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The Rev-erbα gene is regarded as a circadian clock gene and clock-regulated gene which regulates the circadian transcriptional/translational loop in a subtle way. Here, we first detected the circadian oscillation in mature granulosa cells from antral follicles using a real-time monitoring system of Per2 promoter activity with the addition of FSH. Then we used GSK4112, an agonist ligand of Rev-erbα, to investigate the function of Rev-erbα. GSK4112 treatment significantly reduced the Per2-dLuc amplitude and induced the Per2 oscillation phase advance shift. GSK4112 significantly inhibited Bmal1 mRNA expression, whereas it did clearly stimulate expression of StAR mRNA in a dose-dependent manner. Our data are the first to show the Rev-erbα function in the steroid biosynthesis of rat granulosa cells, and to suggest that Rev-erbα may coordinate circadian rhythm and metabolism in rat ovaries., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter-destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation.

Circadian rhythms are modulated in a variety of peripheral tissues, including in the uterus where endometrial stromal cells (UESCs) undergo proliferation and differentiation (decidualization) during gestation. Here the authors focused on circadian rhythms in UESCs during implantation and decidualization in rodents. As revealed by analyses of cultured UESCs from pregnant Per2 promoter-dLuc transgenic rats, Per2 oscillation of ∼24  h was observed in response to dexamethasone. Per2 oscillation was enhanced in UESCs during implantation, whereas they were attenuated during decidualization. In vivo studies showed that PER2 protein in the uteri displayed a peak at zeitberger time 4 (ZT 4) (day 4.50 of gestation) and a trough at ZT 12 (day 4.83), indicating its circadian rhythmicity. Conversely, no significant circadian rhythm of the PER2 protein was observed during decidualization. Fluorescent immunohistochemical studies also supported circadian rhythmicity of the PER2 protein in its intracellular distribution. In accordance with Per2 mRNA expression, a circadian rhythm of vascular endothelial growth factor (Vegf) gene expression, having several E-box or E-box-like sites at the upstream of the transcription start site, was observed during implantation, showing a peak at ZT 0 and a trough at ZT 12. In contrast, Vegf mRNA expression displayed no circadian rhythm during decidualization. Collectively, the present results prove that Per2 oscillation is down-regulated in UESCs during decidualization. It is strongly suggested that cellular differentiation in UESCs interferes with circadian clockwork.

Androgen regulates the proper development and physiological function of the prostate. Here, we investigated the modulation of androgen and androgen receptor (AR) antagonist on circadian oscillations of a clock core gene Period 2 (Per2) in rat prostate mesenchymal cells (PMCs). Circadian oscillations were analyzed with the real-time monitoring system of gene expression using transgenic rats introduced with mouse Per2 promoter fused to a destabilized luciferase (Per2-dLuc) reporter gene. Analyses of circadian oscillations, immunofluorescence, and androgen response element (ARE)-luciferase reporter assay revealed that circadian clocks are operative and the AR protein is functional in PMCs in vitro. Androgen such as testosterone (T) and dihydrotestosterone (DHT) did not cause any changes in circadian Per2-dLuc oscillations of confluent cells. Conversely, flutamide (FL) up-regulated the amplitude of circadian Per2-dLuc oscillations in a dose-dependent manner, whereas T antagonized the action of FL. The PER2 protein was markedly accumulated by FL treatment and localized in both the nucleus and cytoplasm during the first peak period of circadian Per2-dLuc oscillations. Simultaneously, FL treatment increased apoptotic cell death. Collectively, the present study demonstrates that a clock gene Per2 is up-regulated in PMCs during FL-induced apoptotic cell death. Thus, circadian oscillations of Per2 gene expression may be closely linked to the cellular states of PMCs such as apoptotic cell death.

The circadian oscillator is generated within the suprachiasmatic nuclei and synchronizes circadian clocks in numerous peripheral tissues. The molecular basis is composed of a number of genes and proteins that form transcriptional and translational feedback loops. Such molecular oscillators are also operative in peripheral tissues, including in the uterus. Although ovarian steroids regulate the function of uterine endometrial stromal cells, the modulation of ovarian steroids on the circadian rhythms remains unknown. Here we investigate the possibility that estradiol (E2) and progesterone (P4) modulate the circadian oscillator of the stromal cells. The study using transgenic rats constructed with Period 2 (Per2) promoter-destabilized luciferase (Per2-dLuc) gene, with the real-time monitoring system of Per2-dLuc oscillation. The stromal cells displayed constant Per2-dLuc oscillation after treatment with dexamethasone, suggesting that the circadian oscillator is operative. However, the circadian oscillator was disrupted by in vivo administration of human chorionic gonadotropin (hCG) following equine chorionic gonadotropin (eCG), although it was altered into a rhythmic pattern 4 days later following hCG. Chronic treatment with P4 induced constant Per2-dLuc oscillation in the stromal cells from eCG-treated immature and pregnant rats, whereas E2 did not promote such a rhythmic Per2-dLuc oscillation. Collectively, P4 synchronizes the circadian oscillator of the uterus endometrial stromal cells through transcriptional and translational feedback loops of the clockwork system.

It has been established that estrogen can alter circadian rhythms in behavior and endocrine physiology in rodents. The uterus is a reproductive organ that is critically dependent on regulation by ovarian steroids. Here, we examined the expression of Per1 in different compartments of the uterus, and explored whether the ovarian steroids could regulate Per1 expression employing ovariectomized rat uterus. RT-PCR analysis showed that Per1 was cyclically expressed in the uterus. As revealed by in situ hybridization, the staining intensity of Per1 mRNA was stronger at ZT 8 than at ZT 0 in the uterine luminal epithelium (LE), stroma (S), and myometrium (M) compartments, but was not changed in the glandular epithelium (GE). Both in situ hybridization and immunofluorescence analyses revealed that estradiol (E(2)) administration induced high expression of Per1 in the LE, GE, and M, and less expression in the S compartment. Progesterone (P(4)) treatment resulted in an obvious enhancement of Per1 expression in the LE, GE, and S, but unchanged in the M compartment. Furthermore, the E(2)- and P(4)-activated Per1 expression was significantly repressed by their respective antagonists, ICI182 780 and RU486. These findings were further supported by RT-PCR analysis of Per1 expression in cultured uterine stromal cells. Collectively, the present data indicate that E(2) and P(4) might be involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.