Skip to main content
. 2020 Nov 24;9:e54501. doi: 10.7554/eLife.54501

Figure 4. Sexually dimorphic ORs are activated by mature male mouse semiochemicals SBT and MTMT.

(A) Schematic of the pS6-IP-Seq experiment. Litter matched, ~3 week-old (juvenile) mice are used. Mice are habituated to an odor-free environment for 1 hr. One mouse then receives exposure to an odor stimulus, while another receives exposure to the diluent, each for 1 hr. Whole olfactory mucosa is then harvested and immunoprecipitated using an antibody against pS6. (B) The panel of sex-specific and sex-enriched volatiles screened using pS6-IP-Seq. (C) Volcano plot showing the results of pS6-IP-Seq using 0.01% (v/v) SBT diluted in water as stimulus. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (D) Volcano plot showing the results of pS6-IP-Seq using 100 μM MTMT dissolved in ethanol as stimulus. (E) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr910 and pS6 signal induction following exposure of a juvenile mouse to 1% (v/v) SBT diluted in water. Scale bars indicate 20 μm. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr910 following exposure of a juvenile mouse to increasing concentrations of SBT and 1% (v/v) acetophenone. One-way ANOVA with Dunnett’s multiple comparisons test correction reveals only exposure to 0.01% (v/v) SBT, 0.1% (v/v) SBT, and 1% (v/v) SBT leads to significant pS6 induction within OSNs expressing Olfr910 (****p < 0.0001). Data are from n = 3 juvenile mice. (F) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr912 and pS6 signal induction following exposure of a juvenile mouse to 1% (v/v) SBT diluted in water. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr912 following exposure of a juvenile mouse to increasing concentrations of SBT and 1% (v/v) acetophenone (**p < 0.01, ****p < 0.0001). (G) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr1295 and pS6 signal induction following exposure of a juvenile mouse to 10 mM MTMT diluted in ethanol. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr1295 following exposure of a juvenile mouse to increasing concentrations of MTMT and 1% (v/v) acetophenone (****p < 0.0001).

Figure 4—source data 1. Summary statistics for Figure 4.

Figure 4.

Figure 4—figure supplement 1. Sexually dimorphic ORs are not activated by sex-specific or sex-enriched odorants that are not SBT or MTMT.

Figure 4—figure supplement 1.

(A) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) β-caryophyllene dissolved in water as stimulus. Olfr910, Olfr912, and Olfr1295 are highlighted in red and not enriched. The red dashed line indicates an FDR = 0.05. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (B) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) 2-heptanone dissolved in water as stimulus. (C) Volcano plot showing the results of pS6-IP-Seq using 100% (E)-β-farnesene dissolved in water as stimulus. (D) Volcano plot showing the results of pS6-IP-Seq using 77% (v/v) DHB dissolved in water as stimulus. (E) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) 2,5-DMP dissolved in water as stimulus. (F) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) SBT dissolved in water as stimulus. (G) Volcano plot showing the results of pS6-IP-Seq using 100% SBT as stimulus. (H) Volcano plot showing the results of pS6-IP-Seq using 10 mM MTMT dissolved in ethanol as stimulus.
Figure 4—figure supplement 2. Cognate ORs for other sex-specific and sex-enriched volatiles are not sexually dimorphic.

Figure 4—figure supplement 2.

(A) Left: the top five candidate ORs activated by 1% (v/v) 2-heptanone exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 1% (v/v) 2-heptanone exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (B) Left: the top five candidate ORs activated by 100% (E)-β-farnesene exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 100% (E)-β-farnesene exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. (C) Left: the top five candidate ORs activated by 77% (v/v) DHB exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 77% (v/v) DHB exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. (D) Left: the top five candidate ORs activated by 1% (v/v) 2,5-DMP exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 1% (v/v) 2,5-DMP exposure exhibit sexual dimorphism in 43-week-old sex-separated mice.
Figure 4—figure supplement 3. Example in situ stainings showing sexually dimorphic ORs are activated by SBT and MTMT.

Figure 4—figure supplement 3.

(A) Representative images showing in situ mRNA hybridizations probing for Olfr910 expression and pS6 immunostainings. Co-localization events are only seen following SBT exposure as indicated by arrowheads. Scale bars indicate 20 μm. (B) Representative images showing in situ mRNA hybridizations probing for Olfr912 expression and pS6 immunostainings. (C) Representative images showing in situ mRNA hybridizations probing for Olfr1295 expression and pS6 immunostainings.