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Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but few studies have been conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using primary culture of chicken embryonic myoblasts in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1) targeting GDF-8 mRNA was introduced into myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52-kDa GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)w-constructed luciferase reporter assay, a concomitant change in activity was initiated after 24 h. The activity rapidly decreased 30 h after siRNA-1 introduction, whereas high activity was maintained at 30-42 h in the control and siRNA-2-treated myoblasts. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and myotubes with aggregated nuclei were shorter and wider. These results strongly suggest that deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes. GDF-8; GDF-8-mediated promoter activity; multinucleated myotubes doi:10.1152/ajpcell.00543.2005

The role of nitric oxide (NO) in the regulation of epidermal growth factor (EGF) expression during differentiation of rat granulosa cells was investigated. Cultured cells showed a marked increase in guanosine 3',5'-cyclic monophosphate during development, but this was blocked by NO synthase inhibitors. The addition of L-arginine reversed this effect, suggesting that L-arginine is a potent suppressor of the inhibitory effects of NO synthase. Furthermore, exposure of the cells to sodium nitroprusside, an NO donor, increased EGF levels, indicating that NO is involved in EGF expression during cell differentiation.

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.)

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.

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.

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.