Lijia Zhao

Publications (3)10.77 Total impact

• Article: Down-regulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells.

  Huatao Chen, Lijia Zhao, Makoto Kumazawa, Nobuhiko Yamauchi, Yasufumi Shigeyoshi, Seiichi Hashimoto, Masa-Aki Hattori
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  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 CCGs 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 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 siRNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared to non-silencing 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.
  AJP Cell Physiology 04/2013; · 3.54 Impact Factor
• Article: FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.

  Huatao Chen, Lijia Zhao, Guiyan Chu, Gakushi Kito, Nobuhiko Yamauchi, Yasufumi Shigeyoshi, Seiichi Hashimoto, Masa-Aki Hattori
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  ABSTRACT: The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by FSH. Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 d with FSH expressed higher mRNA level of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and Cx43 (Gja1) compared to the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG, and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.
  AJP Endocrinology and Metabolism 01/2013; · 4.75 Impact Factor
• Article: Rev-erbα regulates circadian rhythms and StAR expression in rat granulosa cells as identified by the agonist GSK4112.

  Huatao Chen, Guiyan Chu, Lijia Zhao, Nobuhiko Yamauchi, Yasufumi Shigeyoshi, Seiichi Hashimoto, Masa-aki Hattori
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  ABSTRACT: 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.
  Biochemical and Biophysical Research Communications 03/2012; 420(2):374-9. · 2.48 Impact Factor