Time-Restricted Feeding Regulates Circadian Rhythm of Murine Uterine Clock

Abstract

Background: Skipping breakfast is associated with dysmenorrhea in young women. This suggests that the delay of food intake in the active phase impairs uterine functions by interfering with circadian rhythms.

Objectives: To examine the relation between the delay of feeding and uterine circadian rhythms, we investigated the effects of the first meal occasion in the active phase on the uterine clock.

Methods: Zeitgeber time (ZT) was defined as ZT0 (08:45) with lights on and ZT12 (20:45) with lights off. Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum consumption), group II (time-restricted feeding during ZT12-16, initial 4 h of the active period), and group III (time-restricted feeding during ZT20-24, last 4 h of the active period, a breakfast-skipping model). After 2 wk of dietary restriction, mice in each group were killed at 4-h intervals and the expression profiles of uterine clock genes, Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1), Per1 (period circadian clock 1), Per2, and Cry1 (cryptochrome 1), were examined.

Results: qPCR and western blot analyses demonstrated synchronized circadian clock gene expression within the uterus. Immunohistochemical analysis confirmed that BMAL1 protein expression was synchronized among the endometrium and myometrium. In groups I and II, mRNA expression of Bmal1 was elevated after ZT12 at the start of the active phase. In contrast, Bmal1 expression was elevated just after ZT20 in group III, showing that the uterine clock rhythm had shifted 8 h backward. The changes in BMAL1 protein expression were confirmed by western blot analysis.

Conclusions: This study is the first to indicate that time-restricted feeding regulates a circadian rhythm of the uterine clock that is synchronized throughout the uterine body. These findings suggest that the uterine clock system is a new candidate to explain the etiology of breakfast skipping-induced uterine dysfunction.

Keywords: circadian rhythms; clock gene; dysmenorrhea; meal timing; uterine function.

FIGURE 1

Bmal1 expression in the murine uterus. (A) Bicornuate uterus. (B) Hematoxylin and eosin staining of a horizontal section of the uterus. (C) Negative control. (D) CK-7 was mainly expressed in the endometrium. (E) αSMA was expressed in both the inner circular and outer longitudinal muscle layers. (F) BMAL1 expression was observed in both the endometrium and myometrium. Bars show 500 µm (A), 200 µm (B), and 100 µm (C–F). (G) Western blot analysis detected specific bands at a molecular weight of 75 kDa in the uterus and liver, which correspond to BMAL1. (H) RT-PCR confirmed the mRNA expression of Bmal1, Krt7, and Acta2 in the uterus. Acta2, smooth muscle marker αSMA; BMAL1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1; CK-7, cytokeratin 7; CM, circular muscle; EM, endometrium; Krt7, endometrial epithelial marker CK-7; LM, longitudinal muscle; N.C., negative control; Rplp0, ribosomal protein lateral stalk subunit P0; αSMA, α smooth muscle actin.

FIGURE 2

Circadian rhythms of uterine clock genes. By qPCR analyses, circadian expression profiles of clock genes in the murine uterus were examined. (A) Circadian rhythms of uterine clock genes in group I (ad libitum consumption). The mRNA expression of Bmal1, Per1, Per2, and Cry1 in the uterus showed circadian cycles. Relative expression is presented as fold units per minimal values. The mRNA expression of Bmal1 significantly rises after the start of the dark period (ZT12) and reaches a peak at the end of the dark period (ZT0). (B, C) Although nonsignificant, western blot analysis demonstrated circadian expression profiles of BMAL1 protein in the uterine tissues. The peak of protein expression was observed 8 h after that of mRNA expression. The cyclic changes in mRNA and protein expression of clock genes were analyzed by 1-factor ANOVA followed by the Dunnett test. **P < 0.01, *P < 0.05 between the groups at either end of the bar. αSMA, α-smooth muscle actin; Bmal1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1; Cry1, cryptochrome 1; Per, period circadian clock; ZT, Zeitgeber time.

FIGURE 3

Synchronization of clock gene expression in the uterus. Uterine samples were obtained from 10-wk-old female mice (ad libitum consumption, n = 18) at 4-h intervals, ZT0, ZT4, ZT8, ZT12, and ZT16, and were divided into upper and lower parts for analysis of clock gene expression in each segment. (A) Both segments showed similar periodicity in the expression of clock genes (Bmal1, Per1, Per2, and Cry1) by qPCR. Relative expression is presented as fold units per minimal value of the upper segment. (B) Immunohistochemical analysis demonstrated no differences in the expression intensity of BMAL1 between the upper and lower segments of the uterus. We also observed no difference in Bmal1 expression among the endometrium and inner and outer muscle layers. Bars show 100 µm. (C) The uterus additionally obtained from 10-wk-old female mice (ad libitum consumption, n = 10) at ZT12 was divided into endometrial and myometrial tissues. (D) The mRNA expression of Krt7 and Acta2 was significantly dominant in endometrial and myometrial tissues, respectively. **P < 0.01. (E) qPCR showed that there was no significant difference in the intensity of mRNA expression of clock genes (Per1, Per2, and Cry1) between endometrial and myometrial tissues. Differences in mRNA expression between the endometrium and myometrium were analyzed by the paired t test. Acta2, smooth muscle marker αSMA; Bmal1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1; CK-7, cytokeratin 7; Cry1, cryptochrome 1; Krt7, endometrial epithelial marker CK-7; ns, nonsignificant; Per, period circadian clock; ZT, Zeitgeber time.

FIGURE 4

Circadian rhythms of uterine clock during time restriction of food intake. To examine the relation between the delay of feeding during the active phase and uterine circadian rhythms, the uterine clock gene expression profiles in groups II and III were analyzed by qPCR. (A) Consistent with the results in group I, Bmal1 expression in group II (feeding during ZT12–16) was elevated after ZT12 at the start of the active phase and food intake. In contrast, Bmal1 expression in group III (feeding during ZT20–24) was elevated after ZT20 at the start of food intake, showing a significantly backward shift of circadian expression. Relative expression is presented as fold units per minimal value. This 8-h shift in circadian rhythms was also observed in Per2 and Cry1. (B, C) Western blot analysis confirmed a significant circadian rhythm of BMAL1 protein expression. The peak of protein expression was observed 8 h after that of mRNA expression. The cyclic changes in mRNA and protein expression of clock genes within each group (**P < 0.01; *P < 0.05, among ZTs) and the differences in mRNA expression of each ZT among groups I–III (^##P < 0.01; ^#P < 0.05, group II vs. group III) were analyzed by 1-factor ANOVA followed by the Dunnett test. αSMA, α-smooth muscle actin; Bmal1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1; Cry1, cryptochrome 1; Per, period circadian clock; TRF, time restriction of food intake; ZT, Zeitgeber time.