Abstract
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.
Highlights
In rats, urine-borne male pheromones comprise organic volatile compounds and major urinary proteins (MUPs)
We further examined the activity of recombinant MUPs (rMUPs) by testing whether these proteins elicited behavioural and neuronal responses in female rats
MUP levels were significantly decreased by castration but restored by T treatment (P < 0.001, F = 24.204; T-treated vs. castrated, P < 0.001; T-treated vs. shamoperated control, P = 0.065; sham-operated control vs. castrated, P = 0.002; n = 7 for each group, oneway ANOVA with Tukey’s post hoc honestly significant difference (HSD) test) (Fig. 2b)
Summary
Urine-borne male pheromones comprise organic volatile compounds and major urinary proteins (MUPs). Pheromones play a crucial role in mediating socio-sexual interactions between conspecific members in rodents [1, 2]. Pheromone components comprise both volatile organic compounds and non-volatile proteins, such as exocrine gland-secreted peptide 1 (ESP1) secreted by tear glands [3] and darcin (MUP20), a member of the major urinary proteins (MUPs) [4]. A few organic volatile compounds have been chemically characterized from mouse and rat urine and preputial glands and identified to be male pheromone components [12, 14, 16, 17]. Since the genes of the MUP family share high sequence homology, the purification of MUPs from urine and expression of a single specific protein in vitro is difficult [26], and the role in intraspecific communication of each MUP isoform in male urine, is rarely experimentally verified in rats [25, 27]
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