BACKGROUND
during male reproduction, germ cells are processed from the primordial stage through spermatogenesis occurring in the seminiferous tubules of testis.
the tightly regulated process that occurs in mitotic, meiotic, and post-meiotic phases drives successful male germ cell development or spermatogenesis  <cit> .
spermatogonial stem cells located around the outer region next to the basal lamina surrounding seminiferous tubules undergo mitosis, and some differentiate into later-stage spermatogonia that gradually become primary spermatocytes.
at this stage, the most important event, meiosis, is additionally required.
these cells continue through the first meiotic division to become secondary spermatocytes.
a second meiotic division occurs in rapid succession to produce haploid spermatids that are remodeled into spermatozoa by spermiogenesis.
the tight modulation of this developmental process suggests the expression of a highly organized network of genes.
the regulation of gene expression during spermatogenesis occurs at three levels, namely the intrinsic, interactive, and extrinsic levels  <cit> .
the intrinsic program determines which genes are utilized and when genes are expressed in germ cells.
the interactive process between germ cells and somatic cells is necessary for germ cell proliferation and progression, and is regulated by extrinsic influences, such as steroid and peptide hormones.
notably, the intrinsic genetic program involves germ cell- and stage-specific gene expression.
recently, several studies have focused on the identification of cell- and tissue-specific transcriptomes using high-throughput genomics.
while earlier studies have provided inclusive information about testicular genes, the identities and characteristics of spermatogenic cell-specific genes are largely unknown  <cit> .
the unigene database contains an extensive collection of information about sets of transcript sequences, including ests .
unigene transcripts are organized into clusters composed of fragments of sequences overlapping with at least one other member of the same cluster.
genbank sequences are automatically partitioned into non-redundant sets of gene-oriented clusters  <cit> .
consequently, each cluster contains sequences corresponding to a single gene, and related information, such as gene expression patterns and mapping positions analyzed in silico.
thus, the unigene database provides significant information  to facilitate the prediction of gene expression and function from each cluster.
comprehensive understanding of male germ cell development and fertilization requires discovery and functional characterization of germ cell-specific genes, because they are highly unique processes that do not occur in any other tissue.
previously, we identified and characterized a number of novel genes from the round spermatid unigene library  <cit> .
the spermatocyte stage is also a significant period during which meiosis occurs and haploid daughter cells are produced.
here, as an ongoing study on germ cell-specific genes, we identify  <dig> spermatogenic cell-specific genes, using sequence information from the spermatocyte unigene database, and analyze their characteristics at the gene and transcript levels.
further, we characterized proteins encoded by the genes and predict that a number of them have significant functions in various processes during spermatogenesis and fertilization.
thus, we report the efficient and unique systematic identification and in-depth characterization of unexplored genes specific to spermatogenic cells.
RESULTS
the spermatocyte unigene library and in silico selection of gene candidates
the mccarrey eddy spermatocyte library of mus musculus , one of the largest spermatogenic cell libraries deposited in the unigene database at ncbi  <cit> , was analyzed to classify gene entries into known and unknown genes, or testis-specific and non-testis-specific genes.
as of april  <dig> , the spermatocyte library consisted of  <dig> unigene entries.
we classified the genes from the library on the basis of the following criteria:  genes previously named or assigned with potential functions were classified as 'known', and genes with unassigned functions were regarded as 'unknown'.
if all of ests of a given gene were found only in testis and/or spermatocyte, or the numbers of testis or spermatocyte ests in a cluster were higher  than that of non-testis and/or spermatocyte ests, the gene cluster was selected as 'testis-specific'.
according to these criteria, about three-fourth of the genes were classified as known/named, and  <dig> were testis-specific.
on the other hand, we selected  <dig> clusters as unknown genes, of which  <dig> were testis-specific .
although three quarters of the total gene entries are named/known genes, similar numbers of testis-specific genes were found among the known and unknown candidates, indicating that many of the testis-specific genes remain to be characterized.
taken together, the combination of known and unknown testis-specific genes comprises 11% of the spermatocyte unigene entries.
gene entries from the spermatocyte unigene library were classified into known and unknown genes, or testis-specific and non-testis-specific genes.
genes with >90% testicular ests of total ests, including three non-testicular tissues at most, were classified as testis-specific genes.
one hundred thirty four genes selected from the earlier version of the library were analyzed in vitro to determine whether they are authentic genes with abundant and evident testis-specific expression.
all of the  <dig> genes are listed in table  <dig>
at the beginning of our study , the total number of gene entries within the earlier version of the spermatocyte library was  <dig>  a search for testis-specific genes with unknown or unassigned functions resulted in the selection of  <dig> candidates and these genes were further analyzed in the present study .
gene candidates were subjected to in silico and in vitro analyses.
genes with potential coding sequences were analyzed by rt-pcr in which those expressed  as correctly-sized pcr products were further analyzed to determine whether they are testis-specific .
lastly, to investigate transcript size and expression level, genes with testis-specific expression were subjected to northern blot analysis.
genes with positive and negative results are indicated as + and -, repectively.
testicular expression of genes
to determine whether the candidates selected from the unigene library are true genes with testis-specific expression, various analyses were performed .
in total,  <dig> gene candidates were analyzed with regard to whether their open reading frames contained reliable amino acid coding regions.
we regarded a certain gene candidate as a gene with a reliable amino acid coding region if the size of the longest coding sequence is larger than at least 20% of that of an entire transcript  sequence in the gene.
of these,  <dig> genes encoded possible amino acid sequences, while remaining  <dig> candidates showed that deduced coding sequences are too short, compared to transcript sizes , or displayed unreliable coding regions .
accordingly, these  <dig> genes were eliminated from further analyses.
reverse transcription-polymerase chain reaction  analysis of the  <dig> candidates resulted in the amplification of pcr products with the expected sizes in  <dig> candidates.
however, we observed no or incorrectly sized pcr products of the remaining  <dig> candidates from testes, which were thus excluded from further analysis.
tissue distribution was investigated by pcr using mouse cdna from eight different tissues.
out of the  <dig> candidate genes,  <dig> were identified as testis-specific or predominant .
gene expression data from  <dig> of the  <dig> genes are shown in figure  <dig>  since subsequent northern blot analysis revealed abundant expression only in these genes .
all the gene transcripts were amplified with the correct sizes , and specifically or predominantly expressed in the testis .
spermatogenesis occurs in seminiferous tubules containing a mixture of germ cells and somatic cells, such as sertoli cells.
none of the genes were transcribed in germ cell-lacking testes of w/wv mutant mice .
a the following genes were named previously: mm.
<dig>  tudor domain containing  <dig>   <cit> ; mm <dig>  zinc finger protein  <dig> ; mm.
<dig>  ring finger protein ; mm.
<dig>  spermatogenesis associated  <dig> ; mm.
<dig>  spermatogenesis associated  <dig>   <cit> ; mm.
<dig>  tektin  <dig>   <cit> ; mm.
<dig>  testis expressed gene  <dig>   <cit> ; mm.
<dig>  cysteine-rich perinuclear theca protein  <dig>   <cit> ; mm <dig>  hras-like suppressor family, member  <dig> ; and mm.
<dig>  cation channel, sperm associated  <dig>   <cit> .
transcript analysis of genes
to determine the expression levels and transcript sizes of the gene candidates, we performed northern blot analysis .
for  <dig> of the  <dig> genes, significant signals were detected in total rna samples from testis, but not those from liver tissue .
these results are consistent with tissue distribution data obtained by rt-pcr.
testicular transcript sizes ranged from ~ <dig> kb  to  <dig> kb .
for  <dig> genes, transcript sizes determined by northern blotting were comparable to those estimated from the unigene database, while for the other seven genes, appreciable differences in transcript size  were evident between northern blots and unigene database sequences .
thus, the transcript sequences for the  <dig> genes can be regarded with confidence as full-length cdnas or sequences containing the majority of entire cdna sequences.
the northern blot analysis also revealed that four genes produce transcripts with more than a single size , suggesting the presence of multiple transcript isoforms in these genes by alternative splicing.
developmental expression patterns of genes
to establish the developmental expression patterns of the  <dig> genes during spermatogenesis, rt-pcr analysis was performed using mouse testis cdna obtained at different time-periods after birth .
during spermatogenesis in prepubertal mouse, primordial germ cells proliferate and differentiate increasingly to produce spermatogonia, spermatocytes, and spermatids   <cit> .
if a particular gene is expressed in germ cells during spermatogenesis, the corresponding transcript will appear in the testis at a post-partum time-point corresponding to the specific stage of spermatogenesis.
rt-pcr data disclosed that all the genes are expressed at least after day  <dig>  indicative of germ cell-specific and developmentally regulated expression .
the  <dig> genes could be divided into  <dig> clusters, based on expression patterns.
the first cluster, including two genes, is expressed from the early spermatocyte  stage generated from 12-day-old postnatal mouse testes.
the second and third clusters, the pachytene spermatocyte stage, is generated from 14- and 16-day-old postnatal mouse testes, respectively, and contain  <dig> genes.
two genes comprising the fourth cluster were expressed from the late spermatocyte stage generated from 20-day-old postnatal mouse testes.
the fifth cluster involving the spermatid stage contained four genes.
it should be noted that the majority of the genes are expressed at the spermatocyte stage from which the genes were selected in silico.
genes encoding adam <dig>  and protamine  <dig>  of which expression starts during and after meiosis, respectively, were used for controls .
in silico analysis of genomic, transcript, and protein characteristics
to characterize genomic, transcript and protein natures of the genes, we performed various database searches.
figure  <dig> shows exon organization, chromosomal locations, transcript sizes, numbers of amino acids, specific domain/motif, and gene ontology of the predicted proteins encoded by the genes.
the exon numbers in the genes are variable, ranging from  <dig> to  <dig> exons.
the novel genes are widely distributed on mouse chromosomes.
to extend these findings on mouse genes, we searched the human genome database for orthologs.
human orthologs for  <dig> mouse genes were present in genomic regions of conserved synteny between mice and humans.
the other  <dig> genes did not have human orthologs, suggesting differential expansion in the mouse genome.
the protein-coding region of each gene was defined by selecting the longest amino acid sequence terminating before a polyadenylation signal , and deduced amino acid sequences were subjected to database searches.
nineteen gene products were predicted to contain various domains and motifs, and found to be annotated with gene ontology codes.
thus, based on the in silico information, some of these proteins are predicted to be implicated in transcriptional regulation and/or nuclear activity , metabolic processes  and cell structure  .
eight genes with in vitro protein data  which support in silico information  are functionally categorized.
abbreviations are as follows: tsc, testicular spermatogenic cells; ms, mature sperm; nu, nucleus; cy, cytoplasm; er, endoplasmic reticulum ga, golgi apparatus; n.d., not determined; , tail; , acrosome.
subcellular localization of the proteins
to explore protein characteristics in vitro, we investigated subcellular localization of the gene products  <cit> .
gfp-tagged full-length gene sequences were transiently transfected into gc- <dig> cells.
gc- <dig> cells are immortalized germ cells  of mouse testis  <cit> .
we observed gfp signals from  <dig> out of the  <dig> gene products analyzed.
by contrast, the gfp signals were not detected in the other  <dig> genes, suggesting that the expression of these proteins is highly transient, very low in amount or delayed.
figure  <dig> depicts the subcellular locations identified.
five gene products were found to be localized in the nucleus.
other gene products localized to the endoplasmic reticulum , golgi apparatus , and cytoplasm .
it should be noted that the three genes with cytoplasmic localization displayed a speckled localization pattern .
the localization data support results from in silico prediction  and further protein analysis .
characterization of the proteins in mouse spermatogenic cells and sperm
to further explore the characteristics of proteins encoded by the genes, we generated antibodies against five proteins .
initially, we attempted to generate antibodies to all the proteins, using gst recombinant proteins and synthetic peptides corresponding to the proteins.
however, for the other  <dig> genes, the recombinant proteins were not expressed in a bacterial system or antisera from rabbits immunized with the antigens did not detect corresponding proteins from testis.
the antibodies to the five proteins detected distinctive bands in the extracts of human embryonic kidney  cells transfected with the corresponding cdna sequences, but not in those of cells transcfected with the empty vector .
we examined the presence and localization of five proteins from total protein extracts of testicular spermatogenic cells, testicular sperm or mature sperm from cauda epididymis by western blot analysis .
all the antibodies recognized distinct bands in testicular spermatogenic cells.
they were of the sizes comparable with those predicted from the cdna sequences.
two of the proteins  were present in testicular sperm, but not mature sperm.
the other three proteins  were identified in both testicular and mature sperm.
size differences for mm.
<dig> and mm.
<dig> were evident between testicular spermatogenic cells, testicular sperm and mature sperm, indicating that these proteins undergo changes during sperm development and maturation .
to further establish the subcellular localization of the three proteins in mature sperm, cell surface biotin labeling was performed .
if a certain protein is present on the cell surface, labeling with biotin results in a change in electrophoretic mobility.
however, we observed no changes in mobility of the three proteins in sperm.
adam <dig> was included as a reference protein, since it is known to be processed during sperm maturation and located on the sperm surface.
taken together, our results provide evidence on the localization of these five proteins, both at the developmental stage level  and cellular level  in sperm.
to confirm and further examine the developmental expression and localization of the five proteins in spermatogenic cells or mature sperm, we performed indirect immunofluorescence analysis in paraffin sections of adult mouse testis with the antibodies.
the antibodies to four proteins, corresponding to mm.
<dig>  mm.
<dig>  mm.
<dig> and mm.
<dig>  displayed immunoreactivity in spermatocytes and spermatids .
we were unable to observe signals in testis sections incubated with the antibody to the other protein .
in particular, mm.
<dig> was observed in acrosomes of round spermatids, while mm.
<dig>  mm.
<dig> and mm <dig> were identified in the cytoplasmic region located to the posterior of elongating spermatids .
since the western blot analysis demonstrated the presence of mm.
<dig> and mm.
<dig> in mature sperm , we performed immunofluorescence on mature sperm.
consistent with the result from testis sections, mm <dig> and mm.
<dig> were observed in the acrosomal region  and flagellum  of mature sperm, respectively.
it is important to note that mm.
<dig> is predicted to have a trypsin-like serine protease activity .
table  <dig> summarizes putative reproductive functions of eight genes with both predicted in silico information and supporting in vitro protein data obtained in this study .
discussion
previously, several studies have investigated genes expressed at specific stages or in particular cell type during spermatogenesis  <cit> .
although these studies provided inclusive information about the expression profile of a large number of germ-cell genes, comprehensive understanding of spermatogenesis requires further systematic identification and analysis of uncharacterized genes with germ cell-specific expression.
unigene is a large and widely used transcript sequence database containing a large amount of unexplored information about genes.
the sequences are divided according to tissue type or developmental stage from the unigene database, which provides a resource for identifying novel tissue-, cell-, or stage-specific gene transcripts.
in the present study, analyzing the mouse spermatocyte unigene library , we disclosed that significant proportion  of the spermatocyte genes are testis-specific and about half of the testis-specific genes are unknown or uncharacterized.
previously, a similar approach was applied by our group using the round spermatid unigene library  <cit> , revealing that 22%  of genes expressed in round spermatids are testis-specific and functions of 74% of the testis-specific genes are unexplored.
in the present investigation, the initial number of uncharacterized, testis-specific genes selected from the earlier version of the spermatocyte library is  <dig>  these  <dig> genes were narrowed down to  <dig> authentic genes considered to be abundantly and specifically expressed in the testis by various expression analyses.
the other  <dig> genes were eliminated from consideration because they displayed unreliable coding sequences , were undetected or detected with unexpected sizes in the pcr assay , not specifically or predominantly expressed in the testis  or undetected in the northern blot analysis .
our data provide extensive information on the  <dig> genes at the genomic and transcript levels.
genomic analysis disclosed orthologues for  <dig> mouse genes in the human genome and  <dig> other were identified as mouse-specific genes.
the proportion of mouse genes with a single identifiable ortholog in the human genome is about 80%.
the other 20% of mouse genes lack a strict 1: <dig> relationship, due to differential expansion in at least one of the two genomes  <cit> .
mostly, those mouse-specific genes were involved in reproduction, olfaction and immunity.
similarly, a global view of human and mouse proteases revealed that the mouse degradome is more complex, and several genes in the mouse genome encode proteases involved in reproductive functions  <cit> .
one such example is the testis-specific or predominant adam genes in postmeiotic germ cells  <cit> .
thus, the  <dig> mouse-specific genes identified in our study are related to aspects of reproductive physiology.
at the transcript level, the northern blot analysis revealed that four genes are transcribed into products of more than one size.
in addition, the analysis demonstrated that transcript sizes from the database are consistent with those determined experimentally for most of the genes.
a special feature of genes whose expression is strongly favored in male germ cells is developmentally regulated during meiotic and postmeiotic phases  <cit> .
consistently, the expression patterns of the  <dig> genes during postnatal testicular development, found in the present study, are indicative of developmental regulation.
the pachytene spermatocyte stage is significant during spermatogenesis.
it involves genetic recombination, which occurs only in germ cells through cross-over between paired chromosomes and increases rna and protein synthesis in preparation for the next phase  <cit> .
transcription of more than half  the total genes was found to start from the pachytene spermatocyte stage.
germ cell-specific and developmentally regulated genes could be directly responsible for the spermatogenesis or fertilization.
we also investigated the genes at the protein and cellular levels, providing functional perspectives of the genes.
the proteins encoded by  <dig> out of the  <dig> genes were analyzed in living gc- <dig> cells.
no expression of the other  <dig> genes might be due to their peculiar protein natures, such as instability, translational delay and toxicity to the cells.
cellular localization of the  <dig> genes was divided into nucleus, endoplasmic reticulum, golgi apparatus, and cytoplasm.
to further gain an insight into the characteristics of the proteins, we generated antibodies to five proteins.
the western blot analysis disclosed that two proteins were restricted to testicular spermatogenic cells and testicular sperm, while the others were present at all stages, including testicular spermatogenic cells, testicular sperm, and mature sperm.
results from the immunofluorescence analysis of testis sections and mature sperm corroborate and extend the western blot data.
taken together, our transfection and immuno-analyses provided new information about  <dig> genes at the protein and cellular levels .
among the  <dig> genes with the in vitro data, eight genes have in silico information congruous with the in vitro results .
we attempted to categorize these eight genes based on all the in silico and in vitro data, and relate them to potential functions in reproduction .
three  of the gene products are likely to be involved in transcriptional regulation.
all of these proteins were found to be localized in the nucleus of gc- <dig> cells transfected with the corresponding cdnas.
mm.
<dig>  predicted to be present in a perinuclear region, was targeted to the nucleus of gc- <dig> cells.
this protein might be related to nuclear activity or integrity of spermatogenic cells.
according to a recent report, mm.
<dig> belongs to the cysteine-rich perinuclear theca family with potential functions in the remodelling of the spermatid nucleus  <cit> .
three of the genes seem to encode proteins implicated in sperm structure and motility.
it should be noted that these three genes have been named and reported previously  <cit> .
nonetheless, we did not eliminate them because we have obtained new information about these proteins in this investigation.
mm.
<dig>  named tep <dig>  has been suggested to be involved in the biogenesis of the acrosome and the midpiece of the sperm tail  <cit> .
our western blot analysis newly revealed that the mm.
<dig> protein made as a  <dig> kda-protein in testicular cells is changed to a higher molecular weight form between the stages of testicular sperm and mature sperm, suggesting post-translational modification.
consistent with this, the protein contains several putative glycosylation and phosphorylation sites  <cit> .
mm.
<dig>  identified as catsper <dig>  <cit> , was found localized to the endoplasmic reticulum in gc- <dig> cells in this study.
other catsper family members, catsper <dig> and catsper <dig>  are known to be specifically expressed in sperm and linked to sperm motility  <cit> .
in fact, the expression pattern of the catsper <dig> gene and its essential role in sperm motility and male fertility were reported during the preparation of the present paper  <cit> .
mm.
<dig> has been named tektin <dig> which belongs to the tektin family  <cit> .
tektin <dig> and tektin <dig> are microtubule- or outer dense fiber-associated proteins in sperm flagella  <cit> .
here, we provide the first information about the mm <dig> protein, tektin <dig>  this protein was found to be present at the sperm flagella.
it should be noted that the molecular size of the mm <dig> protein was increased during spermiogenesis, suggestive of post-translational modification.
finally, we also obtained original findings on mm.
<dig>  the protein encoded by mm.
<dig> was targeted to the golgi apparatus in gc- <dig> cells.
the immuno-analysis uncovered that the mm <dig> protein,  <dig> kda, is present in both spermatogenic cells and mature sperm.
in particular, the protein was located in the acrosomal region of mature sperm.
it is important to mention that mm.
<dig> is predicted to contain a trypsin-like serine protease domain.
the acrosome is a golgi-derived secretory granule which is formed during spermiogenesis and positioned at the apex of mature sperm  <cit> .
when sperm reach the egg extracellular coat, the zona pellucida , during fertilization, they bind to it and undergo acrosome reaction, releasing the acrosomal contents at the site of sperm-egg binding.
the hydrolytic and proteolytic enzymes comprising the acrosomal contents digest the zp and, thus, enable sperm to penetrate the zp.
the sperm acrosome contains both unique enzymes and common enzymes present in somatic cells  <cit> .
to date, only a handful of unique enzymes have been identified and enzymes directly responsible for the fertilization process are unknown  <cit> .
thus, the mm.
<dig> protein is a candidate for a type of protease involved in the penetration of the zp during fertilization.
CONCLUSIONS
identification of genes with spermatogenic cell-specific expression is crucial to understanding the molecular basis of spermatogenesis and fertilization.
our in silico analysis indicates that the proportion of testis-specific genes in the spermatocyte unigene library is 11% and half of them has been unexplored.
we have identified and characterized  <dig> authentic genes by systematic and integrative approaches, providing insights to their genomic, transcript and protein characteristics.
in particular, we predict potential functions of the eight genes, based on our in vitro data as well as in silico information.
thus, the data provided by this study provide a large resource for further investigations into molecular mechanisms of mammalian male reproduction.

 during male reproduction, germ cells are processed from the primordial stage through spermatogenesis occurring in the seminiferous tubules of testis. the tightly regulated process that occurs in mitotic, meiotic, and post-meiotic phases drives successful male germ cell development or spermatogenesis. spermatogonial stem cells located around the outer region next to the basal lamina surrounding seminiferous tubules undergo mitosis, and some differentiate into later-stage spermatogonia that gradually become primary spermatocytes. at this stage, the most important event, meiosis, is additionally required. these cells continue through the first meiotic division to become secondary spermatocytes. a second meiotic division occurs in rapid succession to produce haploid spermatids that are remodeled into spermatozoa by spermiogenesis. the tight modulation of this developmental process suggests the expression of a highly organized network of genes. the regulation of gene expression during spermatogenesis occurs at three levels, namely the intrinsic, interactive, and extrinsic levels. the intrinsic program determines which genes are utilized and when genes are expressed in germ cells. the interactive process between germ cells and somatic cells is necessary for germ cell proliferation and progression, and is regulated by extrinsic influences, such as steroid and peptide hormones. notably, the intrinsic genetic program involves germ cell- and stage-specific gene expression. recently, several studies have focused on the identification of cell- and tissue-specific transcriptomes using high-throughput genomics. while earlier studies have provided inclusive information about testicular genes, the identities and characteristics of spermatogenic cell-specific genes are largely unknown. the unigene database contains an extensive collection of information about sets of transcript sequences, including ests. unigene transcripts are organized into clusters composed of fragments of sequences overlapping with at least one other member of the same cluster. genbank sequences are automatically partitioned into non-redundant sets of gene-oriented clusters. consequently, each cluster contains sequences corresponding to a single gene, and related information, such as gene expression patterns and mapping positions analyzed in silico. thus, the unigene database provides significant information to facilitate the prediction of gene expression and function from each cluster. comprehensive understanding of male germ cell development and fertilization requires discovery and functional characterization of germ cell-specific genes, because they are highly unique processes that do not occur in any other tissue. previously, we identified and characterized a number of novel genes from the round spermatid unigene library. the spermatocyte stage is also a significant period during which meiosis occurs and haploid daughter cells are produced. here, as an ongoing study on germ cell-specific genes, we identify spermatogenic cell-specific genes, using sequence information from the spermatocyte unigene database, and analyze their characteristics at the gene and transcript levels. further, we characterized proteins encoded by the genes and predict that a number of them have significant functions in various processes during spermatogenesis and fertilization. thus, we report the efficient and unique systematic identification and in-depth characterization of unexplored genes specific to spermatogenic cells.  the spermatocyte unigene library and in silico selection of gene candidates the mccarrey eddy spermatocyte library of mus musculus, one of the largest spermatogenic cell libraries deposited in the unigene database at ncbi, was analyzed to classify gene entries into known and unknown genes, or testis-specific and non-testis-specific genes. as of april, the spermatocyte library consisted of unigene entries. we classified the genes from the library on the basis of the following criteria: genes previously named or assigned with potential functions were classified as'known', and genes with unassigned functions were regarded as'unknown'. if all of ests of a given gene were found only in testis and/or spermatocyte, or the numbers of testis or spermatocyte ests in a cluster were higher than that of non-testis and/or spermatocyte ests, the gene cluster was selected as'testis-specific'. according to these criteria, about three-fourth of the genes were classified as known/named, and were testis-specific. on the other hand, we selected clusters as unknown genes, of which were testis-specific. although three quarters of the total gene entries are named/known genes, similar numbers of testis-specific genes were found among the known and unknown candidates, indicating that many of the testis-specific genes remain to be characterized. taken together, the combination of known and unknown testis-specific genes comprises 11% of the spermatocyte unigene entries. gene entries from the spermatocyte unigene library were classified into known and unknown genes, or testis-specific and non-testis-specific genes. genes with >90% testicular ests of total ests, including three non-testicular tissues at most, were classified as testis-specific genes. one hundred thirty four genes selected from the earlier version of the library were analyzed in vitro to determine whether they are authentic genes with abundant and evident testis-specific expression. all of the genes are listed in table at the beginning of our study, the total number of gene entries within the earlier version of the spermatocyte library was a search for testis-specific genes with unknown or unassigned functions resulted in the selection of candidates and these genes were further analyzed in the present study. gene candidates were subjected to in silico and in vitro analyses. genes with potential coding sequences were analyzed by rt-pcr in which those expressed as correctly-sized pcr products were further analyzed to determine whether they are testis-specific. lastly, to investigate transcript size and expression level, genes with testis-specific expression were subjected to northern blot analysis. genes with positive and negative results are indicated as + and -, repectively. testicular expression of genes to determine whether the candidates selected from the unigene library are true genes with testis-specific expression, various analyses were performed. in total, gene candidates were analyzed with regard to whether their open reading frames contained reliable amino acid coding regions. we regarded a certain gene candidate as a gene with a reliable amino acid coding region if the size of the longest coding sequence is larger than at least 20% of that of an entire transcript sequence in the gene. of these, genes encoded possible amino acid sequences, while remaining candidates showed that deduced coding sequences are too short, compared to transcript sizes, or displayed unreliable coding regions. accordingly, these genes were eliminated from further analyses. reverse transcription-polymerase chain reaction analysis of the candidates resulted in the amplification of pcr products with the expected sizes in candidates. however, we observed no or incorrectly sized pcr products of the remaining candidates from testes, which were thus excluded from further analysis. tissue distribution was investigated by pcr using mouse cdna from eight different tissues. out of the candidate genes, were identified as testis-specific or predominant. gene expression data from of the genes are shown in figure since subsequent northern blot analysis revealed abundant expression only in these genes. all the gene transcripts were amplified with the correct sizes, and specifically or predominantly expressed in the testis. spermatogenesis occurs in seminiferous tubules containing a mixture of germ cells and somatic cells, such as sertoli cells. none of the genes were transcribed in germ cell-lacking testes of w/wv mutant mice. a the following genes were named previously: mm. tudor domain containing; mm zinc finger protein; mm. ring finger protein; mm. spermatogenesis associated; mm. spermatogenesis associated; mm. tektin; mm. testis expressed gene; mm. cysteine-rich perinuclear theca protein; mm hras-like suppressor family, member; and mm. cation channel, sperm associated. transcript analysis of genes to determine the expression levels and transcript sizes of the gene candidates, we performed northern blot analysis. for of the genes, significant signals were detected in total rna samples from testis, but not those from liver tissue. these results are consistent with tissue distribution data obtained by rt-pcr. testicular transcript sizes ranged from ~ kb to kb. for genes, transcript sizes determined by northern blotting were comparable to those estimated from the unigene database, while for the other seven genes, appreciable differences in transcript size were evident between northern blots and unigene database sequences. thus, the transcript sequences for the genes can be regarded with confidence as full-length cdnas or sequences containing the majority of entire cdna sequences. the northern blot analysis also revealed that four genes produce transcripts with more than a single size, suggesting the presence of multiple transcript isoforms in these genes by alternative splicing. developmental expression patterns of genes to establish the developmental expression patterns of the genes during spermatogenesis, rt-pcr analysis was performed using mouse testis cdna obtained at different time-periods after birth. during spermatogenesis in prepubertal mouse, primordial germ cells proliferate and differentiate increasingly to produce spermatogonia, spermatocytes, and spermatids. if a particular gene is expressed in germ cells during spermatogenesis, the corresponding transcript will appear in the testis at a post-partum time-point corresponding to the specific stage of spermatogenesis. rt-pcr data disclosed that all the genes are expressed at least after day indicative of germ cell-specific and developmentally regulated expression. the genes could be divided into clusters, based on expression patterns. the first cluster, including two genes, is expressed from the early spermatocyte stage generated from 12-day-old postnatal mouse testes. the second and third clusters, the pachytene spermatocyte stage, is generated from 14- and 16-day-old postnatal mouse testes, respectively, and contain genes. two genes comprising the fourth cluster were expressed from the late spermatocyte stage generated from 20-day-old postnatal mouse testes. the fifth cluster involving the spermatid stage contained four genes. it should be noted that the majority of the genes are expressed at the spermatocyte stage from which the genes were selected in silico. genes encoding adam and protamine of which expression starts during and after meiosis, respectively, were used for controls. in silico analysis of genomic, transcript, and protein characteristics to characterize genomic, transcript and protein natures of the genes, we performed various database searches. figure shows exon organization, chromosomal locations, transcript sizes, numbers of amino acids, specific domain/motif, and gene ontology of the predicted proteins encoded by the genes. the exon numbers in the genes are variable, ranging from to exons. the novel genes are widely distributed on mouse chromosomes. to extend these findings on mouse genes, we searched the human genome database for orthologs. human orthologs for mouse genes were present in genomic regions of conserved synteny between mice and humans. the other genes did not have human orthologs, suggesting differential expansion in the mouse genome. the protein-coding region of each gene was defined by selecting the longest amino acid sequence terminating before a polyadenylation signal, and deduced amino acid sequences were subjected to database searches. nineteen gene products were predicted to contain various domains and motifs, and found to be annotated with gene ontology codes. thus, based on the in silico information, some of these proteins are predicted to be implicated in transcriptional regulation and/or nuclear activity, metabolic processes and cell structure. eight genes with in vitro protein data which support in silico information are functionally categorized. abbreviations are as follows: tsc, testicular spermatogenic cells; ms, mature sperm; nu, nucleus; cy, cytoplasm; er, endoplasmic reticulum ga, golgi apparatus; n.d., not determined;, tail;, acrosome. subcellular localization of the proteins to explore protein characteristics in vitro, we investigated subcellular localization of the gene products. gfp-tagged full-length gene sequences were transiently transfected into gc- cells. gc- cells are immortalized germ cells of mouse testis. we observed gfp signals from out of the gene products analyzed. by contrast, the gfp signals were not detected in the other genes, suggesting that the expression of these proteins is highly transient, very low in amount or delayed. figure depicts the subcellular locations identified. five gene products were found to be localized in the nucleus. other gene products localized to the endoplasmic reticulum, golgi apparatus, and cytoplasm. it should be noted that the three genes with cytoplasmic localization displayed a speckled localization pattern. the localization data support results from in silico prediction and further protein analysis. characterization of the proteins in mouse spermatogenic cells and sperm to further explore the characteristics of proteins encoded by the genes, we generated antibodies against five proteins. initially, we attempted to generate antibodies to all the proteins, using gst recombinant proteins and synthetic peptides corresponding to the proteins. however, for the other genes, the recombinant proteins were not expressed in a bacterial system or antisera from rabbits immunized with the antigens did not detect corresponding proteins from testis. the antibodies to the five proteins detected distinctive bands in the extracts of human embryonic kidney cells transfected with the corresponding cdna sequences, but not in those of cells transcfected with the empty vector. we examined the presence and localization of five proteins from total protein extracts of testicular spermatogenic cells, testicular sperm or mature sperm from cauda epididymis by western blot analysis. all the antibodies recognized distinct bands in testicular spermatogenic cells. they were of the sizes comparable with those predicted from the cdna sequences. two of the proteins were present in testicular sperm, but not mature sperm. the other three proteins were identified in both testicular and mature sperm. size differences for mm. and mm. were evident between testicular spermatogenic cells, testicular sperm and mature sperm, indicating that these proteins undergo changes during sperm development and maturation. to further establish the subcellular localization of the three proteins in mature sperm, cell surface biotin labeling was performed. if a certain protein is present on the cell surface, labeling with biotin results in a change in electrophoretic mobility. however, we observed no changes in mobility of the three proteins in sperm. adam was included as a reference protein, since it is known to be processed during sperm maturation and located on the sperm surface. taken together, our results provide evidence on the localization of these five proteins, both at the developmental stage level and cellular level in sperm. to confirm and further examine the developmental expression and localization of the five proteins in spermatogenic cells or mature sperm, we performed indirect immunofluorescence analysis in paraffin sections of adult mouse testis with the antibodies. the antibodies to four proteins, corresponding to mm. mm. mm. and mm. displayed immunoreactivity in spermatocytes and spermatids. we were unable to observe signals in testis sections incubated with the antibody to the other protein. in particular, mm. was observed in acrosomes of round spermatids, while mm. mm. and mm were identified in the cytoplasmic region located to the posterior of elongating spermatids. since the western blot analysis demonstrated the presence of mm. and mm. in mature sperm, we performed immunofluorescence on mature sperm. consistent with the result from testis sections, mm and mm. were observed in the acrosomal region and flagellum of mature sperm, respectively. it is important to note that mm. is predicted to have a trypsin-like serine protease activity. table summarizes putative reproductive functions of eight genes with both predicted in silico information and supporting in vitro protein data obtained in this study. discussion previously, several studies have investigated genes expressed at specific stages or in particular cell type during spermatogenesis. although these studies provided inclusive information about the expression profile of a large number of germ-cell genes, comprehensive understanding of spermatogenesis requires further systematic identification and analysis of uncharacterized genes with germ cell-specific expression. unigene is a large and widely used transcript sequence database containing a large amount of unexplored information about genes. the sequences are divided according to tissue type or developmental stage from the unigene database, which provides a resource for identifying novel tissue-, cell-, or stage-specific gene transcripts. in the present study, analyzing the mouse spermatocyte unigene library, we disclosed that significant proportion of the spermatocyte genes are testis-specific and about half of the testis-specific genes are unknown or uncharacterized. previously, a similar approach was applied by our group using the round spermatid unigene library, revealing that 22% of genes expressed in round spermatids are testis-specific and functions of 74% of the testis-specific genes are unexplored. in the present investigation, the initial number of uncharacterized, testis-specific genes selected from the earlier version of the spermatocyte library is these genes were narrowed down to authentic genes considered to be abundantly and specifically expressed in the testis by various expression analyses. the other genes were eliminated from consideration because they displayed unreliable coding sequences, were undetected or detected with unexpected sizes in the pcr assay, not specifically or predominantly expressed in the testis or undetected in the northern blot analysis. our data provide extensive information on the genes at the genomic and transcript levels. genomic analysis disclosed orthologues for mouse genes in the human genome and other were identified as mouse-specific genes. the proportion of mouse genes with a single identifiable ortholog in the human genome is about 80%. the other 20% of mouse genes lack a strict 1: relationship, due to differential expansion in at least one of the two genomes. mostly, those mouse-specific genes were involved in reproduction, olfaction and immunity. similarly, a global view of human and mouse proteases revealed that the mouse degradome is more complex, and several genes in the mouse genome encode proteases involved in reproductive functions. one such example is the testis-specific or predominant adam genes in postmeiotic germ cells. thus, the mouse-specific genes identified in our study are related to aspects of reproductive physiology. at the transcript level, the northern blot analysis revealed that four genes are transcribed into products of more than one size. in addition, the analysis demonstrated that transcript sizes from the database are consistent with those determined experimentally for most of the genes. a special feature of genes whose expression is strongly favored in male germ cells is developmentally regulated during meiotic and postmeiotic phases. consistently, the expression patterns of the genes during postnatal testicular development, found in the present study, are indicative of developmental regulation. the pachytene spermatocyte stage is significant during spermatogenesis. it involves genetic recombination, which occurs only in germ cells through cross-over between paired chromosomes and increases rna and protein synthesis in preparation for the next phase. transcription of more than half the total genes was found to start from the pachytene spermatocyte stage. germ cell-specific and developmentally regulated genes could be directly responsible for the spermatogenesis or fertilization. we also investigated the genes at the protein and cellular levels, providing functional perspectives of the genes. the proteins encoded by out of the genes were analyzed in living gc- cells. no expression of the other genes might be due to their peculiar protein natures, such as instability, translational delay and toxicity to the cells. cellular localization of the genes was divided into nucleus, endoplasmic reticulum, golgi apparatus, and cytoplasm. to further gain an insight into the characteristics of the proteins, we generated antibodies to five proteins. the western blot analysis disclosed that two proteins were restricted to testicular spermatogenic cells and testicular sperm, while the others were present at all stages, including testicular spermatogenic cells, testicular sperm, and mature sperm. results from the immunofluorescence analysis of testis sections and mature sperm corroborate and extend the western blot data. taken together, our transfection and immuno-analyses provided new information about genes at the protein and cellular levels. among the genes with the in vitro data, eight genes have in silico information congruous with the in vitro results. we attempted to categorize these eight genes based on all the in silico and in vitro data, and relate them to potential functions in reproduction. three of the gene products are likely to be involved in transcriptional regulation. all of these proteins were found to be localized in the nucleus of gc- cells transfected with the corresponding cdnas. mm. predicted to be present in a perinuclear region, was targeted to the nucleus of gc- cells. this protein might be related to nuclear activity or integrity of spermatogenic cells. according to a recent report, mm. belongs to the cysteine-rich perinuclear theca family with potential functions in the remodelling of the spermatid nucleus. three of the genes seem to encode proteins implicated in sperm structure and motility. it should be noted that these three genes have been named and reported previously. nonetheless, we did not eliminate them because we have obtained new information about these proteins in this investigation. mm. named tep has been suggested to be involved in the biogenesis of the acrosome and the midpiece of the sperm tail. our western blot analysis newly revealed that the mm. protein made as a kda-protein in testicular cells is changed to a higher molecular weight form between the stages of testicular sperm and mature sperm, suggesting post-translational modification. consistent with this, the protein contains several putative glycosylation and phosphorylation sites. mm. identified as catsper, was found localized to the endoplasmic reticulum in gc- cells in this study. other catsper family members, catsper and catsper are known to be specifically expressed in sperm and linked to sperm motility. in fact, the expression pattern of the catsper gene and its essential role in sperm motility and male fertility were reported during the preparation of the present paper. mm. has been named tektin which belongs to the tektin family. tektin and tektin are microtubule- or outer dense fiber-associated proteins in sperm flagella. here, we provide the first information about the mm protein, tektin this protein was found to be present at the sperm flagella. it should be noted that the molecular size of the mm protein was increased during spermiogenesis, suggestive of post-translational modification. finally, we also obtained original findings on mm. the protein encoded by mm. was targeted to the golgi apparatus in gc- cells. the immuno-analysis uncovered that the mm protein, kda, is present in both spermatogenic cells and mature sperm. in particular, the protein was located in the acrosomal region of mature sperm. it is important to mention that mm. is predicted to contain a trypsin-like serine protease domain. the acrosome is a golgi-derived secretory granule which is formed during spermiogenesis and positioned at the apex of mature sperm. when sperm reach the egg extracellular coat, the zona pellucida, during fertilization, they bind to it and undergo acrosome reaction, releasing the acrosomal contents at the site of sperm-egg binding. the hydrolytic and proteolytic enzymes comprising the acrosomal contents digest the zp and, thus, enable sperm to penetrate the zp. the sperm acrosome contains both unique enzymes and common enzymes present in somatic cells. to date, only a handful of unique enzymes have been identified and enzymes directly responsible for the fertilization process are unknown. thus, the mm. protein is a candidate for a type of protease involved in the penetration of the zp during fertilization.  identification of genes with spermatogenic cell-specific expression is crucial to understanding the molecular basis of spermatogenesis and fertilization. our in silico analysis indicates that the proportion of testis-specific genes in the spermatocyte unigene library is 11% and half of them has been unexplored. we have identified and characterized authentic genes by systematic and integrative approaches, providing insights to their genomic, transcript and protein characteristics. in particular, we predict potential functions of the eight genes, based on our in vitro data as well as in silico information. thus, the data provided by this study provide a large resource for further investigations into molecular mechanisms of mammalian male reproduction. 
in addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. we analyzed the mouse spermatocyte unigene library containing gene-oriented transcript clusters. our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction. we further analyzed the genes at the protein and cellular levels. transfection assays performed using gc- cells provided information on the cellular characteristics of the gene products. we identify authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein.
 the primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes.  we analyzed the mouse spermatocyte unigene library containing gene-oriented transcript clusters. we predict that 11% of these genes are testis-specific and systematically identified authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. we further analyzed the genes at the protein and cellular levels. transfection assays performed using gc- cells provided information on the cellular characteristics of the gene products. in addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. in particular, we found for the first time that mm. predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein.  we identify authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction. 