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Background Reliable recognition of individuals requires phenotypic identity signatures that are both individually distinctive and appropriately stable over time. Individual-specific vocalisations or visual patterning are well documented among birds and some mammals, whilst odours play a key role in social recognition across many vertebrates and invertebrates. Less well understood, though, is whether individuals are recognised through variation in cues that arise incidentally from a wide variety of genetic and non-genetic differences between individuals, or whether animals evolve distinctive polymorphic signals to advertise identity reliably. As a bioassay to understand the derivation of individual-specific odour signatures, we use female attraction to the individual odours of male house mice (Mus musculus domesticus), learned on contact with a male’s scent marks. Results Learned volatile odour signatures are determined predominantly by individual differences in involatile major urinary protein (MUP) signatures, a specialised set of communication proteins that mice secrete in their urine. Recognition of odour signatures in genetically distinct mice depended on differences in individual MUP genotype. Direct manipulation using recombinant MUPs confirmed predictable changes in volatile signature recognition according to the degree of matching between MUP profiles and the learned urine template. Both the relative amount of the male-specific MUP pheromone darcin, which induces odour learning, and other MUP isoforms influenced learned odour signatures. By contrast, odour recognition was not significantly influenced by individual major histocompatibility complex genotype. MUP profiles shape volatile odour signatures through isoform-specific differences in binding and release of urinary volatiles from scent deposits, such that volatile signatures were recognised from the urinary protein fraction alone. Manipulation using recombinant MUPs led to quantitative changes in the release of known MUP ligands from scent deposits, with MUP-specific and volatile-specific effects. Conclusions Despite assumptions that many genes contribute to odours that can be used to recognise individuals, mice have evolved a polymorphic combinatorial MUP signature that shapes distinctive volatile signatures in their scent. Such specific signals may be more prevalent within complex body odours than previously realised, contributing to the evolution of phenotypic diversity within species. However, differences in selection may also result in species-specific constraints on the ability to recognise individuals through complex body scents. Electronic supplementary material The online version of this article (10.1186/s12915-018-0512-9) contains supplementary material, which is available to authorized users.

The Major Urinary Proteins (MUPs) of the house mouse, Mus musculus domesticus, are 18–19 kDa beta-barrel lipocalins that are involved in chemical communication between individuals. Many of them are excreted in urine where they play multiple roles, including coding of owner identity and transport, and slow release of bound volatile pheromones. One of them, darcin, is a pheromone in its own right and induces long-term memory for the identity and location of the scent mark owner. We have shown that mass spectrometric analysis of intact proteins, and their ion mobility behaviour, is capable of dissecting subtle structural differences between the members of this class of proteins. Moreover, mass spectrometric analysis of the intact proteins can contribute towards molecular phenotyping of MUPs. However, whilst allowing relative quantification, the ionisation propensity or gas phase properties of the individual MUPs may compromise absolute quantification. To solve the challenge of absolute quantification of MUP expression, we have designed and constructed a QconCAT built from endopeptidase LysC peptides that is capable of quantifying MUPs found in laboratory animal strains and some MUPs from wild caught individuals.

A new bio-sensing platform based on major urinaryproteins (MUPs) from the mouse as chemical recognitionelements has been developed. The transducers were surfaceacoustic devices coated with diamond nanoparticles as an intermediatelayer enabling covalent attachment of the proteins.The resulting sensors detected 2,4-Dinitrotoluene, 4-Nitrotoluene,and 2-Isobutyl-3-methoxypyrazine at ppb levels. The best sensorshowed a sensitivity of 24 000 Hz · ppm−1 to 2, 4-DNT whengrafted with the protein MUP20. Trends in the sensitivity ofthe various VOC sensors were compared with the associationconstant values Ka of the proteins to target ligands measured bycompetitive assay in liquid phase. The system is able to detectanalytes both in liquid as well as vapor phase and indicate thatMUPs are robust bio-recognition elements that can be utilized inartificial olfaction applications.

Two different structural classes of chemical signals in mouse urine, i.e., volatile organic compounds (VOCs) and the major urinary proteins (MUPs), interact closely because MUPs sequester VOCs. Although qualitative and/or quantitative differences in each chemical class have been reported, previous studies have examined only one of the classes at a time. No study has analyzed these two sets simultaneously, and consequently binding interactions between volatile ligands and proteins in urines of different strains have not been compared. Here, we compared the release of VOCs in male urines of three different inbred strains (C57BL/6J, BALB/b and AKR) before and after denaturation of urinary proteins, mainly MUPs. Both MUP and VOC profiles were distinctive in the intact urine of each strain. Upon denaturation, each of the VOC profiles changed due to the release of ligands previously bound to MUPs. The results indicate that large amounts of numerous ligands are bound to MUPs and that these ligands represent a variety of different structural classes of VOCs. Furthermore, the degree of release in each ligand was different in each strain, indicating that different ligands are differentially bound to proteins in the urines of different strains. Therefore, these data suggest that binding interactions in ligands and MUPs differ between strains, adding yet another layer of complexity to chemical communication in mice.

Chemosensory information mediates behavior in many rodent genera. Major Urinary Proteins (MUPs) facilitate chemical communication in some species of mice. We sought to demonstrate the importance of MUPs in chemosignaling across a range of rodent genera that live in different habitats and social structures. We analyzed urine from three subterranean rodent genera from different continents, and with diverse social systems: eusocial Zambian mole-rats (Fukomys), solitary Israeli blind mole rats (Spalax), and social Chilean coruros (Spalacopus). 2D gel electrophoresis revealed low levels of protein, with sequences similar to aphrodisin, in Fukomys mole-rat urine, but no MUPs in urine of any of the studied species. Previous research demonstrated that subjects from the tested genera responded differentially to odors indicating transmission of individuality, family/colony or population, species, and reproductive state in secretions and excretions of conspecifics. This extends, to subterranean rodents, the evidence that rodent species can successfully transmit and receive chemosignals without the necessity of MUPs.

Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male-male encounter. Females responded to a male's presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal's odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male's quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine.

The variation of expression of major urinary proteins was studied in laboratory mice to further the understanding of the role of these proteins in social and reproductive contexts. Mouse major urinary proteins (MUPs) are known to carry volatile substances and protect them during their passage from the liver, through the kidneys into the urine. However, most studies on the role of MUPs were carried out on males. Using densitometry analysis of total MUP concentration in the urine, our present study clearly demonstrates that (i) individuals of both sex up-regulate MUPs during social contact, and that (ii) females use these proteins to advertise their reproductive state by varying the concentration of MUPs during the oestrous cycle. As the concentration of MUPs was normalized by the concentration of creatinine -- a marker of glomerular filtration -- the corrected concentration of MUPs represents instantaneous expression on the level of proteins. Cross-correlation analysis between oestrus quantification and MUP expression revealed that the oestrous curve is delayed by 1 day behind the MUP curve so that the expression of MUPs is up-regulated immediately at the beginning of oestrus. To conclude, the regulation of pheromone-carrying MUPs is directly linked to reproduction and, thus, enables female honest signalling.

Hurst, Jane L/0000-0002-3728-9624; Beynon, Robert J/0000-0003-0857-495X; Gunduz, Islam/0000-0002-6436-5397; Searle, Jeremy/0000-0001-7710-5204 WOS: 000244296400014 PubMed: 17268823 Urine from the house mouse, Mus musculus domesticus, contains a high concentration of major urinary proteins (MUPs), which convey olfactory information between conspecifics. In wild populations, each individual expresses a different pattern of around 8 to 14 electrophoretically separable MUP isoforms. To examine whether other Mus species express MUPs and exhibit a similar level of individual heterogeneity, we characterized urinary proteins in urine samples from an aboriginal species, Mus macedonicus, captured from different sites in Turkey. Anion exchange chromatography and electrospray ionization mass spectrometry demonstrated that M. macedonicus urine contained a single major peak of mass 18,742 Da, and in contrast to M. m. domesticus, all individuals were the same. The M. macedonicus masses were not predicted from any known MUP gene sequence. Endoproteinase Lys-C (Lys-C) digestion of the purified M. macedonicus urinary protein followed by matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry demonstrated that it shared considerable, but not complete, sequence homogeneity with M. m. domesticus MUPs. Three M. macedonicus Lys-C peptides differed in mass from their M. m. domesticus counterparts. These three peptides were further characterized by tandem mass spectrometry. The complete sequences of two were determined, and in conjunction with methyl esterification, the amino acid composition of the third was inferred, and the sequence narrowed down to three permutations. The complete M. macedonicus sequence contained a maximum of seven amino acid substitutions, discernible by tandem mass spectrometry, relative to a reference M. m. domesticus sequence. Six of these were on the surface of the molecule. Molecular modeling of the M. macedonicus sequence demonstrated that the amino acid substitutions had little effect on the tertiary structure. The differences in the level of heterogeneity between the two species are discussed in relation to their environment and behavior. In addition, the differences in protein structure allow speculation into molecular mechanisms of MUP function. Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [S19816]

We investigated the specific pattern of major urinary proteins (MUPs) expression in 3-, 4-, and 12-week old mice of CBA/LacY and C57BL/6JY inbred strains using polyacrylamide gel electrophoresis. Quantitative evaluation of 8 protein fractions A-H with regard to sex, age, and genotype of the animals is presented for the first time. Actual problems of genetic control and neuroendocrine regulation of MUPs expression during ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulation information exchange via pheromones, we put forward the idea that the genetically determined structure of the olfactory code of the definitive type is formed at an early ontogenetic stage on the basis of the MUPs combinatorial pattern.

Individual variation in a specialised set of scent communication proteins, the major urinary proteins (MUPs), provides a genetic identity signature that underlies individual and kin recognition, and the assessment of heterozygosity in wild house mice. Here we examine the extent to which MUP variation is retained among 30 classical strains of laboratory mice from three main lineages (Castle, C57, Swiss). Normal wild-type variation in urinary MUP pattern appears to have been lost at an early stage in the derivation of the classical laboratory strains. All strains from the Castle and Swiss lineages shared the same "individual" MUP pattern, consistent with common ancestry from very few founders, while those from the C57 lineage shared a different pattern. Notably, individual variation in MUP pattern was no greater within the Swiss outbred ICR (CD-1) strain than typical for inbred strains. Total urinary protein concentration varied considerably between even closely related substrains, together with minor variation in the relative amount of each MUP isoform expressed, although the functional significance of such quantitative variation in MUP expression has yet to be established. Expression was 2-8 fold higher among males, while a MUP expressed by most male but not female wild mice was expressed by C57 males but variably among Castle and Swiss males and occasionally by females in some strains. The lack of normal variation in MUP patterns within and between strains has important implications for the use of laboratory mice in behavioural or neurophysiological research investigating social recognition or mate choice.

The major urinary proteins are a species-specific complex of proteins excreted by male mice that influence the reproductive behavior and the neuroendocrine condition of female mice through the olfactory system. The aim of this work is to determine their influence on ovulation. The major urinary proteins isolated from the urine of adult male mice were voided of bound odorants, dissolved at a physiological concentration in urine of prepubertal mice, and put on the nostril of reproductively cycling female mice housed in groups, the first day of estrus at 1100. The eggs shed in the oviducts were counted under dissection the morning of the second day of estrus. The results showed that 1) a single stimulus of the major urinary proteins increased ovulation nearly as much as the whole urine of male mice, 2) the effect was not elicited by male rat urine which contains different proteins, 3) a peptide with four residues of the amino-terminal sequence of the major urinary proteins stimulated ovulation, and 4) mice that had been isolated or had the vomeronasal organ (VNO) removed did not respond to the major urinary proteins and had a high spontaneous ovulation. The results suggest that the major urinary proteins activate the neuroendocrine system through the VNO and trigger ovulation.