In most mammals and particularly in mice, chemical communication relies on the detection of ethologically relevant fitness-related cues from other individuals. In mice, urine is the primary source of these signals, so we employed proteomics and metabolomics to identify key components of chemical signalling. We show that there is a correspondence between urinary volatiles and proteins in the representation of genetic background, sex and environment in two house mouse subspecies Mus musculus musculus and M. m. domesticus. We found that environment has a strong influence upon proteomic and metabolomic variation and that volatile mixtures better represent males while females have surprisingly more sex-biased proteins. Using machine learning and combined-omics techniques, we identified mixtures of metabolites and proteins that are associated with biological features.

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Vestec Czech Republic

Department of Zoology Faculty of Science BIOCEV Charles University Vestec Prague Czech Republic

Research Facility Studenec Institute of Vertebrate Biology Czech Academy of Sciences Brno Czech Republic

Zobrazit více v PubMed

Yoon H, Enquist LW, Dulac C. Olfactory inputs to hypothalamic neurons controlling reproduction and fertility. Cell. 2006;123:669–682. doi: 10.1016/j.cell.2005.08.039. PubMed DOI

Marom K, et al. The vomeronasal system can learn novel stimulus response pairings. Cell Rep. 2019;27:676–684.e676. doi: 10.1016/j.celrep.2019.03.042. PubMed DOI

Zala SM, Potts WK, Penn DJ. Scent-marking displays provide honest signals of health and infection. Behav. Ecol. 2004;15:338–344. doi: 10.1093/beheco/arh022. DOI

Zala SM, Bilak A, Perkins M, Potts WK, Penn DJ. Female house mice initially shun infected males, but do not avoid mating with them. Behav. Ecol. Sociobiol. 2015;69:715–722. doi: 10.1007/s00265-015-1884-2. DOI

Mucignat-Caretta C, Cavaggioni A, Caretta A. Male urinary chemosignals differentially affect aggressive behavior in male mice. J. Chem. Ecol. 2004;30:777–791. doi: 10.1023/B:JOEC.0000028431.29484.d7. PubMed DOI

Mucignat-Caretta C, et al. Urinary volatile molecules vary in males of the 2 European subspecies of the house mouse and their hybrids. Chem. Senses. 2010;35:647–654. doi: 10.1093/chemse/bjq049. PubMed DOI

Stopková R, Stopka P, Janotová K, Jedelsky PL. Species-specific expression of major urinary proteins in the house mice (Mus musculus musculus and Mus musculus domesticus) J. Chem. Ecol. 2007;33:861–869. doi: 10.1007/s10886-007-9262-9. PubMed DOI

Pérez-Gómez A, et al. innate predator odor aversion driven by parallel olfactory subsystems that converge in the ventromedial hypothalamus. Curr. Biol. 2015;25:1340–1346. doi: 10.1016/j.cub.2015.03.026. PubMed DOI PMC

Yang J, et al. Landscapes of bacterial and metabolic signatures and their interaction in major depressive disorders. Sci. Adv. 2020;6:eaba8555. doi: 10.1126/sciadv.aba8555. PubMed DOI PMC

Ninkovic V, Markovic D, Rensing M. Plant volatiles as cues and signals in plant communication. Plant Cell Environ. 2020 doi: 10.1111/pce.13910. PubMed DOI PMC

Manoel D, et al. Deconstructing the mouse olfactory percept through an ethological atlas. Curr. Biol. 2021 doi: 10.1016/j.cub.2021.04.020. PubMed DOI PMC

Bansal R, et al. Do all mice smell the same? Chemosensory cues from inbred and wild mouse strains elicit stereotypic sensory representations in the accessory olfactory bulb. BMC Biol. 2021;19:133. doi: 10.1186/s12915-021-01064-7. PubMed DOI PMC

Bergan JF, Ben-Shaul Y, Dulac C. Sex-specific processing of social cues in the medial amygdala. Elife. 2014;3:e02743. doi: 10.7554/eLife.02743. PubMed DOI PMC

Nagel M, et al. A systematic comparison of semiochemical signaling in the accessory olfactory system of wild and lab strain mice. Chem. Senses. 2018;43:E31–E31.

Spehr M, et al. Parallel processing of social signals by the mammalian main and accessory olfactory systems. Cell. Mol. life Sci. 2006;63:1476–1484. doi: 10.1007/s00018-006-6109-4. PubMed DOI PMC

van der Linden C, Jakob S, Gupta P, Dulac C, Santoro SW. Sex separation induces differences in the olfactory sensory receptor repertoires of male and female mice. Nat. Commun. 2018;9:5081. doi: 10.1038/s41467-018-07120-1. PubMed DOI PMC

Santoro SW, Jakob S. Gene expression profiling of the olfactory tissues of sex-separated and sex-combined female and male mice. Sci. Data. 2018;5:180260. doi: 10.1038/sdata.2018.260. PubMed DOI PMC

Moss RL, et al. Urine-derived compound evokes membrane responses in mouse vomeronasal receptor neurons. J. Neurophysiol. 1997;77:2856–2862. doi: 10.1152/jn.1997.77.5.2856. PubMed DOI

Leinders-Zufall T, et al. Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature. 2000;405:792–796. doi: 10.1038/35015572. PubMed DOI

Ibarra-Soria X, Levitin MO, Logan DW. The genomic basis of vomeronasal-mediated behaviour. Mamm. Genome. 2014;25:75–86. doi: 10.1007/s00335-013-9463-1. PubMed DOI PMC

Wynn EH, Sánchez-Andrade G, Carss KJ, Logan DW. Genomic variation in the vomeronasal receptor gene repertoires of inbred mice. BMC Genomics. 2012;13:415. doi: 10.1186/1471-2164-13-415. PubMed DOI PMC

Buck L, Axel R. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell. 1991;65:175–187. doi: 10.1016/0092-8674(91)90418-x. PubMed DOI

Whitten WK, Bronson FH, Greenstein JA. estrus-inducing pheromone of male mice: Transport by movement of air. Science. 1968;161:584–585. doi: 10.1126/science.161.3841.584. PubMed DOI

Whitten WK. Modification of the oestrous cycle of the mouse by external stimuli associated with the male. Changes in the oestrous cycle determined by vaginal smears. J. Endocrinol. 1958;17:307–313. doi: 10.1677/joe.0.0170307. PubMed DOI

Novotny MV, Ma W, Wiesler D, Zídek L. Positive identification of the puberty-accelerating pheromone of the house mouse: The volatile ligands associating with the major urinary protein. Proc. R. Soc. Lond. B. 1999;266:2017–2022. doi: 10.1098/rspb.1999.0880. PubMed DOI PMC

Jemiolo B, Novotny MV. Inhibition of sexual maturation in juvenile female and male mice by a chemosignal of female origin. Physiol. Behav. 1994;55:519–522. doi: 10.1016/0031-9384(94)90110-4. PubMed DOI

Jemiolo B, Harvey S, Novotny M. Promotion of the whitten effect in female mice by synthetic analogs of male urinary constituents. PNAS. 1986;83:4576–4579. doi: 10.1073/pnas.83.12.4576. PubMed DOI PMC

Janotova K, Stopka P. The level of major urinary proteins is socially regulated in wild Mus musculus musculus. J. Chem. Ecol. 2011;37:647–656. doi: 10.1007/s10886-011-9966-8. PubMed DOI

Stopka P, Janotova K, Heyrovsky D. The advertisement role of major urinary proteins in mice. Physiol. Behav. 2007;91:667–670. doi: 10.1016/j.physbeh.2007.03.030. PubMed DOI

Kahan A, Ben-Shaul Y. extracting behaviorally relevant traits from natural stimuli: benefits of combinatorial representations at the accessory olfactory bulb. PLoS Comput. Biol. 2016;12:e1004798. doi: 10.1371/journal.pcbi.1004798. PubMed DOI PMC

Rusu AS, Krackow S, Jedelsky PL, Stopka P, Konig B. A qualitative investigation of major urinary proteins in relation to the onset of aggressive behavior and dispersive motivation in male wild house mice (Mus musculus domesticus) J. Ethol. 2008;26:127–135. doi: 10.1007/s10164-007-0042-3. DOI

Roberts SA, Davidson AJ, McLean L, Beynon RJ, Hurst JL. Pheromonal induction of spatial learning in mice. Science. 2012;338:1462–1465. doi: 10.1126/science.1225638. PubMed DOI

Chamero P, et al. Identification of protein pheromones that promote aggressive behaviour. Nature. 2007;450:899–903. doi: 10.1038/nature05997. PubMed DOI

Sturm T, et al. Mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons. Nat. Commun. 2013;4:1616. doi: 10.1038/ncomms2610. PubMed DOI

Leinders-Zufall T, et al. MHC class I peptides as chemosensory signals in the vomeronasal organ. Science. 2004;306:1003–1037. doi: 10.1126/science.1102818. PubMed DOI

Kwak J, et al. Changes in volatile compounds of mouse urine as it ages: Their interactions with water and urinary proteins. Physiol. Behav. 2013;120:211–219. doi: 10.1016/j.physbeh.2013.08.011. PubMed DOI

Kwak J, et al. Differential binding between volatile ligands and major urinary proteins due to genetic variation in mice. Physiol. Behav. 2012;107:112–120. doi: 10.1016/j.physbeh.2012.06.008. PubMed DOI

Novotny MV, et al. A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice. Chem. Biol. 1999;6:377–383. doi: 10.1016/S1074-5521(99)80049-0. PubMed DOI

Novotny MV. Pheromones, binding proteins and receptor responses in rodents. Biochem. Soc. 2003;31:117–122. doi: 10.1042/bst0310117. PubMed DOI

Zidek L, et al. NMR mapping of the recombinant mouse major urinary protein i binding site occupied by the pheromone 2-sec-Butyl-4,5-dihydrothiazole. Biochemistry. 1999;38:9850–9861. doi: 10.1021/bi990497t. PubMed DOI

Sharrow SD, Vaughn JL, Žídek L, Novotny MV, Stone MJ. Pheromone binding by polymorphic mouse major urinary proteins. Protein Sci. 2002;11:2247–2256. doi: 10.1110/ps.0204202. PubMed DOI PMC

Phelan MM, McLean L, Hurst JL, Beynon RJ, Lian LY. Comparative study of the molecular variation between 'central' and 'peripheral' MUPs and significance for behavioural signalling. Biochem. Soc. Trans. 2014;42:866–872. doi: 10.1042/BST20140082. PubMed DOI

Phelan MM, et al. The structure, stability and pheromone binding of the male mouse protein sex pheromone darcin. PLoS One. 2014;9:e108415. doi: 10.1371/journal.pone.0108415. PubMed DOI PMC

Robertson D, Hurst J, Hubbard S, Gaskell SJ, Beynon R. Ligands of urinary lipocalins from the mouse: Uptake of environmentally derived chemicals. J. Chem. Ecol. 1998;24:1127–1140. doi: 10.1023/A:1022434300449. DOI

Janotová K, Stopka P. Mechanisms of chemical communication: The role of major urinary proteins. Folia Zool. 2009;58:41–55.

Macek P, Novak P, Krizova H, Zidek L, Sklenar V. Molecular dynamics study of major urinary protein-pheromone interactions: A structural model for ligand-induced flexibility increase. FEBS Lett. 2006;580:682–684. doi: 10.1016/j.febslet.2005.12.088. PubMed DOI

Timm DE, Baker LJ, Mueller H, Zidek L, Novotny MV. Structural basis of pheromone binding to mouse major urinary protein (MUP-I) Protein Sci. 2001;10:997–1004. doi: 10.1110/ps.52201. PubMed DOI PMC

Roberts SA, et al. Individual odour signatures that mice learn are shaped by involatile major urinary proteins (MUPs) BMC Biol. 2018;16:48. doi: 10.1186/s12915-018-0512-9. PubMed DOI PMC

Roberts SA, et al. Darcin: A male pheromone that stimulates female memory and sexual attraction to an individual male's odour. BMC Biol. 2010 doi: 10.1186/1741-7007-1188-1175. PubMed DOI PMC

Kaur AW, et al. Murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors. Cell. 2014;157:676–688. doi: 10.1016/j.cell.2014.02.025. PubMed DOI PMC

Papes F, Logan DW, Stowers L. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell. 2010;141:692–703. doi: 10.1016/j.cell.2010.03.037. PubMed DOI PMC

Demir E, et al. The pheromone darcin drives a circuit for innate and reinforced behaviours. Nature. 2020 doi: 10.1038/s41586-020-1967-8. PubMed DOI

Johnson D, Al-Shawi R, Bishop JO. Sexual dimorphism and growth hormone induction of murine phero-binding proteins. J. Mol. Endocrinol. 1995;14:21–34. doi: 10.1677/jme.0.0140021. PubMed DOI

Clissold PM, Hainey S, Bishop JO. Messenger RNAs coding for mouse urinary proteins are differentially induced by testosterone. Biochem. Genet. 1984;22:379–387. doi: 10.1007/BF00484236. PubMed DOI

Shaw PH, Held WA, Hastie ND. The gene family for major urinary proteins: Expression in several secretory tissues of the mouse. Cell. 1983;32:755–761. doi: 10.1016/0092-8674(83)90061-2. PubMed DOI

Zidek L, Novotny MV, Stone MJ. Increased protein backbone conformational entropy upon hydrophobic ligand binding. Nat. Struct. Biol. 1999;6:1118–1121. doi: 10.1038/70057. PubMed DOI

Hurst JL, Robertson DHL, Tolladay U, Beynon RJ. Proteins in urine scent marks of male house mice extend the longevity of olfactory signals. Anim. Behav. 1998;55:1289–1297. doi: 10.1006/anbe.1997.0650. PubMed DOI

Sheehan MJ, Campbell P, Miller CH. Evolutionary patterns of major urinary protein scent signals in house mice and relatives. Mol. Ecol. 2019;28:3587–3601. doi: 10.1111/mec.15155. PubMed DOI

Hurst JL, et al. Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation. Sci. Rep. 2017;7:44992. doi: 10.1038/srep44992. PubMed DOI PMC

Smadja C, Ganem G. Divergence of odorant signals within and between the two European subspecies of the house mouse. Behav. Ecol. 2008;19:223–230. doi: 10.1093/beheco/arm127. DOI

Smadja C, Ganem G. Subspecies recognition in the house mouse: A study of two populations from the border of a hybrid zone. Behav. Ecol. 2002;13:312–320. doi: 10.1093/beheco/13.3.312. DOI

Bímová B, Albrecht T, Macholán M, Piálek J. Signalling components of mate recognition system in the house mouse. Behav. Proc. 2009;80:20–27. doi: 10.1016/j.beproc.2008.08.004. PubMed DOI

Cerna M, Kuntova B, Talacko P, Stopkova R, Stopka P. Differential regulation of vaginal lipocalins (OBP, MUP) during the estrous cycle of the house mouse. Sci Rep. 2017;7:11674. doi: 10.1038/s41598-017-12021-2. PubMed DOI PMC

Thoss M, et al. Regulation of volatile and non-volatile pheromone attractants depends upon male social status. Sci. Rep. 2019;9:489. doi: 10.1038/s41598-018-36887-y. PubMed DOI PMC

Enk VM, et al. Regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods. Mol. Biosyst. 2016;12:3005–3016. doi: 10.1039/c6mb00278a. PubMed DOI PMC

Cox J, et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteomics. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Levy M, Blacher E, Elinav E. Microbiome, metabolites and host immunity. Curr. Opin. Microbiol. 2017;35:8–15. doi: 10.1016/j.mib.2016.10.003. PubMed DOI

Stopková R, Otčenášková T, Matějková T, Kuntová B, Stopka P. Biological roles of lipocalins in chemical communication, reproduction, and regulation of microbiota. Front. Physiol. 2021 doi: 10.3389/fphys.2021.740006. PubMed DOI PMC

Moudra A, et al. Phenotypic and clonal stability of antigen-inexperienced memory-like T cells across the genetic background, hygienic status, and aging. J. Immunol. 2021;206:2109–2121. doi: 10.4049/jimmunol.2001028. PubMed DOI PMC

Kreisinger J, Čížková D, Vohánka J, Piálek J. Gastrointestinal microbiota of wild and inbred individuals of two house mouse subspecies assessed using high-throughput parallel pyrosequencing. Mol. Ecol. 2014;23:5048–5060. doi: 10.1111/mec.12909. PubMed DOI

Gowaty PA, Drickamer LC, Schmid-Holmes S. Male house mice produce fewer offspring with lower viability and poorer performance when mated with females they do not prefer. Anim. Behav. 2003;65:95–103. doi: 10.1006/anbe.2002.2026. DOI

Bimova BV, et al. Reinforcement selection acting on the European house mouse hybrid zone. Mol Ecol. 2011;20:2403–2424. doi: 10.1111/j.1365-294X.2011.05106.x. PubMed DOI

Bianchi F, et al. Vertebrate odorant binding proteins as antimicrobial humoral components of innate immunity for pathogenic microorganisms. PLoS ONE. 2019;14:e0213545. doi: 10.1371/journal.pone.0213545. PubMed DOI PMC

Fluckinger M, Haas H, Merschak P, Glasgow BJ, Redl B. Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores. Antimicrob. Agents Chemother. 2004;48:3367–3372. doi: 10.1128/AAC.48.9.3367-3372.2004. PubMed DOI PMC

Witkowska-Banaszczak E, Długaszewska J. Essential oils and hydrophilic extracts from the leaves and flowers of Succisa pratensis Moench. and their biological activity. J. Pharmacy Pharmacol. 2017;69:1531–1539. doi: 10.1111/jphp.12784. PubMed DOI

Boursot P, Auffray JC, Britton-Davidian J, Bonhomme F. The evolution of house mice. Rev. Ecol. Syst. 1993;24:119–152. doi: 10.1146/annurev.es.24.110193.001003. DOI

Ďureje L, Macholán M, Baird SJ, Piálek J. The mouse hybrid zone in Central Europe: From morphology to molecules. Folia Zool. 2012;61:308–318. doi: 10.25225/fozo.v61.i3.a13.2012. DOI

Stopkova R, Klempt P, Kuntova B, Stopka P. On the tear proteome of the house mouse (Mus musculus musculus) in relation to chemical signalling. PeerJ. 2017;6:e3541. doi: 10.7717/peerj.3541. PubMed DOI PMC

Kuntova B, Stopkova R, Stopka P. Transcriptomic and proteomic profiling revealed high proportions of odorant binding and antimicrobial defense proteins in olfactory tissues of the house mouse. Front. Genet. 2018;9:26. doi: 10.3389/fgene.2018.00026. PubMed DOI PMC

Ibarra-Soria X, Levitin MO, Saraiva LR, Logan DW. The olfactory transcriptomes of mice. Plos Genetics. 2014;10:e1004593. doi: 10.1371/journal.pgen.1004593. PubMed DOI PMC

Strotmann J, Breer H. Internalization of odorant-binding proteins into the mouse olfactory epithelium. Histochem. Cell Biol. 2011;136:357–369. doi: 10.1007/s00418-011-0850-y. PubMed DOI

Trinh K, Storm DR. Detection of odorants through the main olfactory epithelium and vomeronasal organ of mice. Nutr. Rev. 2004;62:S189–S192. doi: 10.1111/j.1753-4887.2004.tb00098.x. PubMed DOI

Stopka P, et al. On the saliva proteome of the Eastern European house mouse (Mus musculus musculus) focusing on sexual signalling and immunity. Sci Rep. 2016;6:32481. doi: 10.1038/srep32481. PubMed DOI PMC

Yip KS, Suvorov A, Connerney J, Lodato NJ, Waxman DJ. Changes in mouse uterine transcriptome in estrus and proestrus. Biol Reprod. 2013;89:13. doi: 10.1095/biolreprod.112.107334. PubMed DOI PMC

Shahan K, Denaro M, Gilmartin M, Shi Y, Derman E. Expression of six mouse major urinary protein genes in the mammary, parotid, sublingual, submaxillary, and lachrymal glands and in the liver. Mol. Cell. Biol. 1987;7:1947–1954. PubMed PMC

Stopková R, et al. Mouse lipocalins (MUP, OBP, LCN) are co-expressed in tissues involved in chemical communication. Front. Ecol. Evol. 2016 doi: 10.3389/fevo.2016.00047. DOI

Garcia M, Ravignani A. Acoustic allometry and vocal learning in mammals. Biol. Let. 2020;16:20200081. doi: 10.1098/rsbl.2020.0081. PubMed DOI PMC

Martinez-Ricos J, Augustin-Pavon C, Lanuza E, Martinez-Garcia F. Role of the vomeronasal system in intersexual attraction in female mice. Neuroscience. 2008;153:383–395. doi: 10.1016/j.neuroscience.2008.02.002. PubMed DOI

Martínez-Ricós J, Agustín-Pavón C, Lanuza E, Martínez-García F. Intraspecific communication through chemical signals in female mice: reinforcing properties of involatile male sexual pheromones. Chem. Senses. 2007;32:139–148. doi: 10.1093/chemse/bjl039. PubMed DOI

Stowers L, Logan DW. Sexual dimorphism in olfactory signaling. Curr. Opin. Neurobiol. 2010;20:770–775. doi: 10.1016/j.conb.2010.08.015. PubMed DOI PMC

Klein SL. The effects of hormones on sex differences in infection: From genes to behavior. Neurosci. Biobehav. Rev. 2000;24:627–638. doi: 10.1016/S0149-7634(00)00027-0. PubMed DOI

Kadel S, Kovats S. Sex hormones regulate innate immune cells and promote sex differences in respiratory virus infection. Front. Immunol. 2018;9:1653. doi: 10.3389/fimmu.2018.01653. PubMed DOI PMC

Caspar KR, et al. Perioral secretions enable complex social signaling in African mole-rats (genus Fukomys) Sci. Rep. 2022;12:22366. doi: 10.1038/s41598-022-26351-3. PubMed DOI PMC

Gregorová S, Forejt J. PWD/Ph and PWK/Ph inbred mouse strains of Mus m. musculus subspecies: A valuable resource of phenotypic variations and genomic polymorphisms. Folia Biologica (Praha) 2000;46:31–41. PubMed

Chang PL, et al. Whole exome sequencing of wild-derived inbred strains of mice improves power to link phenotype and genotype. Mamm. Genome. 2017;28:416–425. doi: 10.1007/s00335-017-9704-9. PubMed DOI PMC

Piálek J, et al. Development of unique house mouse resources suitable for evolutionary studies of speciation. J. Hered. 2007;99:34–44. doi: 10.1093/jhered/esm083. PubMed DOI

Gentleman RC, et al. Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol. 2004;5:R80. doi: 10.1186/gb-2004-5-10-r80. PubMed DOI PMC

Crawley MJ. The R Book. Wiley Publishing; 2007.

Pavelka N, et al. A power law global error model for the identification of differentially expressed genes in microarray data. BMC Bioinform. 2004;5:203. doi: 10.1186/1471-2105-5-203. PubMed DOI PMC

Rodriguez J, Gupta N, Smith RD, Pevzner PA. Does trypsin cut before proline? J Proteome Res. 2008;7:300–305. doi: 10.1021/pr0705035. PubMed DOI

Breiman L. Random Forests. Mach. Learn. 2001;45:5–32. doi: 10.1023/A:1010933404324. DOI

Wickham H. ggplot2: Elegant Graphics for Data Analysis. Chem: Springer-Verlag; 2016.

Deciphering the chemical language of inbred and wild mouse conspecific scents

Microbial, proteomic, and metabolomic profiling of the estrous cycle in wild house mice