Gonadal- and sex-chromosome-dependent sex differences in the circadian system

Abstract

Compelling reasons to study the role of sex in the circadian system include the higher rates of sleep disorders in women than in men and evidence that sex steroids modulate circadian control of locomotor activity. To address the issue of sex differences in the circadian system, we examined daily and circadian rhythms in wheel-running activity, electrical activity within the suprachiasmatic nucleus, and PER2::LUC-driven bioluminescence of gonadally-intact adult male and female C57BL/6J mice. We observed greater precision of activity onset in 12-hour light, 12-hour dark cycle for male mice, longer activity duration in 24 hours of constant darkness for female mice, and phase-delayed PER2::LUC bioluminescence rhythm in female pituitary and liver. Next, in order to investigate whether sex differences in behavior are sex chromosome or gonadal sex dependent, we used the 4 core genotypes (FCG) mouse model, in which sex chromosome complement is independent of gonadal phenotype. Gonadal males had more androgen receptor expression in the suprachiasmatic nucleus and behaviorally reduced photic phase shift response compared with gonadal female FCG mice. Removal of circulating gonadal hormones in adults, to test activational vs organizational effects of sex revealed that XX animals have longer activity duration than XY animals regardless of gonadal phenotype. Additionally, we observed that the activational effects of gonadal hormones were more important for regulating activity levels in gonadal male mice than in gonadal female FCG mice. Taken together, sex differences in the circadian rhythms of activity, neuronal physiology, and gene expression were subtle but provide important clues for understanding the pathophysiology of the circadian system.

Figure 1.

Sex differences in Per2^Luc mouse wheel-running activity rhythms. Representative example of male (left) and female (right) double-plotted actograms in LD (d 1-10) and DD (d 11-20). Male mice have more precise activity onset in LD than females, as indicated by gray circles. Female mice have longer activity duration in DD than males.

Figure 2.

Sex differences in SCN firing rate. Daytime (ZT 4-6) firing rates are higher than nighttime (ZT 14-16) firing rates in male (P < .003) and female (P < .05) dSCN. (A) Daytime firing rates are higher in males than females under baseline conditions (P = .03). (B) GabZ treatment abolishes sex difference in dSCN firing rate (P = .9). (C) No sex difference in early evening vSCN NMDA response. Male and female vSCN neurons firing rate significantly increases after NMDA treatment at ZT 14-16 (male, P = .017; female, P < .00001). The range of firing rates after NMDA treatment is greater in male SCN neurons, but there is no statistically significant interaction between sex and NMDA response (P = .6). Trace examples on the left and corresponding box plots on the right. Bottom and top of box indicate 25th and 75h percentile, respectively, whiskers indicate 10th and 90th percentile, solid line inside box indicates 50th percentile, and dashed lines inside box indicates mean. * indicates day-night difference P < .05, # indicates sex difference P < .05, § indicates effect of NMDA P < .05.

Figure 3.

Sex differences in bioluminescence rhythms of central and peripheral oscillators. (A) No sex difference in bioluminescence phase or amplitude of the SCN. (B) Phase relationship of peripheral oscillators are altered in females relative to male mice. The white/black bar indicates the time of previous lights on and off, respectively. (C) Amplitude of liver and adrenal bioluminescence rhythms are lower in females relative to male mice. *P < .05 between males and females.

Figure 4.

Genetic and hormonal influence on wheel-running activity rhythms of FCG mice in DD. (A) XX chromosome complement confers longer activity duration than XY chromosome complement after GDX. (B) After GDX, phenotypic male FCG mice have greater deficits in wheel-running rhythm power than phenotypic female FCG mice. (C) After GDX, phenotypic male mice have greater activity level decrease than phenotypic female mice. (D) Before GDX, phenotypic female mice respond to CT16 light pulse with greater phase shifts than male mice. After GDX, there is no difference in phase shift. * indicates a significant effect of gonadal sex (P < .05), and ^ indicates a significant effect of sex chromosome (P < .05) as determined by 2-way ANOVA. M refers to gonadal and phenotypic males and F refers to gonadal and phenotypic females.

Figure 5.

AR expression in FCG mice. Grayscale images from fluorescent anti-AR sections are shown with the area positive for DAPI outlined using dotted lines. (A) (left column) XYM (top) and XXM (bottom); (right column) XYF (top) and XXF (bottom). (B) Gonadal male FCG mouse SCN (left; 40 ± 10 cells) expresses more AR⁺ cells than gonadal female FCG mouse SCN (right; 12 ± 11 cells, P < .001). *P < .05 between gonadal sexes. Although there appears to be a sex chromosome difference in AR expression within gonadal females, the analysis was underpowered to draw any conclusions (post hoc t = 11.91, P = .08, power = 0.144).