Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells

Abstract

Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.

Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells

“…Bmal1 control of Ptgs2 transcription has been suggested in other tissues (7,22,25,38). Moreover, our previous data obtained from a DNA microarray in UESCs isolated from day 4.5 of rat pregnancy (49) suggested that the expression level of the Ptgs2 transcript is rhythmic.…”

“…A recent study showed that the Ptgs2 protein is gradually expressed in the rat endometrium in response to deciduogenic stimuli (14). More recently, several reports have indicated that the transcription of the Ptgs2 gene is controlled by the cellular circadian clock (7,22,25,56).A growing number of studies have provided evidence that numerous peripheral circadian clock systems are partially selfoperative and independent in their responses to external and internal stimuli other than the stimuli originating from the suprachiasmatic nucleus, known as the central pacemaker of the circadian clock (17,30,55). Circadian clock systems generate cellular rhythms in physiological functions via identified transcriptional and posttranscriptional regulatory processes, which comprise a core group of clock genes and their protein products.…”

“…A recent study showed that the Ptgs2 protein is gradually expressed in the rat endometrium in response to deciduogenic stimuli (14). More recently, several reports have indicated that the transcription of the Ptgs2 gene is controlled by the cellular circadian clock (7,22,25,56).…”

Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells

“…The cultured UESCs were first plated in 35-mm collagen-coated dishes with 2 ml of DMEM/F12 supplemented with 1ϫ AA, 1ϫ ITS, and 0.1% BSA. After 24 h in culture, the medium was removed, and the Bmal1 siRNA and nonsilencing RNA diluted in Opti-MEM was transfected into cells using Lipofectamine RNAiMAX reagent (Life Technologies), according to the manufacturer's protocol (12). Both the Bmal1 siRNA and nonsilencing RNA were used at a final concentration of 25 nM.…”

Biological rhythms and fertility: the hypothalamus–pituitary–ovary axis

“…40,55 For example, Bmal1 knockdown in rat granulosa cells causes, in addition to the downregulation of core clock gene expression (Per1, Per2, Rev-erbα), the disruption of the circadian expression of several ovarian genes, including the LH receptor gene Lhcgr. 56 Similarly, it is plausible, but to date not fully proven, that a circadian clock system also controls the rhythmic expression of clock genes in the theca cells in the ovary. 40 Similarly to the ovaries, a rhythmic expression of cir cadian clock genes, possibly maintained through ovarian signals, 28 is evident in the uterus.…”