Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels
Introduction
The circadian and reproductive systems communicate bi-directionally through the brain and periphery to coordinate the timing of important reproductive events. The central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus generates daily oscillations [1] responsible for organizing the timing of most behaviors and physiological events in mammals, including the luteinizing hormone (LH) surge during the estrous cycle that triggers ovulation [2], [3]. In cycling female rodents, the LH surge occurs on the day of proestrus at a specific “critical period” in the late afternoon, approximately 2–4 h before lights-off [4], [5]. In female rats, SCN-lesions abolish this LH surge and prevent ovulation [6].
Reproductive events, in turn, have both direct and indirect feedback effects on the temporal organization of behavior and physiology. For example, in humans, menstruation, pregnancy and menopause have all been associated with sleep disorders and changes in body temperature rhythms [7], [8], [9], [10]. Similarly, circadian rhythms of activity significantly change during the estrous cycle in rodents [11], [12], [13], [14]: female rats show phase advances of locomotor activity and higher total activity during stages of proestrus and estrus relative to diestrus [11]. Estrogen itself alters circadian rhythms, as implants shorten the period of locomotor activity of female rats and hamsters while increasing both the amplitude and activity bout length [12], [13], [14]. Interestingly, these estrogenic effects are alleviated in the presence of progesterone, suggesting that complex regulation of the hormonal milieu across the estrous cycle is necessary for the observed behavioral effects.
Our understanding of the specific role of the SCN in both the timing of reproductive events and in the temporal reorganization during these events has been bolstered by our increased understanding of the molecular clock and its constituent clock genes, which drive circadian rhythms of gene expression and physiology with a 24 h period. These genes and their protein products are organized into interlocking positive and negative transcriptional and translational feedback loops that regulate circadian rhythm generation in the SCN [15]. A recent and important finding revealed that the same or similar molecular feedback loops found in SCN cells, are also present in a majority of peripheral organs [16]. Furthermore, evidence suggests that several of these peripheral clocks can continue to oscillate in the absence of the SCN [17]. Therefore, it is possible that peripheral oscillators may locally regulate physiology while also receiving modulatory timing cues from the SCN. Although numerous studies have described the expression of circadian clock genes in reproductive tissues, including the ovary [18], [19] and uterus [20], [21], the physiological function of clock genes in tissues of the reproductive system remains largely unknown. Further, the very nature of the temporal cues carrying timing information from the SCN to peripheral oscillators, and perhaps in the reverse direction, are also poorly understood. Several reports demonstrate reproductive dysfunctions in mice with clock gene mutations [22], [23]. For example, female Clock-mutant mice, which carry a 51-amino acid deletion in the transcriptional activation domain of the CLOCK protein gene, have irregular estrous cycles, do not have a normal LH surge on the day of proestrus [23] and fail to show circadian rhythms of clock gene expression in the uterus [22].
In the present study, our goal was to address two 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 [17β-estradiol (E2) and progesterone (P4)] during the cycle modulate the timing of clock gene expression in target oscillators. To address these hypotheses, we have employed four sets of experiments using multiple levels of analysis. First, using cycling rats, we measured diurnal rhythms of Per1, Per2, and Bmal1 mRNA as a function of estrous cycle stage in both reproductive (uterus and ovary) and non-reproductive tissues (liver). Second, using tissue explants from PER2::LUCIFERASE (PER2::LUC) knockin mice, we compared rhythmic properties of both central and peripheral clocks at different stages of the estrous cycle. To examine the putative role for circulating ovarian steroid hormones, we measured their lasting effects on the period of PER2::LUC expression rhythms in uterine explants from mice. Finally, using the MCF-7 human cancer cell line as a model for endocrine cells containing both steroid hormone receptors and circadian clock genes, we examined the effects of acute E2 or P4-treatment on the level of clock gene expression.
Section snippets
Experiment 1: Effects of the estrous cycle on diurnal rhythms of clock gene expression in peripheral tissues of rats
Intact female Wistar rats were obtained from Charles River Laboratories (Yokohama, Japan) at 7–8 weeks of age, and were maintained under controlled environmental conditions (temperature, 24 ± 1 °C; lights on at 06:00 to 18:00 h) with food and water available ad libitum for at least 2 weeks. Vaginal smears were taken every morning and those rats that exhibited at least two consecutive 4-day estrous cycles were used in the present study. Animals were sacrificed in groups on the same day of the
Experiment 1: Effects of the estrous cycle on diurnal rhythms of clock gene expression in peripheral tissues of rats
To determine whether fluctuating hormone levels during the estrous cycle affect the diurnal rhythm of clock gene expression in tissues of the reproductive system, we examined rhythms of Per1, Per2, and Bmal1 mRNA expression in the uterus, ovary, and liver of female rats at different stages of estrous using quantitative real-time PCR. Although not considered a tissue of the reproductive system, the liver is a well-established peripheral oscillator that displays robust circadian rhythms of clock
Discussion
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
Acknowledgements
We thank Dr. Dawn Loh, Dr. Tsuyoshi Watanabe, Dr. Shigeru Tomida, and Dr. Mayumi Kojima for their help in carrying out the experiments. We would like to express our gratitude to Mr. Andy Vosko for critical reading and comments on the manuscript. This work was supported by NIH Grants RO1 MH062517 (to GDB), Grants-in-Aid for the Promotion of Science for Young Scientists from Japanese Ministry of Education, Science and Culture 14011687 (to TJN), and the Friends of Semel Institute for Neuroscience
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