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Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic

  1. Aashutosh Vihani  Is a corresponding author
  2. Xiaoyang Serene Hu
  3. Sivaji Gundala
  4. Sachiko Koyama
  5. Eric Block
  6. Hiroaki Matsunami  Is a corresponding author
  1. Department of Neurobiology, Neurobiology Graduate Program, Duke University Medical Center, United States
  2. Department of Molecular Genetics and Microbiology, Duke University Medical Center, United States
  3. Department of Chemistry, University at Albany, State University of New York, United States
  4. School of Medicine, Medical Sciences, Indiana University, United States
  5. Duke Institute for Brain Sciences, Duke University, United States
Research Article
Cite this article as: eLife 2020;9:e54501 doi: 10.7554/eLife.54501
12 figures, 1 table and 1 additional file

Figures

Identification of ORs exhibiting sexually dimorphic expression in sex-separated mice.

(A) Schematic of the housing setup. For sex-separation, male mice were co-housed exclusively with male mice. Female mice were co-housed exclusively with female mice. (B) Volcano plot comparing expression of Olfrs between 3-week-old sex-separated male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (C) Volcano plot comparing expression of Olfrs between 9-week-old sex-separated male and female mice. (D) Volcano plot comparing expression of Olfrs between 26-week-old sex-separated male and female mice. (E) Volcano plot comparing expression of Olfrs between 43-week-old sex-separated male and female mice. (F) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr910, Olfr912, and Olfr1295 in sex-separated male and female mice.

Figure 2 with 1 supplement
Sexually dimorphic expression of ORs is consistent with a change in the number of OSNs expressing those ORs.

(A) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr910 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0.0001) between males and females. Data are from n = 3 male and n = 3 female mice. (B) Representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr912 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0 .0001) between males and females. (C) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1295 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference(****p < 0 .0001) between males and females.

Figure 2—figure supplement 1
Architecture of reads uniquely mapping to female-enriched ORs.

(A) Architecture of reads uniquely mapped to Olfr910 in male and female mice. Shown are the defined transcript variants, coding sequence (CDS), uniquely designed riboprobe, and 5′ to 3′ directionality (red arrows). The y-axis shows the number of mapped reads along genomic coordinates described on the x-axis. Reads crossing exon junctions are also quantified. (B) Architecture of reads uniquely mapped to Olfr912. (C) Architecture of reads uniquely mapped to Olfr1295.

Figure 3 with 1 supplement
Sexually dimorphic ORs are activated by mature male mouse odor.

(A) Schematic of exposure experiment. A juvenile mouse (black) was exposed to (in descending order) a clean environment, mature male mice, mature female mice, or 1% (v/v) acetophenone for 1 hr in a sealed container. (B) Left: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events, as indicated by arrowheads, between OSNs expressing Olfr910 and pS6 signal induction following exposure of a juvenile mouse to adult male mice. Scale bars indicate 20 μm. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr910 following exposure of a juvenile mouse to multiple stimuli. One-way ANOVA with Dunnett’s multiple comparisons test correction reveals that only exposure to mature male mice leads to significant (****p < 0.0001) pS6 induction within OSNs expressing Olfr910. Data are from n = 3 juvenile mice. (C) Left: 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 adult male mice. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr912 following exposure of a juvenile mouse to multiple stimuli (****p < 0.0001). (D) Left: 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 adult male mice. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr1295 following exposure of a juvenile mouse to multiple stimuli (****p < 0.0001).

Figure 3—figure supplement 1
Example in situ stainings showing sexually dimorphic ORs are activated by mature male mouse odor.

(A) Representative images showing in situ mRNA hybridizations probing for Olfr910 expression and pS6 immunostainings. Co-localization events are only seen following mature male mouse 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.

Figure 4 with 3 supplements
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—figure supplement 1
Sexually dimorphic ORs are not activated by sex-specific or sex-enriched odorants that are not SBT or MTMT.

(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.

(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.

(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.

Figure 5 with 1 supplement
OSN responses to semiochemicals are not sexually dimorphic between mature male and female mice.

(A) Comparison of responses of OSNs from 26-week-old male and female mice to various stimuli. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 0.1% (v/v) SBT leads to significant pS6 induction in OSNs expressing Olfr910 (***p < 0.001) with no significant differences between males and females (ns; p > 0.05). Data are from n = 3 male and n = 3 female mice. (B) One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 0.1% (v/v) SBT leads to significant pS6 induction in OSNs expressing Olfr912 (**p < 0.01, ****p < 0.0001) with no significant differences between males and females (ns; p > 0.05). (C) One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 10 mM MTMT leads to significant pS6 induction in OSNs expressing Olfr1295 (****p < 0.0001) with no significant differences between males and females (ns; p > 0.05).

Figure 5—figure supplement 1
Example in situ stainings showing responses to semiochemicals are not sexually dimorphic between mature male and female mice.

(A) Representative images from mature male and female mice 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 from mature male and female mice showing in situ mRNA hybridizations probing for Olfr912 expression and pS6 immunostainings. Co-localization events are only seen following SBT exposure. (C) Representative images from mature male and female mice showing in situ mRNA hybridizations probing for Olfr1295 expression and pS6 immunostainings. Co-localization events are only seen following MTMT exposure.

Sex-combined housing leads to the attenuation of the dimorphic OR representations.

(A) Schematic of the housing setup. For sex-combined housing, one male mouse was co-housed with one female mouse. (B) Volcano plot comparing expression of Olfrs between 43-week-old sex-combined male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (C) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr910, Olfr912, and Olfr1295 in sex-separated and sex-combined male and female mice. (D) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in 43-week-old sex-combined male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr910 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr910 (****p < 0.0001). Data are from n = 3 male and n = 3 female mice from each housing condition. (E) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in 43-week-old sex-combined male (top) and female (bottom) mice. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr912 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr912 (****p < 0.0001). (F) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in 43-week-old sex-combined male (top) and female (bottom) mice. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr1295 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr1295 (****p < 0.0001).

Figure 7 with 1 supplement
Olfr1437 and Olfr235 are male-enriched and respond to the macrocyclic musk molecule CPD.

(A) Volcano plot comparing expression of Olfrs between 43-week-old sex-separated male and female mice. Olfr1437 and Olfr235 and are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (B) Volcano plot comparing expression of Olfrs between 43-week-old sex-combined male and female mice. (C) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr1437 and Olfr235 in sex-separated and sex-combined male and female mice. (D) Structure of macrocyclic musk odorant cyclopentadecanone (CPD). (E) Volcano plot showing the results of pS6-IP-Seq using 100 mM CPD diluted in ethanol as stimulus. Olfr1437 and Olfr235 are highlighted in red and 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.

Figure 7—figure supplement 1
Male over-expression is also consistent with a difference in the number of cells expressing Olfr1437 between male and female mice.

(A) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1437 in 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1437 in 43-week-old sex-separated male (top) and female (bottom) mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0.0001) between males and females. Data are from n = 3 male and n = 3 female mice.

Single-cell RNA-Seq analysis reveals an enrichment of activity-associated gene expression in male OSNs expressing Olfr910, Olfr912, and Olfr1295.

(A) Chemosensory receptor-independent UMAP embedding of 17,666 singly sequenced mature OSNs. OSNs expressing Olfr910, Olfr912, Olfr1295, Olfr1437, and Olfr235 are highlighted. (B) Gene expression analysis identified activity-associated genes S100a5, Ptprn, Pcp4l1, and Nrp1 to be enriched amongst OSNs expressing Olfr910, Olfr912, and Olfr1295. (C) UMAP representations of S100a5, Ptprn, Pcp4l1, and Nrp1 show a tendency toward higher expression where OSNs expressing Olfr910, Olfr912, and Olfr1295 are localized.

Figure 9 with 2 supplements
Sex-separated Bax-/- mice fail to generate sexually dimorphic representations of Olfr910Olfr912, and Olfr1295.

(A) Volcano plot comparing expression of Olfrs between 26-week-old Bax-/- sex separated male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (B) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr910 in Bax-/- 43 week-old sex-separated male and female mice. An unpaired two-tailed t-test reveals no statistical difference (ns; p > 0.05) between males and females. Data are from n = 3 male and n = 3 female mice. (C) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr912 in Bax-/- 43-week-old male and female mice. An unpaired two-tailed t-test reveals no statistical difference between males and females (ns; p > 0.05). (D) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1295 in Bax-/- 43-week-old male and female mice. An unpaired two-tailed t-test reveals no statistical difference between males and females (ns; p > 0.05).

Figure 9—figure supplement 1
Sex-separated Bax-/- mice fail to generate sexually dimorphic representations of Olfr1437 and Olfr235. .

(A) Volcano plot comparing expression of Olfrs between 26-week-old Bax-/- sex-separated male and female mice. Olfr1437 and Olfr235 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice.

Figure 9—figure supplement 2
Bax-/- mice exhibit significant changes in their OR repertoire.

(A) Volcano plot comparing expression of Olfrs between 26-week-old wild-type and Bax-/- sex-separated male mice. Olfr910, Olfr912, Olfr1295, Olfr1437, and Olfr235 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 wild-type and n = 3 mutant mice. (B) Volcano plot comparing expression of Olfrs between 26-week-old wild-type and Bax-/- sex-separated female mice.

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Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus C57BL/6J)C57BL/6JJackson Labs000664
Strain, strain background (Mus musculus C57BL/6J)Bax-/-Jackson Labs002994
Commercial assay, kitTRIzolLife Technologies15596026
Commercial assay, kitQUBIT HS RNA Assay KitThermoFisherQ32855
Commercial assay, kitRNase-free DNaseIRoche04 716 728 001
Commercial assay, kitRNeasy Mini KitQiagen74104
Commercial assay, kitSMART-Seq v4 Ultra Low Input RNA KitTakara634898
Commercial assay, kitNexterra XT DNA Library Preparation KitIllumina15032354
Sequence-based reagentOlfr910 UTR FThis paper5'-AAACGCGTGTGAAAATTGTGACAGATCCA-3'
Sequence-based reagentOlfr910 UTR RThis paper5'-AAGCGGCCGCCATTTACAAGAAGGGAATCAG-3'
Sequence-based reagentOlfr912 UTR FThis paper5'-AAACGCGTACTTTGTTCTGATTCAGTTGTT-3'
Sequence-based reagentOlfr912 UTR RThis paper5′-AAGCGGCCGCGTCCACAGAGCAATACAACA-3′
Sequence-based reagentOlfr1295 UTR FThis paper5'-AAACGCGTACTCCTCTCCTAAATCCAAC-3'
Sequence-based reagentOlfr1295 UTR RThis paper5'-AAGCGGCCGCGGCAGCACCACTGATCAA-3'
Commercial assay, kitPhusion DNA PolymeraseNEBF530S
Commercial assay, kitpCI vectorPromegaE1731
Commercial assay, kitDNA polymeraseQiagen203203
Commercial assay, kitMinElute KitQiagen28004
Commercial assay, kitT3 RNA polymerasePromegaP2083
Commercial assay, kitDIG RNA labeling mixRoche11277073910
Commercial assay, kitMicro Bio-Spin P-30 Gel ColumnsBio-Rad732–6223
Commercial assay, kitTissue-Tek O.C.T. CompoundSakura Finetek4583
Commercial assay, kitSuperfrost Plus SlidesFisherbrand1255015
Commercial assay, kitParafilmSigmaP7793
Commercial assay, kitBlocking reagentRoche11096176001
Commercial assay, kitMaleic acidSigmaM0375
AntibodyAnti-DIG antibody (sheep polyclonal)Roche112077339101:1000
Commercial assay, kitTSA-FluoresceinPerkinElmerNEL741B001KT1:400
AntibodypS6 Antibody for IF (rabbit polyclonal)ThermoFisher44–923G1:300
AntibodyAnti-rabbit IgG Cy3 Antibody (donkey polyclonal)Jackson Immuno711-165-1521:200
OtherBisbenzimideSigmaH 332581:100,000
OtherPaper bucketInternational PaperDFM85
OtherBlotting padVWR28298–014
OtherOdor cassetteSakura Finetek0006772–01
Commercial assay, kitLoBind TubeEppendorf22431021
Commercial assay, kitNP40 SubstituteSigma11332473001
Commercial assay, kitDHPCAvanti Polar Lipids850306P
AntibodypS6 Antibody for IP (rabbit monoclonal)Cell SignalingD68F86 μL
Commercial assay, kitDynabeads Protein AInvitrogen10002D
Commercial assay, kitRNeasy Micro KitQiagen74004
Otherβ-CaryophylleneSigmaW225207
Other2,5-DMPSigma175420
Other2-HeptanoneSigmaW254401
Other(E)-β-FarneseneBedoukianP3500-90
OtherCPDSigmaC111201
OtherMTMTLin et al., 2005Synthesized
OtherSBTMeijer et al., 1973; Abrunhosa et al., 2001; Tashiro and Mori, 1999Synthesized
OtherDHBWiesler et al., 1984Synthesized

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