Search

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.

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter-destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation.

Circadian rhythms are modulated in a variety of peripheral tissues, including in the uterus where endometrial stromal cells (UESCs) undergo proliferation and differentiation (decidualization) during gestation. Here the authors focused on circadian rhythms in UESCs during implantation and decidualization in rodents. As revealed by analyses of cultured UESCs from pregnant Per2 promoter-dLuc transgenic rats, Per2 oscillation of ∼24 h was observed in response to dexamethasone. Per2 oscillation was enhanced in UESCs during implantation, whereas they were attenuated during decidualization. In vivo studies showed that PER2 protein in the uteri displayed a peak at zeitberger time 4 (ZT 4) (day 4.50 of gestation) and a trough at ZT 12 (day 4.83), indicating its circadian rhythmicity. Conversely, no significant circadian rhythm of the PER2 protein was observed during decidualization. Fluorescent immunohistochemical studies also supported circadian rhythmicity of the PER2 protein in its intracellular distribution. In accordance with Per2 mRNA expression, a circadian rhythm of vascular endothelial growth factor (Vegf) gene expression, having several E-box or E-box-like sites at the upstream of the transcription start site, was observed during implantation, showing a peak at ZT 0 and a trough at ZT 12. In contrast, Vegf mRNA expression displayed no circadian rhythm during decidualization. Collectively, the present results prove that Per2 oscillation is down-regulated in UESCs during decidualization. It is strongly suggested that cellular differentiation in UESCs interferes with circadian clockwork.

Hasebe, M., Soh, T., Hattori, M. and Fujihara, N. 1998. An attempt to produce transgenic chicken mediating sperm cells as vectors. J. Appl. Anim. Res., 14: 143–150. An attempt was made to produce possible transgenic chicken via sperm cells as vectors. In vitro incubation of ejaculated chicken spermatozoa with exogenous gene (MiwZ DNA) showed a possibility of the incorporation of foreign gene into sperm cells, which was examined by the PCR analysis. The embryos obtained from the eggs inseminated with DNA-treated spermatozoa indicated an existence of transferred exogenous gene in the blastodermal cells of the incubated fertile eggs. Exogenous gene injected into rooster testes was neither detected in the sperm cells nor in the blastodermal cells of fertilized eggs, which were inseminated with spermatozoa obtained from the testes-injected rooster. Sperm head, tail and cell membrane separated from DNA-treated spermatozoa revealed to contain foreign DNA, suggesting a possibility of incorporation of exo...

Zhao, J., Hattori, M.-A. and Fujihara, N. 1998. Effect of Japanese Kampo Medicines on in vitro maturation of bovine oocytes and developmental capacity of embryos. J. Appl. Anim. Res., 13: 93–104. Experiments were conducted to examine the effect on in vitro maturation of bovine oocytes and subsequent developmental capacity of embryos of several Japanese Kampo Medicines (JKMs), which have been reported to be effective in the treatment for reproductive failure in both women and rats. In the first experiment, the most effective JKM and its optimal concentration were determined by adding JKMs to bovine calf serum (CS)-free TCM 199 medium and examining developmental capacities of oocytes after fertilization in vitro. Similarly, in the second experiment, the comparison of the effect of the most effective JKM on in vitro maturation with that of CS was carried out. The morphological characteristics of oocytes after in vitro maturation were also examined using transmission electron microscopy. Keishibukury...

Hanada, K., Hattori, M-A. and Fujihara, N. 1997. Improvement of rat embryo implantation by epidermal growth factor. J. Appl. Anim. Res., 12:71–80. Epidermal Growth Factor (EGF) plays an important role in embryo implantation as well as in growth and differentiation of uterine cells. The present study was undertaken to investigate whether administration of EGF will improve implantation of embryos in rats. The effect of EGF on decidualization was also determined in pseudopregnant rats. The EGF was directly injected into each uterine horn of normal rats on day 5 of pregnancy. The intraluminal injection of EGF was found to be effective in inducing implantation as well as decidualization in both normal ovulation and PMSG- and hCG-induced ovulation in a dose-dependent manner. In addition, to examine the effect of EGF on implantation of embryos transferred into recipient rats, EGF was simultaneously injected into the uterine horns with the embryos (blastocysts) obtained from donor rats. The EGF was signi...

Fujihara, N., Zhao, J. and Hattori, M.-A. 1997. Artificial control of membrane function of rooster spermatozoa. J. Appl. Anim. Res., 11: 137–144. The uptake of non-metabolizable substances such as α-aminoisobutyric acid (AIB), 2-deoxy-D-glucose (2DG), and a cryoprotectant, glycerol, by rooster spermatozoa was compared between two lines, commercial broiler males and genetically selected broiler line males, (called generation 12) to determine whether membrane junction of sperm cells could be controlled by genetical selection of male birds. The freezability of rooster spermatozoa was shown to be controlled by the genetical selection of male birds. The results obtained in the present study suggested that membrane function of rooster spermatozoa could be controlled genetically and possibility of genetical selection of male birds exists for the tolerance of spermatozoa to freezing and thawing.