Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.     

Enzyme immunoassay of 17 beta-estradiol, estrone conjugates, and testosterone in urinary and fecal samples from male and female mice.

ELISA measures of 17 beta-estradiol, estrone conjugates, and testosterone were adapted for fecal and urinary samples from laboratory mice. We will report on validations of these assays and data from interacting males and females. Unconjugated gonadal steroids were consistently measurable in urine and feces of both males and females. Females that were parturient following insemination excreted relatively low levels of urinary testosterone compared to non-parturient females. The results are consistent with evidence that elevated androgens and estrogens are incompatible with intrauterine implantation of fertilized ova, and suggest that steroids in male urine could contribute to pheromonal action. These methods permit repeated noninvasive measurement of steroid activity in this species.     

From sexual attraction to maternal aggression: When pheromones change their behavioural significance

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.     

Pregnancy in female house mice exposed to urinary chemosignals from other females.

Seven experiments were performed to investigate pregnancy termination, urinary chemosignals, and litter sex ratio variation in female house mice. Experiments tested the effects of urine from adult and prepubertal females, housed individually or in groups, on successful insemination and litter production by females treated at different times and for different periods during the 3 weeks before mating and during gestation. Treatment of females with urine from adult females housed eight per cage or with urine pooled from eight adult females housed individually for 2 or 3 weeks before mating resulted in fewer successful pregnancies and significantly more female-biased litters. Treatment with urine from adult or prepubertal females housed eight per cage or with urine pooled from eight mice housed individually for the first 6 days of gestation or throughout pregnancy resulted in a significant increase in the rate of pregnancy termination. These treatments resulted in lower body weights at birth and slower growth rates in all males and in some females. Puberty was delayed in female progeny from urine-treated dams in five of seven experiments, and these young females attained first oestrus at greater mean body weights than mice in other treatments. These findings indicate that, in mice, at high population density, communication via a urinary chemosignal can alter reproduction in recipient females. Availability of, and competition for, resources such as food would be greater at higher densities, possibly lowering the probability of reproductive success. Pregnancy termination and delays in reproduction and attainment of sexual maturity might lead to greater successful reproduction at a later time.     

Pheromone signalling in the mouse: role of urinary proteins and vomeronasal organ.

The lipocalin protein family is characterized in structure by a conserved hydrophobic pocket which can bind small volatile odorants. The Major Urinary Proteins (MUPs) are a class of lipocalins found in the urine of adult male mice which concentrate in the urine odorants which confer a characteristic odor. The behavioural as well as the endocrine effects of mouse urine and MUPs are briefly reviewed, suggesting a complex role is pheromonal communication. Some recent data on the molecular receptors of the vomeronasal organ further suggest a complex interaction with the MUP system.     

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP‐IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP‐IV is highly specific for the male mouse pheromone 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X‐ray crystal structure of MUP‐IV bound to three mouse pheromones: SBT, 2,5‐dimethylpyrazine, and 2‐heptanone. We also obtained the structure of MUP‐IV with 2‐ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water‐mediated hydrogen bond network in the MUP‐I and MUP‐II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP‐IV.     

Wake up and smell the conflict: odour signals in female competition

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.     

Exposure to developing females induces polyuria, polydipsia, and altered urinary levels of creatinine, 17β-estradiol, and testosterone in adult male mice (Mus musculus)

Novel male mice can accelerate reproductive maturation in proximal developing females, an effect mediated by the chemistry of the males' urine. Exogenous estrogens can similarly accelerate female sexual development. In Experiment 1, adult male mice were housed across wire grid from either empty compartments or those containing post-weanling females. Proximity of females caused males to urinate more, progressively over days of exposure, with most urination directed towards females' compartments. Male urine collected after 5 days in these conditions was analyzed by enzyme immunoassay for 17β-estradiol, testosterone, and creatinine. Urinary creatinine of isolated males significantly exceeded that of female-exposed males. Unadjusted urinary steroids also trended toward higher levels in isolates, but creatinine-adjusted estradiol and testosterone of female-exposed males significantly exceeded that of isolated males. In Experiment 2, measurement of water consumption indicated significantly greater drinking by female-exposed as opposed to isolated males. In Experiment 3, males were housed in isolation or beside post-weanling intact (sham-operated) females, ovariectomized females, or intact (sham-operated) males. Male water consumption was elevated in all conditions involving social contact. Urinary creatinine was significantly lower in female-exposed males compared to isolated controls, while unadjusted testosterone was significantly lower in males in all social conditions. Again, creatinine-adjusted estradiol in female-exposed males significantly exceeded that of isolates. These data indicate that adult males drink and urinate more, have more dilute urine, and have a higher ratio of estradiol to creatinine when they are near developing females. These dynamics increase females' exposure to urinary steroids and other urinary constituents that can hasten sexual maturity.     

Male pheromones and their reception by females are co-adapted to affect mating success in two subspecies of brown rats

Pheromonal communication plays a key role in the sociosexual behavior of rodents. The coadaptation between pheromones and chemosensory systems has been well illustrated in insects but poorly investigated in rodents and other mammals. We aimed to investigate whether coadaptation between male pheromones and female reception might have occurred in brown rats Rattus norvegicus. We recently reported that major urinary protein (MUP) pheromones are associated with male mating success in a brown rat subspecies, R. n. humiliatus (Rnh). Here, we discovered that MUPs were less polymorphic and occurred at much lower concentrations in males of a parapatric subspecies, R. n. caraco (Rnc), than in Rnh males, and found no association between pheromones and paternity success. Moreover, the observation of Rnc males that experienced chronic dyadic encounters and established dominance–submission relationships revealed that the dominant males achieved greater mating success than the subordinate males, but their MUP levels did not differ by social status. These findings suggest that male mating success in Rnc rats is related to social rank rather than to pheromone levels and that low concentration of MUPs might not be a reliable signal for mate choice in Rnc rats, which is different from the findings obtained in Rnh rats. In addition, compared with Rnh females, Rnc females exhibited reduced expression of pheromone receptor genes, and a lower number of vomeronasal receptor neurons were activated by MUP pheromones, which imply that the female chemosensory reception of pheromones might be structurally and functionally coadapted with male pheromone signals in brown rats.     

Cues that Elicit Ultrasounds from Adult Male Mice

Adult male mice (Mus musculus) which have a prior history ofexperience with other adult male and adult female mice readilyproduce 70 kHz ultrasonic vocalizations in the presence of urinefrom adult females but not in the presence of urine from adultmales. Urine from immatures of either sex does not elicit ultrasoundsfrom socially experienced adult males. The ultrasound elicitingpotency of adult male urine was not improved substantially followingcastration of adult males, injection of testosterone propionateto castrated adult males, administration of estradiol benzoateto castrated adult males, or neonatal castration. Ovarian hormonesdo not appear to be necessary for either the appearance at puberty,or the maintenance during adulthood, of the ultrasound elicitingcues of female urine. Stage of estrus did not have a major modulatingeffect on urinary cues eliciling male ultrasounds. Treatmentsthat did not substantially reduce the signal value of adultfemale urine include ovariectomy before or after puberty, ovariectomywith adrenalectomy, and neonatal administration of testosterone.The administration of testosterone to ovariectomized adult females,and hypophyseclomy, virtually eliminated the ability of urinefrom adult females to elicit ultrasounds from socially experiencedadult males. The implication of pituitary hormones in the modulationof female urinary cues thai elicit ultrasounds is particularlyinteresting since pituitary factors are also implicated in theproximal causation of postparturient maternal aggression, whichadult male ultrasounds may function to moderate.     

Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice

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 reproductive ecology of the house mouse.

This paper attempts to integrate the physiological and ecological perspectives of the reproductive biology of the house mouse (Mus musculus). The endeavor is made within a larger context to provide a prototype for mammalian reproductive ecology in general. Specifically, the environmental regulation of the reproduction of Mus musculus is examined in relation to its ecological opportunism and, in particular, in relation to its history of global colonization. House mice can live as commensals of man or under totally feral conditions. Stable, high density, commensal populations are characterized by an insular division of the living space into demeterritories, each dominated by a single male. Feral populations typically are characterized by temporal, spatial, and social instability. Territoriality is improbable under such conditions, particularly given the necessity for large home ranges in most feral habitats. In both feral and commensal populations, however, male aggressiveness promotes the large-scale dispersal of young, all of which are potential colonizers. Of the ten or so environmental factors known to influence reproduction in house mice, seven probably are of routine importance in natural populations: diurnal modulation by daily light:dark cycles; caloric intake; nutrition; extreme temperature; agaonistic stimuli; socio-tactile cues; and priming pheronomes. The last two factors named operate directly on the secretion of luteinizing hormone or prolactin; the others act at many points in the reproductive system. Reproduction in the house mouse seems divorced from photoperiodically induced seasonality; indeed, this species breeds well even in constant darkness. Seasonal breeding may or may not then occur, depending upon dietary considerations, with or without a secondary interaction with variation in ambient temperature. There is no evidence for a dependence upon secondary plant compounds. Some of the effects of priming pheromones that have been observed previously in laboratory mice probably play no meaningful role in wild populations. The remaining pheromonal phenomena can be conceptualized as a single cueing system that has three components: (a) urinary cues of socially dominant males can accelerate ovulation in females, adult or prepubertal; (b) female urinary cues may elevate pheromonal potency in adult males, thereby forming a feedback loop by which the females elicit their own ovulation; and (c) the male's action on prepubertal females can be blocked by urinary cues emanating from other females. When all of the above is viewed in toto, the reproductive biology of the house mouse seems uniquely suited to support ecological opportunism. The relatively few environmental inhibitors of reproduction in this species should enhance the ability of dispersing young to colonize an exceptionally wide variety of habitats and climates...     

Biosynthesis of the major urinary proteins in mouse liver: A biochemical genetic study

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 relative levels of the MUPs in urine reflects a similar change in the pattern of MUP synthesis, indicating that the posttranslational processes operate on the quantity, and not the 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.     

Variation in the Major Urinary Protein Multigene Family in Wild-Derived Mice

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.     

Intermittent stimulation and acceleration of puberty by urinary chemosignals in female mice (Mus musculus domesticus)

A sequence of 17 experiments was used to test the effects of intermittent stimulation with urinary chemosignals on the age of puberty in young female mice. The three chemosignals tested all accelerate the age of sexual maturation: urine from adult males, urine from females in estrus, and urine from females that are pregnant or lactating. The basic technique involved presenting the prepubertal females with 'Nestlets' on which the urine was placed. The 'Nestlets' were placed in the cages of the test females for a 15-min period, removed for a variable period, and then replaced in the cage for 15 min. In this manner it was possible to vary the number of exposures, the total length of exposure, and the total time period over which the exposures occurred. Control procedures, involving exposures of young females to cotton squares with water rather than urine placed upon them, resulted in no alterations in puberty relative to untreated females. For mice exposed to the urine-treated cotton squares, acceleration of puberty occurred with less total stimulus-exposure time when the stimulus was presented in short exposures over a number of hours than in previous investigations when the exposure to the urinary chemosignal occurred in a single block of time of one or two hours. For each of the three acceleratory chemosignals, there was a diminution of acceleratory effect when the ratio of total stimulus-exposure time to total exposure time grew smaller. This diminution was more pronounced for urine from pregnant or lactating females than for urine from males or from females in estrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Why do capuchin monkeys urine wash? An experimental test of the sexual communication hypothesis using fMRI

Urine washing (UW) consists of depositing urine on the hands and vigorously rubbing the body. As urine contains chemical and pheromonal cues, UW may convey socially relevant information. Although ritualized UW is observed in many New World primates, including capuchin monkeys, the functional significance of UW remains unclear. In this experiment, we investigated the social signaling hypothesis of UW. Specifically, we hypothesized that UW by males conveys socially relevant signals that females can detect. We used functional magnetic resonance imaging (fMRI) to test whether adult female capuchins show differential brain activation in response to adult male and juvenile male capuchin urine. We expected to see changes in activation of structures involved in olfactory processing, including the piriform cortex, medial preoptic and anterior hypothesis, orbitofrontal cortex, hippocampus, and cerebellum. Data were acquired from four adult female capuchin monkeys. Presentations of odor stimuli (obtained from unfamiliar males) were made during fMRI acquisition using a standard ON-OFF design. All fMRI data were spatially normalized to a template and analyzed using the FMRI Expert Analysis Tool Version 5.98, part of the FMRIB's Software Library (www.fmrib.ox.ac.uk/fsl). Whole brain analyses revealed significant activations in the inferior temporal cortex, parahippocampal gyrus, precuneus, hippocampus, pulvinar, and cerebellum when females were presented with the adult male urine. Notably, significantly greater signal activation was observed in several regions associated with olfactory processing, when subjects were presented with adult male urine as compared with urine from juvenile males. Our results indicate that UW serves a social communicative function in capuchins, providing support for the sexual signaling hypothesis.     

Olfactory stress and modification of phagocytosis in peripheral blood cells of adult male mice]

Data on pheromonal influence on phagocytic activity of leukocytes in peripheral blood of adult randombred and CBA male mice have been obtained. The identified mouse pheromone 2,5-dimethylpyrazine was used, which induces some physiological effects associated with reproduction in both mouse males and females. Significant differences in spontaneous level of phagocytosis were between inbred CBA and randombred animals: the frequency of phagocytic cells was lower in CBA males by 1.4 times. The substance tested here induces phagocytosis in randombred (by 1.7 times) males. A low dose of 2,5-dimethylpyrazine (similar to the natural pheromone concentration) induces a higher increase in phagocytic activity by leukocytes. Possible mechanisms of pheromonal action on phagocytosis are discussed with the perspectives of finding highly effective immunomodulators among mammalian pheromones.     

Chronic exposure of cat odor enhances aggression,urinary attractiveness and sex pheromones of mice

To test whether predator odor exposure negatively affects the behavior of prey, we exposed three groups of male house mice (Mus musculus) to the odors of cat (Felis catus) urine, rabbit (Oryctolagus cuniculus) urine and water (control), respectively, for consecutive 58 days and investigated how the treatments affected the response, aggressiveness, dominance, urinary attractiveness to females and pheromone composition of male mice. Compared to mice exposed to rabbit urine or water, those exposed to cat odor did not show any response habituation to the cat odor and became more aggressive, increased mark urine production and were more attractive to females when the latter were tested with their urine. Furthermore, gas chromatography coupled with mass spectrometry analysis revealed coincident elevations of the well-known male pheromones, E,E-α-farnesene, E-β-farnesene, R,R-dehydro-exo-brevicomin or S-2-sec-butyl-dihydrothiazole. In addition, rabbit urine exposure increased urinary attractiveness to females and pheromonal levels of the males in comparison with the mice exposed to water. This could be related to olfactory enrichment of heterospecific chemosignals, suggesting that predator odors were more beneficial. In light of these anti-intuitional findings in the chemical interaction between cats and mice, we conclude that predator odor affects prey more profoundly than previously believed and that its impact may not always be negative.     

Androgen-dependent proteins in the urine of bank voles (Clethrionomys glareolus)

The concentration of proteins in the urine of adult bank vole males was higher than that in urine of immature males and females. After separation of the urine by polyacrylamide gel electrophoresis in the presence of SDS, the principal urinary protein had a slightly lower mobility than cytochrome C. Urine from females or castrated males contained only trace amounts of this protein. Injection of testosterone into castrated males increased this protein band. We suggest that bank vole males, like those of rats and mice, synthesize and release in their urine an androgen-dependent protein fraction.