Associative learning is necessary for airborne pheromones to activate sexual arousal-linked brain areas of female rats

  • Yao-Hua ZhangEmail author
  • Ming-Ming Tang
  • Xiao Guo
  • Xiao-Rong Gao
  • Jin-Hua Zhang
  • Jian-Xu ZhangEmail author
Original Article


In rodents, urine-borne male pheromones include volatile organic compounds (VOCs) and major urinary proteins (MUPs). In mice, the attraction of females to male odor is reportedly acquired through associative learning with MUPs in some studies. Here, we found that VOC and MUP sex pheromones were differentiated in rats at around 8 weeks of age and that females separated from males at weaning showed no preference for male urine odor after sexual maturity. Olfactory preferences could be gained in females after repeated experience of VOC pheromones alone as well as male urine or a blend of synthetic VOC pheromones and recombinant MUPs. However, differences in acquired olfactory preferences for male urine were further revealed by neuro-immuno-histochemical studies. The blend exhibited neural activation in the main olfactory system (MOS), accessory system (AOS), and the ventromedial hypothalamus (VMH), indicating sexual arousal, whereas the VOC alone only caused neural activation of MOS. We suggest that olfactory preference is generated through repeated experience of either VOCs or a blend of VOCs and MUPs, but the neural activations related to sexual arousal have to be acquired through associative learning with MUPs in female rats.

Significance statement

When adult female rats were separated from males before maturity, they lost their attraction to male urine odor. Female preference to volatiles in male urine could be gained by repeated experience of volatile and protein pheromones. Brain regions related to sexual arousal were activated by male urine in females with experience of VOCs together with MUPs but not in those experienced with VOCs alone. Associative learning between VOC and MUP pheromones is necessary for male urine odor-induced FOS responses in the key regions for sexual arousal/excitement in female rats.


Development of sex pheromones Social learning Olfactory memory Sexual excitement 



We thank BeiJing Zixi Biotechnology Co., Ltd. for expression and purification of the recombinant MUPs. We thank the editors and the two anonymous reviewers for their constructive comments and suggestions which helped to improve this manuscript.

Author contribution

JXZ and YHZ conceived and designed the experiments. YHZ and JHZ performed the behavioral experiments and GC-MS analysis. MMT performed c-Fos immunohistochemistry. XG and XRG performed the SDS-PAGE analysis and prepared the MUPs. YHZ, MMT, and JXZ wrote the paper. All authors read and approved the final manuscript.


This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB11010400), the National Natural Science Foundation of China (Nos. 31572277 and 31672306), and the Foundation of State Key Laboratory of IPM (ChineseIPM1701).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution (Institutional Guidelines for Animal Use and Care at the Institute of Zoology, the Chinese Academy of Sciences, IOZ 2015) or practice at which the studies were conducted.

Data availability statements

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Supplementary material

265_2019_2685_MOESM1_ESM.docx (5.7 mb)
Fig. S1 c-Fos expression in the MOB and AOB after exposure to male urinary volatiles (DOCX 5821 kb)
265_2019_2685_MOESM2_ESM.docx (8.8 mb)
Fig. S2 c-Fos expression in higher brain regions after exposure to male urinary volatiles (DOCX 9034 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture, Institute of ZoologyChinese Academy of SciencesBeijingChina

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