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The circadian system performs an important role in mammalian reproduction with significant effects on hormone secretion. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor in the circadian system and affects granulosa cells (GCs), but how it regulates estrogen synthesis has not been clarified. We investigated the effect of NR1D1 on estrogen synthesis and found that NR1D1 was highly expressed in GCs, mainly in cell nuclei. Additionally, the expression of NR1D1 and estrogen synthesis key genes CYP19A1, CYP11A1 and StAR showed rhythmic changes in porcine ovarian GCs. Activation of NR1D1 enhances its ability to inhibit the transcriptional activity of CYP19A1 by binding to the RORE on the CYP19A1 promoter, resulting in a decrease in estradiol content. Interference with NR1D1 can eliminate the transcriptional inhibition of CYP19A1 and promote the synthesis of estradiol. The results suggest that the hormone secretion of the ovary itself is also regulated by the biological clock, and any factors that affect the circadian rhythm can affect the endocrine and reproductive performance of sows, so the natural rhythm of sows should be maintained in production., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes ( Bmal1 , Per2 , Nr1d1 , and Dbp ) and an autophagy-related gene ( Atg5 ) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of Bmal1 , Per2 , and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1 -/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.

Cellular oscillators in the uterus play critical roles in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes, including growth/differentiation factor (Gdf)10 and Gdf15. The expression of Gdf10 and Gdf15 is significantly increased in the uterus during decidualization, but the mechanism underlying the regulation of Gdf gene expression in the uterus is poorly understood. Here, we focused on the function of the cellular oscillators in the expression of Gdf family by using uterine endometrial stromal cells (UESCs) isolated from pregnant Per2-dLuc transgenic rats. A significant decline of Per2-dLuc bioluminescence activity was induced in in vitro decidualized UESCs, and concomitantly the expression of canonical clock genes was downregulated. Conversely, the expression of Gdf10 and Gdf15 of the Gdf was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Gdf10 and Gdf15 were upregulated. However, Gdf5, Gdf7, and Gdf11 were not significantly affected by Bmal1 silencing. The expression of Gdf10 and Gdf15 was enhanced after treatment with a REV-ERBα antagonist in the presence or absence of progesterone. Chromatin immunoprecipitation-PCR analysis revealed the inhibitory effect of REV-ERBα on the expression of Gdf10 and Gdf15 in UESCs by recognizing their gene promoters. Collectively, our findings indicate that the attenuation of REV-ERBα leads to an upregulation of Gdf10 and Gdf15 in decidual cells, in which cellular oscillators are impaired. Our results provide novel evidence regarding the functions of cellular oscillators regulating the expression of downstream genes during the differentiation of UESCs., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes (Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2-O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E₂ production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE₂ production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization., (Copyright © 2015 the American Physiological Society.)

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family., (Copyright © 2015 the American Physiological Society.)

The nuclear receptor REV-ERBα links circadian rhythms and numerous physiological processes, but its physiological role in ovaries remains largely unknown. The aim of this study was to determine the potential role of REV-ERBα in the regulation of the transcription of its putative target genes in granulosa cells (GCs) prepared from Per2-destablized luciferase (dLuc) reporter gene transgenic rats. Alas1, Ppargc1a, and Il6 were chosen as representatives for genes analysis. A real-time monitoring system of Per2 promoter activity was performed to detect Per2-dLuc circadian oscillations. Two agonists (GSK4112, heme) and an antagonist (SR8278) of REV-ERBα as well as Rev-erbα siRNA knockdown were used to identify its target genes. Clear Per2-dLuc circadian oscillations were generated in matured GCs after synchronization with GSK4112 or SR8278. GSK4112 treatment lengthened and SR8278 treatment shortened the period of circadian oscillations in matured GCs stimulated with or without luteinizing hormone (LH). GSK4112 showed an inhibitory effect on the amplitude of circadian oscillations and caused an arrhythmic expression of canonical clock genes. SR8278 also had a subtle effect on their daily expression profiles, but the treatment resulted only in the arrhythmic expression of Rev-erbα. These findings indicate the functional biological activity of REV-ERBα in response to its ligands. Its natural ligand heme further elongated the period of circadian oscillations and alleviated their amplitudes in GCs cultured with LH. Heme treatment also repressed the expressions of clock genes, Alas1, Il6, and Ppargc1a. Rev-erbα knockdown up-regulated these transcript levels. Collectively, these data extend the recent finding to rat GCs and demonstrate that REV-ERBα represses the expressions of Alas1, Ppargc1a, and Il6, providing novel insights into the physiological significance of REV-ERBα in ovarian circadian oscillators.

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.)