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

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.

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

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.

REV-ERBα inhibits the PTGS2 expression in bovine uterus endometrium stromal and epithelial cells exposed to ovarian steroids.

The nuclear receptor REV-ERBα (encoded by NR1D1) has a critical role in metabolism and physiology as well as circadian rhythm. Here, we investigated the possible contribution of clock genes including NR1D1 to the secretion of prostaglandin F2α (PGF2α) from bovine uterine stromal (USCs) and epithelial cells (UECs) by modulating the expression of PTGS2. The circadian oscillation of clock genes in the cells was weak compared with that reported in rodents, but the expression of BMAL1, PER1, and NR1D1 was changed temporally by treatment with ovarian steroids. Significant expression of clock genes including NR1D1 was detected in USCs exposed to progesterone. NR1D1 was also significantly expressed in UECs exposed to estradiol. The expression of PTGS2 was suppressed in USCs exposed to progesterone, while the expression was initially suppressed in UECs exposed to estradiol and then increased after long-term exposure to estradiol. BMAL1 knockdown with specific siRNA caused a significant decrease in the transcript levels of NR1D1 and PTGS2 in USCs, but not in UECs. The production of PGF2α also decreased in USCs after BMAL1 knockdown, while its level did not significantly change in UECs. The transcript level of PTGS2 was increased by treatment with the antagonist of REV-ERBα in both cell types, but the agonist was ineffective. In these two cell types treated with the agonist or antagonist, the PGF2α production coincided well with the PTGS2 expression. Collectively, these results indicate that REV-ERBα plays an inhibitory role in the expression of PTGS2 in both bovine USCs and UECs treated with ovarian steroids.

The putative promoters of germ cell-specific genes and Nanog are hypomethylated in chicken sperm.

Germ cell-specific genes such as Ddx4, Dnd1, and Dazl play critical roles in the proliferation and survival of germ cells. However, the methylation state of the promoter in mature germ cells is still unknown. Here, we investigated the methylation levels of these genes and the pluripotency marker gene Nanog in chicken sperm as compared with the Alb gene in the liver. CpG islands and/or promoter motifs such as TATA box, GC box and CAAT box were found within the putative promoter regions that we identified. By using the bisulfite reaction, CpG sites in the putative promoters were converted, and they were analyzed by sequencing. The putative promoters of Ddx4, Dnd1, Dazl and Nanog showed very low methylation levels in sperm, but they were highly methylated in the liver. Conversely, the Alb gene promoter was highly methylated in sperm and hypomethylated in the liver. However, no transcripts of Ddx4, Dnd1, Dazl and Nanog were detected in sperm or the liver. Also, no transcripts of Dnmt1 and Dnmt3a were detected in sperm. Our present results may indicate that these germ cell-specific genes and the pluripotency marker gene are ready to express any time after fertilization. Our findings showing that low methylation and selective DNA methylation of specific genes are present in chicken sperm contribute to our understanding of fertilization and embryogenesis of birds.

Expression and regulation of Foxa2 in the rat uterus during early pregnancy.

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.

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

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.

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

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.

Effect of PPARß/δ agonist on the placentation and embryo-fetal development in rats.

BACKGROUND: The present study was conducted to evaluate the developmental toxicity in the endometrium and placenta due to GW501516 administration by gavage to pregnant rats. METHODS: GW501516 was orally administered repeatedly to pregnant rats from gestation day (GD) 6 to 17 at a dose of 0, 30, and 100 mg/kg/day. In next study, GW501516 was also orally administered to pregnant rats on GD 7, 8, 9, 10, or 11 at a single dose of 275 or 350 mg/kg. In these studies, caesarean section was performed to examine the pregnancy outcome on GD21. Additionally, GW501516 was orally administered to pregnant rats on GD 10 at a single dose of 275 mg/kg. Placentae were subjected for temporal histological examinations on GD 11, 13, 15, or 17. RESULTS: Placental malformation was induced by repeated administration of GW501516 at a dose of 100 mg/kg/day. Single oral administration of GW501516 at a dose of 275 and/or 350 mg/kg on GD 8, 9, 10, or 11 induced placental malformation, whereas GW501516 administered on GD 10 was the most effective for increasing placental malformation. Histopathologically, single oral administration of GW501516 on GD 10 induced cystic degeneration associated with cellular lysis of glycogen cells started from GD 15 in the basal zone. CONCLUSIONS: High frequency of placental malformation was observed by the administration of GW501516. From GD 8 to 11, especially GD 10, is more sensitive period to induce the placental malformation.

Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation.

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.

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

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.

Development of a single bovine embryo improved by co-culture with trophoblastic vesicles in vitamin-supplemented medium.

To improve the development of singly cultured bovine embryos, we developed a co-culture method with trophoblastic vesicles. The growth of trophoblastic cells was markedly increased in vitamin-supplemented medium 199 compared with medium 199. Upon co-culture of a single embryo with trophoblastic vesicles in vitamin-supplemented medium 199, embryo development to the blastocyst stage was significantly higher than in embryos co-cultured with trophoblastic vesicles in RPMI 1640 or with cumulus cells in medium 199 (control). In the absence of the vitamin cocktail, co-culture with trophoblastic vesicles in medium 199 did not improve embryo development compared with that of the control. The vitamin cocktail was effective in embryo development when co-cultured with trophoblastic vesicles, but not with cumulus cells. Embryo development was not improved in the absence of co-cultured trophoblastic vesicles, even in the presence of vitamin cocktail. In conclusion, the co-culture system with trophoblastic vesicles in vitamin-supplemented medium 199 efficiently enhances the development of singly cultured embryos.

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy.

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARß/δ were strongly detected in the endometrial stroma on days 4.5-6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARß/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARß/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARß/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.

Upregulation of P-glycoprotein activity in porcine oocytes and granulosa cells during in vitro maturation.

Multidrug resistance P-glycoprotein (Pgp), coded by the multidrug resistance type I (MDR1/ABCB1) gene, is an energy-dependent efflux pump and functions in systemic detoxification processes. In the present study, the expression and development of Pgp were evaluated in the porcine oocyte during in vitro maturation to compare with the expression of Pgp in cultured granulosa cells. As revealed by Western blotting using anti-human Pgp antibody, a single band of Pgp with an apparent molecular size of 170 kDa was detected in the germinal vesicle stage oocytes. The surface of GV oocyte was positively labeled by immunostaining. In the second metaphase oocyte after culture in the maturation medium containing porcine follicular fluid and human chorionic gonadotropin, the level of Pgp was increased. The elevation of the oocyte Pgp level was associated with increased activity of rhodamine 6G efflux from the oocyte, and its efflux was suppressed by verapamil, an inhibitor of Pgp. Removal of porcine follicular fluid from the maturation medium resulted in little alteration of the oocyte Pgp level. Expression of Pgp was also elevated in cultured porcine granulosa cells during cell maturation when stimulated with follicle-stimulating hormone or luteinizing hormone for 24-48 h. Collectively, the present results indicate that the transporting activity of P-glycoprotein upregulates in porcine oocytes and granulosa cells during exposure to gonadotropins or prior to ovulation.

Steroidal regulation of Ihh and Gli1 expression in the rat uterus.

Ovarian steroid hormones, progesterone (P4), and estradiol (E2) strictly regulate the endometrial tissue remodeling required for successful embryo implantation. Indian hedgehog (Ihh) is up-regulated by P4 and critically mediates uterine receptivity in the mouse. However, the regulation of Ihh expression during the implantation period still remains unclear. The present study was conducted to elucidate the mechanism of the steroidal regulation in the expression of Ihh and Gli1, the mediator of the Ihh pathway. Ihh mRNA was expressed in the rat uterus on 3.5-5.5 days post-coitus (dpc), while Gli1 expression transiently increased at 3.5 dpc but decreased significantly on 5.5 dpc (P < 0.001). In delayed implantation, the expression of Ihh was induced by the implantation-induced E2 treatment in the primed rat uterus. In contrast, expression of Gli1 was significantly decreased by E2 treatment (P = 0.016). In the case of ICI182.780 (ICI) treatment, Ihh expression was eliminated by ICI, whilst Gli1 expression increased. These results suggest that Ihh expression is maintained at a high level until the initiation of implantation, while the expression of Gli1 is decreased just prior to the initiation of implantation depending on the E2 action. This observation aids in the understanding of the Ihh signaling pathway mediating uterine remodeling for implantation.

Up-regulation of circadian clock gene Period 2 in the prostate mesenchymal cells during flutamide-induced apoptosis.

Androgen regulates the proper development and physiological function of the prostate. Here, we investigated the modulation of androgen and androgen receptor (AR) antagonist on circadian oscillations of a clock core gene Period 2 (Per2) in rat prostate mesenchymal cells (PMCs). Circadian oscillations were analyzed with the real-time monitoring system of gene expression using transgenic rats introduced with mouse Per2 promoter fused to a destabilized luciferase (Per2-dLuc) reporter gene. Analyses of circadian oscillations, immunofluorescence, and androgen response element (ARE)-luciferase reporter assay revealed that circadian clocks are operative and the AR protein is functional in PMCs in vitro. Androgen such as testosterone (T) and dihydrotestosterone (DHT) did not cause any changes in circadian Per2-dLuc oscillations of confluent cells. Conversely, flutamide (FL) up-regulated the amplitude of circadian Per2-dLuc oscillations in a dose-dependent manner, whereas T antagonized the action of FL. The PER2 protein was markedly accumulated by FL treatment and localized in both the nucleus and cytoplasm during the first peak period of circadian Per2-dLuc oscillations. Simultaneously, FL treatment increased apoptotic cell death. Collectively, the present study demonstrates that a clock gene Per2 is up-regulated in PMCs during FL-induced apoptotic cell death. Thus, circadian oscillations of Per2 gene expression may be closely linked to the cellular states of PMCs such as apoptotic cell death.

Effects of ectopic expression of human telomerase reverse transcriptase on immortalization of feather keratinocyte stem cells.

Normal somatic cells possess a finite life span owing to replicative senescence. Telomerase functions as a potential regulator of senescence in various cells. Expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and cellular immortalization. In this study, we investigated the effects of ectopic expression of hTERT on proliferation potential of chicken feather keratinocyte stem cells (FKSCs). We established FKSCs transduced with hTERT catalytic subunit fused with EGFP marker gene (hTERT-EGFP-FKSCs). hTERT-EGFP-FKSCs had the great potential of proliferation in vitro and expressed kerainocyte stem cell markers integrin beta1 and CD49c. Keratin 15 and keratin 19, as native FKSCs, were also detected in hTERT-EGFP-FKSCs. By the analysis of fluorescent RT-PCR, western blotting and TRAP assay, hTERT-EGFP-FKSCs were positive for telomerase activity, in comparison with native FKSCs showing no telomerase activity. We demonstrated that ectopic expression of hTERT could result in immortalization of FKSCs. Tumorigenecity of hTERT-EGFP-FKSCs were examined by soft agar assay and transplantation into NOD-SCID mice. Results showed that hTERT-EGFP-FKSCs sustained the cellular characteristics of native FKSCs and had no transforming activity. In vivo differentiation multipotentials of hTERT-EGFP-FKSCs were confirmed by transplantation into developing chicken embryos and in situ hybridization analysis. These data provide a novel framework for understanding human telomerase activity in different species and suggest a new insight for manipulating hTERT for therapeutic purposes in treating tissue injury and aging.

In vitro decidualization of rat endometrial stromal cells.

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes.

Monitor of the myostatin autocrine action during differentiation of embryonic chicken myoblasts into myotubes: effect of IGF-I.

Myogenesis is regulated through the proliferation and differentiation of myoblasts expressing myostatin which functions as a negative regulator by generating Smad signals. Here, we monitored the autocrine action of myostatin in quiescent chicken myoblasts transfected with the Smad-mediated promoter reporter vector to evaluate the modulation of several growth factors. During differentiation of myoblasts into myotubes, stretched and spherical types of myoblasts were observed at 12 h after induction, at which the promoter activity began to increase. Maximal promoter activity was observed at approximately 30 h. Multinucleated myotubes were markedly formed at 72 h, but the activity was very low. IGF-I, known as a positive regulator of myogenesis, increased the promoter activity, but the increase was rather small at its high concentration (100 ng/ml). IGF-I significantly increased the level of myostatin transcript in myoblasts and newly formed myotubes at 24 h, but not at 36 h. However, the cell fusion of myoblasts was not accelerated in the presence of IGF-I. Consequently, this study indicates that the autocrine action of myostatin is partially enhanced by IGF-I through increasing its expression.

Progesterone, but not estradiol, synchronizes circadian oscillator in the uterus endometrial stromal cells.

The circadian oscillator is generated within the suprachiasmatic nuclei and synchronizes circadian clocks in numerous peripheral tissues. The molecular basis is composed of a number of genes and proteins that form transcriptional and translational feedback loops. Such molecular oscillators are also operative in peripheral tissues, including in the uterus. Although ovarian steroids regulate the function of uterine endometrial stromal cells, the modulation of ovarian steroids on the circadian rhythms remains unknown. Here we investigate the possibility that estradiol (E2) and progesterone (P4) modulate the circadian oscillator of the stromal cells. The study using transgenic rats constructed with Period 2 (Per2) promoter-destabilized luciferase (Per2-dLuc) gene, with the real-time monitoring system of Per2-dLuc oscillation. The stromal cells displayed constant Per2-dLuc oscillation after treatment with dexamethasone, suggesting that the circadian oscillator is operative. However, the circadian oscillator was disrupted by in vivo administration of human chorionic gonadotropin (hCG) following equine chorionic gonadotropin (eCG), although it was altered into a rhythmic pattern 4 days later following hCG. Chronic treatment with P4 induced constant Per2-dLuc oscillation in the stromal cells from eCG-treated immature and pregnant rats, whereas E2 did not promote such a rhythmic Per2-dLuc oscillation. Collectively, P4 synchronizes the circadian oscillator of the uterus endometrial stromal cells through transcriptional and translational feedback loops of the clockwork system.

Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2.

Implantation serine protease (ISP) was first identified in the uteri of pregnant mice. It is thought that ISP may have an important role in the initiation of implantation. However, the expression status and detailed functions of ISP remain unclear. In this study, the expression of ISP was investigated in the rat uterus. The analysis of two rat genes registered in GenBank, accession nos. XM_220240 and XM_577076, exhibited high identities to the mouse ISP2 genes, respectively at an mRNA level. We labeled the former as rISP2a and the latter as rISP2b. Using RT-PCR, we found that both genes were expressed in the uterus. Specifically, rISP2a mRNA was detected in the uterus throughout pregnancy, whereas rISP2b mRNA was only expressed in the uterus from day 5 of pregnancy until the end of gestation. Expression of both genes was observed specifically within the endometrial gland epithelium. Furthermore, rISP2a was also observed to be expressed in the fetus and placenta, whereas rISP2b expression was observed in the fetus but not in the placenta. An expressional signal of the rISP2a gene was observed in the spongiotrophoblasts, giant cells and decidual endometrium in the placenta. In the embryo, the ventral specific region was positive in rISP2a and rISP2b gene expression. These findings indicate the possibility that the presently examined genes with high identity to mouse ISP2 may play some role not only during the implantation phase, but also in the development of the placenta and embryo.