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The major urinary proteins (MUPs) of house mice (Mus musculus) bind and stabilize the release of pheromones and other volatile organic compounds (VOCs) from urinary scent marks, which mediate chemical communication. Social status influences MUP and VOC excretion, and the urinary scent of dominant males is attractive to females. Urinary pheromones influence the sexual behavior and physiology of conspecifics, and yet it is not known whether they also affect reproductive success. We monitored the excretion of urinary protein and VOCs of wild-derived house mice living in large seminatural enclosures to compare the sexes and to test how these compounds correlate with reproductive success. Among males, urinary protein concentration and VOC expression correlated with reproductive success and social status. Territorial dominance also correlated with reproductive success in both sexes; but among females, no urinary compounds were found to correlate with social status or reproductive success. We found several differences in the urinary protein and volatile pheromones of mice in standard cages versus seminatural enclosures, which raises caveats for conventional laboratory studies. These findings provide novel evidence for chemical signals that correlate with male reproductive success of house mice living in competitive conditions.

BALB/cJ mice exhibit considerable phenotypic differences with other BALB/c substrains. Some of these traits involve the liver, including persistent postnatal expression of genes that are normally expressed only in the fetal liver and reduced expression of major urinary proteins. These traits are due to a mutation that dramatically reduces expression of the gene encoding the transcription factor Zinc fingers and homeoboxes 2 (Zhx2). BALB/cJ mice also exhibit reduced serum lipid levels and resistance to atherosclerosis compared to other mouse strains when placed on a high fat diet. This trait is also due, at least in part, to the Zhx2 mutation. Microarray analysis identified many genes affecting lipid homeostasis, including Lipoprotein lipase, that are dysregulated in BALB/cJ liver. This led us to investigate whether hepatic lipid levels would be different between BALB/cJ and BALB/c mice when placed on a normal chow or a high fat chow diet. On the high fat chow, BALB/cJ mice had increased weight gain, increased liver/body weight ratio, elevated hepatic lipid accumulation and markers of liver damage when compared to BALB/c mice. These traits in BALB/cJ mice were only partially reversed by a hepatocyte-specific Zhx2 transgene. These data indicate that Zhx2 reduces liver lipid levels and is hepatoprotective in mice on a high fat diet, but the partial rescue by the Zhx2 transgene suggests a contribution by both parenchymal and non-parenchymal cells. A model to account for the cardiovascular and liver phenotype in mice with reduced Zhx2 levels is provided.

In this study, we found that loss of the circadian clock gene Bmal1 causes disruptions throughout the growth hormone (GH) axis, from hepatic gene expression to production of urinary pheromones and pheromone-dependent behavior. First, we show that Bmal1 knockout (KO) males elicit reduced aggressive responses from wild-type (WT) males and secrete lower levels of major urinary proteins (MUPs); however, we also found that a liver-specific KO of Bmal1 (liver-Bmal1-KO) produces a similar reduction in MUP secretion without a defect in aggressive behavior, indicating that the decrease in elicited aggression arises from another factor. We then shifted our investigation to determine the cause of MUP dysregulation in Bmal1 KO animals. Because the pulse pattern of GH drives sexually dimorphic expression of hepatic genes including MUPs, we examined GH pulsatility. We found that Bmal1 KO males have a female-like pattern of GH release, whereas liver-Bmal1-KO mice are not significantly different from either WT or Bmal1 KO. Since differential patterns of GH release regulate the transcription of many sexually dimorphic genes in the liver, we then examined hepatic gene transcription in Bmal1 KO and liver-Bmal1-KO mice. We found that while some female-predominant genes increase in the Bmal1 KO, there was no decrease in male-predominant genes, and little change in the liver-Bmal1-KO. We also found disrupted serum insulin growth factor 1 (IGF-1) and liver Igf1 messenger RNA in the Bmal1 KO mice, which may underlie the disrupted GH release. Overall, our findings differentiate between GH-pulse–driven and circadian-driven effects on hepatic genes, and the functional consequences of altered GH pulsatility.

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.

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.

Mature male mice produce a particularly high concentration of major urinary proteins (MUPs) in their scent marks that provide identity and status information to conspecifics. Darcin (MUP20) is inherently attractive to females and, by inducing rapid associative learning, leads to specific attraction to the individual male’s odour and location. Other polymorphic central MUPs, produced at much higher abundance, bind volatile ligands that are slowly released from a male’s scent marks, forming the male’s individual odour that females learn. Here, we show that infection of C57BL/6 males with LCMV WE variants (v2.2 or v54) alters MUP expression according to a male’s infection status and ability to clear the virus. MUP output is substantially reduced during acute adult infection with LCMV WE v2.2 and when males are persistently infected with LCMV WE v2.2 or v54. Infection differentially alters expression of darcin and, particularly, suppresses expression of a male’s central MUP signature. However, following clearance of acute v2.2 infection through a robust virus-specific CD8 cytotoxic T cell response that leads to immunity to the virus, males regain their normal mature male MUP pattern and exhibit enhanced MUP output by 30 days post-infection relative to uninfected controls. We discuss the likely impact of these changes in male MUP signals on female attraction and mate selection. As LCMV infection during pregnancy can substantially reduce embryo survival and lead to lifelong infection in surviving offspring, we speculate that females use LCMV-induced changes in MUP expression both to avoid direct infection from a male and to select mates able to develop immunity to local variants that will be inherited by their offspring.

Summary The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors.

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.

Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.

Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species.

This article is part of a Special Issue “Chemosignals and Reproduction”. This paper reviews the role of chemosignals in the socio-sexual interactions of female mice, and reports two experiments testing the role of pup-derived chemosignals and the male sexual pheromone darcin in inducing and promoting maternal aggression. Female mice are attracted to urine-borne male pheromones. Volatile and non-volatile urine fractions have been proposed to contain olfactory and vomeronasal pheromones. In particular, the male-specific major urinary protein (MUP) MUP20, darcin, has been shown to be rewarding and attractive to females. Non-urinary male chemosignals, such as the lacrimal protein ESP1, promote lordosis in female mice, but its attractive properties are still to be tested. There is evidence indicating that ESP1 and MUPs are detected by vomeronasal type 2 receptors (V2R). When a female mouse becomes pregnant, she undergoes dramatic changes in her physiology and behaviour. She builds a nest for her pups and takes care of them. Dams also defend the nest against conspecific intruders, attacking especially gonadally intact males. Maternal behaviour is dependent on a functional olfactory system, thus suggesting a role of chemosignals in the development of maternal behaviour. Our first experiment demonstrates, however, that pup chemosignals are not sufficient to induce maternal aggression in virgin females. In addition, it is known that vomeronasal stimuli are needed for maternal aggression. Since MUPs (and other molecules) are able to promote intermale aggression, in our second experiment we test if the attractive MUP darcin also promotes attacks on castrated male intruders by lactating dams. Our findings demonstrate that the same chemosignal, darcin, promotes attraction or aggression according to female reproductive state

Rats are predators of mice in nature. Nevertheless, it is a common practice to house mice and rats in a same room in some laboratories. In this study, we investigated the behavioral and physiological responsively of mice in long-term co-species housing conditions. Twenty-four male mice were randomly assigned to their original raising room (control) or a rat room (co-species-housed) for more than 6 weeks. In the open-field and light-dark box tests, the behaviors of the co-species-housed mice and controls were not different. In a 2-choice test of paired urine odors [rabbit urine (as a novel odor) vs. rat urine, cat urine (as a natural predator-scent) vs. rabbit urine, and cat urine vs. rat urine], the co-species-housed mice were more ready to investigate the rat urine odor compared with the controls and may have adapted to it. In an encounter test, the rat-room-exposed mice exhibited increased aggression levels, and their urines were more attractive to females. Correspondingly, the levels of major urinary proteins were increased in the co-species-housed mouse urine, along with some volatile pheromones. The serum testosterone levels were also enhanced in the co-species-housed mice, whereas the corticosterone levels were not different. The norepinephrine, dopamine, and 5-HT levels in the right hippocampus and striatum were not different between the 2. Our findings indicate that chronic co-species housing results in adaptation in male mice; furthermore, it appears that long-term rat-odor stimuli enhance the competitiveness of mice, which suggests that appropriate predator-odor stimuli may be important to the fitness of prey animals.

Oxidative stress (an overproduction of reactive oxygen species in relation to defense mechanisms) may restrict investment in life history traits, such as growth, reproduction, lifespan, and the production of sexual signals to attract mates. The constraint on sexual signaling by oxidative stress is of particular interest because it has been proposed as a mechanism ensuring that only good-quality males produce the most attractive sexual signals. Despite these predictions, evidence supporting this theory is, at best, equivocal. We used a superoxide dismutase knockout mouse to demonstrate that oxidative stress directly impairs investment in morphological (preputial glands) and molecular (major urinary proteins) components of olfactory signaling essential for mate attraction. By maintaining males in a much more competitive environment than usual for mouse laboratory experiments, we also revealed a range of phenotypes of superoxide dismutase deficiency not observed in previous studies of this mouse model. This range included impaired bioenergetic function, which was undetectable in the control environment of this study. We urge further examination of model organisms in seminatural conditions and more competitive laboratory environments, as important phenotypes can be exposed under these more demanding conditions.

Mice release a variety of chemical signals, particularly through urine, which mediate social interactions and endocrine function. Studies have been conducted to investigate the stability of urinary chemosignals in mice. Neuroendocrine and behavioral responses of mice to urine samples of male and female conspecifics which have aged for different amounts of time have been examined, demonstrating that the quality and intensity of signaling molecules in urine change over time. In this study, we monitored changes in volatile organic compounds (VOCs) released from male and female mouse urine following aging the urine samples. Substantial amounts of some VOCs were lost during the aging process of urine, whereas other VOCs increased. Considerable portions of the VOCs which exhibited the increased release were shown to have previously been dissolved in water and subsequently released as the urine dried. We also demonstrated that some VOCs decreased slightly due to their binding with the major urinary proteins (MUPs) and identified MUP ligands whose headspace concentrations increased as the urine aged. Our results underscore the important role of MUPs and the hydration status in the release of VOCs in urine, which may largely account for the changes in the quality and intensity of urinary signals over time.

Background The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms. Methods We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A. Results Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56 °C, able to reversibly unfold and it exhibits an enhanced allergenicity. Conclusions The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications. General significance We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface.

Spots to Remember Scent marking is an essential component of communication for most mammals. Individuals remember the location of scent marks and regularly revisit marked sites, presumably to assess the condition and status of the animal doing the marking. It is known that individuals can follow odor or pheromone gradients to locate another individual, but relocating scent marks is a much more difficult task given the small amount of volatile compounds deposited, and their static nature. Roberts et al. (p. 1462 ) show that a nonvolatile component of male urine, the protein pheromone darcin, stimulates spatial preference and learning in mice. Female mice preferred locations where male urine (or synthesized darcin) had been found, and remembered these spatial locations for 2 weeks post-exposure.

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.

Krüppel-associated box-zinc finger proteins (KRAB-ZFPs) are the largest class of transcriptional regulators in mammals, yet few have been assigned biological roles. Cloning the genes underlying the regulator of sex-limitation (rsl) phenotype, in which the normally male-specific sex-limited protein (SLP) is expressed in female mice, identified two KRAB-ZFPs, Rsl1 and Rsl2, as influencing sexually dimorphic liver gene expression. Combined absence of both repressors in rsl mice leads to increased expression in female liver of major urinary proteins (MUPs) and certain enzymes of steroid metabolism, as well as SLP. We hypothesized that this altered gene expression might affect reproductive physiology in rsl females. Urinary MUP (uMUP) concentration varied with the estrous cycle in both wt and rsl females but was consistently higher in rsl urine. A behavioral odor test revealed that wild-type (wt) males preferred rsl to wt females, possibly due to elevated uMUPs providing greater pheromone presentation. To ascribe activity to Rsl1, Rsl2, or both, the genes were individually expressed as liver-specific transgenes. RSL2 overexpression accentuated uMUP fluctuations across the estrous cycle, whereas RSL1 overexpression did not. In addition, puberty onset, as indicated by vaginal opening (VO), occurred 2 days earlier in rsl females, and excess RSL2, but not RSL1, restored VO timing to wt. Hence, transcriptional repression by RSL in liver modifies female mouse reproduction via targets that likely impact both hormonal and pheromonal cues. The large and rapidly diversifying KRAB-ZFP family may modulate biological processes, including reproduction, to confer individual differences that may isolate populations and ultimately lead to speciation.

Scents play key roles in mediating sexual behaviour in many vertebrates, both in the recognition of opposite sex conspecifics and in assessing the suitability of different individuals as potential mates. The recognition and assessment that underlies female attraction to male scents involves an important interaction between the main and accessory (vomeronasal) olfactory systems. Female mice gain information through the vomeronasal system on nasal contact with a scent source that is essential to stimulate attraction to an individual male's scent. Three highly polymorphic multigene families contribute involatile proteins and peptides to mouse scents that are detected through specific vomeronasal receptors during contact with scent. Major urinary proteins (MUPs) provide an individual genetic identity signature that underlies individual recognition and assessment of male competitive ability, kin recognition to avoid inbreeding, and genetic heterozygosity assessment. Familiar mates are recognised in the context of pregnancy block using MHC peptides, while exocrine-gland secreting peptides (ESPs) are likely to play additional roles in sexual assessment. By associating this involatile information in individual male scents, gained on initial scent contact, with the individual male's airborne volatile signature detected simultaneously through the main olfactory system, females subsequently recognise and are attracted by the individual male's airborne volatile signature alone. This allows much more rapid recognition of scents from familiar animals without requiring physical contact or processing through the vomeronasal system. Nonetheless, key information that induces attraction to a male's scent is held in involatile components detected through the vomeronasal system, allowing assessment of the genetic identity and attractiveness of each individual male.

Studies on chemosensory communication generally rely on using samples of emanations that have been frozen for convenience, and assume that freezing has no detrimental effects. Female urine triggers courtship ultrasonic vocalizations (USVs) in male mice, and in this study, we examined whether freezing urine affects males' USV responses. We used wild-derived house mice (Mus musculus musculus), and recorded males' USVs after being presented samples of fresh versus frozen urine. We found that males emitted significantly fewer USVs when presented with frozen versus fresh urine, and furthermore, males were no longer able to discriminate the scent of familiar versus unfamiliar females after samples were frozen. Our findings indicate that freezing alters the bioactivity of urinary compounds both by reducing its potential to elicit behavioral responses from receivers (male courtship USVs) and eliminating important information about senders (familiarity). Freezing may denature the major urinary proteins (MUPs) released in rodent urine that play an important role in chemical communication. Our results raise concerns about the negative results of studies utilizing frozen samples for studying chemical signals.

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.

Summary Animals might be able to use highly polymorphic genetic markers to recognize very close relatives and avoid inbreeding [1, 2]. The major histocompatibility complex (MHC) is thought to provide such a marker [1, 3–6] because it influences individual scent in a broad range of vertebrates [6–10]. However, direct evidence is very limited [1, 6, 10, 11]. In house mice ( Mus musculus domesticus ), the major urinary protein (MUP) gene cluster provides another highly polymorphic scent signal of genetic identity [8, 12–15] that could underlie kin recognition. We demonstrate that wild mice breeding freely in seminatural enclosures show no avoidance of mates with the same MHC genotype when genome-wide similarity is controlled. Instead, inbreeding avoidance is fully explained by a strong deficit in successful matings between mice sharing both MUP haplotypes. Single haplotype sharing is not a good guide to the identification of full sibs, and there was no evidence of behavioral imprinting on maternal MHC or MUP haplotypes. This study, the first to examine wild animals with normal variation in MHC, MUP, and genetic background, demonstrates that mice use self-referent matching of a species-specific [16, 17] polymorphic signal to avoid inbreeding. Recognition of close kin as unsuitable mates might be more variable across species than a generic vertebrate-wide ability to avoid inbreeding based on MHC.

SummaryThe major histocompatibility complex (MHC) is widely assumed to be a primary determinant of individual-recognition scents in many vertebrates [1–6], but there has been no functional test of this in animals with normal levels of genetic variation. Mice have evolved another polygenic and highly polymorphic set of proteins for scent communication, the major urinary proteins (MUPs) [7–12], which may provide a more reliable identity signature ([13, 14] and A.L. Sherborne, M.D.T., S. Paterson, F.J., W.E.R.O., P. Stockley, R.J.B., and J.L.H., unpublished data). We used female preference for males that countermark competitor male scents [15–17] to test the ability of wild-derived mice to recognize individual males differing in MHC or MUP type on a variable genetic background. Differences in MHC type were not used for individual recognition. Instead, recognition depended on a difference in MUP type, regardless of other genetic differences between individuals. Recognition also required scent contact, consistent with detection of involatile components through the vomeronasal system [6, 18]. Other differences in individual scent stimulated investigation but did not result in individual recognition. Contrary to untested assumptions of a vertebrate-wide mechanism based largely on MHC variation, mice use a species-specific [12] individual identity signature that can be recognized reliably despite the complex internal and external factors that influence scents [2]. Specific signals for genetic identity recognition in other species now need to be investigated.

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.

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 IgG(4) antibodies.

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.

Individual recognition is an important component of behaviors, such as mate choice and maternal bonding that are vital for reproductive success. This article highlights recent developments in our understanding of the chemosensory cues and the neural pathways involved in individuality discrimination in rodents. There appear to be several types of chemosensory signal of individuality that are influenced by the highly polymorphic families of major histocompatibility complex (MHC) proteins or major urinary proteins (MUPs). Both have the capability of binding small molecules and may influence the individual profile of these chemosignals in biological fluids such as urine, skin secretions, or saliva. Moreover, these proteins, or peptides associated with them, can be taken up into the vomeronasal organ (VNO) where they can potentially interact directly with the vomeronasal receptors. This is particularly interesting given the expression of major histocompatibility complex Ib proteins by the V2R class of vomeronasal receptor and the highly selective responses of accessory olfactory bulb (AOB) mitral cells to strain identity. These findings are consistent with the role of the vomeronasal system in mediating individual discrimination that allows mate recognition in the context of the pregnancy block effect. This is hypothesized to involve a selective increase in the inhibitory control of mitral cells in the accessory olfactory bulb at the first level of processing of the vomeronasal stimulus.

Mouse urine contains a complex mixture of chemosignals including a variety of small volatile molecules that are bound to major urinary proteins. In addition to signalling maleness, male urine also conveys information about individuality, which allows recently mated female mice to distinguish the urinary chemosignals of the mating male from those of an unfamiliar male. The highly polymorphic nature of the major urinary proteins makes them a likely candidate for conveying individuality information in the context of the pregnancy block effect. This was investigated by comparing the pregnancy-blocking effectiveness of a high molecular weight urinary fraction, containing major urinary proteins, with that of a low molecular weight fraction containing volatile ligands. Not only was the high molecular weight fraction ineffective in blocking pregnancy, but it also appeared to be less important in signalling individuality than the low molecular fraction. The high molecular weight fraction was ineffective in inducing expression of the immediate early gene product egr-1 in the accessory olfactory bulb. In contrast, the low molecular weight fraction induced egr-1 expression in the mitral/tufted neurons in the anterior subregion of the accessory olfactory bulb, suggesting that they activate the V1R class of vomeronasal receptor neuron.

The genomes of rats and mice both contain a cluster of multiple genes that encode small (18–20 kDa) eight-stranded β-barrel lipocalins that are expressed in multiple secretory tissues, some of which enter urine via hepatic biosynthesis. These proteins have been given different names, but are mostly generically referred to as MUPs (major urinary proteins). The mouse MUP cluster is increasingly well understood, and, in particular, a number of roles for MUPs in chemical communication between conspecifics have been established. By contrast, the literature on the rat orthologues is much less well developed and is fragmented. In the present review, we summarize current knowledge on the MUPs from the Norway (or brown) rat, Rattus norvegicus . Abbreviations: MUP, major urinary protein; RGSC, Rat Genome Sequence Consortium

SummaryDuring social interactions, an individual’s behavior is largely governed by the subset of signals emitted by others. Discrimination of “self” from “other” regulates the territorial urine countermarking behavior of mice. To identify the cues for this social discrimination and understand how they are interpreted, we designed an olfactory-dependent countermarking assay. We find major urinary proteins (MUPs) sufficient to elicit countermarking, and unlike other vomeronasal ligands that are detected by specifically tuned sensory neurons, MUPs are detected by a combinatorial strategy. A chemosensory signature of “self” that modulates behavior is developed via experience through exposure to a repertoire of MUPs. In contrast, aggression can be elicited by MUPs in an experience-independent but context-dependent manner. These findings reveal that individually emitted chemical cues can be interpreted based on their combinatorial permutation and relative ratios, and they can transmit both fixed and learned information to promote multiple behaviors.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10(-11) M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Male mice excrete large quantities of major urinary proteins that have been proposed to have an important pheromonal role either alone or by way of their bound ligands. We have found that these major urinary proteins are not only likely to mediate the pregnancy blocking effects of male urine, but that they also convey the strain recognition signal of the male pheromone. Recent molecular biological investigations have characterized two classes of pheromonal receptor in the vomeronasal organ that appear to project separately to anterior and posterior regions of the accessory olfactory bulb. However, it is not known whether these separate pathways handle fundamentally different types of pheromonal information. We have attempted to investigate this question using the expression of the immediate-early gene egr-1 as a marker for activity of neurons in the accessory olfactory bulb of female mice in response to putative pheromonal constituents. Exposure to 2,3 dihydro-exo-brevicomin and 2-sec-butyl-4,5-dihydro-thiazole, the main ligands bound to the major urinary proteins, elicited expression of egr-1 in clusters of presumed mitral neurons at the medial and lateral margins of the posterior accessory olfactory bulb. Whole male urine and a preparation of major urinary proteins that had been stripped of their ligands induced egr-1 expression in mitral cells of the anterior half of the accessory olfactory bulb in addition to the posterior clusters. This would suggest that the anterior and posterior halves of the accessory olfactory bulb are processing different aspects of the male pheromone signal with the anterior region, which responds preferentially to major urinary proteins, being principally concerned with the strain recognition component.

Mouse urinary protein (MUP) and alpha 2u-globulin are structurally homologous proteins that belong to a superfamily of ligand-binding proteins and represent the major urinary proteins excreted by adult male mice and rats, respectively. Although a variety of xenobiotics bind to alpha 2u-globulin and produce a male rat-specific hyaline droplet nephropathy, no endogenous ligand for this protein has been identified. Despite extensive sequence homology. MUP does not bind to hyaline droplet-inducing agents. While performing experiments with purified MUP, we observed that it presented with a strong, distinctive odor reminiscent of mouse urine. To determine whether this odor was the result of contamination or degradation or was attributed to an endogenous ligand bound to the protein, the protein was subjected to thermal desorption and any released volatile compounds were detected with a gas chromatograph equipped with an external sniff port and mass spectrometer. With this approach, two odorous compounds were detected at the sniff port by a human observer, but only one was present in sufficient mass to allow identification. This compound, which presented with the characteristic odor, was subsequently identified as 2-sec butyl-4,5-dihydrothiazole (DHT) by GC/MS/matrix isolation IR and NMR analyses. The identification of DHT was confirmed by comparing the chromatographic and spectral properties to those of the synthesized authentic compound. In direct contrast, purified urinary alpha 2u-globulin did not present with an obvious odor, and no volatile ligands were detected on this protein. Although DHT is a major endogenous ligand for MUP, it was also found to competitively inhibit the binding of [14C]d-limonene-1,2-epoxide to alpha 2u-globulin with relatively high affinity (Ki = 2.3 microM). When dosed orally to F344 rats, DHT (1 mmol/kg for 3 days) caused the characteristic exacerbation of hyaline droplets in male rat kidneys and increased renal levels of immunoreactive alpha 2u-globulin about threefold over control levels. These results indicate that despite structural homology, MUP and alpha 2u-globulin are distinguished by the presence of a volatile endogenous ligand only on the former, a distinction that may reflect differences in the physiological functions of the two proteins. Furthermore, although DHT can bind to both MUP and alpha 2u-globulin, renal toxicity was only observed in rats, thereby emphasizing the unique toxicological properties of alpha 2u-globulin in the development of hyaline droplet nephropathy.

Competition between adult males for limited resources such as food and receptive females is shaped by the male pattern of pituitary growth hormone (GH) secretion that determines body size and the production of urinary pheromones involved in male-to-male aggression. In the brain, dopamine (DA) provides incentive salience to stimuli that predict the availability of food and sexual partners. Although the importance of the GH axis and central DA neurotransmission in social dominance and fitness is clearly appreciated, the two systems have always been studied unconnectedly. Here we conducted a cell-specific genetic dissection study in conditional mutant mice that selectively lack DA D2 receptors (D2R) from pituitary lactotropes (lacDrd2KO) or neurons (neuroDrd2KO). Whereas lacDrd2KO mice developed a normal GH axis, neuroDrd2KO mice displayed fewer somatotropes; reduced hypothalamic Ghrh expression, pituitary GH content, and serum IGF-I levels; and exhibited reduced body size and weight. As a consequence of a GH axis deficit, neuroDrd2KO adult males excreted low levels of major urinary proteins and their urine failed to promote aggression and territorial behavior in control male challengers, in contrast to the urine taken from control adult males. These findings reveal that central D2Rs mediate a euroendocrineexocrine cascade that controls the maturation of the GH axis and downstream signals that are critical for fitness, social dominance, and competition between adult males. Fil: Noain, Daniela Maria Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina Fil: Pérez Millán, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Bello Gay, Estefania Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Casas Cordero, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina Fil: Gelman, Diego Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina Fil: Peper, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina Fil: Isabel Garcia-tornadu. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Malcolm J Low. Michigan State University; Estados Unidos; Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina

Odour signals used in competitive and aggressive interactions between males are well studied in the context of sexual selection. By contrast, relatively little is known about comparable signals used by females, despite current interest in the evolution of female ornaments and weaponry. Available evidence suggests that odour signals are important in competitive interactions between female mammals, with reductions or reversals of male-biased sexual dimorphism in signalling where female competition is intense. Scent marking is often associated with conflict between females over access to resources or reproductive opportunities. Female scent marks may therefore provide reliable signals of competitive ability that could be used both by competitors and potential mates. Consistent with this hypothesis, we report that aggressive behaviour of female house mice is correlated with the amount of major urinary protein (MUP) excreted in their urine, a polymorphic set of proteins that are used in scent mark signalling. Under semi-natural conditions, females with high MUP output are more likely to produce offspring sired by males that have high reproductive success, and less likely to produce offspring by multiple different sires, suggesting that females with strong MUP signals are monopolized by males of particularly high quality. We conclude that odour signals are worthy of more detailed investigation as mediators of female competition.

The hypothalamic-growth hormone (GH)-liver axis represents a new concept in endocrine regulation of drug toxicity. Preponderant sex differences are found in liver gene expression, mostly dependent on the sexually dimorphic pattern of GH secretion which is set during the neonatal period by gonadal steroids. We tested if GH-dependent sexually dimorphic liver enzymes and proteins was perturbed by neonatal Bisphenol A (BPA) treatment in female rats. Female rats were sc injected with BPA (50 or 500 μg/50 μl) or castor oil vehicle from postnatal day 1 to 10. At five months serum prolactin, pituitary GH, and serum and liver insulin growth factor-I (IGF-I) were measured by RIA. Major urinary proteins (MUPs) were determined by electrophoresis. Liver Cyp2c11, Cyp2c12, Adh1, Hnf6, and Prlr mRNA levels were determined by real time PCR. Pituitary GH content and liver IGF-I concentration were increased by neonatal BPA treatment, indicating partial masculinization of the GH axis in treated females. GH-dependent female predominant liver enzyme genes (Cyp2c12 and Adh1) and a transcription factor (Hnf6) were downregulated or defeminized, while there were no changes in a male predominant gene (Cyp2c11) or protein (MUP). Our findings indicate that perinatal exposure to BPA may compromise the sexually dimorphic capacity of the liver to metabolize drugs and steroids.

Vomeronasal stem cells are generated throughout the life of a mouse and differentiate into neurons that express one vomeronasal type 1 (V1r), one or two vomeronasal type 2 (V2r), or one olfactory receptor. Vomeronasal stem cells can be induced to differentiate into neurons by treatment with lipocalins from mouse urine or by epigenetic modification following treatment with histone deacetylase inhibitors. An important question is, do chemosensory signals, modify the detection capabilities of the vomeronasal organ and affect behaviour. Rearing mice in the presence of urine (and its pheromonal signals) derived from a different mouse strain, affected the behavioural preference for non-kin which were accompanied by changes in vomeronasal receptor expression. Significant changes in the expression of vomeronasal V1r, V2r and olfactory receptors, major urinary proteins, and a number of genes thought to be involved in transcriptional regulation were also observed following urine treatment. These results suggest that modification of a mouse’s urinary environment may exert epigenetic effects on developing vomeronasal neurons, which modify the type of vomeronasal receptors that are expressed. This may provide a mechanism by which environmental changes are able to modify the detection capabilities of the vomeronasal organ to respond optimally to the most likely social environment that a mouse will encounter when mature.

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.

The rodent vomeronasal organ (VNO) mediates the regulation of species-specific and interspecies social behaviors. We have used gene targeting to examine the role of the G protein Gαo, encoded by the gene Gnao1 , in vomeronasal function. We used the Cre- loxP system to delete Gαo in those cells that express olfactory marker protein, which includes all vomeronasal sensory neurons of the basal layer of the VNO sensory epithelium. Using electrophysiology and calcium imaging, we show that the conditional null mice exhibit strikingly reduced sensory responses in V2R receptor-expressing vomeronasal sensory neurons to specific molecular cues, including MHC1 antigens, major urinary proteins, and exocrine gland-secreting peptide. Gαo is also vital for vomeronasal sensing of two N -formylated mitochondrially encoded peptides derived from NADH dehydrogenase 1. Furthermore, we show that Gαo is an essential requirement for the display of male–male territorial aggression as well as maternal aggression in mice. Finally, we show that Gαo-dependent maternal aggression can be induced by major urinary proteins. These cellular and behavioral phenotypes identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein pheromones by sensory neurons of the VNO.

Many rodents are now known to exhibit an obligate proteinuria that delivers urine-mediated chemosignals. In this paper, we explore the urinary proteins of the Roborovski hamster (Phodopus roborovskii). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of urine from individual male and female Roborovski hamsters revealed 2 proteins, with approximate masses of 6 and 17 kDa, the expression pattern of which showed little variation between individuals or between sexes. Peptide mass fingerprints obtained from these 2 proteins revealed a number of features: 1) the proteins of a given mass were the same in all individuals regardless of sex, 2) the 6 kDa protein was not a fragment of the 21 kDa protein, and 3) neither protein was a fragment of a larger, conserved protein such as serum albumin. Electrospray mass spectrometry of purified protein preparations established the mass of the larger protein as invariant, at 17144 � 2 Da in all samples. This protein has been termed roborovskin. The primary structure of roborovskin was determined by tandem mass spectrometry of peptides derived from independent and overlapping digestion with 3 proteases, supported by Edman degradation of the protein N-terminus. Roborovskin shared significant homology with olfactory-binding proteins from Myodes glareolus (bank vole) and with aphrodisin and submandibular protein from the golden hamster Mesocricetus auratus, all of which belong to the lipocalin superfamily. Lower levels of homology were also indicated between a variety of other lipocalins including the major urinary proteins from house mice and Norway rats. A model of the tertiary structure of roborovskin was constructed from the primary sequence by homology modeling. This model structure resembled other 8-stranded beta barrel lipocalins. Thus, the Roborovski hamster may demonstrate another variant of urinary lipocalin expression, as for the animals studied here, there appears to be no polymorphism in expression either between sexes or individuals.

Background Chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. Efficient communication via this protein - pheromone complex is essential for triggering multiple responses including aggression, mate choice, copulatory behaviour, and onset and synchronization of oestrus. The roles of lipocalins in communication were studied in many organisms and especially in mice (i.e. Mus musculus domesticus) which excrete Major Urinary Proteins (Mup) in excessive amounts in saliva and urine. Other mammals, however, often lack the genes for Mups or their expression is very low. Therefore, we aimed at characterization of candidate lipocalins in Myodes glareolus which are potentially linked to chemical communication. One of them is Aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males. Results Here we show that Aphrodisin-like proteins exist in other species, belong to a group of Odorant Binding Proteins (Obp), and contrary to the expression of Aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of M. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus. On the level of mRNA, we have detected three different gene variants. To assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. On the protein level we confirmed the presence of Obp2 and Obp3 in both saliva and urine. Appropriate bands in the range of 17-20 kDa from vaginal discharge were, however, beyond the MS detection limits. Conclusion Our results demonstrate that three novel Obps (Obp1, Obp2, and Obp3) are predominant lipocalins in Myodes urine and saliva. On the protein level we have detected further variants and thus we assume that similarly as Major Urinary Proteins in mice, these proteins may be important in chemical communication in this Cricetid rodent.

SummarySegregation of sperm abnormality level and the pattern of major urinary proteins (MUPs) were investigated in F2 and B1 hybrid males obtained from crosses involving two contrasting inbred strains of mice: CBA/Kw (Mup-1a1a, 3·3% abnormal sperm) and C57BL/Kw (Mup-1b1b, 21·9% abnormal sperm). In the progeny of both crosses mean levels of abnormal spermatozoa were significantly higher for males typed as Mup-1b1b than for heterozygous Mup-1a1b males. Moreover, all F2 hybrid males showing very high percentages of abnormal sperm were Mup-1b1bhomozygotes. Similarly, among B1 males with a high level of deformed spermatozoa, a statistically significant majority were Mup-1b1b genotypes. Our results suggest that at least two genes which influence sperm abnormality level are segregating in these crosses. Both appear to be recessive for high sperm abnormality level, and one shows weak linkage to Mup-1 on chromosome 4.

In mating systems that involve competing males and choosy females, males are expected to advertise their genetic quality to discriminating females. Most examples have focused on visual or acoustic signals, such as ornamentation or song; yet arguably, olfactory communication may be more important to the majority of vertebrates with the possible exception of birds. Fortunately, attention has begun to shift to the role of odours in mate choice, with most of that attention being directed at the major histocompatibility complex or more recently at the major urinary proteins. The study of male ring-tailed lemurs presented by Charpentier and colleagues in this issue adds a new dimension to investigations of the influence of genes on mate choice via odour production. By comparing genetic heterozygosity to the production of semiochemicals in the scrotal scent gland, they provide a link between genetic composition and scent-marking behaviour as a potential advertisement of male quality.

SummaryThe role of individual genetic heterozygosity in mate choice is the subject of much current debate [1–6]. Several recent studies have reported female preference for more heterozygous males [7–9], but the mechanisms underlying heterozygote preference remain largely unknown. Females could favor males that are more successful in intrasexual competition [10–15], but they could also assess male heterozygosity directly at specific polymorphic genetic markers [9, 16–18]. Here, we use a breeding program to remove the intrinsic correlation between genome-wide heterozygosity and two highly polymorphic gene clusters that could allow direct assessment of heterozygosity through scent in mice: the major histocompatibility complex (MHC) [19–21] and the major urinary proteins (MUPs) [22–24]. When other sources of variation are controlled and intrasexual competition is minimized, female mice prefer to associate with MUP heterozygous over MUP homozygous males. MHC heterozygosity does not influence preference, and neither does heterozygosity across the rest of the genome when intrasexual competition between males is restricted. Female mice thus assess male heterozygosity directly through multiple MUP isoforms expressed in scent signals, independently of the effects of genome-wide heterozygosity on male competitiveness. This is the first evidence that animals may use signals of genetic heterozygosity that have no direct association with individual vigour.

Two terpenic constituents, E,E,-alpha-farnesene and E-beta-farnesene, were found to be elevated in dominant male urine when compared to subordinate or control males. These two urinary compounds were absent in the bladder urine of males; however, they were the most prominent constituents of the perputial gland's aliquots. The results of a two-choice preference test, conducted on ICR/Alb subordinate males, gave a strong indication that these two terpenic constituents introduced into the previously attractive stimulus significantly discouraged prolonged investigations by male mice. The compounds, whether present in the urine matrix or water, rendered the stimulus with a quality behaviorally similar to the urine of dominant males. It appears that they may be synonymous with the previously described aversion signal produced by dominant males. We suggest that these compounds may play a wide-ranging role in the territorial marking behavior of male mice.

The ability of animals to distinguish individual con-specifics influences many aspects of their behaviour, including choice of mate, territorial marking and mother-offspring interactions. In rodents, information about individuality is conveyed by chemical cues in their urine and body secretions. Hence, mice can be trained to discriminate the urine odour of congenic mice that differ only in genes of their major histocompatibility complex (MHC). This ability is likely to be based on MHC-related differences in the profile of urinary volatiles (Singer et al., 1997), which are sensed by the main olfactory system and generate statistically different patterns of activity across large populations of neurons in the main olfactory bulb (Schaefer et al., 2002). In contrast, individual recognition underlying urine countermarking behaviour of male mice depends on the profile of major urinary proteins rather than MHC type (Hurst et al., 2001), suggesting that mice use different chemosignals for signalling individual identity in different behavioural contexts. Furthermore, information about the individual identity of mice can be conveyed by both the main olfactory and vomeronasal systems. Whereas the main olfactory system is adapted to learn and discriminate small differences in the profile of airborne volatiles, the vomeronasal system responds to a more limited range of non-volatile stimuli taken up following direct contact. Moreover, the representation of individual identity in vomeronasal system appears to differ from that in the main olfactory system, as individual neurons in the accessory olfactory bulb (AOB), respond highly selectively to the strain identity of anaesthetized conspecifics (Luo et al., 2003).

The genomic locus on mouse chromosome 13 called Regulator of sex-limitation (Rsl) accentuates sex differences in hepatic gene expression. Females homozygous for variant rsl alleles express some otherwise male-specific liver proteins, such as sex-limited protein (Slp), major urinary proteins (MUPs), and members of the cytochrome P450 (cyp) 2d subfamily. We recently identified mutations in two genes, Rsl1 and Rsl2, accounting for the rsl phenotype. These genes encode KRAB zinc-finger proteins (KRAB-ZFPs) and are embedded within a cluster of over 20 similar genes. Mammalian genomes contain over 200 KRAB-ZFP genes, which act biochemically to repress transcription, but the Rsl genes are the first to have their biological functions elucidated. Here we compare Rsl1 and Rsl2 with their neighboring genes, tracing a series of duplication, inversion, and gene conversion events that have created subfamilies within the locus. Polymorphisms among inbred mouse strains and feral species suggest that mutations responsible for the rsl phenotype arose during the creation of inbred strains. Comparisons among mouse, rat, and human sequences show that the Rsl genes, like members of certain other multigene families, have diversified in a species-specific manner. The targets of Rsl regulation also vary between species, occurring in gene families with functions in steroid and xenobiotic metabolism (Cyp2d), reproduction (MUPs), and immunity (Slp). This suggests that the Rsl locus in mouse, and comparable KRAB-ZFP genes in other mammals, may play a role in speciation via modulation of expression of genes influencing reproductive fitness or behavior.