Two predominant MUPs, OBP3 and MUP13, are male pheromones in rats

Abstract

Background: In 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.

Results: We 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.

Conclusions: The 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.

Keywords: Activation of neural pathways; Female attraction; MUPs; Male pheromones.

Fig. 1

Comparison of MUP levels between males and females. a Concentrations of total urine proteins in males and females were assayed using the Bradford protein assay (**P < 0.01, n = 7, independent-sample t-test). b MUP levels in the urine (2 μL of diluted urine samples) from male and female Lewis rats (n = 7 for each sex) were detected using SDS-PAGE with a marker of molecular weight (10.5 kDa-175 kDa, CW0986S, Beijing ComWin Biotech Co., Ltd., China). One male sample was loaded on every gel as a standard to normalize the intensities of samples on different gels. The gel shown is a representative sample of four females and four males. c MUP abundance was quantified in SDS-PAGE gels using the ImageJ program, and the data were shown as the mean ± standard error (SE), n = 7 (**P < 0.01, independent-sample t-test). d Differences in MUP levels of urine (1 μL of a 1:10 dilution) between males and females (n = 7 for each sex) were assessed using IEF with a marker of isoelectric point (pI) in 1D gel (pI 5-6, 42,967, SERVA, Germany). The gel shown is a representative sample of four females and four males. The bands 1-3 represented the three most abundant MUPs

Fig. 2

MUP levels in males were regulated by androgen. a SDS-PAGE showed the amount of MUPs in sham-operated, castrated and T-treated male rats (the molecular weight of marker: 10.5 kDa-175 kDa, CW0986S, Beijing ComWin Biotech Co., Ltd., China). The gel shown has 12 urine samples from three groups (2 μL of 2-fold dilution for each lane); remaining samples were on another gel which is not shown. One sham-operated male urine sample was run as a standard on every gel, and its intensity was used to normalize all values. b The relative abundance of MUPs on SDS-PAGE gels was analysed using the ImageJ program (mean ± SE, n = 7, **P < 0.01, one-way ANOVA followed by Tukey’s post hoc HSD test). c Each MUP in the urine samples (1 μL of a 1 in 10 dilution) from different groups was detected using IEF with a marker of pI values in 1D gel. (pI 5-6, 42,967, SERVA, Germany. S: sham-operated, C: castrated, C + T: castrated + T-treated). The bands 1-3 were the three most abundant proteins in MUPs

Fig. 3

MUP sequences were identified by nLC-MS/MS in male urine; (a), (b) and (c) represent the sequence coverage of the three identified proteins: MUP13, Alpha-2u globulin PGCL2 and OBP3, respectively. d Sequence alignment of the three proteins using DANMAN software. Light blue shading shows several amino acids that differ among the three proteins. P02761, Q8K1Q6 and Q78E14 respectively represented the accession number of MUP13, Alpha-2u globulin PGCL2 and OBP3 in UniProt database. e Similarity analysis of the three proteins using DANMAN

Fig. 4

Comparison of the hepatic expression of the Mup13 and Obp3 mRNAs between the two sexes and among the three groups in the castration experiments. a Differences in Mup13 and Obp3 expression in female and male rats (mean ± SE, n = 5 for each sex, **P < 0.01, independent-sample t-test). b Expression patterns of Mup13 and Obp3 among groups in the castration experiment (mean ± SE, n = 5 for each group, *P < 0.05, ** P < 0.01, one-Way ANOVA followed by Tukey’s post hoc HSD test) (S: sham-operated, C: castrated, C + T: castrated + T-treated)

Fig. 5

Recombinant proteins were assessed by nLC-MS/MS and SDS-PAGE. a Molecular weights of purified rMUP13 and rOBP3 were determined using SDS-PAGE (the molecular weight of marker: 12 kDa-120 kDa, DR201-01, Beijing TransGen Biotech Co., Ltd., China). b and c Sequence coverage of rOBP3 and rMUP13, respectively

Fig. 6

Behavioural tests were analysed by determining the time females spent investigating the samples. a Comparisons of the time females spent investigating samples from sham-operated and castrated males, castrated and castrated + rOBP3-treated or castrated + rMUP13-treated males, sham-operated and castrated + rOBP3-treated or castrated + rMUP13-treated males and castrated + rMUP13-treated males and castrated + rOBP3-treated males (n = 12 for each group, mean ± SE, **P < 0.01, *P < 0.05, Paired t-test or Wilcoxon signed-rank test)

Fig. 7

Male urine treated with rOBP3 induced c-Fos expression in females. The c-Fos-ir cells were distributed in the AOB (a) and higher brain areas (d). (b), (c) and (e) showed the statistical analyses of the number of c-Fos-ir cells in different brain regions (n = 6, mean ± SE, **P < 0.01, *P < 0.05, independent-sample t-test for five brain areas, AOB, aAOB and pAOB between the PBS-treated control and experimental groups; paired t-test or Wilcoxon signed-rank test for aAOB and pAOB in PBS-treated control or experimental rats. Scale bars = 25 μm)

Fig. 8

Increased c-Fos expression in females after sniffing rMUP13. Expression of c-Fos in the AOB (a) and five different higher brain centres (d); relative statistical analyses of the data presented in (b), (c) and (e) (n = 6, mean ± SE, **P < 0.01, *P < 0.05, independent-sample t-test for brain areas, aAOB, pAOB and AOB in PBS-treated control and treated groups, paired t-test or Wilcoxon signed-rank test for aAOB and pAOB from the PBS-treated control and treated groups. Scale bars = 25 μm)