Fig 1.
Expression level of steroid receptors detected by RNA-Seq.
Heat Map showing the transcript expression level of steroid receptors in ORNs and OE [36]. Deeper colors indicate a higher expression level.
Fig 2.
Expression of membrane progestin and estradiol receptors in the OE.
Protein expression was assessed by staining coronal sections of OMP-GFP mice with anti-Paqr8, anti-Paqr9 and anti-Gpr30 antibodies. Left: GFP fluorescence (green) served to identify mature ORNs. Center: Paqr9 immunoreactivity (red) was observed in the soma membrane of a subset of mature ORNs, while Paqr8 and Gpr30 immunoreactivity was detected in the cilia membrane as indicated by the arrows. Right: Overlay of the GFP signal and antibody staining. Scale bar, 5 μm. Control staining with the secondary antibody showed low background (S3 Fig).
Fig 3.
Progesterone decreased Henkel 100-induced responses in a dose-dependent manner in the OE of female mice.
(A) Representative submerged EOG esponses to Henkel 100 (1:5,000) were recorded from the surface of the septum from female mouse OE. The first response was recorded as a control, the second response was recorded after a 4-min progesterone preincubation (1 μM) and the third response was recorded after progesterone washout. The amplitude value was decreased after application of progesterone, but recovered after progesterone washout. (B) The graph shows the relative amplitude of the response to Henkel 100, Henkel 100 after progesterone preincubation and after progesterone washout. The response was significantly decreased after preincubation with 1 μM progesterone to 72.8 ± 3.6%, but it recovered to 90.8 ± 2.3% of the starting value after progesterone washout (n = 8). (C) Representative submerged EOG measurements of the responses to Henkel 100 (1:5,000) recorded from the surface of the septum of female mice during (red) and without progesterone (black) incubation. After 2 min of progesterone preincubation, the response decreased. After 3–4 min of preincubation, the response was maximally decreased. (D) Representative submerged EOG responses to Henkel 100 (1:5,000) under control conditions (black) and 4 min after progesterone preincubation at different concentrations (red). (E) Relative reduction in peak response to Henkel 100 after progesterone preincubation at different concentrations (0.1 nM: 11.9 ± 2.1% (n = 3), 1 nM: 18.4 ± 3.1% (n = 3), 10 nM: 22.0 ± 11.4% (n = 4), 100 nM: 31.4 ± 7.3% (n = 4), 1 μM: 37.9 ± 2.2% (n = 4), and 10 μM: 48.8 ± 2.1% (n = 4)). Progesterone reduced the peak response in a dose-dependent manner. Significant data are labeled: *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.
Fig 4.
Progesterone decreased benzaldehyde- and vanillin-induced responses in the OE of mice.
(A) Representative submerged EOG responses to 200 μM benzaldehyde were recorded from the surface of the septum of male mice. Preincubation with 10 nM progesterone reversibly reduced the benzaldehyde response. (B) The amplitude was significantly decreased after progesterone incubation (37.9 ± 12.3%) and recovered to 89.5 ± 11.6% after progesterone washout (n = 3). (C) Representative submerged EOG responses to 1 mM vanillin were recorded from the surface of the septum of female mice. (D) Preincubation with 1 μM progesterone reversibly reduced the vanillin response. The amplitude was decreased after progesterone incubation (56.0 ± 9.0%) and recovered to 79.4 ± 3.9%) after progesterone washout (n = 4). Significant data are labeled: *p ≤ 0.05, ***p ≤ 0.001.
Fig 5.
Progesterone decreased vanillin-induced responses in the ORNs of p5 mice.
(A) Representative responses to 100 μM vanillin were obtained from Olfr73 positive ORNs in tissue slices using patch-clamp recordings in a voltage-clamp configuration (Vh = -56 mV). The amplitude was decreased after a 1-min preincubation with 1 μM progesterone. Application time was 1 s as indicated by the application bar. (B) As a control, neurons were challenged with two subsequent applications of 100 μM vanillin. To characterize the effect of progesterone, 100 μM vanillin was applied followed by a progesterone preincubation and a subsequent second application of vanillin. The bar diagram shows the second response with or without progesterone preincubation relative to the first vanillin application. Relative to the second application of vanillin after 1 min in control neurons, the response of Olfr73 positive neurons was significant decreased in neurons preincubated with 1 μM progesterone for 1 min (n = 7). Significant data are labeled: *p ≤ 0.05.
Fig 6.
Pregnenolone decreased the effect of Henkel 100-induced responses in the OE.
(A) Representative submerged EOG responses to Henkel 100 (1:5,000) were recorded from the surface of the septum of female mice. Pregnenolone preincubation (1 μM) temporarily reduced the response amplitude. (B) The response was significantly decreased after pregnenolone preincubation (31.9 ± 4.8%) and recovered to 86.4 ± 10.2% after washout (n = 3). Significant data are labeled: *p ≤ 0.05.
Fig 7.
Mifepristone (RU-486) decreased the Henkel 100-induced response in the OE.
(A) Representative submerged EOG responses to Henkel 100 (1:5,000) were recorded from the surface of the septum of female mice. The first response was recorded as a control, the second response was recorded after a 4-min RU-486 preincubation (1 μM), the third response was recorded after preincubation with RU-468 and progesterone (1 μM) and the fourth response was recorded after washout. (B) The response to Henkel 100 was decreased after RU-468 preincubation (32.7 ± 9.6%). This effect was further decreased after preincubation with RU-486 and progesterone (49.0 ± 10.4%). The response recovered to 76.9 ± 15.7% after washout (n = 3). Significant data are labeled: *p ≤ 0.05.
Fig 8.
Estradiol affected Henkel 100-induced responses in the OE.
(A) Representative submerged EOG responses to Henkel 100 (1:5,000) were recorded from the surface of the septum of female mice. The amplitude was lowered after preincubation with 1 μM estradiol and recovered after washout. (B) The graph shows the relative response amplitudes to Henkel 100, Henkel 100 after estradiol preincubation and after estradiol washout. The response was significantly decreased after estradiol preincubation (39.4 ± 8.3%) and then recovered to 82.0 ± 9.5% after washout (n = 4). (C) Representative submerged EOG responses to Henkel 100 (1:5,000) were recorded under control conditions, after a 4-min preincubation with 1 nM estradiol and after washout. The amplitude value was reduced after preincubation with 1 nM estradiol and recovered after washout. (D) The graph shows the relative response to Henkel 100, Henkel 100 after estradiol preincubation and after estradiol washout. The response was significantly decreased after estradiol preincubation (42.2 ± 3.8%) and recovered to 72.2 ± 2.2% of the starting value after washout (n = 3). Significant data are labeled: *p ≤ 0.05, **p ≤ 0.01.
Fig 9.
Estradiol decreased vanillin-induced responses in the ORNs of p5 mice.
(A) Representative responses to 100 μM vanillin from Olfr73 positive ORNs in tissue slices were obtained from patch-clamp recordings set to a voltage-clamp configuration (Vh = -56 mV). The amplitude was decreased after 1-min preincubation with 1 nM estradiol. Application time was 1 s as indicated by the application bar. B) As a control, neurons were challenged with two subsequent applications of 100 μM vanillin. To characterize the effect of progesterone, 100 μM vanillin was applied followed by a estradiol preincubation and a subsequent second application of vanillin. The bar diagram shows the second response with or without estradiol preincubation relative to the first vanillin application. Relative to the second application of vanillin after 1 min in control neurons, the response of Olfr73 positive neurons was significant decreased in neurons preincubated with 1 nM estradiol for 1 min (n = 8). Significant data are labeled: **p ≤ 0.01.
Fig 10.
Progesterone decreased odorant-induced cAMP levels in dissociated cells of the OE.
Bar diagrams show the percent increase in cAMP compared to control cells after Henkel 100 stimulation (1:100,000). Progesterone preincubation reduced the effect of the Henkel 100 stimulation (n = 4 independent pools of the OE from 10 mice (male and female)). Significant data are labeled: **p ≤ 0.01.
Fig 11.
Estradiol decreased odorant-induced cAMP levels.
The bar charts represent the relative cAMP levels compared to the negative control in dissociated cells of the OE after Henkel 100 stimulation (1:100,000), Henkel 100 after preincubation with estradiol and Henkel 100 after preincubation with estradiol and the Gpr30-specific antagonist G15 (1 μM, stimulation (n = 3 independent pools of the OE from 10 mice (male and female))). Significant data are labeled: *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.
Fig 12.
Effect of the Gpr30-specific antagonist G15 on estradiol modulated odorant-evoked responses in submerged EOG recordings from female mice.
Bar charts show the relative peak response to Henkel 100 (1:5,000), Henkel 100 after estradiol preincubation, after G15 preincubation, after G15 and estradiol preincubation and after washout. Estradiol preincubation significantly reduced the Henkel 100 response to 42.0 ± 6.1%. The estradiol effect was significantly reduced in the presence of the antagonist G15 (1 μM, 68.9 ± 7.8%). After washout, the response significantly recovered to 83.6 ± 5.3% of control values (n = 6). Significant data are labeled: *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.