Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system


Animals display a repertoire of different social behaviours. Appropriate behavioural responses depend on sensory input received during social interactions. In mice, social behaviour is driven by pheromones, chemical signals that encode information related to age, sex and physiological state1. However, although mice show different social behaviours towards adults, juveniles and neonates, sensory cues that enable specific recognition of juvenile mice are unknown. Here we describe a juvenile pheromone produced by young mice before puberty, termed exocrine-gland secreting peptide 22 (ESP22). ESP22 is secreted from the lacrimal gland and released into tears of 2- to 3-week-old mice. Upon detection, ESP22 activates high-affinity sensory neurons in the vomeronasal organ, and downstream limbic neurons in the medial amygdala. Recombinant ESP22, painted on mice, exerts a powerful inhibitory effect on adult male mating behaviour, which is abolished in knockout mice lacking TRPC2, a key signalling component of the vomeronasal organ2,3. Furthermore, knockout of TRPC2 or loss of ESP22 production results in increased sexual behaviour of adult males towards juveniles, and sexual responses towards ESP22-deficient juveniles are suppressed by ESP22 painting. Thus, we describe a pheromone of sexually immature mice that controls an innate social behaviour, a response pathway through the accessory olfactory system and a new role for vomeronasal organ signalling in inhibiting sexual behaviour towards young. These findings provide a molecular framework for understanding how a sensory system can regulate behaviour.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: ESP22 is secreted into juvenile tear fluid.
Figure 2: ESP22 activates the vomeronasal system.
Figure 3: Trpc2−/− males display increased sexual behaviour towards juveniles.
Figure 4: ESP22 inhibits male sexual behaviour.


  1. Tirindelli, R., Dibattista, M., Pifferi, S. & Menini, A. From pheromones to behavior. Physiol. Rev. 89, 921–956 (2009)

    Article  CAS  Google Scholar 

  2. Leypold, B. G. et al. Altered sexual and social behaviors in trp2 mutant mice. Proc. Natl Acad. Sci. USA 99, 6376–6381 (2002)

    Article  ADS  CAS  Google Scholar 

  3. Stowers, L., Holy, T. E., Meister, M., Dulac, C. & Koentges, G. Loss of sex discrimination and male-male aggression in mice deficient for TRP2. Science 295, 1493–1500 (2002)

    Article  ADS  CAS  Google Scholar 

  4. Ferrero, D. M. & Liberles, S. D. The secret codes of mammalian scents. Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 23–33 (2010)

    Article  CAS  Google Scholar 

  5. Nodari, F. et al. Sulfated steroids as natural ligands of mouse pheromone-sensing neurons. J. Neurosci. 28, 6407–6418 (2008)

    Article  CAS  Google Scholar 

  6. Novotny, M. V. Pheromones, binding proteins and receptor responses in rodents. Biochem. Soc. Trans. 31, 117–122 (2003)

    Article  CAS  Google Scholar 

  7. Touhara, K. Sexual communication via peptide and protein pheromones. Curr. Opin. Pharmacol. 8, 759–764 (2008)

    Article  CAS  Google Scholar 

  8. Karn, R. C. & Laukaitis, C. M. The roles of gene duplication, gene conversion and positive selection in rodent esp and mup pheromone gene families with comparison to the abp family. PLoS ONE 7, e47697 (2012)

    Article  ADS  CAS  Google Scholar 

  9. Kimoto, H., Haga, S., Sato, K. & Touhara, K. Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons. Nature 437, 898–901 (2005)

    Article  ADS  CAS  Google Scholar 

  10. Logan, D. W., Marton, T. F. & Stowers, L. Species specificity in major urinary proteins by parallel evolution. PLoS ONE 3, e3280 (2008)

    Article  ADS  Google Scholar 

  11. Chamero, P. et al. G protein Gαo is essential for vomeronasal function and aggressive behavior in mice. Proc. Natl Acad. Sci. USA 108, 12898–12903 (2011)

    Article  ADS  CAS  Google Scholar 

  12. Papes, F., Logan, D. W. & Stowers, L. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell 141, 692–703 (2010)

    Article  CAS  Google Scholar 

  13. Haga, S. et al. The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor. Nature 466, 118–122 (2010)

    Article  ADS  CAS  Google Scholar 

  14. Kimoto, H. et al. Sex- and strain-specific expression and vomeronasal activity of mouse ESP family peptides. Curr. Biol. 17, 1879–1884 (2007)

    Article  CAS  Google Scholar 

  15. Li, Q. et al. Synchronous evolution of an odor biosynthesis pathway and behavioral response. Curr. Biol. 23, 11–20 (2013)

    Article  Google Scholar 

  16. Mandiyan, V. S., Coats, J. K. & Shah, N. M. Deficits in sexual and aggressive behaviors in Cnga2 mutant mice. Nature Neurosci. 8, 1660–1662 (2005)

    Article  CAS  Google Scholar 

  17. Wang, Z. et al. Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium. J. Neurosci. 26, 7375–7379 (2006)

    Article  CAS  Google Scholar 

  18. He, J. et al. Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ. J. Neurosci. 30, 7473–7483 (2010)

    Article  CAS  Google Scholar 

  19. Dulac, C. & Wagner, S. Genetic analysis of brain circuits underlying pheromone signaling. Annu. Rev. Genet. 40, 449–467 (2006)

    Article  CAS  Google Scholar 

  20. Choi, G. B. et al. Lhx6 delineates a pathway mediating innate reproductive behaviors from the amygdala to the hypothalamus. Neuron 46, 647–660 (2005)

    Article  CAS  Google Scholar 

  21. Yang, C. F. et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153, 896–909 (2013)

    Article  CAS  Google Scholar 

  22. Chamero, P. et al. Identification of protein pheromones that promote aggressive behaviour. Nature 450, 899–902 (2007)

    Article  ADS  CAS  Google Scholar 

  23. Novotny, M., Harvey, S., Jemiolo, B. & Alberts, J. Synthetic pheromones that promote inter-male aggression in mice. Proc. Natl Acad. Sci. USA 82, 2059–2061 (1985)

    Article  ADS  CAS  Google Scholar 

  24. Liberles, S. D. & Buck, L. B. A second class of chemosensory receptors in the olfactory epithelium. Nature 442, 645–650 (2006)

    Article  ADS  CAS  Google Scholar 

  25. Montmayeur, J. P., Liberles, S. D., Matsunami, H. & Buck, L. B. A candidate taste receptor gene near a sweet taste locus. Nature Neurosci. 4, 492–498 (2001)

    Article  CAS  Google Scholar 

  26. Liberles, S. D. et al. Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ. Proc. Natl Acad. Sci. USA 106, 9842–9847 (2009)

    Article  ADS  CAS  Google Scholar 

  27. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996)

    Article  CAS  Google Scholar 

  28. Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001)

    Article  ADS  CAS  Google Scholar 

  29. Eng, J. K., Mccormack, A. L. & Yates, J. R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994)

    Article  CAS  Google Scholar 

  30. Hagendorf, S., Fluegge, D., Engelhardt, C. & Spehr, M. Homeostatic control of sensory output in basal vomeronasal neurons: activity-dependent expression of ether-a-go-go-related gene potassium channels. J. Neurosci. 29, 206–221 (2009)

    Article  CAS  Google Scholar 

  31. Spehr, J. et al. Ca2+-calmodulin feedback mediates sensory adaptation and inhibits pheromone-sensitive ion channels in the vomeronasal organ. J. Neurosci. 29, 2125–2135 (2009)

    Article  CAS  Google Scholar 

  32. Franklin, K. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates Fig. 44 (Academic, 2008)

    Google Scholar 

Download references


We thank M. Albers and S. R. Datta for reading the manuscript, J. Yang, C. Mark Fletcher and Y. Tachie-Baffour for experimental assistance, and the Taplin Mass Spectrometry Facility for mass spectrometry analysis. This work was supported by a grant from the National Institutes of Health (to S.D.L., award number R01 DC010155) and in part by a Grant-in-Aid for Young Scientists (S) from the Japan Society for the Promotion of Science, and by ERATO Touhara Chemosensory Signal Project from the Japan Science and Technology Agency (to K.T.). N.H. is supported by a Grant-in-Aid for JSPS Fellows, M.S. is a Lichtenberg-Professor of the Volkswagen Foundation and D.M.F. is supported by a Boehringer Ingelheim Fonds PhD Fellowship.

Author information

Authors and Affiliations



D.M.F., S.D.L., M.S. and K.T. conceived the project, designed the experiments and wrote the manuscript. D.M.F. performed molecular biology, biochemistry and behaviour experiments. D.S.R. and Q.L. performed in situ hybridization analysis. L.M.M., A.C., T.O. and N.H. performed electrophysiological analysis. T.O. performed cFos analysis.

Corresponding author

Correspondence to Stephen D. Liberles.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 RNA in situ hybridization to characterize expression of Esp genes in the lacrimal gland.

ac, Colorimetric analysis in tissue from animals indicated using cRNA riboprobes for (a) Esp15 and (b) Esp22, and two-colour fluorescence analysis (c) in juvenile lacrimal gland with cRNA riboprobes for Esp22 (red) and a marker for acinal secretory cells, Rab3D (green). cRNA riboprobes for Esp15 are expected to cross-hybridize with Esp16 mRNA. Some images used in b are identical to panels in Fig. 1d, and are included for reference. Dashed boxes (c) indicate regions magnified below. Arrows, acinar cells; arrowheads, ductal cells; scale bars, 100 μm (a, b, c top), 20 μm (c bottom).

Extended Data Figure 2 qPCR analysis of gene expression.

a, Esp22 qPCR primers specifically detect a plasmid containing cloned Esp22, but not plasmids containing other Esps with greater than 60% identity to Esp22. bf, cDNA was derived from lacrimal gland (be), submaxillary gland (e) or other tissues (f) of animals indicated. In f, abundance is calculated by normalization to amounts of Gapdh. C57BL/6 mice were used (bd) unless otherwise indicated (b). Experiments where sex is not indicated involved equal numbers of males and females; olfactory epithelium (OE), olfactory bulb (OB), harderian gland (HG), submaxillary gland (SMG), parotid gland (PG), sublingual gland (SLG) (n = 6–12, averages ± s.e.m., **P < 0.01, two-way ANOVA followed by Tukey’s HSD post hoc tests).

Extended Data Figure 3 Quantification of protein concentrations in tear fluid by western blot analysis using an anti-ESP22 antibody.

a, b, A standard curve based on signal intensity was generated using different concentrations of recombinant ESP22 (a, left panel; b). The arrow indicates the intensity level of the band in the juvenile tear sample (a, right panel). c, Entire western blot analysis of tear fluid using anti-ESP22 antibody.

Extended Data Figure 4 ESP22-derived tryptic peptides identified by mass spectrometry.

a, The amino-acid sequence of immature ESP22 is depicted, along with a predicted signal peptide and the epitope used for antibody generation. Four tryptic peptides were identified by mass spectrometry (highlighted in red), including one peptide containing the first amino acid after the predicted signal sequence and another containing the encoded carboxy (C)-terminal residue. Trypsin does not efficiently cleave amino (N)-terminal lysines or arginines, consistent with R23 being the first amino acid in mature ESP22. b, Mass spectrum of an high-performance liquid chromatography fraction of juvenile tear fluid showing the ESP22-derived tryptic peptide GIVFNTIK, with sequence identity confirmed by tandem mass spectrometry analysis.

Extended Data Figure 5 Electrophysiological responses to ESP22 in VNO sensory neurons.

a, Single-unit extracellular loose-seal recording from a single VNO sensory neuron repeatedly exposed to different stimuli indicates reproducibility of responses. b, The percentage of basal VNO sensory neurons responsive to 20 pM (n = 383) and 2 nM (n = 749) ESP22.

Extended Data Figure 6 cFos responses to ESP22 in the amygdala.

a, ESP22 and juvenile tear fluid, but not MBP, induce cFos expression in the postero-ventral MeA. Dashed lines and arrows indicate boundaries of MeA regions. b, Similar responses were not observed in other amygdala nuclei that receive olfactory input, including the postero-medial cortical amygdala (PMCo), anterior cortical amygdala (CoA) and postero-lateral cortical amygdala (PLCo) (mean ± s.e.m., n = 3).

Extended Data Figure 7 Trpc2−/− males show increased sexual behaviour towards wild-type juveniles.

a, b, Histograms of mounts by minute of social interaction and intermount intervals shown towards juveniles byTrpc2+/+ and Trpc2−/− males (sum, n = 12). Inset depicts average intermount intervals (mean ± s.e.m., *P < 0.05, **P < 0.01, Mann–Whitney U-test). c, Analysis of adult male sexual behaviour during simultaneous interaction with juvenile and adult oestrous females. Trpc2+/+ and Trpc2−/− males show similar amounts of sexual behaviour towards adult oestrous females, but Trpc2−/− males show increased sexual behaviour towards juveniles (n = 10, averages ± s.e.m., *P < 0.05, **P < 0.01, one-way multivariate ANOVA).

Extended Data Figure 8 Trpc2−/− males show sexual behaviour towards juvenile males.

a, Raster plots depicting individual mounting displays of adult Trpc2+/+ and Trpc2−/− males towards juvenile males (C57BL/6, postnatal day 17) during social interaction (30 min). Each tick indicates onset of one mount. b, Quantitative analysis of parameters associated with sexual behaviour towards juvenile males shown by Trpc2+/+ and Trpc2−/− males (n = 11 or 12, averages ± s.e.m., *P < 0.05, **P < 0.01, Mann–Whitney U-test).

Extended Data Figure 9 ESP22 did not decrease social investigation time.

Wild-type C57BL/6 males were introduced to C3H juvenile females painted with stimuli indicated. Social investigation time of the male was recorded as time spent with the nose in direct contact with the female. These data were extracted from the same experiments reported in Fig. 4c, d, with additional experiments involving TMT (100 μl, 155 mM, n = 11 or 12, averages ± s.e.m., **P < 0.01, one-way ANOVA followed by Tukey’s HSD post hoc tests).

Extended Data Figure 10 ESP22 (10 μg) inhibits sexual behaviour of C3H males.

a, Raster plots of sexual behaviour shown by C3H males towards C3H juvenile females (postnatal day 17) painted with indicated stimuli (30 min social interaction). Each tick indicates onset of one mount. b, Quantitative analysis of parameters associated with sexual behaviour towards juvenile females shown by C3H males (n = 11, averages ± s.e.m., *P < 0.05, **P < 0.01, one-way ANOVA followed by Tukey’s HSD post hoc tests).

Supplementary information

A Trpc2+/+ adult male interacts with a juvenile female

Video showing a social interaction between a wild type adult male mouse and a juvenile female mouse (C57BL/6, p17). Animals in the video display active social investigation but not sexual behavior. (MOV 4084 kb)

A Trpc2-/- adult male displays sexual behavior towards a juvenile female

Video showing a social interaction between a Trpc2-/- adult male mouse and a juvenile female mouse (C57BL/6, p17). Frequent mounting displays characteristic of mouse sexual behavior are observed. (MOV 3320 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ferrero, D., Moeller, L., Osakada, T. et al. A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system. Nature 502, 368–371 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing