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

BackgroundIn rats, urine-borne male pheromones comprise organic volatile compounds and major urinary proteins (MUPs). A number of volatile pheromones have been reported, but no MUP pheromones have been identified in rat urine.ResultsWe used sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) after in gel digestion of the proteins and quantitative real-time PCR (qRT-PCR) and showed that the levels of two MUPs, odorant-binding protein 3 (OBP3) (i.e. PGCL4) and MUP13 (i.e. PGCL1), in urine and their mRNAs in liver were higher in males than in females and were suppressed by orchidectomy and restored by testosterone treatment (T treatment). We then generated recombinant MUPs (rMUPs) and found that the sexual attractiveness of urine from castrated males to females significantly increased after the addition of either recombinant OBP3 (rOBP3) or recombinant MUP13 (rMUP13). Using c-Fos immunohistochemistry, we further examined neuronal activation in the brains of female rats after they sniffed rOBP3 or rMUP13. Both rOBP3 and rMUP13 activated the accessory olfactory bulb (AOB), medial preoptic area (MPA), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), posteromedial cortical amygdala (PMCo) and ventromedial nucleus of the hypothalamus (VMH), which participate in the neural circuits responsible for pheromone-induced sexual behaviours. In particular, more c-Fos-immunopositive (c-Fos-ir) cells were observed in the posterior AOB than in the anterior AOB.ConclusionsThe expression of OBP3 and MUP13 was male-biased and androgen-dependent. They attracted females and activated brain areas related to sexual behaviours in female rats, suggesting that both OBP3 and MUP13 are male pheromones in rats. Particularly, an OBP excreted into urine was exemplified to be a chemical signal.

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.