Sign up to take part. A Nature Research Journal. We investigated the regulation of chemical signals of house mice living in seminatural social conditions. We found that male mice more than doubled the excretion of major urinary proteins MUPs after they acquired a territory and become socially dominant.
MUPs bind and stabilize the release of volatile pheromone ligands, and some MUPs exhibit pheromonal properties themselves. We conducted olfactory assays and found that female mice were more attracted to the scent of dominant than subordinate males when they were in estrus.
Regulation of volatile and non-volatile pheromone attractants depends upon male social status
Yet, when male status was controlled, females were not attracted to urine with high MUP concentration, despite being comparable to levels of dominant males. To determine which compounds influence female attraction, we conducted additional analyses and found that dominant males differentially upregulated the excretion of particular MUPs, including the pheromone MUP20 darcin , and a volatile pheromone that influences female reproductive physiology and behavior.
Our findings show that once male house mice become territorial and socially dominant, they upregulate the amount and types of excreted MUPs, which increases the intensities of volatiles and the attractiveness of their urinary scent to sexually receptive females. House mice Mus musculu s excrete large quantities of major urinary proteins MUPs that bind and transport hydrophobic ligands, including several volatile pheromones 1 , 2 , 3.
Upon excretion, MUPs slow down the release of volatiles from scent marks 4 , 5 , 6 , 7 , 8 , which may prolong their attraction and influence on conspecifics. MUPs are often suggested to show high individual diversity and thereby mediate individual and kin recognition 9 , They are encoded by 21 paralogous loci, but MUP genes are highly homologous 11 , 12 and no individual variation has been detected in wild populations Interestingly, MUP excretion is sexually dimorphic 5 , 14 , 15 , under endocrine control 16 , 17 , 18 , and dynamically regulated 19 depending upon health 20 , 21 , 22 , nutritional status 23 , and social interactions 24 , Our aims here were to test whether house mice regulate the excretion of MUPs or volatile pheromone ligands depending upon their social status, and whether such regulation influences the attractiveness of their odor to potential mates Male house mice are territorial and much evidence indicates that intra- and inter-sexual selection are mediated by chemosensory signals.
Male reproductive success correlates with scent-marking when male social status is controlled and females are free to select their mates These findings might help explain how females discriminate winners from losers 32 , 34 , 35 and how dominant, territorial males achieve greater reproductive success than subordinates 47 , 48 , These studies have provided valuable insights into chemosensory-mediated sexual selection, however, several questions still need to be addressed. First, studies on chemical communication in mice are primarily conducted with laboratory strains Mus laboratorius 50 , but their ecological relevance should not be assumed 51 , 52 especially since male laboratory mice are not territorial and most strains are not as aggressive as wild mice 53 , These assays are also used to study social defeat 55 , 56 , 57 , which results in behavioral 58 and physiological changes 55 , 57 , including detrimental health effects Indeed, a recent study on laboratory mice indicates that winning dyadic agonistic assays did not predict social dominance in more complex social environments Third, it is unclear whether MUP excretion is regulated according to social status, or vice versa, as previously suggested 44 and it is also unclear whether winners upregulate or losers down-regulate MUPs and volatile pheromones.
Longitudinal studies are needed to examine pheromone excretion in social interactions and with controls for comparison. Finally, the regulation of MUPs and volatiles have mainly been investigated independently, with few exceptions 61 and studies are needed to examine how MUP regulation influences odor, as well as the excretion of volatiles. We conducted a study on social behavior and regulation of MUPs and other pheromones in wild-derived F1 from wild-caught house mice Mus musculus musculus to address the following aims: 1 We tested whether MUP production provides a reliable indicator of social status in seminatural conditions over 12 weeks, and we also analyzed protein excretion from controls kept in standard housing conditions during the same time.
We expected dominant males to upregulate, or subordinates to down-regulate social defeat protein excretion, or both. To determine whether regulation is temporary or long-lasting, we continued measuring MUP excretion after social interactions were terminated. We expected females to have lower levels of protein excretion than males, and to regulate protein excretion depending upon their social status, as previously suggested We expected that females would discriminate and prefer the urine of dominant over that of subordinate males, and that they would prefer the scent of males with high MUP concentration when social status is controlled as observed in rats Finally, we expected that mice would show differential regulation of particular MUP proteoforms 43 , volatile pheromones 39 , or both, depending upon their social status.
Males that became socially dominant DOM in the seminatural enclosures upregulated urinary protein excretion, as expected, whereas subordinates SUB and controls CTRL showed no significant changes during this time Fig. Subordinate males did not down-regulate urinary protein excretion and had levels similar to caged control males SUB: 1.
Dominant males upregulate urinary protein excretion in seminatural conditions. Male urinary protein excretion PC ratio before, during, and after housing in seminatural enclosures for socially dominant solid line and subordinate dot-dashed line males compared to caged control males dashed line. Mice were repeatedly sampled before two urine collections and during enclosure phase three urine collections. Unlike males, female mice did not regulate their urinary protein excretion according to social status Fig.
Female urinary protein excretion increases over time.
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Female urinary protein excretion PC ratio before, during, and after housing in seminatural enclosures for socially dominant solid line and subordinate dot-dashed line females compared to caged control females dashed line. Mice were repeatedly sampled before two collections and during three urine collections the enclosure phases. To determine how females discriminate dominant from subordinate males, we conducted analyses to examine how social status influenced the excretion of particular MUP proteoforms and volatile pheromones, using high-resolution proteomics and GC-MS analyses, respectfully.
On the contrary, fold changes in protein amount and relative intensity of MUPs in subordinate males did not differ from the null expectation. Volatile intensity is correlated with total urinary protein concentration.
Correlation of total ion chromatogram intensity measured by GC-MS and total urinary protein concentration in A intact and B denatured urine samples of females circles and males triangles. Panels A,B have different y-axes. Our findings show that male house mice upregulated urinary protein excretion once they acquired a territory and became socially dominant. Females showed more attraction to the scent of dominant over subordinate males, though only during estrus Contrary to our expectations, male urinary protein concentration had no influence on female preferences when male social status was controlled.
This finding suggests that female attraction to dominant males was due to changes in the urine other than the amount of urinary protein. MUP excretion was correlated with an overall intensity of the VOCs in urine, and MUP upregulation may therefore increase and prolong the volatile signals in male scent marks. These findings show that territorial male mice upregulate the excretion of both volatile and non-volatile urinary chemical compounds that attract and influence the reproductive behavior of female mice 42 , 64 , 65 , Below we address our main results in more detail.
We found that males upregulated MUP excretion after acquiring a territory, which is consistent with previous studies 24 , 43 , 44 , 45 and our results provide several additional findings. First, the differences in MUP excretion between dominant and subordinate males emerged after and not before social status was established.
Interestingly, this finding is consistent with growing evidence that social behavior can affect gene expression 67 , which contrasts with the usual assumption that gene expression can influence behavior, but not vice versa. Second, subordinate males did not significantly decrease urinary protein excretion, contrary to what we expected from laboratory studies on social defeat.
Third, body mass had no influence on male urinary protein excretion, indicating that MUP excretion is not an index of size.
Neither body mass nor age correlated with social status, unlike some rodent studies 48 , 68 , though such correlations may be a consequence of social rank influencing access to food, rather than vice versa 69 , Our results align with other studies that find no relationship between body mass and social status or urinary protein excretion 44 , 45 , though the correlation between size and social rank may occur in Mus musculus domesticus , and not Mus musculus musculus Fourth, MUP upregulation by territorial males was relatively stable over the 12 weeks of living in seminatural conditions Supplementary Fig.
S12 , and it was reversible, as it returned to levels similar to caged-controls after removing social interactions.
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Females did not regulate protein excretion according to social status, contrary to a previous suggestion 62 , though our findings confirm that protein excretion is sexually dimorphic in seminatural as well as laboratory conditions 5 , 14 , Sex differences in MUP excretion were less pronounced in the enclosure mice than controls in standard conditions during the enclosure phase male:female was in enclosures versus ca. This result is explained by female enclosure mice showing an increase in total urinary protein excretion under seminatural conditions. A previous study observed an increase in protein excretion PC ratio of females housed in mixed-sex groups and subjected to brief social interactions with unfamiliar individuals Thus, our results confirm sex differences in urinary MUP excretion, though they were not as large in laboratory conditions here or in previous studies in Mus m.
Future studies on chemosensory sex recognition 73 should therefore consider the possibility that sex differences might be inflated in the laboratory. The mechanisms controlling the regulation of MUP excretion and their adaptive functions are unclear. Urinary MUPs are transcribed in the liver and expression is under complex endocrine control, involving several hormones and pulsatile secretion 16 , 17 , Therefore, behaviors associated with social dominance, such as winning fights and scent-marking, may upregulate MUP expression by triggering pulsatile hormonal secretions.
Unfortunately, previous studies have focused on physiological and behavioral changes following social defeat rather than winning 57 , 75 , whereas our findings cannot be explained by subordinates losing fights 26 , having poorer health, nutrition, or condition 21 , 22 , These hypotheses are not mutually exclusive, and we investigated the first hypothesis.
Our olfactory experiments showed that estrous female mice were more attracted to the scent of dominant than subordinate males, which is consistent with previous studies using dyadic agonistic assays as proxies for social status 33 , Female olfactory preferences were expressed only during estrus, suggesting a mating function. We presented females with samples of male urine collected from the same individual that differed in protein concentration — at levels comparable to the differences between dominant and subordinate males — and surprisingly, females showed no preference for high protein concentration.
This result means that differences in the amount of urinary protein concentration per se are not sufficient to explain female olfactory preferences for dominant males even though females can discriminate larger differences in MUP quantities, such as for sex recognition Therefore, we conducted proteomic and GC-MS analyses to test for differential regulation of particular MUP proteoforms and changes in known pheromones.
We applied high-resolution mass spectrometry 25 to determine whether urinary protein regulation was consistent for all MUP proteoforms, or whether only specific MUPs were regulated depending on social status. However, differences between dominant and subordinate males became evident when we compared within-individual fold changes of MUP proteoform production.
These results indicate that dominant male house mice show differential upregulation of specific MUP proteoforms. Upregulating particular MUP proteoforms may function as a mechanism for dominant males to elicit specific information or responses from conspecifics. Adding recombinant MUP3 to male urine, for example, promotes aggression from male conspecifics Furthermore, MUP20 is predominately expressed in male urine though it is also found in utero-vaginal secretions 84 , it is a female attractant 66 , and it mediates spatial learning 85 by stimulating neurogenesis MUP20 also stimulates male-male aggression 83 and maternal aggression from lactating females Our findings support previous studies of Mus musculus domesticus showing increased MUP20 production in relation to social status after a three-day 44 and a day enclosure experience 45 in highly male-biased populations.
Taken together, these studies suggest that upregulation of specific MUP proteoforms provides a reliable indicator of dominant male social status, which may deter challenges from subordinate rivals, as well as attract potential mates. MUPs control the binding of volatile male pheromones, but there are surprisingly few studies on the regulation of MUPs on volatiles — and none in natural or seminatural conditions. Our previous analyses verified the presence of male pheromones in our enclosure mice 80 , and we expected that elevating MUP excretion would increase the intensities of volatile pheromones and other ligands in urine.
Indeed, we found a strong, positive correlation between urinary protein concentration and intensity of VOCs, and denaturing the proteins increased the overall ion intensities of all VOCs detected and analyzed peak heights in the TIC, Fig. This result supports the hypothesis that increasing MUP concentration increases the amount of pheromones and other VOCs in the urine.
It suggests that by increasing MUP excretion, dominant males should be able to prolong the attachment of their pheromones to substrate and subsequently extend the release of volatiles over time. We expected that we might detect differences in the amount of unbound VOCs, and particularly, pheromones previously found to correlate with male rank 39 , 44 , However, male social status did not affect urinary VOC profiles, except for hydroxymethylheptanone HMH , which showed the highest relative intensity in dominant males.
HMH is an androgen-dependent volatile compound previously found to accelerate female puberty in some laboratory strains 42 , 64 , Thus, elevated HMH levels in dominant males might function to attract adult females and accelerate sexual maturity in peripubertal females. The intensity of SBT was higher in caged controls than enclosure males, supporting results that compared isolated versus group-living mice in laboratory conditions These findings raise additional caveats about studying chemosignals in standard laboratory conditions.
We found no evidence for female-specific VOCs denaturation of female urine released mainly flavor and fragrance compounds which were potentially derived from food , and VOC profiles of dominant and subordinate females were almost indistinguishable. It is surprising that we did not detect more compounds that differ between dominant and subordinate males, but MUPs show extremely strong binding affinity for male pheromones, and so some ligands remain bound, even after extensive purification Thus, it is possible that GC-MS will fail to detect actual differences in the amounts of these bound ligands.
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