Chronic stress alters adrenal clock function in a sexually dimorphic manner

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   Figure 1

   Chronic restraint stress influences adrenal clock function. (A) Schematic illustrating in vivo chronic restraint stress protocol and adrenal collection for PER2::LUC recording. Exposure to restraint stress for 7 days decreased the gain in body weight in both sexes (stress:: F(1,18) 22.8, P < 0.0005; sex: F(1,18) = 0.05, P > 0.8; stress * sex: F(1,18) = 0.6, P > 0.5). Adrenals were collected from non-stressed controls (Control) and stressed mice either 1 day (Chronic) or 7 days (Recovery) after the last day of restraint. *DSXN: day and time of dissection for indicated group. (B) Representative PER2::LUC time series from male and female adrenals from the non-stressed control, chronic stress and recovery groups. (C) Chronic restraint stress eliminated the sex difference in the amplitude of adrenal PER2::LUC rhythms (repeated-measures ANOVA, non-stressed controls: sex: F(1,27) = 39.7, P < 0.0001; time: F(4,24) = 109.0, P < 0.0001; sex * time: F(4,24) = 12.05, P < 0.0001; chronic: sex: F(1,28) = 2.5, P > 0.1; time: F(4,25) = 25.7, P < 0.0001; sex * time: F(4,25) = 1.0, P > 0.4; recovery: sex: F(1,19) = 10.1, P < 0.0001; time: F(4,16) = 41.1, P < 0.0001; sex * time: F(4,16) = 10.3, P < 0.0001). (D) Chronic restraint stress reduced the amplitude of PER2::LUC rhythms in males and females (repeated-measures ANOVA, males: stress: F(2,40) = 11.6, P < 0.0001; time: F(4,37) = 67.8, P < 0.0001; stress * time: F(8,74) = 4.2, P < 0.0001; females: stress: F(2,34) = 2.9, P = 0.07; time: F(4,31) = 90.8, P < 0.0001; stress * time: F(8,62) = 3.1, P < 0.01). Note that data in (D) are re-plotted from (C) to facilitate visualization of effects of chronic stress in each sex. Number of cultures per group = 9–16 (Table 1). *LSM contrasts, P < 0.01, color-coded in (D) to represent comparisons between each stress group and non-stressed controls.
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   Figure 2

   A single exposure to 2-h restraint stress does not influence adrenal clock function. (A) Schematic illustrating in vivo acute restraint stress protocol and adrenal collection for PER2::LUC recording. Adrenals were collected from non-stressed controls (Control) and stressed mice either immediately after the 2-h restraint (Acute) or the next day (Recovery). *DSXN: day and time of dissection for indicated group. (B) Representative PER2::LUC time series from male and female adrenals from the non-stressed control, acute stress and recovery groups. (C) Acute restraint stress did not eliminate the sex difference in adrenal amplitude (repeated-measures ANOVA, non-stressed controls: sex: F(1,14) = 16.6, P < 0.005; time: F(4,11) = 20.89, P < 0.0001; sex * time: F(4,11) = 5.18, P < 0.05; acute: sex: F(1,14) = 22.0, P < 0.0005; time: F(4,11) = 29.6, P < 0.0001; sex * time: F(4,11) = 5.76, P < 0.05; recovery: sex: F(1,14) = 3.87, P = 0.07; time: F(4,11) = 25.5, P < 0.0001; sex * time: F(4,11) = 5.9, P < 0.01). (D) Acute restraint stress did not reduce the amplitude of PER2::LUC rhythms in males or females (repeated-measures ANOVA, males: stress: F(2,21) = 0.8, P > 0.4; time: F(4,18) = 49.7, P < 0.0001; stress * time: F(8,36) = 1.4, P > 0.2; females: stress: F(2,21) = 0.9, P > 0.4; time: F(4,18) = 27.8, P < 0.0001; stress * time: F(8,36) = 2.1, P = 0.06). Note that data in (D) are re-plotted from (C) to illustrate the lack of effect of acute stress in both sexes. Number of cultures per group = 8. *LSM contrasts, P < 0.01.
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   Figure 3

   Sex differences in the response of the adrenal clock to a chronic stress model due to increased amplitude of PER2::LUC rhythms in female adrenals. (A) Schematic illustrating in vivo chronic stress protocol and adrenal collection for PER2::LUC recording. Adrenals were collected from non-stressed controls (Control) and stressed mice either 1 day (Stress) or 7 days (Recovery) after single prolonged stress. *DSXN: day and time of dissection for indicated group. (B) Male and female mice displayed similar fear behavior over consecutive days of FC training. (C) Male and female mice displayed similar duration of immobility during forced swim on the day of SPS. (D) Representative PER2::LUC time series from male and female adrenals collected from non-stressed control, stress, and recovery mice. (E) Stress increased PER2::LUC amplitude in female adrenals (repeated-measures ANOVA stress: F(2,21) = 12.4, P < 0.0005; time: F(4,18) = 95.5, P < 0.0001; stress * time: F(8,36) = 5.1, P < 0.0005), but did not influence PER2::LUC amplitude in males (repeated-measures ANOVA, males: stress: F(2,21) = 1.2, P > 0.3; time: F(4,18) = 69.7, P < 0.0001; stress * time: F(8,36) = 1.6, P > 0.1). (F) Exposure to the stress manipulation did not eliminate sex differences in the amplitude of PER2::LUC rhythms in any group (repeated-measures ANOVA, non-stressed controls: sex: F(1,14) = 19.8, P < 0.001; time: F(4,11) = 45.5, P < 0.0001; sex * time: F(4,11) = 7.2, P < 0.005; stress: sex: F(1,14) = 28.1, P < 0.0005; time: F(4,11) = 56.1, P < 0.0001; sex * time: F(4,11) = 4.8, P < 0.05; recovery: sex: F(1,14) = 10.7, P < 0.01; time: F(4,11) = 38.1, P < 0.0001; sex * time: F(4,11) = 2.8, P = 0.08). Number of cultures per group = 8. *LSM contrasts, P < 0.01, color-coded in (E) to represent comparisons between each stress group and non-stressed controls.
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   Figure 4

   Sex differences in the intrinsic response of the adrenal clock to ACTH. (A) Representative PER2::LUC time series from male and female adrenals treated with ACTH at CT 0 or CT8. Each ACTH-treated sample is plotted along with a same-sex vehicle-treated control (black trace). Arrowhead indicates time of treatment. (B) Phase resetting responses of male and female adrenals treated with ACTH or vehicle. Male and females adrenals differed in their response to ACTH treatment (sex: F(1,151) = 4.43, P < 0.05, time: F(5,151) = 405.5, P < 0.0001, sex * time: F(5,151) = 19.2, P < 0.0001), but not vehicle treatment (sex: F(1,146) = 1.2, P > 0.2, time: F(5,146) = 18.4, P < 0.0001, sex * time: F(5,146) = 0.9, P > 0.5). Note that the response of unstimulated samples did not differ by sex (data plotted at time ‘–’, t(1,14) = −0.05, P > 0.9, see ‘Methods’ section for more information). Number of cultures per group = 8–12/group. (C) ACTH-induced amplitude potentiation was larger in female adrenals than male adrenals (sex: F(1,151) = 132.6, P < 0.0001, time: F(5,151) = 21.6, P < 0.0001, sex * time: F(5,151) = 9.8, P < 0.0001). (D) ACTH induces a drug-specific pattern of sex differences in resetting that was not observed with acetylcholine (ACh), dexamethasone (DEX) or vehicle treatment (ACTH: sex: F(1,18) = 0.1, P > 0.7, time: F(2,18) = 139.2, P < 0.0001, sex * time: F(2,18) = 13.3, P < 0.0005; ACh: sex: F(1,23) = 1.5, P > 0.2, time: F(2,23) = 42.0, P < 0.0001, sex * time: F(2,23) = 0.2, P > 0.8; DEX: sex: F(1,22) = 10.2, P < 0.005, time: F(2,22) = 60.0, P < 0.0001, sex * time: F(2,22) = 0.3, P > 0.7; vehicle: sex: F(1,19) = 1.4, P > 0.2, time: F(2,19) = 8.5, P < 0.005, sex * time: F(2,19) = 1.02, P > 0.3). (E) Adrenal amplitude was increased by ACTH, but not by ACh, DEX or vehicle (ACTH: sex: F(1,18) = 27.7, P < 0.0001, time: F(2,18)= 9.1, P < 0.005, sex * time: F(2,18) = 3.2, P = 0.06; ACh: sex: F(1,23) = 0.5, P > 0.4, time: F(2,23) = 2.2, P > 0.1, sex * time: F(2,23) = 0.5, P > 0.8; DEX: sex: F(1,22) = 0.2, P > 0.6, time: F(2,22) = 1.2, P > 0.3, sex * time: F(2,22) = 2.4 P > 0.1; vehicle: sex: F(1,19) = 0.03, P > 0.8, time: F(2,19) = 0.4, P > 0.6, sex * time: F(2,19) = 0.3, P > 0.3). *LSM contrasts, ACTH-stimulated male vs female, P < 0.01.
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   Figure 5

   Reversing the sex difference in adrenal clock amplitude inverted the dimorphism in ACTH responsiveness. (A) Testosterone (T) administration to females in vivo increased the amplitude (AMP) of the PER2::LUC rhythm on the third cycle in vitro, whereas 7 days of chronic restraint stress in vivo decreased amplitude in male adrenals on the third cycle in vitro (sex: F(1,82) = 7.5, P < 0.01; group: F(1,82) = 0.2, P > 0.6; sex * group: F(1,82) = 77.2, P < 0.0001). (B) Regardless of sex, adrenals with low amplitude displayed higher resetting responses when pulsed with ACTH at bib8 on the fourth cycle in vitro (sex: F(1,36) = 0.04, P > 0.8; group: F(1,36) = 0.5, P > 0.4; sex * group: F(1,36) = 52.4, P < 0.0001). (C) Adrenals with low amplitude displayed higher amplitude responses when pulsed with ACTH at bib8 on the fourth cycle in vitro (sex: F(1,36) = 0.6, P > 0.4; group: F(1,36) = 0.9, P > 0.3; sex * group: F(1,36) = 19.1, P = 0.0001). Groups that do not share letters in common differ significantly, Tukey’s HSD, P < 0.05.

• © 2018 Society for Endocrinology