We observed that the rhythms of Per1, Per2
, and Bmal1
expression varied with the stage of the estrous cycle in both reproductive and non-reproductive tissues of female rats. Focusing on Per2
rhythms in the uterus, more robust daily rhythms with an advanced peak phase were observed on proestrus compared with other stages. This in vivo
result was also seen in the ex vivo
experiment as uterine explants exhibited a larger amplitude and phase advanced rhythm in PER2::LUC expression when cultures were prepared on proestrus relative to metestrus. These data strongly support our hypothesis that the estrous cycle drives changes in the timing of the circadian clocks in reproductive tissues. However, the impact of the estrous cycle varied across tissues. In previous work, we found evidence that estrogen may be an important regulator of clock gene expression. Eliminating the estrous-driven changes in estrogen and progesterone by ovariectomy and E2 implants altered circadian rhythms of Per1
mRNA expression in peripheral tissues including the liver, kidney, and uterus. These data suggest that E2 differentially regulates the rhythm of clock gene expression in these tissues [21
]. In the central nervous system, there is evidence that PER2 expression in the bed nucleus of the stria terminalis and central nucleus of the amygdala, but not the SCN, varies across the estrous cycle in female rats [29
]. In the present study, we also found that the rhythms of PER2::LUC expression in SCN explants were unaffected by the estrous stage. These data indicate significant tissue-specific effects of the estrous cycle on clock gene expression in both neural and non-neural oscillators, but not the SCN. It may be that these tissue-specific effects of the estrous cycle are mediated by cycle dependent changes in the distribution of hormone receptors and other proteins, such as coactivators and corepressors in the target tissue. For instance, in situ
hybridization, immunocytochemistry, and RT-PCR analyses indicate that the SCN contains ERβ [30
], whereas the liver and the kidney express abundant levels of ERα [33
] and ERα is predominantly expressed in the uterus [34
]. Further studies using additional molecular techniques will be required to determine how the estrous cycle modifies clock gene expression at the cellular level, with a focus on both steroid hormone binding to consensus sequences on target genes and pituitary peptide hormone receptor signaling mechanisms within target tissues.
Treatment of uterine explants with estrogen and progesterone concentrations intended to mimic the steroid hormone background of proestrus increased the amplitude and delayed the peak of PER2::LUC expression in the uterus. This effect is consistent with the response of the uterine rhythm of Per2 expression on proestrus that we observed in our in vivo and ex vivo experiments. Further, the phase-delay observed on the next treatment day matches the phase-delay of Per2 mRNA rhythm on estrus in vivo. These data support our second hypothesis, which suggests that fluctuating levels of circulating ovarian steroid hormones during the estrous cycle modulate the timing of clock gene expression in target oscillators.
We also found that P4, but not E2, acutely induces clock gene expression in MCF-7 human cancer cells. The MCF-7 expresses functional ERs and PRs [26
] and has been extensively used as a model to study steroid hormone dependent signaling mechanisms. While they do not intrinsically represent a model for the direct effects of the estrus cycle on clock gene expression, the fact that they express functional steroid receptors and clock genes makes them an excellent model for the study of steroid effects on the molecular clock. Therefore, by extension, we propose that fluctuating steroid hormone levels in the cycling female can affect the molecular clock in target cells (e.g. uterus, ovary, etc.) that are known to express the components of the molecular clock. We observed a dose dependent stimulatory effect of P4 on Per1
expression that was attenuated, but not abolished, by co-treatment with the P4-receptor antagonist RU-486. These data suggest that the molecular clock can be modulated by progesterone, acting via the PR in target cells. Further, 1 h treatment with P4 induced Per1
mRNA expression, but failed to stimulate either Per2
mRNA expression in MCF-7 cells. P4 and E2 are known to exert their effects by binding to cytoplasmic nuclear hormone receptors that in turn bind to unique DNA sequences referred to as PRE (progesterone response elements) or ERE (estrogen response elements) [37
]. There is a PRE-half sequence in the first intron and some ERE-half sites in the 5’ flanking regulatory regions of mouse Per1
gene (GeneBank accession number: AB030818). Taken together, it is possible that P4 directly enhances Per1
transcription following direct binding and activation of these target elements in the Per1
promoter. Although further studies using ER and PR positive or negative-cell lines will be required to determine how the classical nuclear steroid receptors regulate the molecular circadian clock, our results indicate that MCF-7 cells are a good model to analyze how the signaling from steroid hormones affects molecular clocks. Unlike in previous reports [38
], we did not observe a significant effect of E2 on clock gene expression in these cells. This difference is likely to due treatment parameters as in the previous work. That is, E2 was applied at a considerably higher concentration (1 µM) and duration (16 h) in that study relative to our present work (10 nM, 1 or 4 h). Another report also showed that 6 h of stimulation with 10 nM E2 induced Per1
mRNA expression in cultured rat uterine stroma cells [28
], the induction level in that study was ≤ 1.5-fold. Our previous ex vivo
study revealed that 10 nM – 10 µM E2 significantly shortened the period of PER2::LUC expression in cultured uterine explants, whereas P4 did not [24
]. These data support the notion that E2 may directly affect the period and phase of the circadian clock in ER-positive tissues, without affecting the amplitude of clock gene expression. Further, it is reasonable to speculate, based on the current data and previous reports, that E2 and P4 act differentially but in concert to modulate circadian rhythms of clock gene expression within target tissues.
In summary, we have identified estrous cycle dependent changes in clock gene expression rhythms in both reproductive and non-reproductive tissues. The influence of fluctuating hormone levels during the estrous cycle on rhythms of clock gene expression in reproductive tissues was confirmed by bioluminescence recordings of cultured uterine explants. We have also shown that progesterone treatment causes acute up-regulation of Per1, Per2, and Bmal1 expression in human breast cancer MCF-7 cells that are known to express nuclear hormone receptors. The effect of P4 in these cells was attenuated by the addition of a selective receptor antagonist, implicating classic PR-dependent signaling in the response of the clock to P4-treatment. Taken together, our results strongly support our hypotheses: 1) that the estrous cycle drives changes in the timing of the circadian clocks in reproductive tissues and 2) that fluctuating levels of circulating ovarian steroid hormones during the cycle modulate the timing of clock gene expression in target oscillators. Finally, our present and previous data strongly suggest that ovarian steroid hormones mediate these effects by acting on their cognate receptors. By adjusting the timing of the circadian clock in tissues such as the uterus across the days of the estrous cycle, ovarian steroid hormones may play a critical role in the stable homeostasis of female reproductive function.