BACKGROUND
lipocalins are known to be involved in many important biological processes, of which one of them is chemical communication.
due to a conservative tertiary structure forming typical beta barrel all lipocalins share ability to bind various ligands of different size and structure.
for some of the lipocalin members there already exists evidence for causing significant behavioural effects by their specific pheromonal ligands.
the main lipocalin proteins studied to date in association with chemical communication are the mouse major urinary proteins  secreted predominantly in the urine and saliva and odorant binding proteins  with predominant site of expression in nasal tissue.
in mice, urine is an important source of chemical information, frequently investigated by conspecifics and having the ability to elicit various behavioural responses including aggression, dominance and mate choice  <cit> .
the major components of mouse urine are the major urinary proteins, constituting approximately 90% of all proteins in the urine.
the amount of mups excreted to urine is sexually dimorphic, with males expressing more mups than females on both mrna and protein level  <cit> .
however even the lower amount of mups in female urine may be sufficient as a signal of reproductive status, advertising the beginning of oestrus  <cit> .
various substances with pheromonal effect bound to major urinary proteins were identified  <cit>  and many of studies have brought the evidence that these mup-ligand complexes  <cit> , unbounded ligands and potentially mup itself  <cit>  have effect upon the reproductive physiology of the signal/pheromone receiver.
mups are coded by high number of genes which have probably evolved by recent gene duplication and form a cluster of  <dig> intact genes and  <dig> pseudogenes on chromosome  <dig>  <cit> .
such variability leads to the hypothesis that urine marks may serve for individual odour discrimination in mice  <cit> .
however, high variability of mups is clearly demonstrated only in particular subspecies of the house mouse mus musculus domesticus and derived laboratory strains and is completely missing in some other rodent species.
contrary to mup sexual dimorphism biased to males there exists another lipocalin in hamsters, called aphrodisin, of which the expression is attributed only to females.
aphrodisin is a lipocalin that was isolated from hamster vaginal discharge by singer et al.
<cit>  and is detected  via the vomeronasal organ of male hamsters.
the term aphrodisin originates from its functional property to stimulate copulatory behaviour in male hamsters which were exposed to vaginal odour of females  <cit> .
analysis of various fractions of female vaginal discharge led to the conclusion that this lipocalin itself is responsible for various behavioural and physiological effects  <cit> .
however later studies revealed that recombinant aphrodisin produced in e. coli was clearly less active than aphrodisin isolated from vaginal discharge  <cit> .
it, however, remains to be determined whether 3d structure of lipocalins produced in bacteria corresponds to the structure detected in mammals that naturally produce aphrodisin.
the effect of aphrodisin may be enhanced by various ligands that were detected in hamster vaginal smears by briand et al.
<cit>  who described five specific compounds which are also present in insect pheromone blends.
analysis of the aphrodisin expression sites revealed that the main tissue expressing this gene is vagina with the bartholin's glands  <cit> , cervix uteri, low level of expression was detected also in ovaries  <cit> , and moreover in the parotid glands of females  <cit> .
expression of aphrodisin is attributed only to females, nevertheless there is weak evidence for the absence of aphrodisin in males, because there is only one paper  <cit>  from available literature where the researchers analyzed male saliva and parotid glands and had found no expression.
to our knowledge, there is as yet no published information on the expression of aphrodisin in male bulbourethral glands which are homologous to female bartholin's glands.
according to the protein sequence analysis and of exon-intron organization, hamster aphrodisin falls within the monophyletic group of lipocalins  including i.a.
mouse obps, rat obp1f and probasin  <cit> .
protein sequence comparison of aphrodisin shows the highest similarity with the mouse obp1a and obp1b   <cit>  and ~40% similarity with the rat obp1f  <cit> .
our presented novel sequences on the translated protein level share 59% similarity with this hamster lipocalin.
in mice odorant binding protein forms heterodimer containing  <dig> kda and  <dig> kda subunits called obp1a and obp1b, which are expressed mainly in the main olfactory epithelium  <cit> .
in our previous paper  <cit>  we have analyzed public databases  and proposed that the mouse obp forms a gene cluster of more than only two obp variants.
on the x chromosome in the range of  <dig>  mb there are at least  <dig> obp genes, including one pseudogene and these genes can be further divided into three structural groups  <cit> .
lipocalins linked to the monophyletic cluster x  <cit> , including all mouse obp genes, homologous rat obp genes from x chromosome and aphrodisin share typical sequence characterized by extra two cystein residues forming cxxxc motif  <cit> .
in addition to the conservative disulfide bond tightening the beta-barel to the c-terminal tail  <cit>  this extra two cystein residues form a second disulfide bridge characteristic for this group of lipocalins  <cit> .
interestingly, this motif also occurs in some other lipocalin genes, namely probasin, with chromosomal location next to the obp cluster, and recently described hamster's male specific salivary protein  and female lacrimal protein   <cit> .
it suggests that despite the different names, these lipocalins belong to the obp group and may also act as odorant/pheromone carriers, however, this has to be further studied.
obps are known to bind various odorant molecules  <cit>  and are found through the wide spectrum of animals  <cit> .
the suggested role of obp in olfaction predicts the transport of hydrophobic odorants across the aqueous mucous layer to the odorant receptors  <cit> , termination of olfactory signals by removing the odorants from the receptor  <cit>  or enhancing the signal by maintain the active conformation of olfactory receptors and thus contribute to signal amplification  <cit> .
boudjelal et al.
<cit>  pointed out that the receptor for obp is presented in number of tissues unrelated to olfaction and therefore suggested that the function of this lipocalin may not be limited only to odorant transport.
another feature of lipocalins is their ability to scavenge for toxic substances or act as an antibacterial defence.
the scavenger function of porcine and bovine obp was suggested  <cit>  in an experiment which clearly showed that obp binds with high affinity hne , a toxic compound derived from lipid peroxidation, and therefore can protect living cells from damage caused by oxidative stress by removing cytotoxic compounds from the nasal mucus.
the antibacterial defence arises from the ability of some lipocalins  to bind bacterial siderophores and thus prevent the bacterial growth  <cit> .
there are few candidate siderocalins in mammals , however it is possible that due to the conservative tertiary structure some other lipocalins may also share this ability  <cit> .
the main goal of this paper was to identify dominant transcripts/proteins in myodes glareolus that are secreted to urine, saliva and vaginal secrets and propose potential roles for these proteins.
bank voles are a target species in various ecological studies and our previous analysis revealed that individuals of both sex contain candidate proteins of approximately 17- <dig> kda in the urine.
however, there is little available information on the mechanisms that bank voles use to mark their territories, attract individuals of opposite sex and how is this information transmitted to other individuals.
therefore, this study employed gene expression and gene characterization techniques to uncover a potential group of proteins that may play a role in chemical communication in this species.
RESULTS
m. glareolus voles express three novel obp genes/transcripts in different secretory tissues
rt-pcr was conducted using mrna from secretory tissues .
the samples were derived from wild caught m. glareolus to ascertain whether m. glareolus express transcripts for candidate lipocalins that are known to carry volatiles facilitating chemical communication.
use of a hamster aphrodisin specific primer set generated clear bands in the expected area.
m. glareolus amplicons were purified and cloned with ta topo kit and consequently the total of  <dig> clones were each forward and reverse sequenced.
this analysis revealed three unique cdna amplicons described as obp <dig> , obp <dig> , and obp <dig>   which were similar to hamster aphrodisin  and mouse obp1a and obp1b  from public ncbi database.
sequencing further revealed that  <dig> sequences - identified as obp <dig> - were present in individuals of both sex and across all tissues analyzed except male prostate.
three nucleotide polymorphisms were revealed in two of the female samples  causing 1% difference  from obp <dig> for which we use the term obp1b.
however, this could be a result of heterozygous effect.
the obp <dig> transcripts were detected in male prostate  whilst obp <dig> was detected from a single clone using cdna from submandibular glands.
more detailed analysis of translated transcripts revealed that the novel obp genes have typical lipocalin signatures including gxw motif  <cit> , two conservative cystein residues characteristic for most of the lipocalins, and extra two cysteins forming the cxxxc motif .
this motif is a specific feature of hamster aphrodisin described as a position of the second disulphide bond in this lipocalin  <cit> .
to add, all mouse obps, rat obp1f, and other lipocalins compared in figure  <dig> share the same feature.
contrary to hamsters, the expression of novel genes is not female specific but is present in individuals of both sex and across different tissues studied.
although, these novel sequences were highly similar to hamster aphrodisin  we use more general term obps  with which they share 72% identity  because the term aphrodisin presupposes its aphrodisiac function which was not assessed for novel proteins in this study.
2-d electrophoresis and ms analyses confirmed the presence of detected obps in the urine and saliva but not in vaginal flushes
image analysis of 2d gels of urinary, salivary and vaginal samples revealed protein spots in the predicted region of pi  and in the mw range of 18- <dig> kda.
after tryptic digestion protein spots were subjected to maldi-ms/ms and lc-ms/ms analysis.
ms/ms data were primarily searched against to the obp library containing dna- derived sequences.
subsequently, the data were searched against ncbi protein database to avoid random false positives caused by small size of the obp library.
ms/ms data of peptides which have not matched to obp library in mascot database search were manually evaluated.
based on ms/ms data three obp proteins were unambiguously identified: obp <dig> and two obp <dig> isoforms .
these proteins correspond to the most intensive spots in 2d gel of the salivary sample.
only obp <dig> and obp3-a were identified in the urinary sample .
the transcript corresponding to obp <dig> gene was abundant on the level of mrna, however, remained undetected on the level of protein.
reasons for this failure may involve modifications that changed its pi or that the concentration of obp <dig> protein was low and thus below ms identification limits.
furthermore, the obp proteins were present also in less intense spots but their identification was based on sequence regions identical for all variants not allowing correct determination of the particular variant and they are not further discussed in this study.
results of ms/ms analysis of three main obp variants in salivary samples are summarized in figure  <dig>
sequence coverage ranged from 63%  to 77%  when non-specific trypsin cleavage were taken in account .
while detected parts of obp <dig> sequence were completely in agreement with cdna-derived sequence, obp <dig> protein sequence showed several differences from cdna-derived sequence indicating the presence of two obp <dig> variants .
the amino acid changes were tentatively identified and their confirmation on the mrna and protein level is a subject of ongoing projects.
complete results of mascot searches against obp sequences library including amino acid corrections for obp <dig> are available in additional file  <dig>
discussion
wild caught m. glareolus voles transcribe  three novel obp genes highly similar to those previously described as hamster aphrodisin.
exon  <dig>  encoding the first sheet of beta barrel, includes a specific signature in amino acid sequence  that places these novel genes to a family of proteins called lipocalins.
as in mouse and hamster these myodes obp genes also include specific amino acid feature cxxxc which is known to form disulphide bond in hamster aphrodisin.
for its ability to form a pheromone-carrying beta barrel this protein is important as it may have overtaken the role of major urinary proteins  in those species where mups are not present or are expressed in low quantities.
this argument is further supported by studies that show mups being important in individual recognition and mate choice  <cit> .
if lipocalins are generally important in these processes they must by substituted by a similar sort of proteins in those species where the expression of mups is lost.
m. glareolus obp  mrna derived nucleotide sequences  were aligned against the corresponding consensus sequences from the mus, rattus, and cricetus sequences from ncbi library.
the analyses of replicate trees in which the associated taxa/proteins clustered together revealed that novel myodes obps cluster together with cricetus aphrodisin, thus, forming a parallel cluster to mouse obp1a, obp1b and remaining mus obps  previously described as o4-o <dig> in stopková et al.
<cit> .
to add, both cricetus aphrodisin and myodes obp form a group of proteins that, due to a high structural similarity may also serve similar functions where a capacity to bind and transport pheromones by cricetus aphrodisin has already been documented  <cit> .
furthermore, the method of clustering in figure  <dig> shows that the divergence of obp-like lipocalins caused by gene duplication preceded a mouse - rat split that occurred  <dig>  millions of years ago as well as muroid and cricetid divergence that most likely occurred  <dig>  millions years ago  <cit> .
this is seen in prb  with a branching pattern where a mouse - rat split was preceded by a split of the mouse - rat common ancestor from cricetid rodents .
in contrast to mouse major urinary proteins with  <dig> genes and  <dig> pseudogenes, obps in myodes glareolus were detected as only three unique transcripts.
however, our 2-de and consequent ms/ms analyses indicate that the number of potential protein products depicted in figure  <dig> may be higher and that further analysis is required to uncover other potential genes and/or allelic variants.
on the level of proteins the number of detected variants exceeded the number of detected transcripts in case of obp <dig>  and several other obp proteins were detected in low abundant spots.
to add myodes glareolus obp is highly similar to cricetus and mesocricetus aphrodisin, however, their expression pattern is different.
contrary to hamsters, m. gareolus obp was detected in both sexes and in all tested tissues whilst it was missing in vaginal flushes or its expression was low.
its absence might have been caused by the fact that the main site of obp production in this area are prepucial glands which secret this protein in to the vaginal lumen under specific stimuli which our experimental sample collection method did not satisfy.
CONCLUSIONS
myodes glareolus voles express three novel aphrodisin-like genes here named as obp <dig>  obp <dig>  and obp <dig>  proteomic analysis further revealed that there may be further variants of this genes and that they are secreted into urine and saliva.
the comparative analysis provided evidence that these transcripts are expressed in all studied tissues and by individuals of both sex.
all this evidence - along with the fact that almost identical obps in other species have binding affinity to pheromones - suggests that these proteins may be used in m. glareolus for chemical communication similarly as major urinary proteins in mice.

 lipocalins are known to be involved in many important biological processes, of which one of them is chemical communication. due to a conservative tertiary structure forming typical beta barrel all lipocalins share ability to bind various ligands of different size and structure. for some of the lipocalin members there already exists evidence for causing significant behavioural effects by their specific pheromonal ligands. the main lipocalin proteins studied to date in association with chemical communication are the mouse major urinary proteins secreted predominantly in the urine and saliva and odorant binding proteins with predominant site of expression in nasal tissue. in mice, urine is an important source of chemical information, frequently investigated by conspecifics and having the ability to elicit various behavioural responses including aggression, dominance and mate choice. the major components of mouse urine are the major urinary proteins, constituting approximately 90% of all proteins in the urine. the amount of mups excreted to urine is sexually dimorphic, with males expressing more mups than females on both mrna and protein level. however even the lower amount of mups in female urine may be sufficient as a signal of reproductive status, advertising the beginning of oestrus. various substances with pheromonal effect bound to major urinary proteins were identified and many of studies have brought the evidence that these mup-ligand complexes, unbounded ligands and potentially mup itself have effect upon the reproductive physiology of the signal/pheromone receiver. mups are coded by high number of genes which have probably evolved by recent gene duplication and form a cluster of intact genes and pseudogenes on chromosome. such variability leads to the hypothesis that urine marks may serve for individual odour discrimination in mice. however, high variability of mups is clearly demonstrated only in particular subspecies of the house mouse mus musculus domesticus and derived laboratory strains and is completely missing in some other rodent species. contrary to mup sexual dimorphism biased to males there exists another lipocalin in hamsters, called aphrodisin, of which the expression is attributed only to females. aphrodisin is a lipocalin that was isolated from hamster vaginal discharge by singer et al. and is detected via the vomeronasal organ of male hamsters. the term aphrodisin originates from its functional property to stimulate copulatory behaviour in male hamsters which were exposed to vaginal odour of females. analysis of various fractions of female vaginal discharge led to the conclusion that this lipocalin itself is responsible for various behavioural and physiological effects. however later studies revealed that recombinant aphrodisin produced in e. coli was clearly less active than aphrodisin isolated from vaginal discharge. it, however, remains to be determined whether 3d structure of lipocalins produced in bacteria corresponds to the structure detected in mammals that naturally produce aphrodisin. the effect of aphrodisin may be enhanced by various ligands that were detected in hamster vaginal smears by briand et al. who described five specific compounds which are also present in insect pheromone blends. analysis of the aphrodisin expression sites revealed that the main tissue expressing this gene is vagina with the bartholin's glands, cervix uteri, low level of expression was detected also in ovaries, and moreover in the parotid glands of females. expression of aphrodisin is attributed only to females, nevertheless there is weak evidence for the absence of aphrodisin in males, because there is only one paper from available literature where the researchers analyzed male saliva and parotid glands and had found no expression. to our knowledge, there is as yet no published information on the expression of aphrodisin in male bulbourethral glands which are homologous to female bartholin's glands. according to the protein sequence analysis and of exon-intron organization, hamster aphrodisin falls within the monophyletic group of lipocalins including i.a. mouse obps, rat obp1f and probasin. protein sequence comparison of aphrodisin shows the highest similarity with the mouse obp1a and obp1b and ~40% similarity with the rat obp1f. our presented novel sequences on the translated protein level share 59% similarity with this hamster lipocalin. in mice odorant binding protein forms heterodimer containing kda and kda subunits called obp1a and obp1b, which are expressed mainly in the main olfactory epithelium. in our previous paper we have analyzed public databases and proposed that the mouse obp forms a gene cluster of more than only two obp variants. on the x chromosome in the range of mb there are at least obp genes, including one pseudogene and these genes can be further divided into three structural groups. lipocalins linked to the monophyletic cluster x, including all mouse obp genes, homologous rat obp genes from x chromosome and aphrodisin share typical sequence characterized by extra two cystein residues forming cxxxc motif. in addition to the conservative disulfide bond tightening the beta-barel to the c-terminal tail this extra two cystein residues form a second disulfide bridge characteristic for this group of lipocalins. interestingly, this motif also occurs in some other lipocalin genes, namely probasin, with chromosomal location next to the obp cluster, and recently described hamster's male specific salivary protein and female lacrimal protein. it suggests that despite the different names, these lipocalins belong to the obp group and may also act as odorant/pheromone carriers, however, this has to be further studied. obps are known to bind various odorant molecules and are found through the wide spectrum of animals. the suggested role of obp in olfaction predicts the transport of hydrophobic odorants across the aqueous mucous layer to the odorant receptors, termination of olfactory signals by removing the odorants from the receptor or enhancing the signal by maintain the active conformation of olfactory receptors and thus contribute to signal amplification. boudjelal et al. pointed out that the receptor for obp is presented in number of tissues unrelated to olfaction and therefore suggested that the function of this lipocalin may not be limited only to odorant transport. another feature of lipocalins is their ability to scavenge for toxic substances or act as an antibacterial defence. the scavenger function of porcine and bovine obp was suggested in an experiment which clearly showed that obp binds with high affinity hne, a toxic compound derived from lipid peroxidation, and therefore can protect living cells from damage caused by oxidative stress by removing cytotoxic compounds from the nasal mucus. the antibacterial defence arises from the ability of some lipocalins to bind bacterial siderophores and thus prevent the bacterial growth. there are few candidate siderocalins in mammals, however it is possible that due to the conservative tertiary structure some other lipocalins may also share this ability. the main goal of this paper was to identify dominant transcripts/proteins in myodes glareolus that are secreted to urine, saliva and vaginal secrets and propose potential roles for these proteins. bank voles are a target species in various ecological studies and our previous analysis revealed that individuals of both sex contain candidate proteins of approximately 17- kda in the urine. however, there is little available information on the mechanisms that bank voles use to mark their territories, attract individuals of opposite sex and how is this information transmitted to other individuals. therefore, this study employed gene expression and gene characterization techniques to uncover a potential group of proteins that may play a role in chemical communication in this species.  m. glareolus voles express three novel obp genes/transcripts in different secretory tissues rt-pcr was conducted using mrna from secretory tissues. the samples were derived from wild caught m. glareolus to ascertain whether m. glareolus express transcripts for candidate lipocalins that are known to carry volatiles facilitating chemical communication. use of a hamster aphrodisin specific primer set generated clear bands in the expected area. m. glareolus amplicons were purified and cloned with ta topo kit and consequently the total of clones were each forward and reverse sequenced. this analysis revealed three unique cdna amplicons described as obp, obp, and obp which were similar to hamster aphrodisin and mouse obp1a and obp1b from public ncbi database. sequencing further revealed that sequences - identified as obp - were present in individuals of both sex and across all tissues analyzed except male prostate. three nucleotide polymorphisms were revealed in two of the female samples causing 1% difference from obp for which we use the term obp1b. however, this could be a result of heterozygous effect. the obp transcripts were detected in male prostate whilst obp was detected from a single clone using cdna from submandibular glands. more detailed analysis of translated transcripts revealed that the novel obp genes have typical lipocalin signatures including gxw motif, two conservative cystein residues characteristic for most of the lipocalins, and extra two cysteins forming the cxxxc motif. this motif is a specific feature of hamster aphrodisin described as a position of the second disulphide bond in this lipocalin. to add, all mouse obps, rat obp1f, and other lipocalins compared in figure share the same feature. contrary to hamsters, the expression of novel genes is not female specific but is present in individuals of both sex and across different tissues studied. although, these novel sequences were highly similar to hamster aphrodisin we use more general term obps with which they share 72% identity because the term aphrodisin presupposes its aphrodisiac function which was not assessed for novel proteins in this study. 2-d electrophoresis and ms analyses confirmed the presence of detected obps in the urine and saliva but not in vaginal flushes image analysis of 2d gels of urinary, salivary and vaginal samples revealed protein spots in the predicted region of pi and in the mw range of 18- kda. after tryptic digestion protein spots were subjected to maldi-ms/ms and lc-ms/ms analysis. ms/ms data were primarily searched against to the obp library containing dna- derived sequences. subsequently, the data were searched against ncbi protein database to avoid random false positives caused by small size of the obp library. ms/ms data of peptides which have not matched to obp library in mascot database search were manually evaluated. based on ms/ms data three obp proteins were unambiguously identified: obp and two obp isoforms. these proteins correspond to the most intensive spots in 2d gel of the salivary sample. only obp and obp3-a were identified in the urinary sample. the transcript corresponding to obp gene was abundant on the level of mrna, however, remained undetected on the level of protein. reasons for this failure may involve modifications that changed its pi or that the concentration of obp protein was low and thus below ms identification limits. furthermore, the obp proteins were present also in less intense spots but their identification was based on sequence regions identical for all variants not allowing correct determination of the particular variant and they are not further discussed in this study. results of ms/ms analysis of three main obp variants in salivary samples are summarized in figure sequence coverage ranged from 63% to 77% when non-specific trypsin cleavage were taken in account. while detected parts of obp sequence were completely in agreement with cdna-derived sequence, obp protein sequence showed several differences from cdna-derived sequence indicating the presence of two obp variants. the amino acid changes were tentatively identified and their confirmation on the mrna and protein level is a subject of ongoing projects. complete results of mascot searches against obp sequences library including amino acid corrections for obp are available in additional file discussion wild caught m. glareolus voles transcribe three novel obp genes highly similar to those previously described as hamster aphrodisin. exon encoding the first sheet of beta barrel, includes a specific signature in amino acid sequence that places these novel genes to a family of proteins called lipocalins. as in mouse and hamster these myodes obp genes also include specific amino acid feature cxxxc which is known to form disulphide bond in hamster aphrodisin. for its ability to form a pheromone-carrying beta barrel this protein is important as it may have overtaken the role of major urinary proteins in those species where mups are not present or are expressed in low quantities. this argument is further supported by studies that show mups being important in individual recognition and mate choice. if lipocalins are generally important in these processes they must by substituted by a similar sort of proteins in those species where the expression of mups is lost. m. glareolus obp mrna derived nucleotide sequences were aligned against the corresponding consensus sequences from the mus, rattus, and cricetus sequences from ncbi library. the analyses of replicate trees in which the associated taxa/proteins clustered together revealed that novel myodes obps cluster together with cricetus aphrodisin, thus, forming a parallel cluster to mouse obp1a, obp1b and remaining mus obps previously described as o4-o in stopková et al.. to add, both cricetus aphrodisin and myodes obp form a group of proteins that, due to a high structural similarity may also serve similar functions where a capacity to bind and transport pheromones by cricetus aphrodisin has already been documented. furthermore, the method of clustering in figure shows that the divergence of obp-like lipocalins caused by gene duplication preceded a mouse - rat split that occurred millions of years ago as well as muroid and cricetid divergence that most likely occurred millions years ago. this is seen in prb with a branching pattern where a mouse - rat split was preceded by a split of the mouse - rat common ancestor from cricetid rodents. in contrast to mouse major urinary proteins with genes and pseudogenes, obps in myodes glareolus were detected as only three unique transcripts. however, our 2-de and consequent ms/ms analyses indicate that the number of potential protein products depicted in figure may be higher and that further analysis is required to uncover other potential genes and/or allelic variants. on the level of proteins the number of detected variants exceeded the number of detected transcripts in case of obp and several other obp proteins were detected in low abundant spots. to add myodes glareolus obp is highly similar to cricetus and mesocricetus aphrodisin, however, their expression pattern is different. contrary to hamsters, m. gareolus obp was detected in both sexes and in all tested tissues whilst it was missing in vaginal flushes or its expression was low. its absence might have been caused by the fact that the main site of obp production in this area are prepucial glands which secret this protein in to the vaginal lumen under specific stimuli which our experimental sample collection method did not satisfy.  myodes glareolus voles express three novel aphrodisin-like genes here named as obp obp and obp proteomic analysis further revealed that there may be further variants of this genes and that they are secreted into urine and saliva. the comparative analysis provided evidence that these transcripts are expressed in all studied tissues and by individuals of both sex. all this evidence - along with the fact that almost identical obps in other species have binding affinity to pheromones - suggests that these proteins may be used in m. glareolus for chemical communication similarly as major urinary proteins in mice. 
on the protein level we confirmed the presence of obp and obp in both saliva and urine. on the protein level we have detected further variants and thus we assume that similarly as major urinary proteins in mice, these proteins may be important in chemical communication in this cricetid rodent. therefore, we aimed at characterization of candidate lipocalins in myodes glareolus which are potentially linked to chemical communication. the roles of lipocalins in communication were studied in many organisms and especially in mice which excrete major urinary proteins in excessive amounts in saliva and urine. one of them is aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males. chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. to assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. our results demonstrate that three novel obps are predominant lipocalins in myodes urine and saliva. here we show that aphrodisin-like proteins exist in other species, belong to a group of odorant binding proteins, and contrary to the expression of aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of m. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus.
 chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. efficient communication via this protein - pheromone complex is essential for triggering multiple responses including aggression, mate choice, copulatory behaviour, and onset and synchronization of oestrus. the roles of lipocalins in communication were studied in many organisms and especially in mice which excrete major urinary proteins in excessive amounts in saliva and urine. other mammals, however, often lack the genes for mups or their expression is very low. therefore, we aimed at characterization of candidate lipocalins in myodes glareolus which are potentially linked to chemical communication. one of them is aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males.  here we show that aphrodisin-like proteins exist in other species, belong to a group of odorant binding proteins, and contrary to the expression of aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of m. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus. on the level of mrna, we have detected three different gene variants. to assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. on the protein level we confirmed the presence of obp and obp in both saliva and urine. appropriate bands in the range of 17- kda from vaginal discharge were, however, beyond the ms detection limits.  our results demonstrate that three novel obps are predominant lipocalins in myodes urine and saliva. on the protein level we have detected further variants and thus we assume that similarly as major urinary proteins in mice, these proteins may be important in chemical communication in this cricetid rodent. 