Downregulation 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

Research output: Contribution to journalArticle

30 Citations (Scopus)

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-erb, 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-erb, 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.

Original languageEnglish
Pages (from-to)C1131-C1140
JournalAmerican Journal of Physiology - Cell Physiology
Volume304
Issue number12
DOIs
Publication statusPublished - Jun 25 2013

Fingerprint

Granulosa Cells
Prostaglandins
Progesterone
Down-Regulation
Luciferases
Genes
Luteinizing Hormone
Small Interfering RNA
Fertility
Transgenic Rats
Gene Expression
Computer Systems
RNA Interference
Reporter Genes
Dinoprostone
Mammals
Steroids
Hormones
RNA
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells. / Chen, Huatao; Zhao, Lijia; Kumazawa, Makoto; Yamauchi, Nobuhiko; Shigeyoshi, Yasufumi; Hashimoto, Seiichi; Hattori, Masa Aki.

In: American Journal of Physiology - Cell Physiology, Vol. 304, No. 12, 25.06.2013, p. C1131-C1140.

Research output: Contribution to journalArticle

Chen, Huatao ; Zhao, Lijia ; Kumazawa, Makoto ; Yamauchi, Nobuhiko ; Shigeyoshi, Yasufumi ; Hashimoto, Seiichi ; Hattori, Masa Aki. / Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells. In: American Journal of Physiology - Cell Physiology. 2013 ; Vol. 304, No. 12. pp. C1131-C1140.
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AU - Chen, Huatao

AU - Zhao, Lijia

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AU - Yamauchi, Nobuhiko

AU - Shigeyoshi, Yasufumi

AU - Hashimoto, Seiichi

AU - Hattori, Masa Aki

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AB - 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-erb, 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-erb, 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.

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