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Male house mice excrete large amounts of protein in their urinary scent marks, mainly composed of Major Urinary Proteins (MUPs), and these lipocalins function as pheromones and pheromone carriers. Here, we review studies on sexually dimorphic MUP expression in house mice, including the proximate mechanisms controlling MUP gene expression and their adaptive functions. Males excrete 2 to 8 times more urinary protein than females, though there is enormous variation in gene expression across loci in both sexes. MUP expression is dynamically regulated depending upon a variety of factors. Males regulate MUP expression according to social status, whereas females do not, and males regulate expression depending upon health and condition. Male-biased MUP expression is regulated by pituitary secretion of growth hormone (GH), which binds receptors in the liver, activating the JAK2-STAT5 signaling pathway, chromatin accessibility, and MUP gene transcription. Pulsatile male GH secretion is feminized by several factors, including caloric restriction, microbiota depletion, and aging, which helps explain condition-dependent MUP expression. If MUP production has sex-specific fitness optima, then this should generate sexual antagonism over allelic expression (intra-locus sexual conflict) selectively favoring sexually dimorphic expression. MUPs influence the sexual attractiveness of male urinary odor and increased urinary protein excretion is correlated with the reproductive success of males but not females. This finding could explain the selective maintenance of sexually dimorphic MUP expression. Producing MUPs entails energetic costs, but increased excretion may reduce the net energetic costs and predation risks from male scent marking as well as prolong the release of chemical signals. MUPs may also provide physiological benefits, including regulating metabolic rate and toxin removal, which may have sex-specific effects on survival. A phylogenetic analysis on the origins of male-biased MUP gene expression in Mus musculus suggests that this sexual dimorphism evolved by increasing male MUP expression rather than reducing female expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Penn, Zala and Luzynski.)

Mouse urine contains major urinary proteins (MUPs) that are not found in human urine. Therefore, even healthy mice exhibit proteinuria, unlike healthy humans, making it challenging to use mice as models for human diseases. It was also unknown whether dipsticks for urinalysis could measure protein concentrations precisely in urine containing MUPs. To resolve these problems, we produced MUP-knockout (Mup-KO) mice by removing the Mup gene cluster using Cas9 proteins and two guide RNAs and characterized the urinary proteins in these mice. We measured the urinary protein concentrations in Mup-KO and wild-type mice using a protein quantitation kit and dipsticks. We also examined the urinary protein composition using SDS-PAGE and two-dimensional electrophoresis (2DE). The urinary protein concentration was significantly lower (P<0.001) in Mup-KO mice (17.9 ± 1.8 mg/dl, mean ± SD, n=3) than in wild-type mice (73.7 ± 8.2 mg/dl, n=3). This difference was not reflected in the dipstick values, perhaps due to the low sensitivity to MUPs. This suggests that dipsticks have limited ability to measure changes in MUPs with precision. SDS-PAGE and 2DE confirmed that Mup-KO mice, like humans, had no MUPs in their urine, whereas wild-type mice had abundant MUPs in their urine. The absence of the masking effect of MUPs in 2DE would enable clear comparisons of urinary proteins, especially low-molecular-weight proteins. Thus, Mup-KO mice may provide a useful model for human urinalysis.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays an important role, not only in endothelium-dependent vasodilation but also in lipid and glucose homeostasis in the liver and exerts beneficial effects on mitochondrial biogenesis and respiration. Thus, the aim of our study was to use iTRAQ-based quantitative proteomics to investigate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of the double (apolipoprotein E (apoE) and eNOS) knockout (apoE/eNOS-DKO) mice as compared to apoE KO mice (apoE -/- ) - an animal model of atherosclerosis and hepatic steatosis. Collectively, the deficiency of eNOS resulted in increased expression of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the decreased expression of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, one of the most downregulated proteins were major urinary proteins (MUPs), which are abundantly expressed in the liver and were shown to be involved in the regulation of lipid and glucose metabolism. The exact functional consequences of the revealed alterations require further investigation., (© 2018 John Wiley & Sons Australia, Ltd.)

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.

When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal.

Major urinary proteins (MUPs) are often suggested to be highly polymorphic, and thereby provide unique chemical signatures used for individual and genetic kin recognition; however, studies on MUP variability have been lacking. We surveyed populations of wild house mice (Mus musculus musculus), and examined variation of MUP genes and proteins. We sequenced several Mup genes (9 to 11 loci) and unexpectedly found no inter-individual variation. We also found that microsatellite markers inside the MUP cluster show remarkably low levels of allelic diversity, and significantly lower than the diversity of markers flanking the cluster or other markers in the genome. We found low individual variation in the number and types of MUP proteins using a shotgun proteomic approach, even among mice with variable MUP electrophoretic profiles. We identified gel bands and spots using high-resolution mass spectrometry and discovered that gel-based methods do not separate MUP proteins, and therefore do not provide measures of MUP diversity, as generally assumed. The low diversity and high homology of Mup genes are likely maintained by purifying selection and gene conversion, and our results indicate that the type of selection on MUPs and their adaptive functions need to be re-evaluated.

Major urinary proteins (MUPs) are highly homologous proteoforms that function in binding, transporting and releasing pheromones in house mice. The main analytical challenge for studying variation in MUPs, even for state-of-the-art proteomics techniques, is their high degree of amino acid sequence homology. In this study we used unique peptides for proteoform-specific identification. We applied different search engines (ProteinPilot™vs. PEAKS®) and protein databases (MUP database vs. SwissProt + unreviewed MUPs), and found that proteoform identification is influenced by addressing background proteins (unregulated urinary proteins, non-MUPs) during the database search. High resolution Q-TOF mass spectrometry was used to identify and precisely quantify the regulation of MUP proteoforms in male mice that were reared in standard housing and then transferred to semi-natural enclosures (within-subject design). By using a designated MUP database we were able to distinguish 19 MUP proteoforms, with A2CEK6 (a Mup11 gene product) being the most abundant based on spectral intensities. We compared three different quantification strategies based on MS1- (from IDA and SWATH™ spectra) and MS2 (SWATH™) data, and the results of these methods were correlated. Furthermore, three data normalization methods were compared and we found that increased statistical significance of fold-changes can be achieved by normalization based on urinary protein concentrations. We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs (differential regulation), e.g., A2ANT6, a Mup6 gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database. Finally, we show that 85 ± 7% of total MS intensity can be attributed to MUP-derived peptides, which supports the assumption that MUPs are the primary proteins in mouse urine. Our results provide new tools for assessing qualitative and quantitative variation of MUPs and suggest that male mice regulate the expression of specific MUP proteoforms, depending upon social conditions.

Major urinary proteins (MUPs) have been proven to be non-volatile male pheromones in mice. Here, we aimed to elucidate the relationship between MUPs and dominance hierarchy, and the underlying molecular mechanisms. Dominance-submission relationship was established by chronic dyadic encountering. We found that at the urinary protein level and hepatic mRNA level, the expression of major MUPs, including Mup20, was enhanced in dominant males compared with subordinate males, indicating that MUPs might signal the social status of male mice. Meanwhile, the mRNA level of hepatic corticotropin releasing hormone receptor 2 (CRHR2) was higher in subordinate male mice than in dominant male mice. Castration also enhanced the expression of CRHR2, but suppressed that of MUPs. CRHR2 agonist treatment reduced the expression of MUPs in liver. However, male social status failed to exert significant influence on serum testosterone and corticosterone as well as the mRNA expression of their receptors. These findings reveal that some MUPs, especially Mup20, might constitute potential dominance pheromones and could be downregulated by hepatic CRHR2, which is possibly independent of androgen or corticosterone systems., (© 2015 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and Wiley Publishing Asia Pty Ltd.)

Female rats show a distinct attraction for males. This attraction remains consistent without the necessity for the physical presence of the male. However, the identity of the olfactory cues contributing to attraction in rats remains unknown. Rat urine contains copious amounts of major urinary proteins (MUPs). Here, we investigated the hypothesis that MUPs mediate sexual attractiveness in rats. We first demonstrated that a member of a male dyad receiving greater copulatory opportunities in competitive mate choice tests excrete greater amounts of MUPs. Furthermore, the amount of male MUPs positively correlated with both copulatory opportunities received and female exploration of the urine. Using females and a two-choice olfactory attraction test, we demonstrated that urinary fractions containing MUPs were sufficient to induce attraction and that male MUPs activated neurons in the posterodorsal medial amygdala in female rats. Taken together, these results suggest that olfactory cues associated with MUPs act as an attractant to female rats in estrus., (© 2014 by the Society for the Study of Reproduction, Inc.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1) diversity, to enable the signaling of multiple behaviors, 2) dynamic regulation, to indicate age and dominance, and 3) species-specificity. Recently, the major urinary proteins (Mups) have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues.Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species-specific function in mammals.

Background: Recombinant allergens are preferred over natural allergen extracts in measuring antibodies. We tested the use of recombinant variants of the major mouse allergen Mus m 1 in detection of mouse-specific antibodies in sera of laboratory animal workers and children., Methods: Six recombinant major urinary proteins (MUPs) were produced and antibody-binding capacity was compared to natural Mus m 1 and to mouse urine extract. In a specific subset, cross-reactivity of MUP with Mus m 1 and between the different recombinant MUPs was determined., Results: For IgE antibodies, MUP8 showed high cross-reactivity with Mus m 1. MUP8-specific IgE was found in 55% of the mouse urine IgE-positive sera. Specific IgG and IgG4 antibodies against natural Mus m 1 correlated strongly with antibodies against recombinant MUP8 and were cross-reactive. IgG4 levels against MUP8 and mouse urine extract correlated, but detection of mouse urine-specific IgG4 in the absence of MUP-specific IgG4 was not uncommon. Cross-reactivity of IgG antibodies between MUP8 and Mus m 1 as well as between the different MUPs was high and inhibition varied between 54 and 99%., Conclusion: The mouse allergen Mus m 1 can be replaced in antibody testing by recombinant MUP8. Other MUPs, except MUP4, are interchangeable with MUP8. However, mouse urine extract showed better detection of both mouse-specific IgE and IgG4 levels. Other components in the mouse urine, like mouse albumin and other yet unidentified components, also induce IgE and IgG4 antibodies., (Copyright 2007 S. Karger AG, Basel.)

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.

The MUPs (major urinary proteins) of the house mouse, Mus domesticus, are lipocalins that bind and slowly release male-specific pheromones in deposited scent marks. However, females also express these proteins, consistent with a second role in encoding individual signatures in scent marks. We have purified and characterized an atypical MUP from the urine of male C57BL/6J inbred mice, which is responsible for the binding of most of the male pheromone, 2-sec-butyl-4,5-dihydrothiazole, and which is also responsible for the slow release of this pheromone from scent marks. This protein is absent from the urine of female mice of the same strain. The protein has been characterized by MS, leading to unequivocal identification as a previously uncharacterized gene product, providing compelling evidence for the expression of this gene in liver and manifestation in urine. These properties contrast strongly with those of the other MUPs in the same urine sample, and suggest that the requirement to manifest a male-specific pheromone has been met by evolution of a cognate protein specifically adapted to the binding and release of this ligand. This atypical MUP is also present in a random sample of wild-caught male mice, confirming that this protein is not specific to the inbred mouse strain but is present in natural populations also.

The urine of the house mouse, Mus domesticus, contains large amounts of proteins that are specifically synthesized in the liver to be secreted in the urine. These proteins, termed major urinary proteins (MUPs), have multiple roles in the communication of information in urine-derived scent marks. They bind low-molecular-mass volatile pheromones, and effect their delivery to the scent mark, followed by a slow release that is controlled by the rate of dissociation from the MUPs. However, this family of proteins is extremely polymorphic, more than might be expected for a simple role of ligand binding and release. We have analysed the polymorphism in wild mice, and have now shown that the pattern of MUPs in the urine acts as a type of individuality 'bar code' that signals the identity of the owner of the scent mark. This multiplicity of function, from a generic ligand-binding property to an extremely specific individuality, sets the MUPs apart from other lipocalin family proteins that are involved in chemical signalling.

Mouse major urinary proteins (MUPs) have been proposed to play a role in regulating the release and capture of pheromones. Here, we report affinity measurements of five recombinant urinary MUP isoforms (MUPs-I, II, VII, VIII, and IX) and one recombinant nasal isoform (MUP-IV) for each of three pheromonal ligands, (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (+/-)dehydro-exo-brevicomin (DHB). Dissociation constants for all MUP-pheromone pairs were determined by isothermal titration calorimetry, and data for SBT were corroborated by measurements of intrinsic protein fluorescence. We also report the isolation of MUP-IV protein from mouse nasal extracts, in which MUP-IV mRNA has been observed previously. The affinity of each MUP isoform for SBT (K(d) approximately 0.04 to 0.9 micro M) is higher than that for DHB (K(d) approximately 26 to 58 micro M), which in turn is higher than that for HMH (K(d) approximately 50 to 200 micro M). Isoforms I, II, VIII, and IX show very similar affinities for each of the ligands. MUP-VII has approximately twofold higher affinity for SBT but approximately twofold lower affinity for the other pheromones, whereas MUP-IV has approximately 23-fold higher affinity for SBT and approximately fourfold lower affinity for the other pheromones. The variations in ligand affinities of the MUP isoforms are consistent with structural differences in the binding cavities of the isoforms. The data indicate that the concentrations of available pheromones in urine may be influenced by changes in the expression levels of urinary MUPs or the excretion levels of other MUP ligands. The variation in pheromone affinities of the urinary MUP isoforms provides only limited support for the proposal that MUP heterogeneity plays a role in regulating profiles of available pheromones. However, the binding data support the proposed role of nasal MUPs in sequestering pheromones and possibly transporting them to their receptors.

Major urinary proteins (MUPs) are present in high levels in the urine of mice, and the specific profile of MUPs varies considerably among wild-caught individuals. We have conducted a detailed study of the polymorphic variation within a geographically constrained island population, analyzing the MUP heterogeneity by isoelectric focusing and analytical ion exchange chromatography. Several MUPs were purified in sufficient quantities for analysis by electrospray ionization mass spectrometry and MALDI-TOF mass spectrometry of endopeptidase Lys-C peptide maps. The results of such analyses permitted the identification of three new MUP allelic variants. In each of these proteins, the sites of variation were located to a restricted segment of the polypeptide chain, projecting to a patch on the surface of the protein, and connected to the central lipocalin calyx through the polypeptide backbone. The restriction of the polymorphic variation to one segment of the polypeptide may be of functional significance, either in the modulation of ligand release or in communication of individuality signals within urinary scent marks.

The ability to recognize individuals is essential to many aspects of social behaviour, such as the maintenance of stable social groups, parent-offspring or mate recognition, inbreeding avoidance and the modulation of competitive relationships. Odours are a primary mediator of individuality signals among many mammals. One source of odour complexity in rodents, and possibly in humans, resides in the highly polymorphic major histocompatibility complex (MHC). The olfactory acuity of mice and rats allows them to distinguish between the urinary odours of congenic strains differing only in single genes within the MHC, although the chemical mediators or odorants are unknown. However, rodent urine also contains a class of proteins, termed major urinary proteins (MUPs), that bind and release small volatile pheromones. We have shown that the combinatorial diversity of expression of MUPs among wild mice might be as great as for MHC, and at protein concentrations a million times higher. Here we show in wild house mice (Mus domesticus) that urinary MUPs play an important role in the individual recognition mechanism.

The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.

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.

The analysis of expression of pheromone-carrying major urinary proteins (MUPs) from two subspecies of house mice (Mus m. musculus, Mus m. domesticus) was studied. It has been previously shown that commensal populations of the two subspecies can discriminate on the basis of urinary signals. MUPs are predominant urinary proteins that protect pheromones from rapid degradation in a hydrophilic environment, and individuals of M. m. musculus tend to rely on these urinary cues in the process of subspecies discrimination more than M. m. domesticus individuals. Although it is not precisely known what triggers phenotypic and epigenetic changes of MUP expression, our results show that in the subspecies M. m. musculus, sex is a significant factor influencing variations in the regulation of selected MUPs in the liver. Furthermore, male M. m. musculus individuals expressed all the studied MUPs' mRNA significantly more than females or individuals of either sex in M. m. domesticus. Correspondingly, the pattern of mRNA abundance was corroborated with the level of total MUP concentration in the urine, such that the level of sexual dimorphism was also significant and species-specific. Our finding introduces a hypothesis that quantitative variation of these proteins may be an essential part of a subspecies recognition system that maintains homospecific mixing.

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.

The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as alpha(2U)-globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded beta-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 10(4)-10(5) M(-1) range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics.

Young male CBA/LacStoRap mice for 2 h were exposed to pheromones that are transferred by major urinary proteins of sexually mature males of the same line. The treatment was conducted using either a complete set of the major urinary proteins typical of the CBA mice or incomplete set of protein fractions detected in some animals. The effect of pheromones was estimated 24 h after treatment by cytogenetic analysis for disturbances in dividing bone marrow cells at anaphase--telophase and in germ cells at metaphase I. The frequency of both mitotic and meiotic disturbances was significantly increased after exposure to pheromones associated with the complete spectrum of the major urinary proteins. Conversely, no cytogenetic effect was observed in the absence of particular protein fractions. Possible consequences of the pheromonal effect on the genomes of somatic and germ cells are discussed as well as the relationships between pheromones and the major urinary proteins.

Major Urinary Proteins (MUPs) from different inbred strains of mouse have been analysed by high-resolution ion-exchange chromatography and mass spectrometry. MUPs from six strains were resolved chromatographically into four major protein peaks which characterized two distinct phenotypes, typified by the profiles obtained from the Balb/c and C57BL/6 inbred strains. A combination of ion-exchange chromatography and electrospray ionization mass spectrometry analysis of the MUPs from each strain identified five proteins, only one of which was common to both strains. The charge and mass data, together with N-terminal sequence analyses, were correlated with the masses of the proteins inferred from published cDNA sequences. Several members of the family of MUP sequences differ in only four positions, and in some circumstances the substitutions elicit a minimal change in protein mass (Lys/Gln; Lys/Glu). Peptide mapping with endopeptidase Lys-C, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry permitted identification of new MUPs that were correlated with partial cDNA sequence data. In the two strains there are at least 13 different MUPs, either observed or predicted, indicating the heterogeneity of expression of this group of proteins.

Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx-binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed.

Mixtures of a specific group of proteins, the major urinary proteins (MUPs), believed to have a role in odorant binding, have been analysed by electrospray ionization mass spectrometry. The data obtained from conventional transformed electrospray data confirm that the molecular weights of the protein mixtures, deduced from published sequences, lie in the molecular weight range 18,600 to 19,000 Da. Application of maximum-entropy analysis to the raw electrospray data has confirmed a heterogeneity in MUP composition, consistent with allelic similarities (and differences) between the different mouse strains. This work demonstrates the use of maximum entropy in the assessment of protein content and in the subsequent resolution enhancement of naturally occurring protein mixtures containing components of closely similar molecular mass.

We have investigated the sexual dimorphism of the mouse major urinary proteins (MUPs) by isoelectric focusing (IEF). In each of two inbred strains which have different male patterns (C57BL and BALB/c), the females show a simpler pattern with fewer prominent components. The main female component is different in each strain, and these may be the products of allelic structural genes. Treatment with testosterone induces the excretion of MUPs with the male pattern. The in vitro translation of hybrid-selected MUP mRNA was studied in the same way. Again, the female patterns were simpler than the male, and the patterns obtained with female mRNA from the two inbred strains were different. When the females were treated with testosterone, a male or male-like pattern was obtained. We argue that some MUP structural genes are quite actively transcribed in females and that the transcription of others is dependent to different degrees upon induction by testosterone.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.

The differences in electrophoretic patterns of major urinary proteins (MUP's) due to sex, strain, and subspecies of the mouse (Mus musculus) were examined by the combined methods of agarose gel electrophoresis and immunofixation. The MUP's from the male of common laboratory inbred mice (M. m. domesticus) showed a component that was absent in females of the same strain, in addition to a strain-specific component that was determined by the Mup-1 allele carried by the strain. In MOA mice, an inbred strain from M. m. molossinus (Japanese wild mice), there was no apparent sex difference in the constitution of the components. The MUP's from this strain contained a major component that was virtually absent in those from laboratory mice, but lacked the strain-specific components of laboratory mice. In addition, the present methods revealed that both liver and serum contained MUP's consisting of components similar to those found in the urine from the same sex and strain.

The major urinary proteins (MUPs) of the mouse are encoded by a multigene family located at the Mup a locus on chromosome 4. Previous investigations have shown that the MUPs are synthesized in the liver, secreted and then excreted in the urine. We have found significant levels of MUP mRNA in several secretory tissues: the liver and the submaxillary, lachrymal and mammary glands. There are striking differences in hormonal and developmental regulation of MUP gene expression in these tissues. Furthermore, each tissue appears to express a characteristic pattern of MUP mRNAs. In particular, the lachrymal glands appear to express an entirely different set of MUP mRNAs. These results are discussed in relation to the organization of the MUP gene cluster and a possible function of the MUPs.

The present study showed that the presence or absence of a new component of major urinary proteins (Mups), which is found in MOA mice, an inbred strain of Mus musculus molossinus (Japanese wild mice), is controlled by a single codominant gene locus. The linkage analysis shows that the locus is on chromosome 4, where the Mup-1 locus is assigned; its alleles, Mup-1a, and Mup-1b determine two phenotypic forms of MUPs in laboratory mice (M. m. domesticus). Recombination values between the locus and other loci on chromosome 4, such as brown (b), Pgm-2, and Gpd-1 are compatible with the gene order, Mup-1-b-Pgm-2-Gpd-1, on chromosome 4. Thus, it is concluded that the locus is identical to Mup-1 and it is proposed that Mup-1c be designated as the allele that determines a third phenotypic form of MUPs in MOA mice.

Sequence analysis of the pyrazine-binding protein from bovine olfactory mucosa reveals marked homology with a family of proteins of unknown function found in the urine of the adult male mouse and rat. In view of the dramatic biological responses to odorants transmitted in male rodent urines, it is proposed that these proteins play important roles in some aspects of odor transmission and reception.

We mapped the proteins of human urine by high-resolution two-dimensional electrophoresis, utilizing the ISO-DALT system. Wide-range pH gradients and narrow-range acid gradients were both used in the first-dimension separations. The patterns revealed proteins ranging in relative molecular mass from 10 000 to 90 000. Proteins identified in the map included transferrin, albumin, hemopexin, alpha 2-HS glycoprotein, alpha 1-antitrypsin. Gc globulin, alpha 1-acid glycoprotein, Zn alpha 2-glycoprotein, retinol binding protein, beta 2-microglobulin, the immunoglobulin light chains, and MAUP (most acid urinary protein). The use and utility of internal-charge and molecular-mass standards are described. We used electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological methods to identify some proteins.

A survey of major urinary proteins (MUPs) from eight BALB/c mouse substrains by isoelectric focusing identified a common pattern with about 10 protein bands in males. One substrain, BALB/cJPt, differed in that it expressed two variant MUP patterns, designated 4.1lo and null. To find the chromosomal location of the gene which determines the 4.1lo phenotype, BALB/cJPt-MUP-4.1lo was crossed with a wild-derived Mus musculus domesticus inbred strain (CLA) that expresses the common BALB/c MUP pattern. The F1 phenotype revealed that the gene(s) controlling the MUP-4.1lo trait was recessive. A restriction fragment polymorphism between these strains found with a MUP cDNA probe allowed us to establish that a gene determining the MUP-4.1lo trait was not linked to the MUP structural genes on chromosome 4. Assays for other chromosomal marker loci revealed that a gene determining the MUP-4.1lo trait, designated Mupm-1, was closely linked to Myc-1 on chromosome 15. To determine the genetic basis of the null trait, BALB/cJPt-MUP-null mice were crossed with BALB/cJPt-MUP-4.1lo mice. A MUP restriction fragment polymorphism between these two lines was tightly linked to a gene or genes involved in determining the MUP-null phenotype. The two variant MUP phenotypes in BALB/cJ mice are determined by separate genes, one of which is located on chromosome 4 and the other on chromosome 15. The chromosomal location of Mupm-1 suggests that it produces a trans-acting factor which regulates MUP expression.

We have mapped the multiple (15-25) genes coding for the hormonally regulated major urinary proteins (MUPs) of the mouse by using a cloned cDNA probe. By Southern blot analysis of DNA from Chinese hamster-mouse somatic cell hybrids, all of the MUP genes were found to be on chromosome 4. Different inbred mouse strains showed DNA polymorphism in their MUP Southern hybridization pattern. Analysis of recombinant inbred strains derived from these parent strains has shown that all the polymorphisms are linked to the MUP-a locus on chromosome 4. The combination of these mapping techniques should be applicable to many cloned DNA sequences.

By labeling liver protein in vivo with [3H] leucine, the relative biosynthetic rate has been measured for the major urinary proteins (MUPs), three closely related, androgen-regulated proteins that are synthesized in mouse liver, secreted into the bloodstream, and excreted into the urine. In livers from females of strain C57BL/6J, total MUP synthesis represents about 0.6-0.9% of the total protein synthesis; in males and testosterone-treated females of the same strain, synthesis increases to about 3.5-4.0% of the total. This 4- to 6-fold induction of total MUP synthesis is similar to the androgen-mediated increase in MUP-specific messenger RNA reported by others, and indicates that the previously observed 20- to 25-fold induction of total MUP excretion into urine is generated partly at the posttranslational level. By measuring the ratio of synthesis of the individual MUPs, it was determined that the testosterone-mediated change in the pattern of MUP synthesis, indicating nature, of MUPs excreted. A survey of seven inbred mouse strains revealed polymorphism for the rate of total MUP synthesis in untreated females. Two classes could be distinguished on the basis of a 3-to 5-fold difference in the rate. This variation does not correlate with variation at Mup-a, a locus that controls the ratio of the three MUPs in urine from androgen-induced mice. These findings are consistent with the notion that MUP expression is controlled by a variety of independently assorting genes.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.

Pheromones and olfactory communication play vital roles in sex recognition and mate choice in rodents. Asian house rats (Rattus tanezumi) (RT) often startle easily, making behavioral measurements difficult to carry out accurately in the laboratory. Here, the behavioral and olfactory preferences of the female RT between males and females were not observed using a conventional two-choice device; we then explored the neuro-immunohistochemical evidence in the brains of RT females. We found that male urine elicited significantly higher c-fos expression in the accessory olfactory system and sex-related brain regions in females than female urine did. On the other hand, the differences of volatile compounds and major urinary proteins (MUPs) in both voided urine and preputial glands (PGs) of the RT were detected using gas chromatography-mass spectrometer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and liquid chromatography-electrospray ionization mass spectrometry. We found that PG-derived 1-(4,5-dihydro-2-thiazolyl)-ethanone and total MUPs were more abundant in males versus females, suggesting these sexually divergent components might activate the female's accessory olfactory system. In conclusion, the neuro-immunohistochemical evidence indicated that potential sex pheromones might exist in RT; however, the strength of the chemical signal might be too weak to cause behavioral responses in females., (© 2023 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Reproductive success in mice depends on sexually dimorphic major urinary proteins (Mup) that facilitate interactions between females and males. Deletion of cystathionine β-synthase (Cbs) gene, a metabolic gene important for homeostasis of one-carbon metabolism, impairs reproduction by causing female infertility in mice. Here, we examined Mup biogenesis and sexual signaling in Cbs -/- versus Cbs +/- mice. We found significantly reduced levels of total urinary Mup protein in male and female Cbs -/- versus Cbs +/- mice. SDS-PAGE/Western blot, ESI-MS, and RT-qPCR analyses of the liver, plasma, and urinary proteins identified a male-specific Mup20 in Cbs -/- , but not in Cbs +/- females. The 18 893 Da Mup20 became the most abundant in urine of Cbs -/- females and males. Effects of Cbs genotype on 18 645 Da, 18 693 Da, and 18 709 Da Mup species abundance were Mup- and sex-specific. Cbs genotype-dependent changes in hepatic Mups and Mup20 expression were similar at the protein and mRNA level. Changes in Mups, but not in Mup20, can be explained by downregulation of hepatic Zhx2 and Ghr receptors in Cbs -/- mice. Behavioral testing showed that Cbs +/- females ignored Cbs -/- male urine but were attracted to Cbs +/- male urine. Cbs +/- males ignored urine of Cbs -/- males but countermarked urine of other Cbs +/- males and were attracted to urines of Cbs -/- as well as Cbs +/- females. Cbs -/- males did not countermark urine of Cbs +/- males but were attracted to urines of Cbs +/- females. Taken together, these findings show that Cbs, a metabolic gene, interacts with the processes involved in Mup biogenesis that are essential for the maintenance of sexual dimorphism and signaling and suggest that dysregulation of these interactions impairs reproductive fitness in mice., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

For individuals to survive and function in society, it is essential that they recognize, interact with, and learn from other conspecifics. Observational fear (OF) is the well-conserved empathic ability of individuals to understand the other's aversive situation. While it is widely known that factors such as prior similar aversive experience and social familiarity with the demonstrator facilitate OF, the neural circuit mechanisms that explicitly regulate experience-dependent OF (Exp OF) were unclear. In this review, we examine the neural circuit mechanisms that regulate OF, with an emphasis on rodent models, and then discuss emerging evidence for the role of fear memory engram cells in the regulation of Exp OF. First, we examine the neural circuit mechanisms that underlie Naive OF, which is when an observer lacks prior experiences relevant to OF. In particular, the anterior cingulate cortex to basolateral amygdala (BLA) neural circuit is essential for Naive OF. Next, we discuss a recent study that developed a behavioral paradigm in mice to examine the neural circuit mechanisms that underlie Exp OF. This study found that fear memory engram cells in the BLA of observers, which form during a prior similar aversive experience with shock, are reactivated by ventral hippocampal neurons in response to shock delivery to the familiar demonstrator to elicit Exp OF. Finally, we discuss the implications of fear memory engram cells in Exp OF and directions of future research that are of both translational and basic interest., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

To be social, the ability to recognize and discriminate conspecific individuals is indispensable in social animals, including primates, rodents, birds, fish, and social insects which live in societies or groups. Recent studies using molecular biology, genetics, in vivo and in vitro physiology, and behavioral neuroscientific approaches have provided detailed insights into how animals process and recognize the information of individuals. Here, we review the most distinct sensory modalities for individual recognition in animals, namely, olfaction and vision. In the case of rodents, two polymorphic gene complexes have been identified in their urine as the key and essential pheromonal components for individual recognition: the major histocompatibility complex (MHC) and the major urinary protein (MUP). Animals flexibly utilize MHC and/or MUP, which are detected by the main olfactory epithelium (MOE) and/or the vomeronasal organ (VNO) for various types of social recognition, such as strain recognition, kin recognition, and individual recognition. In contrast, primates, including humans, primarily use facial appearance to identify others. Face recognition in humans and other animals is naturally unique from genetic, cognitive, developmental, and functional points of view. Importantly note that nurture effects during growth phase such as social experience and environment can also shape and tune this special cognitive ability, in order to distinguish subtle differences between individuals. In this review, we address such unique nature and nurture mechanisms for individual recognition., (© 2020 Japanese Society of Developmental Biologists.)

Scent marks are important mediators of territorial behaviour and sexual selection, especially among mammals. The evolution of compounds used in scent marks has the potential to inform our understanding of signal evolution in relation to social and sexual selection. A major challenge in studies of chemical communication is that the link between semiochemical compounds and genetic changes is often unclear. The major urinary proteins (MUPs) of house mice provide information on sex, status and individual identity. Importantly, MUPs are a direct protein product of genes, providing a clear link between genotype and phenotype. Here, we examine the evolution of urinary protein signals among house mice and relatives by examining the sequences and patterns of mRNA expression of Mup genes related to urinary scent marks. MUP patterns have evolved among mouse species both by gene duplication and variation in expression. Notably, protein scent signals that are male specific in well-studied inbred laboratory strains vary in sex-specificity among species. Our data reveal that individual identity signals in MUPs evolved prior to 0.35 million years ago and have rapidly diversified through recombining a modest number of amino acid variants. Amino acid variants are much more common on the exterior of the protein where they could interact with vomeronasal receptors, suggesting that chemosensory perception may have played a major role in shaping MUP diversity. These data highlight diverse processes and pressures shaping scent signals, and suggest new avenues for using wild mice to probe the evolution of signals and signal processing., (© 2019 John Wiley & Sons Ltd.)

Major urinary proteins (MUPs), members of the broader lipocalin protein family, are classified as pheromones that are excreted in male rodent urine to define conspecific territoriality. In screening for differentially regulated mRNA transcripts in a mouse model of type 1 experimental diabetes mellitus (DM), we identified an unexpected upregulation of several closely related MUP transcripts within diabetic sensory dorsal root ganglia (DRG). Both sexes expressed overall MUP protein content as identified by an antibody widely targeting these upregulated family members, and immunohistochemistry identified expression within neurons, satellite glial cells, and Schwann cells. In dissociated adult sensory neurons, knockdown by an siRNA targeting upregulated MUP mRNAs, enhanced neurite outgrowth, indicating a growth-suppressive role, an impact that was synergistic with subnanomolar insulin neuronal signaling. While MUP knockdown did not generate rises in insulin signaling transcripts, the protein did bind to several mitochondrial and glial targets in DRG lysates. Analysis of a protein closely related to MUPs but that is expressed in humans, lipocalin-2, also suppressed growth, but its impact was unrelated to insulin. In a model of chronic type 1 DM, MUP siRNA knockdown improved electrophysiological and behavioral abnormalities of experimental neuropathy. MUPs have actions beyond pheromone signaling in rodents that involve suppression of growth plasticity of sensory neurons. Its hitherto unanticipated actions overlap with those of lipocalin-2 and may identify a common and widely mediated impact on neuron growth properties by members of the lipocalin family. Knockdown of MUP supports the trophic actions of insulin as a strategy that may improve features of type 1 experimental diabetic neuropathy. NEW & NOTEWORTHY New molecular mechanisms are important to unravel and understand diabetic polyneuropathy, a disorder prevalent in over half of persons with diabetes mellitus (DM). MUPs, members of the lipocalin family of molecules, have an unexpected impact on the plasticity of sensory neurons that are targeted in type 1 experimental diabetic neuropathy. This work explores this potential target in neuropathy in the context of the lipocalin family of molecules.