Publications by authors named "Lijia Zhao"

16 Publications

  • Page 1 of 1

Glyphosate exposure attenuates testosterone synthesis via NR1D1 inhibition of StAR expression in mouse Leydig cells.

Sci Total Environ 2021 Sep 25;785:147323. Epub 2021 Apr 25.

Northwest A&F University, Yangling 712100, Shaanxi, China; Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China. Electronic address:

Glyphosate is a broad-spectrum herbicide that impairs testosterone synthesis in mammals. Leydig cells (LCs), the primary producers of testosterone, demonstrate rhythmic expression of circadian clock genes both in vivo and in vitro. The nuclear receptor NR1D1 is an important clock component that constitutes the subsidiary transcriptional/translational loop in the circadian clock system. Nr1d1 deficiency resulted in diminished fertility in both male and female mice. However, whether NR1D1 is involved in the glyphosate-mediated inhibition of testosterone synthesis in LCs remains unclear. Here, the involvement of NR1D1 in glyphosate-mediated inhibition of testosterone synthesis was investigated both in vitro and in vivo. Glyphosate exposure of TM3 cells significantly increased Nr1d1 mRNA levels, but decreased Bmal1, Per2, StAR, Cyp11a1, and Cyp17a1 mRNA levels. Western blotting confirmed elevated NR1D1 and reduced StAR protein levels following glyphosate exposure. Glyphosate exposure also reduced testosterone production in TM3 cells. In primary LCs, glyphosate exposure also upregulated Nr1d1 mRNA levels and downregulated the mRNA levels of other clock genes (Bmal1 and Per2) and steroidogenic genes (StAR, Cyp17a1, Cyp11a1, and Hsd3b2), and inhibited testosterone synthesis. Moreover, glyphosate exposure significantly reduced the amplitude and shortened the period of PER2::LUCIFERASE oscillations in primary LCs isolated from mPer2 knock-in mice. Four weeks of oral glyphosate upregulated NR1D1 at both the mRNA and protein levels in mouse testes, and this was accompanied by a reduction in StAR expression. Notably, serum testosterone levels were also drastically reduced in mice treated with glyphosate. Moreover, dual-luciferase reporter and EMSA assays revealed that in TM3 cells NR1D1 inhibits the expression of StAR by binding to a canonical RORE element present within its promoter. Together, these data demonstrate that glyphosate perturbs testosterone synthesis via NR1D1 mediated inhibition of StAR expression in mouse LCs. These findings extend our understanding of how glyphosate impairs male fertility.
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http://dx.doi.org/10.1016/j.scitotenv.2021.147323DOI Listing
September 2021

Different Phenomena in Magnetic/Electrical Properties of Co(II) and Ni(II) Isomorphous MOFs.

ACS Omega 2021 Apr 26;6(13):9213-9221. Epub 2021 Mar 26.

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Research & Development Center for Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming 650091, P. R. China.

Two unprecedented and stable metal-organic frameworks, {[Co(HO)(L)(OH)]·2.5HO·0.5DMF} () and {[Ni(HO)(L)(OH)]·1.75HO} (), have been synthesized (HL = 5-(5-carboxy-pyridin-3-yloxy)-isophthalic acid, DMF = ,-dimethylformamide). Structural analysis shows that and are heteronuclear isomorphous, possessing a three-dimensional (3D) (4,8)-connected flu/fluorite topological framework formed through the interconnection of tetranuclear butterfly {M(COO)(OH)} clusters and the ligands. Although the frameworks of these two compounds are similar, their magnetic properties are different. Compound exhibits an antiferromagnetic interaction in the high-temperature region, while shows a weak ferromagnetic interaction in the whole-temperature region. Furthermore, considering the presence of hydroxyl groups and water molecules in the frameworks, we tested their proton conductivity. The efficient proton transfer pathway in the framework endowed and with excellent proton conductivities of 9.07 × 10 and 1.29 × 10 S·cm at 363 K and 98% relative humidity (RH), respectively.
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http://dx.doi.org/10.1021/acsomega.1c00574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028129PMC
April 2021

Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells.

J Cell Physiol 2021 Sep 17;236(9):6706-6725. Epub 2021 Feb 17.

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0-5 IU/ml) and 22R-OH-cholesterol (0-30 μM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.
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http://dx.doi.org/10.1002/jcp.30334DOI Listing
September 2021

promotes prostaglandin E synthesis by upregulating transcription in response to increasing estradiol levels in pregnant mice.

Am J Physiol Endocrinol Metab 2021 04 8;320(4):E747-E759. Epub 2021 Feb 8.

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, China.

Prostaglandin G/H synthase 2 (PTGS2) is a rate-limiting enzyme in prostaglandin synthesis. The present study assessed the role of the uterine circadian clock on transcription in response to steroid hormones during early pregnancy. We demonstrated that the core clock genes (, , , and ), , and and their encoded proteins, have rhythmic expression in the mouse uterus from to () of pregnancy. Progesterone (P) treatment of cultured uterus endometrial stromal cells (UESCs) isolated from reporter gene knock-in mice on D4 induced a phase shift in oscillations. This P-induced phase shift of oscillations was significantly attenuated by the P antagonist RU486. Additionally, the amplitude of oscillations was increased by estradiol (E) treatment in the presence of P. Consistently, the mRNA levels of clock genes ( and ), , and were markedly increased by E treatment of UESCs in the presence of P. Treatment with E also promoted prostaglandin E (PGE) synthesis by UESCs. Depletion of in UESCs by small-interfering RNA (siRNA) decreased the transcript levels of clock genes ( and ), , and compared with nonsilencing siRNA treatment. knockdown also inhibited PGE synthesis. Moreover, the mRNA expression levels of clock genes ( and ), , and , and their respective proteins were significantly decreased in the uterus of mice. Thus, these data suggest that in mice promotes PGE synthesis by upregulating in response to increases in E on D4 of pregnancy. Rhythmic expression of Bmal1 and Ptgs2 was observed in the uterus isolated from of pregnant mice. E increased the expression of Bmal1 and Ptg2 in UESCs isolated from mice on D4. The expression of Ptgs2 was significantly decreased in Bmal1-siRNA treated UESCs. knockdown also inhibited PGE synthesis. Thus, these data suggest that Bmal1 in mice promotes PGE synthesis by upregulating Ptgs2 in response to increases in E on D4 of pregnancy.
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http://dx.doi.org/10.1152/ajpendo.00466.2020DOI Listing
April 2021

Bisphenol A attenuates testosterone production in Leydig cells via the inhibition of NR1D1 signaling.

Chemosphere 2021 Jan 23;263:128020. Epub 2020 Aug 23.

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China. Electronic address:

Bisphenol A (BPA) is an endocrine-disrupting compound that impairs testosterone synthesis in male mammals. A circadian clock gene deficiency leads to diminished fertility and even infertility in male mice. However, whether circadian clock signaling pathways mediate the suppressive effect of BPA on testosterone synthesis in Leydig cells (LCs) remains unknown. The present study aims to detect the effect of BPA on cellular circadian clock and testosterone synthesis in mouse LCs, and examine the mechanisms underlying NR1D1 signaling. BPA treatment significantly attenuated the transcription levels of Nr1d1 and steroidogenic genes (Hsd3b2 and Hsd17b3) in TM3 cells, but increased other circadian clock gene levels (Per2 and Dbp). BPA treatment also significantly downregulated NR1D1 and StAR protein expression, but upregulated BMAL1 protein expression in TM3 cells. Furthermore, there was a marked decline in testosterone production in BPA-treated TM3 cells. Intraperitoneal injection of BPA profoundly reduced NR1D1 and StAR protein levels and steroidogenic gene transcription levels (Cyp11a1, Hsd3b2, and Hsd17b3), while enhancing BMAL1 protein and other circadian clock gene (Per2 and Dbp) levels in mouse testes. Notably, serum testosterone levels were also drastically reduced in BPA-treated mice. Moreover, SR9009, an NR1D1 agonist, augmented testosterone production in TM3 cells via elevated expression of steroidogenic genes (StAR, Cyp11a1 and Hsd17b3). Conversely, Nr1d1 knockdown inhibited testosterone accumulation and attenuated steroidogenic gene expression. Moreover, treatment with SR9009 partially reversed the BPA effect on the circadian clock and testosterone production. Taken together, our study demonstrates that BPA perturbs testosterone production, at least partially, via inhibiting NR1D1 signaling in LCs.
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http://dx.doi.org/10.1016/j.chemosphere.2020.128020DOI Listing
January 2021

Zearalenone perturbs the circadian clock and inhibits testosterone synthesis in mouse Leydig cells.

J Toxicol Environ Health A 2021 Feb 4;84(3):112-124. Epub 2020 Nov 4.

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University , Yangling, China.

Zearalenone (ZEA), a mycotoxin, is known to impair reproductive capability by disrupting the synthesis and secretion of testosterone by Leydig cells (LCs), although the mechanism is unknown. Robust rhythmicity of circadian clock and steroidogenic genes were identified in LCs. The aim of this study was to examine whether ZEA significantly attenuated the transcription of core clock genes (, and ) as well as steroidogenic genes (, and ) in mouse testis Leydig cell line (TM3). Western blotting confirmed declines in BMAL1, NR1D1, and StAR protein levels. ZEA also suppressed secreted testosterone levels. In primary LCs, isolated from PER2::LUCIFERASE reporter gene knock in mice, ZEA diminished the amplitude of expression, and induced a phase shift and period extension. In primary LCs, ZEA also suppressed the expression levels of core clock and steroidogenic genes, reduced protein levels of BMAL1, and decreased testosterone secretion. expression of core clock and steroidogenic genes were reduced in testes of mice exposed to ZEA for 1 week leading to decreased serum testosterone levels. In summary, data suggest that ZEA may impair testosterone synthesis through attenuation of the circadian clock in LCs culminating in reproductive dysfunction in male mammals .
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http://dx.doi.org/10.1080/15287394.2020.1841699DOI Listing
February 2021

Focused Ion Beam-Induced Displacive Phase Transformation From Austenite to Martensite during Fabrication of Quenched and Partitioned Steel Micro-Pillar.

J Alloys Compd 2020 ;812

Graduate Institute of Ferrous Technology, Pohang University of Science and Technology, Pohang, 37673, South Korea.

We report evidence of a displacive phase transformation from retained austenite to martensite during preparation of quenched and partitioned steel micro-pillars by using a focused ion beam (FIB) technique. The BCC phase produced by the FIB damage was identified as martensite. The invariant-plane strain surface relief associated with the martensitic transformation was observed in the retained austenite phase immediately after a FIB scan of the surface with the Ga ion beam. Use of a low acceleration voltage appears to lower the probability of the phase transformation, while a decrease of the acceleration voltage will result in an increase of the total milling time required to prepare a micro-pillar. This report addresses challenges related to the preparation of austenite micro-pillars by a conventional FIB technique.
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http://dx.doi.org/10.1016/j.jallcom.2019.152061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047741PMC
January 2020

Combination of dynamic transformation and dynamic recrystallization for realizing ultrafine-grained steels with superior mechanical properties.

Sci Rep 2016 12 14;6:39127. Epub 2016 Dec 14.

Department of Materials Science and Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan.

Dynamic recrystallization (DRX) is an important grain refinement mechanism to fabricate steels with high strength and high ductility (toughness). The conventional DRX mechanism has reached the limitation of refining grains to several microns even though employing high-strain deformation. Here we show a DRX phenomenon occurring in the dynamically transformed (DT) ferrite, by which the required strain for the operation of DRX and the formation of ultrafine grains is significantly reduced. The DRX of DT ferrite shows an unconventional temperature dependence, which suggests an optimal condition for grain refinement. We further show that new strategies for ultra grain refinement can be evoked by combining DT and DRX mechanisms, based on which fully ultrafine microstructures having a mean grain size down to 0.35 microns can be obtained without high-strain deformation and exhibit superior mechanical properties. This study will open the door to achieving optimal grain refinement to nanoscale in a variety of steels requiring no high-strain deformation in practical industrial application.
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http://dx.doi.org/10.1038/srep39127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155429PMC
December 2016

The nuclear receptor REV-ERBα represses the transcription of growth/differentiation factor 10 and 15 genes in rat endometrium stromal cells.

Physiol Rep 2016 Feb;4(2)

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan

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.
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http://dx.doi.org/10.14814/phy2.12663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760387PMC
February 2016

Integration of the nuclear receptor REV-ERBα linked with circadian oscillators in the expressions of Alas1, Ppargc1a, and Il6 genes in rat granulosa cells.

Chronobiol Int 2015 23;32(6):739-49. Epub 2015 Jun 23.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University , Fukuoka , Japan .

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.
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http://dx.doi.org/10.3109/07420528.2015.1042582DOI Listing
April 2016

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

Am J Physiol Endocrinol Metab 2015 Apr 3;308(8):E650-61. Epub 2015 Feb 3.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan;

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.
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http://dx.doi.org/10.1152/ajpendo.00533.2014DOI Listing
April 2015

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

Am J Physiol Cell Physiol 2015 Apr 14;308(7):C528-38. Epub 2015 Jan 14.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan;

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.
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http://dx.doi.org/10.1152/ajpcell.00220.2014DOI Listing
April 2015

Profiling of circadian genes expressed in the uterus endometrial stromal cells of pregnant rats as revealed by DNA microarray coupled with RNA interference.

Front Endocrinol (Lausanne) 2013 8;4:82. Epub 2013 Jul 8.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University , Fukuoka , Japan.

The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation by a DNA microarray. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations. As revealed by the biological pathway analysis using the database for annotation, visualization, and integrated discovery online annotation software, genes were involved in cell cycle, glutathione metabolism, MAPK signaling pathway, fatty acid metabolism, ubiquitin mediated proteolysis, focal adhesion, and PPAR signaling pathway. The clustering of clock genes were mainly divided into four groups: the first group was Rorα, Timeless, Npas2, Bmal1, Id2, and Cry2; the second group Per1, Per2, Per3, Dec1, Tef, and Dbp; the third group Bmal2, Cry1, E4bp4, Rorβ, and Clock; the fourth group Rev-erbα. Eleven implantation-related genes and 24 placenta formation-related genes displayed significant alterations, suggesting that these genes involved in implantation and placenta formation are controlled under circadian clock. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa was significantly increased. During active oscillation of circadian clock, the apoptosis-related genes Fas and Caspase3 remained no significant changes, but they were significantly increased by knockdown of Bmal1 mRNA. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation.
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http://dx.doi.org/10.3389/fendo.2013.00082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703733PMC
July 2013

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

Am J Physiol Cell Physiol 2013 Jun 17;304(12):C1131-40. Epub 2013 Apr 17.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1152/ajpcell.00008.2013DOI Listing
June 2013

FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.

Am J Physiol Endocrinol Metab 2013 Mar 8;304(6):E566-75. Epub 2013 Jan 8.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.

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 follicle-stimulating hormone (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 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with 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.
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http://dx.doi.org/10.1152/ajpendo.00432.2012DOI Listing
March 2013

Rev-erbα regulates circadian rhythms and StAR expression in rat granulosa cells as identified by the agonist GSK4112.

Biochem Biophys Res Commun 2012 Apr 8;420(2):374-9. Epub 2012 Mar 8.

Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1016/j.bbrc.2012.02.164DOI Listing
April 2012