf were upregulated in males, whereas the go terms differentially expressed between the sexes at dpf were upregulated in females at dpf. some enriched go categories were shared between different developmental time points and between different genotypes with some terms switching between upregulation in males and upregulation in females. for example structural molecular activity was upregulated in males at dpf in samples from osu/cw, but upregulated in females at dpf in osu/cw. however, some go terms that were shared between the two genotypes showed similar patterns of expression such as cellular biosynthesis process, which was upregulated in males in samples from osu/cw at dpf and the same result was found in samples from osu/osu at dpf. enrichment analysis on the clusters shown in figure failed to identify any go terms that were differentially expressed compared to both the complete and sex-biased datasets. this suggests that patterns of similar expression within clusters are not due to shared functionality. thirty-eight go terms were found to be significantly under expressed in clusters but this is almost certainly due to small cluster size rather than a cluster of genes with shared function. qrt-pcr of candidate sex genes we further examined whether genes with a potential role in sex determination were differentially expressed between male and female embryos. these genes were identified using two approaches: 1) based on go term analyses of differentially expressed genes on the microarray, candidate sex determining genes were identified for further study. of these only amplified with qrt-pcr methods. 2) of candidate sex genes described in baron et al we investigated genes. all genes amplified product of the expected size. note that cyp19a1a was the only candidate sex gene identified from both the microarray and was in baron et al's study. both sets of genes were examined in the original three developmental time points, and a subset of six genes from baron et al. as well as zonadhesin and cyp19a1a identified from the microarray were investigated at three additional developmental times points. there is a common pattern in six of the genes which show a higher peak of expression in dpf samples than the other developmental time points. at dpf, the expression was higher in the males for ovol wt foxl2b and dmrt and higher in the females for zonadhesin and cyp19a1a. looking at each time point only ovol shows a significant difference in expression between the sexes with higher expression in males than in females. sex was significant when it was considered as an interaction term with dpf and with qtl. qtl was a significant factor in ovol zonadhesin and cyp19a1a although in all cases it was only marginally significant. the other sex genes did not show a significant relationship with sex either as a main or interaction term except solt where the sex*dpf interaction was significant. most of these genes showed very similar levels of expression across all three developmental time periods. most of the candidate sex genes except for the ones discussed above show constant low levels of expression throughout all developmental time points and in both sexes. scatter plots shown in figure plot the mean relative expression values of the male and female samples per gene averaged between the early developmental time points and the late developmental time points. of note, nr5a appears to be highly expressed in male samples from early developmental time points, whereas the other samples seem roughly similarly expressed between males and females. ovol has a two fold increase in expression in males than in females in later stages in development. cyp19a1a and nr0b show higher expression in females than in males at later stages of development. candidate gene linkage mapping where possible, candidate sex genes were mapped to determine if any map to the putative sex determining region on the rainbow trout sex chromosomes. four of the candidate sex determining genes have already been mapped in rainbow trout, and so were not mapped here; none of these map to the sex chromosome. of the remaining candidate sex determining genes, three failed to produce clean sequence product, despite primer redesign and optimization. this suggests that these genes are duplicated in the rainbow trout. of the eight remaining genes all had at least one polymorphic base within the sequence and were genotyped in the osu × cw doubled haploid mapping population. one of these genes did not produce significant linkage with any other marker and remains in an unmapped region of the genome. none of the remaining seven genes mapped to the sex linkage group. discussion patterns of gene expression between the sexes repeatedly, microarray experiments have found that a substantial portion of the transcriptome is differentially expressed between the sexes. most of these studies have focused on model organisms such as drosophila, c. elegans and mus, with relatively few studies on non-model species that lack a complete genome sequence. moreover, very few studies have included individuals that have not yet undergone sexual differentiation. our study mined data from a microarray project to find a total of genes that showed a significant difference in expression between the sexes before the completion of sexual differentiation. in so doing, it is not only one of the few studies to evaluate sex bias in the transcriptome in a non-model species, but is also one of the first studies to examine expression patterns between the genotypic sexes in embryos before the completion of gonad differentiation. most genes differentially expressed between the sexes in this study are involved in processes such as cell cycle function and general structure, and are not necessarily associated with sexual development. this suggests that there are inherent differences in the transcriptome of male and female rainbow trout, and that these differences are present early in development. other microarray studies in ovarian o. mykiss samples also found a wide range of genes differentially expressed in the ovaries, many of which had no obvious sex function. these included transcripts that encode various elastases, cathepsins, proteases and immune function transcripts, suggesting that rainbow trout are exhibiting broad differences in transcriptome expression between the sexes throughout life. an expression quantitative trait loci study in an f cross between "dwarf" and "normal" lake whitefish found strong evidence for more eqtls in females than in males. however, the genes underlying these eqtls were found not to be differentially expressed between the sexes, strongly suggesting pleiotropic sex-linked effects in the transcriptome, at least within white muscle, the tissue used for microarray analysis. here we find a relatively low proportion of the transcriptome that exhibits sex bias in gene expression when compared to other studies, but it is important to consider the developmental stages of the samples. in our study, % of the features showed sex-biased gene expression in at least one of the three developmental time points in one of the two studied qtl genotypes. these numbers are low when compared to results in drosophila, where a much higher percentage of sex-biased expression in mature adults with most studies reporting a number near 50%. studies from whole individual adult tribolium and adult zebra fish report around 20% and 38%, respectively. similar numbers have been found in mice; although note that like in drosophila, sex-biased expression is very variable depending on tissue type with up to 72% of genes from liver to 13% of genes from brain. taken together these studies suggest that sex-biased gene expression is commonplace, however it seems to be variable both within and among species and variable with regard to tissue type and developmental stage. there are a number of potential reasons for why we see fewer genes showing sex-biased expression compared to prior studies. firstly, most other studies have focused on developmental stages that have occurred after sex differentiation, so presumably the samples used in such studies were actively expressing genes linked to sex and gamete functions. in salmonids gonad differentiation tends to occur soon after the onset of hatching which is around our oldest time point of sampling. so it is perhaps surprising that we did not find more sex-biased genes, and genes with an obvious sex function in samples from our oldest developmental point. secondly, the absolute numbers of genes that are being expressed in embryos are less compared to the number of genes that are expressed in adults. many of the sex and gamete function processes do not begin at such early stages of development and transcripts of such genes, presumably, would not be present. thirdly, the microarray is not a complete survey of the transcriptome and so there will be sex genes and sex-biased genes that were not surveyed. for example, none of the candidate sex genes chosen for qrt-pcr study were on the microarray. although these genes did not show significant differences in expression between the sexes there are many other genes that have been shown to be differentially expressed at the onset of exogenous feeding in o. mykiss that were not studied here. these reasons are not mutually exclusive, and all three undoubtedly help explain our results. other studies have also found low levels of sex-bias expression in embryos, for example roughly % of the chicken embryo gonad transcriptome is female biased and % male biased. this number increases to % and % in the adult gonads of females and males, respectively. although the proportions of the transcriptome that was differentially expressed in chicken is similar to this study in rainbow trout, it is important to note key differences in the study designs. in our study, whole embryos were used from developmental time points prior to gross morphological differentiation of the gonads, while in gonad tissue was used from embryos after sex differentiation. previous studies have examined sex-bias in gene expression to test hypotheses about the influence of sexual selection on gene expression. as other authors have suggested, a male bias in gene expression could provide support for a higher rate of functional evolution in genes important for sex and reproduction in males, where selective pressure on males in the form of male-male competition for mates is high. we however, found roughly equal expression between the number of male biased and female biased genes, with only slightly higher number of male biased genes in samples from both lines at dpf and slightly higher number of female biased genes in osu/osu at dpf. moreover, our enrichment analysis failed to identify any key functional processes with sex bias in gene expression and moreover within and among genes there was no consistent directionality to sex-biased in expression across time points. all of the sex-biased genes and candidate sex determining genes showed spiky patterns of expression, suggesting rapid change in the transcriptome of the developing embryo. many other studies using adult individuals report large differences in the number of sex-biased genes with, frequently, many more male-biased than female-biased genes, supporting the idea that sex bias towards males could be a product of sexual selection. however, studies in both drosophila and zebra fish have found more female-biased genes than male-biased genes. these studies reinforce the idea that results can be very dependent on which transcriptomes are being investigated. for example, studies in the gonads and whole body transcriptome of drosophila produce different results. in a hypothetical situation where sexual selection arises through sperm competition, sex bias may be expected to occur only at the level of the gonad and potentially swamped by signals from other tissues in whole body analyses. in contrast, sex bias in gene expression arising from selection in the form of male-male competition through alternative behaviors, ornamentation, or other'phenotypes' outside of the gonads themselves could be found in the transcriptome of multiple tissues or at the level of the whole body. moreover, the way in which animals used for transcriptome studies are maintained prior to tissue sampling can have a large effect. for example, separated male and female zebra fish whereas kept both sexes together immediately prior to sampling and in so doing, may have modified the expression of sex related genes. in other words, it is clear that the way individuals are reared prior to sampling can have an effect on the results of the experiment. this potentially could lead to misleading interpretations of the effects of sexual selection on sex-bias expression, as behavioral and physiological interactions among members of the opposite sex can elicit changes in gene expression. finally, the degree to which gene expression early in life is genetically correlated to gene expression late in life when sexual selection is prominent also remain as questions to be explored in future studies of model and non-model organisms where sexual selection is prevalent. candidate sex determination genes studies on post hatch rainbow trout indicate that genes known to be involved in the sex determination process are differentially expressed between the sexes. however, we are still unclear as to when expression of these genes is initiated as the majority of the candidate sex determination genes were differentially expressed at the earliest point of sampling. the o. mykiss embryos in our study were reared at 11°c and hatched at dpf, and therefore, our sampling procedure should span the point of sex determination. it has been found that differences in expression between the sexes in these genes tend to spike more at specific time points, rather than show consistent differences in multiple development points. our results show a similar trend in that out of candidate sex genes only ovol showed a statistically significant difference in expression between male and female samples across all developmental time points sampled, although many genes showed differences in expression at specific time points. in the fruit fly ovol is required for female germ line determination and differentiation. ovol is also expressed in sheep ovaries immediately prior to gonad differentiation. here we find upregulation of ovol in males at 15dpf and females at dpf. of the other genes that showed a significant difference between the sexes, nr5a expression has been shown to be directly affected by sox in mammals and wt in reptiles and is turned on after male determination. with so few candidate genes showing differences between the sexes, it is important to give pause as to why this might be. results from studies in mouse suggest that the window of differential expression of sex determining genes is small. perhaps we have missed the specific time point when these genes are upregulated? increasing sampling effort just before the onset of hatch to the point of first exogenous feeding, and using a whole-transcriptome approach, could prove fruitful. mapping of candidate sex differentiation genes the fact that none of the candidate sex genes mapped to the sex chromosome in this study further suggests that a master control gene for the determination of sex in o. mykiss remains elusive. linkage mapping of other candidate sex determining genes in other populations of o. mykiss also found no genes mapping to the sex chromosome. a total of candidate sex genes have been mapped prior to this study, including different transcripts of wt, three sox loci and sox genes, amh, dmrt and nrob. the addition of fst, ovo zonadhesin, igf foxl2a, cyp19a1a and wt brings the total number of loci to the sex chromosomes of fishes are at the early stages of differentiation and this in part helps explain why only one sex determining gene is known in any fish. in order to identify the master sex gene in rainbow trout a concentrated sequencing effort of the sex chromosome is needed.  here we found evidence that a proportion of the transcriptome is differentially expressed between the sexes at early stages of development in rainbow trout, prior to the morphological differentiation of the gonads. most of the features were not connected with obvious sex function suggesting that there is sexual dimorphism in gene expression even prior to sex differentiation, and the processes that are differentially regulated are varied and complex. the early age of our sampling precluded us from looking at the transcriptome of the gonads; however the fact that so many genes are differentially expressed so early in development is a novel and interesting find. in addition, we found evidence that several candidate sex determining genes showed a significant difference in expression between males and females. in all cases, this difference was seen at one or at most two time points only, suggesting that the transcriptome of o. mykiss embryos goes through rapid changes early in development. it is possible that our sampling scheme has missed the key window of sexual differentiation in o. mykiss, to that end a more thorough sampling effort to include embryos after dpf and before with dpf as well as earlier points in development could further our understanding of embryonic development in o. mykiss. 
here we use data from a microarray and qrt-pcr to identify genes differentially expressed between the sexes at three time points in pre-hatch embryos, prior to the known timing of sexual differentiation in this species. these differences are especially noticeable once gonad differentiation and maturation has occurred, but less is known at earlier stages of development. in rainbow trout qrt-pcr studies have found significant differences in expression of several candidate sex determining genes. the microarray study revealed differentially expressed features between the sexes with roughly equal numbers of male and female upregulated features across time points. candidate gene analysis revealed sox dmrt nr5a and wt were upregulated in males at some time points and foxl ovol fst and cyp19a1a were upregulated in females at some time points. there are large differences between the sexes at the genetic level; these differences include heterogametic sex chromosomes and/or differences in expression of genes between the sexes. most of the differentially expressed genes on the microarray were not related to sex function, suggesting large scale differences in gene expression between the sexes are present early in development.
 there are large differences between the sexes at the genetic level; these differences include heterogametic sex chromosomes and/or differences in expression of genes between the sexes. in rainbow trout qrt-pcr studies have found significant differences in expression of several candidate sex determining genes. however, these genes represent a very small fraction of the genome and research in other species suggests there are large portions of the transcriptome that are differentially expressed between the sexes. these differences are especially noticeable once gonad differentiation and maturation has occurred, but less is known at earlier stages of development. here we use data from a microarray and qrt-pcr to identify genes differentially expressed between the sexes at three time points in pre-hatch embryos, prior to the known timing of sexual differentiation in this species.  the microarray study revealed differentially expressed features between the sexes with roughly equal numbers of male and female upregulated features across time points. most of the differentially expressed genes on the microarray were not related to sex function, suggesting large scale differences in gene expression between the sexes are present early in development. candidate gene analysis revealed sox dmrt nr5a and wt were upregulated in males at some time points and foxl ovol fst and cyp19a1a were upregulated in females at some time points.  this is the first study to identify sexual dimorphism in expression of the genome during embryogenesis in any fish and demonstrates that transcriptional differences are present before the completion of gonadogenesis. sex-biased expressionovarytestismicroarraycandidate genes 