Estrogen directly modulates circadian rhythms of PER2 expression in the uterus

Abstract

Fluctuations in circulating estrogen and progesterone levels associated with the estrous cycle alter circadian rhythms of physiology and behavior in female rodents. Endogenously applied estrogen shortens the period of the locomotor activity rhythm in rodents. We recently found that estrogen implants affect Period (Per) gene expression in the suprachiasmatic nucleus (SCN; central clock) and uterus of rats in vivo. To explore whether estrogen directly influences the circadian clock in the SCN and/or tissues of the reproductive system, we examined the effects of 17beta-estradiol (E(2)) on PER2::LUCIFERASE (PER2::LUC) expression in tissue explant cultures from ovariectomized PER2::LUC knockin mice. E(2) applied to explanted cultures shortened the period of rhythmic PER2::LUC expression in the uterus but did not change the period of PER2::LUC expression in the SCN. Raloxifene, a selective estrogen receptor modulator and known E(2) antagonist in uterine tissues, attenuated the effect of E(2) on the period of the PER2::LUC rhythm in the uterus. These data indicate that estrogen directly affects the timing of the molecular clock in the uterus via an estrogen receptor-mediated response.

Fig. 1.

Effects of estrogen on the period of PER2::LUCIFERASE (PER2::LUC) expression in the suprachiasmatic nucleus (SCN) of ovariectomized (OVX) mice. A: representative bioluminescence traces showing the effects of 17β-estradiol (E₂) on circadian rhythms of PER2::LUC expression in cultured SCN explants from OVX mice. The SCN was treated with vehicle [100 μM β-cyclodextrin (βCD)], 10 nM E₂, or 10 μM E₂. B: mean period values (±SE) for each group. Numbers in parentheses indicate the number of animals in each treatment group.

Fig. 2.

Estrogen shortens the period of PER2::LUC expression in the uterus of OVX mice. A–E: representative bioluminescence traces showing the effects of E₂ on circadian rhythms of PER2::LUC expression in cultured uterine explants from OVX mice. The uterus was treated with vehicle (100 μM β-cyclodextrin) or 1 nM (A), 10 nM (B), 100 nM (C), 1 μM (D), or 10 μM E₂ (E). The black trace in each graph is the same vehicle control trace. F: mean period values (±SE) for each group. Numbers in parentheses indicate the number of animals in each group. **P < 0.01 and *P < 0.05 when compared with vehicle group (Tukey's test).

Fig. 3.

Effects of progesterone (P₄) on the period of PER2::LUC expression in the uterus of OVX mice. A–D: representative bioluminescence traces showing the effects of P₄ on circadian rhythms of PER2::LUC expression in cultured uterine explants from OVX mice. The uterus was treated with vehicle (100 μM βCD) or 10 nM (A), 100 nM (B), 1 μM (C), or 10 μM P₄ (D). The black trace in each graph is the same vehicle control trace. E: mean period values (±SE) for each group. Numbers in parentheses indicate the number of animals in each group.

Fig. 4.

Raloxifene (RLX) attenuates the effect of estrogen on the period of PER2::LUC expression in the uterus of OVX mice. A, C, D, and E: representative bioluminescence traces showing the effects of RLX on circadian rhythms of PER2::LUC expression in untreated and E₂-treated uterine explants from OVX mice. A: uterine cultures treated with vehicle (DMSO) or 2 concentrations (10 nM or 10 μM) of RLX in DMSO. B: Mean period values (±SE) for cultures treated with RLX alone. C: uterine cultures treated with vehicle containing both DMSO and 100 μM βCD. D and E: cultures treated with 10 nM E₂ + 10 nM RLX (D) or 10 nM E₂ + 10 μM RLX (E). The plot of vehicle control data is duplicated in C–E. F: mean period values (±SE) for vehicle-, E₂-, and E₂ + RLX-treated cultures. Numbers in parentheses indicate the number of animals in each group. *P < 0.05 when compared with DMSO + 10 nM E₂ treatment group (Tukey's test).