Sex differences in circadian food anticipatory activity are not altered by individual manipulations of sex hormones or sex chromosome copy number in mice

Abstract

Recent studies in mice have demonstrated a sexual dimorphism in circadian entrainment to scheduled feeding. On a time restricted diet, males tend to develop food anticipatory activity (FAA) sooner than females and with a higher amplitude of activity. The underlying cause of this sex difference remains unknown. One study suggests that sex hormones, both androgens and estrogens, modulate food anticipatory activity in mice. Here we present results suggesting that the sex difference in FAA is unrelated to gonadal sex hormones. While a sex difference between males and females in FAA on a timed, calorie restricted diet was observed there were no differences between intact and gonadectomized mice in the onset or magnitude of FAA. To test other sources of the sex difference in circadian entrainment to scheduled feeding, we used sex chromosome copy number mutants, but there was no difference in FAA when comparing XX, XY-, XY-;Sry Tg, and XX;Sry Tg mice, demonstrating that gene dosage of sex chromosomes does not mediate the sex difference in FAA. Next, we masculinized female mice by treating them with 17-beta estradiol during the neonatal period; yet again, we saw no difference in FAA between control and masculinized females. Finally, we observed that there was no longer a sex difference in FAA for older mice, suggesting that the sex difference in FAA is age-dependent. Thus, our study demonstrates that singular manipulations of gonadal hormones, sex chromosomes, or developmental patterning are not able to explain the difference in FAA between young male and female mice.

Fig 1. Sex differences in food anticipatory activity.

(A) Mean (+/- SEM) seconds of high activity behaviors (hanging, jumping, rearing, and walking) seven days prior to initiating scheduled CR feeding in male (green) and female (blue) mice. (B) Normalized mean high activity of data shown in A. Mean seconds of high activity behaviors after (C) four or (E) five weeks of timed CR feeding. (D,F) Normalized mean +/- SEM high activity of data shown in C and E, respectively. (G) Mean seconds of high activity data in the three hours preceding scheduled feeding across 42 days of the experiment. (H) Mean normalized high activity data in the three hours preceding scheduled feeding across the experiment. (I) Mean body weights for males and female mice on a CR diet for five weeks. (J) Percentage weight loss calculated by normalizing to day 0 weight. n = 8 per group. ** P<0.01; ***P<0.001, Mann-Whitney. The arrow indicates the time of expected food delivery (ZT7).

Fig 2. Lack of effect of gonadectomy on food anticipatory activity.

Mean (+/- SEM) normalized high activity behaviors seven days prior to initiating scheduled CR feeding in (A) male (solid green) and castrated male (dashed green) and (B) female (solid blue) and ovariectomized (dashed blue) mice. Normalized mean high activity of (C) male and castrated males on day 28, (D) female and ovariectomized females on day 28, (E) male and castrated males on day 35, and (F) female and ovariectomized females on day on day 35. Mean normalized high activity data in the three hours preceding scheduled feeding across 35 days of the experiment for (G) intact versus castrated males and H) intact versus ovariectomized females. (I) Mean body weights loss for intact and castrated males. (J) Mean body weight loss for intact and ovariectomized female mice on a CR diet for five weeks. ** P<0.01; *** P<0.001, Student T test. n = 7–8 per group. The arrow indicated the time of expected food delivery (ZT7).

Fig 3. Sex chromosome copy number does not alter food anticipatory activity.

(A) Mean (+/- SEM) seconds of high activity behaviors seven days prior to initiating scheduled CR feeding in XX (purple), XX; Sry Tg+ (red), XY- (green), and XY-; Sry Tg+ (blue) mice. (B) Normalized mean high activity of data shown in A. Mean seconds of high activity behaviors after (C) three or (E) four weeks of timed CR feeding. (D,F) Normalized mean high activity of data shown in E and F, respectively. (G) Mean seconds of high activity data in the three hours preceding scheduled feeding across 28 days of the experiment. (H) Mean normalized high activity data in the three hours preceding scheduled mealtime. (I) Mean body weights for males and female mice one week pre-CR and four weeks on a CR diet. (J) Percentage weight loss calculated by normalizing to day 0. * P<0.05, **P<0.01, *** P<0.0001, One-way ANOVA, Tukey-Kramer multiple comparisons. n = 9 for XX mice, n = 12 for XX;Sry Tg mice, n = 11 for XY- mice, and n = 11 for XY-;Sry Tg mice. The arrow indicated the time of expected food delivery (ZT6).

Fig 4. Neonatal 17-beta estradiol brain masculinization does not alter food anticipatory activity.

(A) Mean (+/- SEM) seconds of high activity data in the three hours preceding scheduled feeding across 28 days of the experiment. (B) Mean normalized high activity in the three hours preceding scheduled feeding across 28 days of the experiment. (C) Mean body weights for control and 17-beta estradiol -injected mice across the duration of the experiment. (D) Percentage weight loss calculated by normalizing to day 0. * P<0.05; ** P<0.01, Student T test.

Fig 5. Aged mice do not have a sex difference in food anticipatory activity.

(A) Mean (+/- SEM) seconds of high activity behaviors (hanging, jumping, rearing, and walking) on the first day of scheduled CR feeding in male (green) and female (blue) mice. (B) Normalized mean high activity of data shown in A. (C) Mean seconds of high activity behaviors after five weeks of timed CR feeding. (D)Normalized mean high activity of data shown in C. (E) Mean seconds of high activity data in the three hours preceding scheduled feeding across 42 days of the experiment. (F) Mean normalized high activity data in the three hours preceding scheduled feeding across 35 days of the experiment. (G) Mean body weights for aged males and female mice on a CR diet for five weeks. (H) Percentage weight loss calculated by normalizing to day 0. n = 3–6 for females and n = 6–8 for males * P<0.05, ***P<0.001, Mann-Whitney. The arrow indicated the time of expected food delivery (ZT7).