BACKGROUND
from the selfish dna perspective  <cit> , gonads are fundamentally important organs of an organism.
during the first meiotic division of gametogenesis, crossing-over enhances the re-assortment of information carried in parental dna molecules and virtually immortal genetic information is then transferred to next generations of mortal individuals via the final products of gametogenesis, spermatozoa and eggs.
moreover, testes and ovaries are the only niches where the paternal and maternal dna interacts with a different environment.
the dissimilar gonadal environment enables sex-dependent epigenetic modifications of paternal and maternal dna such as reactivation of the x chromosome in female germ cells  <cit> , inactivation of a single x chromosome in pachytene spermatocytes  <cit>  or differential establishment of imprinting marks on paternally or maternally imprinted genes  <cit> .
spermatogenesis also serves as an important checkpoint filtering out many de novo occurring gene mutations  <cit>  and chromosomal rearrangements  <cit>  by making their carriers sterile.
a special form of meiotic checkpoint is represented by hybrid sterility, which facilitates creation of new species.
obeying the haldane's rule, hybrid sterility preferentially affects gametogenesis in testis in species with heterogametic  sex  <cit> .
molecular analyses of these phenomena are hindered by the fact that testis is a complex organ with many types of intimately intermingled somatic and germline cells.
moreover, the spermatogenic differentiation is almost impossible to achieve ex vivo, in a cell culture system.
the main cell types can be fractionated, via gravity sedimentation, centrifugal elutriation or fluorescence activated cell sorting, but the time required can be fairly long to exclude possible artificial changes of mrna levels.
in the present work we used serial analysis of gene expression   <cit>  to characterize the transcriptome of mouse total testis.
we created a catalogue of genes expressed in the adult mouse testis of the c57bl/6j  inbred strain.
the b <dig> inbred strain has been chosen because its genome has been recently sequenced  <cit>  and since it has been selected as a recipient strain for creation of two sets of chromosome substitution strains, c57bl/6j-chr#a/j  <cit>  and c57bl/6j -chr#pwd/ph .
except for the characterization of the total testis transcriptome, we compared our data with the publicly available sage library from adult testis somatic cells  <cit>  and other sage libraries constructed from normal mouse tissues.
furthermore, we were interested in the organization of testicular genes in the mouse genome and we present here a detailed bioinformatic analysis of the distribution of testicular genes between the x chromosome and autosomes, and the positional clustering of genes with preferential expression in testis.
RESULTS
characterization of the sage libraries of b <dig> mouse testis
we have constructed two high-quality sage libraries, tt  <dig> and tt  <dig>  from the total mouse testis of adult b <dig> males .
the libraries contain  <dig>  <dig>  and  <dig>  <dig>  tags corresponding to  <dig>  <dig> and  <dig>  <dig> unique tags, respectively.
the tags with abundance >  <dig> comprise  <dig>  <dig>  and  <dig>  <dig>  of the total tag mass but only  <dig>  <dig>  and  <dig>  <dig>  of the unique tags, respectively.
the high average number of tags per clone  and low contamination with linker-derived tags  and duplicated ditags  indicate that the sage libraries are of high quality.
both total testis sage libraries provided similar gene expression profiles , which suggests a good reproducibility of sage data.
however, a certain variation was observed in the tag abundances when  <dig>  <dig> unique tags found in both total testis sage libraries were compared by monte carlo simulations.
three hundred thirteen tags exhibited significant differences in their frequency between tt  <dig> and tt  <dig> libraries at p <  <dig>   representing non-hereditary variations in transcription profiles and variations introduced by the experimental process.
the fold factor value  for  <dig>  % of the compared tags ranged between - <dig>  and  <dig>  .
dot plot comparison and fold factor distribution graphs  depict the similarity of both total testis libraries.
despite this variation, the sage method produced reproducible gene expression profiles and the libraries could be combined into the total testis sage library  with the total of  <dig>  <dig> tags and  <dig>  <dig> unique tags.
the raw data from the total testis sage libraries are deposited in the geo repository  <cit>  under accession numbers gsm <dig>  and gsm <dig> .
the set of tags with abundance >  <dig> in tt 1+ <dig> sage library with reliable tag identification is listed in additional file  <dig>  the testis sage libraries are also freely available for interactive exploration and analysis in the mouse sage site database  <cit> .
* excluding tags from duplicated ditags and linker-derived tags
tag-to-gene identification in the b <dig> testis transcriptome
tag-to-gene identification in the tt 1+ <dig> sage library was evaluated using three different criteria applied to the sagemap database.
the first was the most commonly used sagemap reliable mapping  <cit> .
the second was a modified approach based on the sagemap full mapping file and implemented in the mouse sage site database  <cit> .
in this approach, the tag-to-gene associations were considered reliable if supported by tags extracted from at least one mrna sequence  or at least  <dig> ests with a poly signal or at least  <dig> ests with no poly signal  <cit> .
the third approach  was also based on the sagemap full mapping file.
tag-to-gene associations were considered reliable if supported by tags extracted from at least one mrna sequence.
the  <dig>  <dig> tags with tag count >  <dig> in the tt 1+ <dig> library were subjected to the sagemap reliable mapping that could identify  <dig>  % tags to unigene clusters .
when a more restricted reliable mapping from the mouse sage site was used, only 63% tags were identified to unigene clusters  and about  <dig>  % tags had unreliable identification to one or more unigene clusters.
based only on the tags extracted from mrna sequences, the rna evidence mapping identified  <dig>  % tags to unigene clusters  leaving  <dig> % tags with unreliable identification.
using any of the tag identification methods,  <dig>  % tags could not be identified to unigene clusters and may be associated with novel genes.
further in this work, we used mouse sage site or rna evidence mapping appropriately for a particular analysis .
functional categories of genes expressed in total testis
we associated genes and their corresponding tag counts to functional categories from the biological process ontology of go database  <cit>  .
in the total testis transcriptome, we observed more than  <dig> genes involved in metabolism, particularly in the protein metabolism  and nucleic acid metabolism .
as expected, the genes associated with spermatogenesis , chromosome organization, cell cycle and cell differentiation were highly expressed.
notably represented gene functions also included transport , signal transduction  anchor protein  <dig>  pdz domain containing  <dig>  wd repeat domain 12), cytoskeleton organization  and apoptosis .
from the mitochondrial genome, atp synthase  <dig>  cytochrome c oxidase i and iii were the most highly expressed genes.
comparing the transcriptomes of total testis and adult testis somatic cells
the mouse testis is composed of two main cell types with principally different origin and functions, the germ cells that differentiate from spermatogonia to mature spermatozoa and the somatic cells that carry out all supportive functions to make the spermatogenesis and reproduction possible.
seminiferous tubules of the adult testis consist of approximately 88% germ cells and 12% somatic cells including myoid and sertoli cells  <cit> .
we compared our total testis sage library  with a sage library constructed from the somatic cells of adult testis .
this library was created from testes largely devoid of germ cells  <dig> days after busulphan treatment  <cit> .
the sage library sizes are similar for tt 1+ <dig> and the adult testis somatic cells  comprising  <dig>  <dig> and  <dig>  <dig> tags, respectively.
the number of unique tags  as well as the proportions of tags with abundance >  <dig> to the total tag mass  and to the number of unique tags  are also comparable .
as anticipated, comparison of tt 1+ <dig> and atsc sage libraries using monte carlo simulations revealed extensive differences in gene expression between total testis and somatic cells of adult testis.
out of the  <dig>  <dig> unique tags in tt 1+ <dig> and atsc libraries, the simulations detected significantly different tag abundances in  <dig>  <dig> tags at p <  <dig> .
concerning the fold factor,  <dig> % of the compared tags stretch in the range between - <dig>  and  <dig>  .
at the extreme ends,  <dig> tags reach > 10-fold increase in tag counts in the atsc library  and  <dig> tags reach > 10-fold increase in the tt 1+ <dig> library  .
dot plot comparison and fold factor distribution graphs for tt 1+ <dig> and atsc transcriptomes illustrate their dramatic dissimilarities .
genes with predominant expression in the germinal or somatic component of testis
to sort out subsets of genes with predominant expression in either germinal or somatic cells of testis we applied tentative criteria to account for the presence of somatic cells in tt 1+ <dig> and for residues of germ cells in atsc.
predominant expression of a gene was considered if the corresponding tag was significantly more frequent in one of the libraries  and exhibited at least fivefold enrichment of tag counts .
according to this criterion a set of  <dig> genes is expressed predominantly in germ cells and  <dig> genes are expressed mainly from the somatic part of the testis .
moreover, we identified  <dig> tags corresponding to  <dig> genes encoded in the mitochondrial genome .
a gene coding for cytochrome c oxidase iii  displayed two tags separated by  <dig> bp in mt-co <dig> gene mrna.
one isoform was predominantly present in the atsc library and the other was observed exclusively in the tt 1+ <dig> library.
substantial over-expression of mitochondrial cytochrome c oxidase complexes i, ii, iii and nadh dehydrogenase  <dig> and  <dig> was noted in testicular somatic cells .
exploring the dissimilarity of testis transcriptomes and transcriptomes of other mouse tissues
we examined the similarity of b <dig> testis transcriptomes to other available mouse sage transcriptomes created from normal and diseased bulk tissues by hierarchical clustering.
thirty-two sage libraries containing  <dig>  <dig> unique tags  were used as input in this analysis .
we computed pair-wise library distances based on differences between normalized tag counts  <cit>  and used the average agglomerative method for hierarchical clustering due to the highest cophenetic correlation .
in the dendrogram of dissimilarities the two total testis sage libraries, tt  <dig> and tt  <dig>  cluster together in contrast to the library from somatic cells of the adult testis .
the atsc library is located separately and close to the libraries created from heart, liver and kidney in accord with the somatic origin of all these tissues.
interestingly, another sage library created from somatic cells of the fetal testis did not cluster with the atsc library, but was placed close to the libraries from developing limbs, juvenile retina and whole brains.
another cluster consists of the six libraries generated from the whole adult kidneys.
several specialized brain tissues form a cluster with a brain tumor tissue .
an additional small cluster groups three libraries created from whole brain samples .
nonrandom representation of testis-expressed genes on the x chromosome
previous works have shown a significant enrichment of prostate- and spermatogonia-specific genes on the x chromosome when compared to autosomes  <cit> .
we asked what proportion of testis-expressed genes maps to the x chromosome and compared it with the proportion of x-linked genes expressed in somatic  tissues.
furthermore, we examined whether the proportion of testis-specific genes on the x chromosome differs from the proportion of x-linked tissue-specific genes in somatic tissues.
out of the  <dig>  <dig> genes expressed in sage libraries from total testis, adult testis somatic cells and  <dig> somatic tissues   we considered only genes identified by corresponding tag count >  <dig>  the proportion of genes expressed from the x chromosome in a pool of  <dig> somatic tissues was  <dig>  % .
although the proportions of x-linked genes in somatic tissues were uneven, there were no significant differences among the tissues .
in testicular somatic cells, we observed  <dig> % x-linked genes , while in total testis only  <dig>  % genes  were expressed from the x chromosome .
we can conclude that the number of expressed x-linked genes is underrepresented in the transcriptome of total testis.
the same set of  <dig>  <dig> genes was examined for the distribution of tissue-specific genes on autosomes and the x chromosome.
we compared the genes specific for either total testis  or adult testis somatic cells  in conjunction with somatic  tissue-specific genes.
a gene was considered to be tissue-specific if it was expressed only in one tissue type .
moreover, the corresponding tag count >  <dig> was required to guarantee that the gene is truly expressed.
the tissue-specific genes were assigned to chromosomes according to the locuslink database and the significance of their chromosomal distribution was evaluated by permutations  and confirmed by fisher's exact test .
out of the  <dig> genes specific for total testis  <dig> % mapped to the x chromosome .
essentially the same proportion of x-linked genes was found for genes specific for  <dig> somatic  tissues.
in testicular somatic cells, we detected only  <dig> tissue-specific genes, but  <dig> % were x-linked .
this is a  <dig> -fold increase in the proportion of testis somatic cell-specific genes on the x chromosome and represents their significant enrichment  in comparison to the genes specific for other tissues.
all the x-linked testis-specific genes were subjected to blast against the whole x chromosome, which revealed no duplicated genes.
the results from the permutation analysis indicate a significantly increased amount of testis-specific genes on the x chromosome in somatic cells of the testis when compared to autosomal testis-specific genes.
the genes specific for  <dig> somatic tissues did not show a significant preference for the x chromosome.
the list of x-linked genes expressed in total testis and testicular somatic cells with indicated testis-specific genes is available in additional file  <dig>
chromosomal clustering of genes with preferential expression in testis
based on the data from testis and other publicly available sage libraries  we identified genes with preferential expression in testis by preferential expression measure   <cit> .
pem score controls for the genes that are highly expressed in many tissues  and reports positive values for over-expressed genes and negative values for under-expressed genes in a given tissue.
large positive pem scores for a gene in a particular tissue indicate that the gene is unusually highly expressed in that tissue, relative to its expression in other tissues  <cit> .
we considered a gene to be preferentially expressed if the pem score reached at least  <dig> % of the maximum pem value encountered in that tissue.
using this criterion, we scored expression of genes in total testis or testicular somatic cells in conjunction with their expression in  <dig> other tissues .
further we analyzed the genome organization of genes preferentially expressed in testis.
we evaluated the expression of  <dig>  <dig> genes among the studied tissues and for  <dig>  <dig> genes we were able to assign a genomic position according to the ncbi mouse genome assembly .
the genomic position was resolved for  <dig>  <dig> and  <dig>  <dig> genes expressed in total testis and testicular somatic cells, respectively, including  <dig>  <dig>  and  <dig>  <dig>  preferentially expressed genes, respectively .
to evaluate the gene order of preferentially expressed genes in testis and to eliminate the effect of tandem duplications we purged the whole mouse genome of tandemly duplicated genes .
the tandem duplicate-free genome resulted in total of  <dig>  <dig> locuslink genes and preserved  <dig>  <dig> and  <dig>  <dig> genes preferentially expressed in total testis and testicular somatic cells, respectively.
using a search with a sliding window  we localized chromosomal clusters containing at least three adjacent preferentially expressed genes .
similarly, we searched for clusters with at least three preferentially expressed genes among the six adjacent genes  to include genes that could be preferentially expressed but did not pass the above criterion for preferential expression or their expression was not detected by sage.
by definition, the tight clusters form a subset of the loose clusters.
the chromosomal distribution of clusters with preferentially expressed genes in testis is illustrated in figure  <dig>  we observed  <dig> and  <dig> genes preferentially expressed in total testis and testicular somatic cells located in  <dig> and  <dig> tight clusters, respectively.
two hundred and thirty and  <dig> genes preferentially expressed in total testis and testicular somatic cells resided in  <dig> and  <dig> loose clusters, respectively .
two of the tight clusters and eight of the loose clusters shared preferentially expressed genes between total testis and testicular somatic cells.
statistical analysis revealed that the observed number of preferentially expressed genes located in tight clusters was  <dig> -fold and  <dig> -fold higher for total testis and testicular somatic cells, respectively, than the average number of preferentially expressed genes located in clusters in randomized genomes .
although only slighly higher  than the average in randomized genomes, the observed number of preferentially expressed genes in testis located in loose clusters was still significant in case of total testis and nearly significant in case of testicular somatic cells .
not surprisingly, the most highly expressed genes detected in total testis and involved in spermatogenesis  formed one of the tight clusters on chromosome  <dig>  the results indicate a nonrandom distribution of the genes preferentially expressed in total testis and testicular somatic cells into chromosomal clusters, which did not arise from tandem duplications.
comparing the b <dig> and bdf <dig> total testis transcriptomes
in a recent study focused on senescence changes in testis, a modified sage method was used to generate digital gene expression profiles of total testis from 3- and 29-month-old mice of the bdf <dig> strain and 14-month-old mice of the samp <dig> strain that exhibits an accelerated senescence  <cit> .
because of the different anchoring enzyme  used in construction of the libraries and the limited availability of data from the bdf <dig> testis transcriptome, we could perform only a rough manual comparison of our b <dig> testis transcriptome  and the combined bdf <dig> testis transcriptome from 3- and 29-month-old bdf <dig> mice .
we focused on the most highly expressed testicular genes in gnf mouse atlas v <dig>  <cit>  that were detected by affymetrix genechips.
a set of  <dig> highly expressed genes in testis  was organized with sage tag counts from b <dig> and bdf <dig> testis .
in the b <dig> total testis, we detected  <dig> out of  <dig> genes .
in contrast, only  <dig> genes were detected in the bdf <dig> testis library,  <dig> genes were missing due to the absence of rsai restriction site in the transcript and for  <dig> other genes the expression data from bdf <dig> testis were not publicly available.
furthermore, out of the  <dig> highly expressed genes in testis,  <dig> genes were among the top  <dig> most expressed genes in the b <dig> total testis library, but only  <dig> genes were among the top  <dig> most expressed genes in the bdf <dig> total testis library.
it appears that our sage data from the b <dig> testis transcriptome shows better correspondence to the microarray data than the data from the transcriptome of bdf <dig> testis.
discussion
serial analysis of gene expression is a high-throughput method for building a catalogue of expressed genes and their expression levels of "normal" as well as diseased or genetically variant tissues and organs  <cit> .
the digital character of sage data enables addition and direct comparison of different sage libraries, provided they were built with the same anchoring enzyme and originated from individuals of the same species.
the utilization of such global transcriptome databases is multifold, including positional cloning of mutations or quantitative trait loci  <cit> , functional genome annotation  <cit>  or analysis of a nonrandom gene order  <cit> .
admittedly, the sage, as used in this work, has several limitations, including a significant proportion of repetitive and low complexity tags.
the sage is obviously more labor-intensive than transcriptome analysis based on microarrays.
at present, some of these inconveniencies can be solved by applying longsage or massively parallel signature sequencing technologies  <cit> .
in this study we constructed a sage library of the total testis of the c57bl/6j  mouse inbred strain, compared it with other public available mouse sage libraries and analyzed localization of testis-expressed genes within the mouse genome.
the b <dig> strain was favored for the availability of its high-quality draft genomic sequence  <cit>  and because series of congenics and recently also consomic strains have used the b <dig> strain as a background strain  <cit>  .
the combined total testis sage library, tt 1+ <dig>  consisted of  <dig>  <dig> total tags representing  <dig>  <dig> unique tags.
the tag-to-gene reliable identification method used in mouse sage site  <cit>  was applied to tags with frequency ≥  <dig>  out of these tags,  <dig> %  revealed a reliable match to single and  <dig> %  to multiple unigene clusters.
considering the size of the total testis sage library, medium to highly expressed genes are present in the expression profile.
the library size is comparable to the recently published sage library of somatic cells of the mouse testis  <cit>  and almost twice the size of a library constructed from the total testis of bdf <dig> hybrid mice using a modified sage method  <cit> .
contrary to microarrays, sage data are platform independent, which permits the use of unrelated datasets coming from various sources to compare gene expression patterns.
we analyzed the mouse testis transcriptome by comparing our total testis sage library to the adult testis somatic cells library  <cit>  and to additional publicly available sage libraries from  <dig> different tissues.
we recognized three different modes of differential expression.
predominant expression of genes in the germinal or somatic part of the testis, which did not consider expression in other tissues.
preferential expression in testis that was defined by comparing the expression of testis to  <dig> somatic tissues for which sage data were available.
testis-specific expression that was defined by null expression  in sage libraries of seven tissues or organs other than testis.
complete lists of genes predominantly expressed in germinal or testis-somatic cells, as well as the catalogues of genes preferentially expressed in testis and testis-specific genes are available online in additional file  <dig> ,  <dig> and additional file  <dig>
conflicting results have been reported on the representation of male-biased genes on the x chromosome in various species.
spermatogonia-specific genes were found to be an order of magnitude more abundant on the mouse x chromosome  <cit> .
in human, the prostate-specific genes were twice more frequent on the x chromosome, but the female mammary gland- and ovary-specific x-linked genes were not enriched in respective sage libraries  <cit> .
on the contrary, under-representation or absence of male-biased genes on the x chromosome was reported in caenorhabditis elegans  <cit>  and in drosophila  <cit> .
in the mouse, an under-representation of testis-expressed and testis-enriched genes on the x chromosome was also revealed by the analysis of microarray and est data  <cit> .
our present data favor under-representation of x-linked genes in the total testis transcriptome but not in testis-somatic cells.
because the germ cells in different stages of differentiation constitute about 90% of the total cell mass of testis, the data indicate that the deficit of x-linked testis-expressed genes may reflect the lack of transcription from the x chromosome in meiotic cells.
these results are in agreement with the idea of x-chromosome silencing during the first meiotic division, the phenomenon based mostly on circumstantial evidence in flies and mice  <cit> .
thus, transcription at the haploid stage of spermatogenesis is expected for most of the x-linked genes expressed in total testis.
the meiotic x chromosome inactivation seems to be restricted to primary spermatocytes, but sertoli cells, which form the somatic part of seminiferous tubules, may have the x chromosome in the active state.
indeed, in the transcriptome of adult testis somatic cells the proportion of expressed x-linked genes  was more than twice higher than in total testis  and did not differ from the proportion of x-linked genes expressed in non-testis  tissues.
testis-specific genes belong to a wider category of sex-biased genes, which according to the hypothesis of sexually antagonistic genes are more likely to spread on the x chromosome than on autosomes  <cit> .
this is because on the x chromosome they will express their favorable effect in the hemizygous state  while their deleterious effect will be masked by their recessivity in the other sex .
consequently, accumulation of male-specific genes on the x chromosome will be possible by the effect of modifiers that narrow the expression of sex-biased genes only to the male sex  <cit> .
thus, the evolution of sexually antagonistic genes and x inactivation may act as opposing forces on the germline lineage of testis while accumulation of male-specific genes could be expected in somatic cells of testis.
in accord with these assumptions the proportion of x-linked genes specific for total testis did not significantly differ from the proportion of genes specific for other tissues, while we observed a significant  <dig> -fold enrichment of the proportion of x-linked genes specific for testicular somatic cells.
the eukaryotic gene order is nonrandom obviously not only due to shifting of sex-biased genes to and from the x chromosome, but also owing to a nonrandom clustering of genes within chromosomes.
this somewhat unexpected conclusion  is gaining gradual support from global transcriptome analyses of various eukaryotic species   <cit> .
the observed examples of clustering are apparently a mixture of several unrelated phenomena, including large domains of similarly expressed genes in drosophila and humans  <cit> , clustering of housekeeping genes  <cit> , clustering of highly expressed genes  <cit>  or genes with similar expression breadth in regions of similar gc content  <cit> .
in drosophila melanogaster one third of testes-specific genes occur in clusters  <cit> , a phenomenon not reported in any other species.
using pem  <cit>  to define preferentially expressed genes we were able to demonstrate that in the mouse, the genes preferentially expressed in germ cells as well as in somatic cells of testis occur in tight clusters with a frequency  <dig> -fold and  <dig> -fold higher than the expected average frequency in randomized genomes.
moreover, our results indicate that this phenomenon is not merely a consequence of tandem duplications.
further analysis of clustering of testis-expressed genes may reveal new insights into the functional organization of the mammalian genome.
CONCLUSIONS
we identified chromosomal clusters of adjacent genes with preferential expression in testis that contain a significantly higher number of genes than expected by chance.
this phenomenon is not merely a consequence of tandem duplication.
the genes with specific expression in testicular somatic cells are more abundant on the x chromosome, which favors the theory of accumulation of male-biased genes on the x chromosome.
in contrast, the x-linked genes are under-represented in the transcriptome of total testis, which is in accordance with the idea of x-chromosome inactivation during the first meiotic division.

 from the selfish dna perspective, gonads are fundamentally important organs of an organism. during the first meiotic division of gametogenesis, crossing-over enhances the re-assortment of information carried in parental dna molecules and virtually immortal genetic information is then transferred to next generations of mortal individuals via the final products of gametogenesis, spermatozoa and eggs. moreover, testes and ovaries are the only niches where the paternal and maternal dna interacts with a different environment. the dissimilar gonadal environment enables sex-dependent epigenetic modifications of paternal and maternal dna such as reactivation of the x chromosome in female germ cells, inactivation of a single x chromosome in pachytene spermatocytes or differential establishment of imprinting marks on paternally or maternally imprinted genes. spermatogenesis also serves as an important checkpoint filtering out many de novo occurring gene mutations and chromosomal rearrangements by making their carriers sterile. a special form of meiotic checkpoint is represented by hybrid sterility, which facilitates creation of new species. obeying the haldane's rule, hybrid sterility preferentially affects gametogenesis in testis in species with heterogametic sex. molecular analyses of these phenomena are hindered by the fact that testis is a complex organ with many types of intimately intermingled somatic and germline cells. moreover, the spermatogenic differentiation is almost impossible to achieve ex vivo, in a cell culture system. the main cell types can be fractionated, via gravity sedimentation, centrifugal elutriation or fluorescence activated cell sorting, but the time required can be fairly long to exclude possible artificial changes of mrna levels. in the present work we used serial analysis of gene expression to characterize the transcriptome of mouse total testis. we created a catalogue of genes expressed in the adult mouse testis of the c57bl/6j inbred strain. the b inbred strain has been chosen because its genome has been recently sequenced and since it has been selected as a recipient strain for creation of two sets of chromosome substitution strains, c57bl/6j-chr#a/j and c57bl/6j -chr#pwd/ph. except for the characterization of the total testis transcriptome, we compared our data with the publicly available sage library from adult testis somatic cells and other sage libraries constructed from normal mouse tissues. furthermore, we were interested in the organization of testicular genes in the mouse genome and we present here a detailed bioinformatic analysis of the distribution of testicular genes between the x chromosome and autosomes, and the positional clustering of genes with preferential expression in testis.  characterization of the sage libraries of b mouse testis we have constructed two high-quality sage libraries, tt and tt from the total mouse testis of adult b males. the libraries contain and tags corresponding to and unique tags, respectively. the tags with abundance > comprise and of the total tag mass but only and of the unique tags, respectively. the high average number of tags per clone and low contamination with linker-derived tags and duplicated ditags indicate that the sage libraries are of high quality. both total testis sage libraries provided similar gene expression profiles, which suggests a good reproducibility of sage data. however, a certain variation was observed in the tag abundances when unique tags found in both total testis sage libraries were compared by monte carlo simulations. three hundred thirteen tags exhibited significant differences in their frequency between tt and tt libraries at p < representing non-hereditary variations in transcription profiles and variations introduced by the experimental process. the fold factor value for % of the compared tags ranged between - and. dot plot comparison and fold factor distribution graphs depict the similarity of both total testis libraries. despite this variation, the sage method produced reproducible gene expression profiles and the libraries could be combined into the total testis sage library with the total of tags and unique tags. the raw data from the total testis sage libraries are deposited in the geo repository under accession numbers gsm and gsm. the set of tags with abundance > in tt 1+ sage library with reliable tag identification is listed in additional file the testis sage libraries are also freely available for interactive exploration and analysis in the mouse sage site database. * excluding tags from duplicated ditags and linker-derived tags tag-to-gene identification in the b testis transcriptome tag-to-gene identification in the tt 1+ sage library was evaluated using three different criteria applied to the sagemap database. the first was the most commonly used sagemap reliable mapping. the second was a modified approach based on the sagemap full mapping file and implemented in the mouse sage site database. in this approach, the tag-to-gene associations were considered reliable if supported by tags extracted from at least one mrna sequence or at least ests with a poly signal or at least ests with no poly signal. the third approach was also based on the sagemap full mapping file. tag-to-gene associations were considered reliable if supported by tags extracted from at least one mrna sequence. the tags with tag count > in the tt 1+ library were subjected to the sagemap reliable mapping that could identify % tags to unigene clusters. when a more restricted reliable mapping from the mouse sage site was used, only 63% tags were identified to unigene clusters and about % tags had unreliable identification to one or more unigene clusters. based only on the tags extracted from mrna sequences, the rna evidence mapping identified % tags to unigene clusters leaving % tags with unreliable identification. using any of the tag identification methods, % tags could not be identified to unigene clusters and may be associated with novel genes. further in this work, we used mouse sage site or rna evidence mapping appropriately for a particular analysis. functional categories of genes expressed in total testis we associated genes and their corresponding tag counts to functional categories from the biological process ontology of go database. in the total testis transcriptome, we observed more than genes involved in metabolism, particularly in the protein metabolism and nucleic acid metabolism. as expected, the genes associated with spermatogenesis, chromosome organization, cell cycle and cell differentiation were highly expressed. notably represented gene functions also included transport, signal transduction anchor protein pdz domain containing wd repeat domain 12), cytoskeleton organization and apoptosis. from the mitochondrial genome, atp synthase cytochrome c oxidase i and iii were the most highly expressed genes. comparing the transcriptomes of total testis and adult testis somatic cells the mouse testis is composed of two main cell types with principally different origin and functions, the germ cells that differentiate from spermatogonia to mature spermatozoa and the somatic cells that carry out all supportive functions to make the spermatogenesis and reproduction possible. seminiferous tubules of the adult testis consist of approximately 88% germ cells and 12% somatic cells including myoid and sertoli cells. we compared our total testis sage library with a sage library constructed from the somatic cells of adult testis. this library was created from testes largely devoid of germ cells days after busulphan treatment. the sage library sizes are similar for tt 1+ and the adult testis somatic cells comprising and tags, respectively. the number of unique tags as well as the proportions of tags with abundance > to the total tag mass and to the number of unique tags are also comparable. as anticipated, comparison of tt 1+ and atsc sage libraries using monte carlo simulations revealed extensive differences in gene expression between total testis and somatic cells of adult testis. out of the unique tags in tt 1+ and atsc libraries, the simulations detected significantly different tag abundances in tags at p <. concerning the fold factor, % of the compared tags stretch in the range between - and. at the extreme ends, tags reach > 10-fold increase in tag counts in the atsc library and tags reach > 10-fold increase in the tt 1+ library. dot plot comparison and fold factor distribution graphs for tt 1+ and atsc transcriptomes illustrate their dramatic dissimilarities. genes with predominant expression in the germinal or somatic component of testis to sort out subsets of genes with predominant expression in either germinal or somatic cells of testis we applied tentative criteria to account for the presence of somatic cells in tt 1+ and for residues of germ cells in atsc. predominant expression of a gene was considered if the corresponding tag was significantly more frequent in one of the libraries and exhibited at least fivefold enrichment of tag counts. according to this criterion a set of genes is expressed predominantly in germ cells and genes are expressed mainly from the somatic part of the testis. moreover, we identified tags corresponding to genes encoded in the mitochondrial genome. a gene coding for cytochrome c oxidase iii displayed two tags separated by bp in mt-co gene mrna. one isoform was predominantly present in the atsc library and the other was observed exclusively in the tt 1+ library. substantial over-expression of mitochondrial cytochrome c oxidase complexes i, ii, iii and nadh dehydrogenase and was noted in testicular somatic cells. exploring the dissimilarity of testis transcriptomes and transcriptomes of other mouse tissues we examined the similarity of b testis transcriptomes to other available mouse sage transcriptomes created from normal and diseased bulk tissues by hierarchical clustering. thirty-two sage libraries containing unique tags were used as input in this analysis. we computed pair-wise library distances based on differences between normalized tag counts and used the average agglomerative method for hierarchical clustering due to the highest cophenetic correlation. in the dendrogram of dissimilarities the two total testis sage libraries, tt and tt cluster together in contrast to the library from somatic cells of the adult testis. the atsc library is located separately and close to the libraries created from heart, liver and kidney in accord with the somatic origin of all these tissues. interestingly, another sage library created from somatic cells of the fetal testis did not cluster with the atsc library, but was placed close to the libraries from developing limbs, juvenile retina and whole brains. another cluster consists of the six libraries generated from the whole adult kidneys. several specialized brain tissues form a cluster with a brain tumor tissue. an additional small cluster groups three libraries created from whole brain samples. nonrandom representation of testis-expressed genes on the x chromosome previous works have shown a significant enrichment of prostate- and spermatogonia-specific genes on the x chromosome when compared to autosomes. we asked what proportion of testis-expressed genes maps to the x chromosome and compared it with the proportion of x-linked genes expressed in somatic tissues. furthermore, we examined whether the proportion of testis-specific genes on the x chromosome differs from the proportion of x-linked tissue-specific genes in somatic tissues. out of the genes expressed in sage libraries from total testis, adult testis somatic cells and somatic tissues we considered only genes identified by corresponding tag count > the proportion of genes expressed from the x chromosome in a pool of somatic tissues was %. although the proportions of x-linked genes in somatic tissues were uneven, there were no significant differences among the tissues. in testicular somatic cells, we observed % x-linked genes, while in total testis only % genes were expressed from the x chromosome. we can conclude that the number of expressed x-linked genes is underrepresented in the transcriptome of total testis. the same set of genes was examined for the distribution of tissue-specific genes on autosomes and the x chromosome. we compared the genes specific for either total testis or adult testis somatic cells in conjunction with somatic tissue-specific genes. a gene was considered to be tissue-specific if it was expressed only in one tissue type. moreover, the corresponding tag count > was required to guarantee that the gene is truly expressed. the tissue-specific genes were assigned to chromosomes according to the locuslink database and the significance of their chromosomal distribution was evaluated by permutations and confirmed by fisher's exact test. out of the genes specific for total testis % mapped to the x chromosome. essentially the same proportion of x-linked genes was found for genes specific for somatic tissues. in testicular somatic cells, we detected only tissue-specific genes, but % were x-linked. this is a -fold increase in the proportion of testis somatic cell-specific genes on the x chromosome and represents their significant enrichment in comparison to the genes specific for other tissues. all the x-linked testis-specific genes were subjected to blast against the whole x chromosome, which revealed no duplicated genes. the results from the permutation analysis indicate a significantly increased amount of testis-specific genes on the x chromosome in somatic cells of the testis when compared to autosomal testis-specific genes. the genes specific for somatic tissues did not show a significant preference for the x chromosome. the list of x-linked genes expressed in total testis and testicular somatic cells with indicated testis-specific genes is available in additional file chromosomal clustering of genes with preferential expression in testis based on the data from testis and other publicly available sage libraries we identified genes with preferential expression in testis by preferential expression measure. pem score controls for the genes that are highly expressed in many tissues and reports positive values for over-expressed genes and negative values for under-expressed genes in a given tissue. large positive pem scores for a gene in a particular tissue indicate that the gene is unusually highly expressed in that tissue, relative to its expression in other tissues. we considered a gene to be preferentially expressed if the pem score reached at least % of the maximum pem value encountered in that tissue. using this criterion, we scored expression of genes in total testis or testicular somatic cells in conjunction with their expression in other tissues. further we analyzed the genome organization of genes preferentially expressed in testis. we evaluated the expression of genes among the studied tissues and for genes we were able to assign a genomic position according to the ncbi mouse genome assembly. the genomic position was resolved for and genes expressed in total testis and testicular somatic cells, respectively, including and preferentially expressed genes, respectively. to evaluate the gene order of preferentially expressed genes in testis and to eliminate the effect of tandem duplications we purged the whole mouse genome of tandemly duplicated genes. the tandem duplicate-free genome resulted in total of locuslink genes and preserved and genes preferentially expressed in total testis and testicular somatic cells, respectively. using a search with a sliding window we localized chromosomal clusters containing at least three adjacent preferentially expressed genes. similarly, we searched for clusters with at least three preferentially expressed genes among the six adjacent genes to include genes that could be preferentially expressed but did not pass the above criterion for preferential expression or their expression was not detected by sage. by definition, the tight clusters form a subset of the loose clusters. the chromosomal distribution of clusters with preferentially expressed genes in testis is illustrated in figure we observed and genes preferentially expressed in total testis and testicular somatic cells located in and tight clusters, respectively. two hundred and thirty and genes preferentially expressed in total testis and testicular somatic cells resided in and loose clusters, respectively. two of the tight clusters and eight of the loose clusters shared preferentially expressed genes between total testis and testicular somatic cells. statistical analysis revealed that the observed number of preferentially expressed genes located in tight clusters was -fold and -fold higher for total testis and testicular somatic cells, respectively, than the average number of preferentially expressed genes located in clusters in randomized genomes. although only slighly higher than the average in randomized genomes, the observed number of preferentially expressed genes in testis located in loose clusters was still significant in case of total testis and nearly significant in case of testicular somatic cells. not surprisingly, the most highly expressed genes detected in total testis and involved in spermatogenesis formed one of the tight clusters on chromosome the results indicate a nonrandom distribution of the genes preferentially expressed in total testis and testicular somatic cells into chromosomal clusters, which did not arise from tandem duplications. comparing the b and bdf total testis transcriptomes in a recent study focused on senescence changes in testis, a modified sage method was used to generate digital gene expression profiles of total testis from 3- and 29-month-old mice of the bdf strain and 14-month-old mice of the samp strain that exhibits an accelerated senescence. because of the different anchoring enzyme used in construction of the libraries and the limited availability of data from the bdf testis transcriptome, we could perform only a rough manual comparison of our b testis transcriptome and the combined bdf testis transcriptome from 3- and 29-month-old bdf mice. we focused on the most highly expressed testicular genes in gnf mouse atlas v that were detected by affymetrix genechips. a set of highly expressed genes in testis was organized with sage tag counts from b and bdf testis. in the b total testis, we detected out of genes. in contrast, only genes were detected in the bdf testis library, genes were missing due to the absence of rsai restriction site in the transcript and for other genes the expression data from bdf testis were not publicly available. furthermore, out of the highly expressed genes in testis, genes were among the top most expressed genes in the b total testis library, but only genes were among the top most expressed genes in the bdf total testis library. it appears that our sage data from the b testis transcriptome shows better correspondence to the microarray data than the data from the transcriptome of bdf testis. discussion serial analysis of gene expression is a high-throughput method for building a catalogue of expressed genes and their expression levels of "normal" as well as diseased or genetically variant tissues and organs. the digital character of sage data enables addition and direct comparison of different sage libraries, provided they were built with the same anchoring enzyme and originated from individuals of the same species. the utilization of such global transcriptome databases is multifold, including positional cloning of mutations or quantitative trait loci, functional genome annotation or analysis of a nonrandom gene order. admittedly, the sage, as used in this work, has several limitations, including a significant proportion of repetitive and low complexity tags. the sage is obviously more labor-intensive than transcriptome analysis based on microarrays. at present, some of these inconveniencies can be solved by applying longsage or massively parallel signature sequencing technologies. in this study we constructed a sage library of the total testis of the c57bl/6j mouse inbred strain, compared it with other public available mouse sage libraries and analyzed localization of testis-expressed genes within the mouse genome. the b strain was favored for the availability of its high-quality draft genomic sequence and because series of congenics and recently also consomic strains have used the b strain as a background strain. the combined total testis sage library, tt 1+ consisted of total tags representing unique tags. the tag-to-gene reliable identification method used in mouse sage site was applied to tags with frequency ≥ out of these tags, % revealed a reliable match to single and % to multiple unigene clusters. considering the size of the total testis sage library, medium to highly expressed genes are present in the expression profile. the library size is comparable to the recently published sage library of somatic cells of the mouse testis and almost twice the size of a library constructed from the total testis of bdf hybrid mice using a modified sage method. contrary to microarrays, sage data are platform independent, which permits the use of unrelated datasets coming from various sources to compare gene expression patterns. we analyzed the mouse testis transcriptome by comparing our total testis sage library to the adult testis somatic cells library and to additional publicly available sage libraries from different tissues. we recognized three different modes of differential expression. predominant expression of genes in the germinal or somatic part of the testis, which did not consider expression in other tissues. preferential expression in testis that was defined by comparing the expression of testis to somatic tissues for which sage data were available. testis-specific expression that was defined by null expression in sage libraries of seven tissues or organs other than testis. complete lists of genes predominantly expressed in germinal or testis-somatic cells, as well as the catalogues of genes preferentially expressed in testis and testis-specific genes are available online in additional file, and additional file conflicting results have been reported on the representation of male-biased genes on the x chromosome in various species. spermatogonia-specific genes were found to be an order of magnitude more abundant on the mouse x chromosome. in human, the prostate-specific genes were twice more frequent on the x chromosome, but the female mammary gland- and ovary-specific x-linked genes were not enriched in respective sage libraries. on the contrary, under-representation or absence of male-biased genes on the x chromosome was reported in caenorhabditis elegans and in drosophila. in the mouse, an under-representation of testis-expressed and testis-enriched genes on the x chromosome was also revealed by the analysis of microarray and est data. our present data favor under-representation of x-linked genes in the total testis transcriptome but not in testis-somatic cells. because the germ cells in different stages of differentiation constitute about 90% of the total cell mass of testis, the data indicate that the deficit of x-linked testis-expressed genes may reflect the lack of transcription from the x chromosome in meiotic cells. these results are in agreement with the idea of x-chromosome silencing during the first meiotic division, the phenomenon based mostly on circumstantial evidence in flies and mice. thus, transcription at the haploid stage of spermatogenesis is expected for most of the x-linked genes expressed in total testis. the meiotic x chromosome inactivation seems to be restricted to primary spermatocytes, but sertoli cells, which form the somatic part of seminiferous tubules, may have the x chromosome in the active state. indeed, in the transcriptome of adult testis somatic cells the proportion of expressed x-linked genes was more than twice higher than in total testis and did not differ from the proportion of x-linked genes expressed in non-testis tissues. testis-specific genes belong to a wider category of sex-biased genes, which according to the hypothesis of sexually antagonistic genes are more likely to spread on the x chromosome than on autosomes. this is because on the x chromosome they will express their favorable effect in the hemizygous state while their deleterious effect will be masked by their recessivity in the other sex. consequently, accumulation of male-specific genes on the x chromosome will be possible by the effect of modifiers that narrow the expression of sex-biased genes only to the male sex. thus, the evolution of sexually antagonistic genes and x inactivation may act as opposing forces on the germline lineage of testis while accumulation of male-specific genes could be expected in somatic cells of testis. in accord with these assumptions the proportion of x-linked genes specific for total testis did not significantly differ from the proportion of genes specific for other tissues, while we observed a significant -fold enrichment of the proportion of x-linked genes specific for testicular somatic cells. the eukaryotic gene order is nonrandom obviously not only due to shifting of sex-biased genes to and from the x chromosome, but also owing to a nonrandom clustering of genes within chromosomes. this somewhat unexpected conclusion is gaining gradual support from global transcriptome analyses of various eukaryotic species. the observed examples of clustering are apparently a mixture of several unrelated phenomena, including large domains of similarly expressed genes in drosophila and humans, clustering of housekeeping genes, clustering of highly expressed genes or genes with similar expression breadth in regions of similar gc content. in drosophila melanogaster one third of testes-specific genes occur in clusters, a phenomenon not reported in any other species. using pem to define preferentially expressed genes we were able to demonstrate that in the mouse, the genes preferentially expressed in germ cells as well as in somatic cells of testis occur in tight clusters with a frequency -fold and -fold higher than the expected average frequency in randomized genomes. moreover, our results indicate that this phenomenon is not merely a consequence of tandem duplications. further analysis of clustering of testis-expressed genes may reveal new insights into the functional organization of the mammalian genome.  we identified chromosomal clusters of adjacent genes with preferential expression in testis that contain a significantly higher number of genes than expected by chance. this phenomenon is not merely a consequence of tandem duplication. the genes with specific expression in testicular somatic cells are more abundant on the x chromosome, which favors the theory of accumulation of male-biased genes on the x chromosome. in contrast, the x-linked genes are under-represented in the transcriptome of total testis, which is in accordance with the idea of x-chromosome inactivation during the first meiotic division. 
an extensive bioinformatic analysis and comparison of sage transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the x chromosome was tested. the genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis sage transcriptomes to the available transcriptomes of seven non-testis tissues. we uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. we observed a significant -fold enrichment of the proportion of x-linked genes specific for testicular somatic cells, while the proportions of x-linked genes specific for total testis and for other tissues were comparable. in contrast to the tissue-specific genes, an under-representation of x-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. our results provide new evidence in favor of the theory of male-biased genes accumulation on the x chromosome in testicular somatic cells and indicate the opposite action of the meiotic x-inactivation in testicular germ cells. two high-quality sage libraries containing a total of tags were constructed. we sorted out genes predominantly expressed from the germinal part and genes from the somatic part of the testis. we generated the gene expression profile of the total testis from the adult c57bl/6j male mice using serial analysis of gene expression.
 we generated the gene expression profile of the total testis from the adult c57bl/6j male mice using serial analysis of gene expression. two high-quality sage libraries containing a total of tags were constructed. an extensive bioinformatic analysis and comparison of sage transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the x chromosome was tested.  we sorted out genes predominantly expressed from the germinal part and genes from the somatic part of the testis. the genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis sage transcriptomes to the available transcriptomes of seven non-testis tissues. we uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. we observed a significant -fold enrichment of the proportion of x-linked genes specific for testicular somatic cells, while the proportions of x-linked genes specific for total testis and for other tissues were comparable. in contrast to the tissue-specific genes, an under-representation of x-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected.  our results provide new evidence in favor of the theory of male-biased genes accumulation on the x chromosome in testicular somatic cells and indicate the opposite action of the meiotic x-inactivation in testicular germ cells. 