117 results on '"Hattori, Masa-Aki"'
Search Results
102. Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing
- Author
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Hattori, Masa-aki, primary and Wakabayashi, Katsumi, additional
- Published
- 1989
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103. Different profiles of isoelectric avian luteinizing hormone components in biological activity and immunoreactivity.
- Author
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HATTORI, MASA-AKI, primary and WAKABAYASHI, KATSUMI, additional
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- 1983
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104. In Vitro Decidualization of Rat Endometrial Stromal Cells.
- Author
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Matsumoto, Kenji, Watanabe, Ryo, Oozono, Shinji, Kubota, Kaiyu, Nishimura, Kyouhei, Soh, Tomoki, Yamauchi, Nobuhiko, and Hattori, Masa-aki
- Abstract
During embryo implantation in rodents and human, the endometrial stromal cells proliferate and differenciate into decidual cells. This process ultimately results in the formation of the maternal component of the placenta. Induction of decidualization of endometrial stromal cells is possible in an in vitro cell culture system. This could be a powerful tool for investigating the complex process of the decidualization in vitro. However, the methods to induce in vitro decidualization differ according to speicies or cell types and a more stable or universal system has not yet been developed. The purpose of present study is to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). In order to find the most effective in vitro decidualization system, several decidual treatments were compared by their resulting gene expression and morphological change. Then, using the in vitro decidualization system, the effects of gonadotropins on decidualization, and the changes of MMP production in RES were investigated. The RES, derived from the uterus at implantation period (day 5 of pregnancy), were treated with 1) medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (db-cAMP) (MPA treatment), 2) estradiol and progesterone (E+P) or 3) arachidonic acid (AA). Morphological changes in RES were introduced at 2 days (MPA) and 4 days (E+P and AA) after each treatment. After 24 h of the treatments, cells responded to all of the stimulations by expressing desmin mRNA. However, dPRP mRNA was only expressed in the MPA treated cells. Immunocytochemical study shows that desmin was expressed in MPA treated cells. We concluded that MPA treatment in RES was the most effective method for introducing decidualization. Therefore, we used the MPA treatment to induce in vitro decidualization of RES in further studies. Morphological changes introduced by decidual treatment occurred 2 days after treatment in mature rat derived RES, whilst, the changes in immature rat derived RES took a longer period (4 days). Furthermore, desmin and dPRP mRNA did not express in the RES derived from immature rat uteri. Similar to the RES from the implantation period, RES from the diestrous stage responded to stimulation by expressing desmin and dPRP mRNA. Injection of gonadotropins (eCG and hCG) to the immature rats (3 weeks) did not affect morphological changes of RES, before and after the decidual treatment. However, desmin and dPRP mRNA were expressed in RES derived from gonadotropin injected immature rats after the tratment with MPA. Expression of MMP-2 and -9 mRNA did not change after decidual treatment of RES examined by real-time PCR. However, the results of gelatinzymography showed that the active form of MMP-2 and -9 significantly increased after in vitro decidualization (P<0.05). We clarified that MPA + db-cAMP treatment is the most effective to induce in vitro decidualization in RES. Using this system, it was suggested that sexual maturation and gonadotropins are important in RES for their ability for decidualization. Furthermore, the activity of MMP-2 and -9 is suggested to be increased during decidualization without a corresponding increase of the expression of those genes. This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).
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- 2008
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105. Effect of bullfrog LH and FSH on newt testes under different temperatures
- Author
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Tanaka, Shigeyasu, Sakai, Makoto, Hattori, Masa-aki, Kikuyama, Sakae, Wakabayashi, Katsumi, and Hanaoka, Yoichi
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GLYCOPROTEIN hormones , *SCROTUM , *TESTOSTERONE , *SALAMANDRIDAE - Abstract
Environmental temperature plays important roles for amphibian gonadal function. In this study, we examined the responses of testicular tissue of adult male newts (Cynops pyrrhogaster) to amphibian gonadotropins both in vitro and in vivo under different temperatures. When minced testes were incubated in vitro at different temperatures (8–37 °C) under an atmosphere of 95% O2–5% CO2 for 3 h with bullfrog luteinizing hormone (LH) or follicle-stimulating hormone (FSH), LH stimulated testosterone production more than FSH. The testosterone production increased as the incubation temperature increased. Hypophysectomized newts were injected with bullfrog LH or FSH and maintained at 8 or 18 °C. In the 18 °C group, the testicular weight of the hypophysectomized control decreased when compared with that of with the intact control. The testicular weight of the LH-treated hypophysectomized group decreased more than that of the hypophysectomized control, indicating that LH induced the evacuation of mature spermatozoa from the testes of LH-treated hypophysectomized newts. In the FSH-treated newts, the testicular weight was greater than that in the hypophysectomized control, and was maintained at a value similar to that of the intact control. In the 8 °C group, there was no significant difference in testicular size among the intact control, hypophysectomized control, and FSH-treated newts. LH strongly induced spermiation as it did at 18 °C. The plasma testosterone level in the hypophysectomized newts decreased dramatically, but LH was effective in restoring it. Its effect was more potent at 8 °C than at 18 °C. On the other hand, FSH did not induce a significant increase in the plasma testosterone levels at either temperatures. The results indicate a temperature-dependent difference in responsiveness of the testis both in vitro and in vivo to LH and FSH. [Copyright &y& Elsevier]
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- 2004
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106. The nuclear receptor REV-ERBα represses the transcription of growth/differentiation factor 10 and 15 genes in rat endometrium stromal cells.
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Zhao L, Isayama K, Chen H, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
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- Animals, Cell Differentiation, Chromatin Immunoprecipitation, Endometrium cytology, Female, Growth Differentiation Factor 10 genetics, Growth Differentiation Factor 15 genetics, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Pregnancy, RNA, Small Interfering, Rats, Rats, Transgenic, Stromal Cells cytology, Stromal Cells metabolism, Transcription, Genetic, Transfection, Endometrium metabolism, Gene Expression Regulation physiology, Growth Differentiation Factor 10 metabolism, Growth Differentiation Factor 15 metabolism, Nuclear Receptor Subfamily 1, Group D, Member 1 metabolism
- Abstract
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.)
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- 2016
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107. Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells.
- Author
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Isayama K, Zhao L, Chen H, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
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- 5' Untranslated Regions, ARNTL Transcription Factors antagonists & inhibitors, ARNTL Transcription Factors genetics, ARNTL Transcription Factors metabolism, Animals, Cells, Cultured, Cyclooxygenase 2 genetics, Endometrium cytology, Endometrium enzymology, Female, Nuclear Receptor Subfamily 1, Group D, Member 1 antagonists & inhibitors, Nuclear Receptor Subfamily 1, Group D, Member 1 genetics, Nuclear Receptor Subfamily 1, Group F, Member 1 genetics, Nuclear Receptor Subfamily 1, Group F, Member 1 metabolism, Pregnancy, Prolactin analogs & derivatives, Prolactin genetics, Prolactin metabolism, RNA Interference, RNA, Small Interfering, Rats, Rats, Transgenic, Response Elements, Stromal Cells cytology, Stromal Cells enzymology, Circadian Clocks, Cyclooxygenase 2 metabolism, Endometrium metabolism, Gene Expression Regulation, Enzymologic, Nuclear Receptor Subfamily 1, Group D, Member 1 metabolism, Placentation, Stromal Cells metabolism
- Abstract
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.)
- Published
- 2015
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108. Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells.
- Author
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Tasaki H, Zhao L, Isayama K, Chen H, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
- Subjects
- Animals, Cells, Cultured, Female, Gene Expression Regulation, Pregnancy, Rats, Rats, Transgenic, Stromal Cells metabolism, Uterus cytology, Uterus metabolism, Bone Morphogenetic Proteins antagonists & inhibitors, Bone Morphogenetic Proteins biosynthesis, Endometrium cytology, Endometrium metabolism, Nuclear Receptor Subfamily 1, Group D, Member 1 biosynthesis
- Abstract
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.)
- Published
- 2015
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109. Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells.
- Author
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Chen H, Zhao L, Kumazawa M, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
- Subjects
- ARNTL Transcription Factors biosynthesis, ARNTL Transcription Factors genetics, Animals, CLOCK Proteins antagonists & inhibitors, CLOCK Proteins biosynthesis, Cells, Cultured, Female, Gene Expression Regulation, Mice, Progesterone biosynthesis, Progesterone genetics, Prostaglandins biosynthesis, Rats, Rats, Transgenic, ARNTL Transcription Factors antagonists & inhibitors, CLOCK Proteins genetics, Down-Regulation genetics, Luteal Cells metabolism, Progesterone antagonists & inhibitors, Prostaglandins genetics
- Abstract
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.
- Published
- 2013
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110. FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.
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Chen H, Zhao L, Chu G, Kito G, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
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- Animals, Cell Differentiation drug effects, Cells, Cultured, Chorionic Gonadotropin metabolism, Circadian Rhythm Signaling Peptides and Proteins biosynthesis, Circadian Rhythm Signaling Peptides and Proteins genetics, Circadian Rhythm Signaling Peptides and Proteins metabolism, Connexin 43 antagonists & inhibitors, Connexin 43 genetics, Dexamethasone pharmacology, Female, GABA-A Receptor Antagonists pharmacology, Gap Junctions drug effects, Genes, Reporter drug effects, Glucocorticoids pharmacology, Granulosa Cells cytology, Granulosa Cells drug effects, Membrane Transport Modulators pharmacology, Mice, Period Circadian Proteins biosynthesis, Period Circadian Proteins genetics, Period Circadian Proteins metabolism, Rats, Rats, Transgenic, Receptors, LH biosynthesis, Receptors, LH genetics, Receptors, LH metabolism, Circadian Clocks drug effects, Connexin 43 metabolism, Follicle Stimulating Hormone metabolism, Gap Junctions metabolism, Granulosa Cells metabolism, Up-Regulation drug effects
- Abstract
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.
- Published
- 2013
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111. Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation.
- Author
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Chu G, Misawa I, Chen H, Yamauchi N, Shigeyoshi Y, Hashimoto S, and Hattori MA
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- Animals, Circadian Rhythm Signaling Peptides and Proteins biosynthesis, Circadian Rhythm Signaling Peptides and Proteins genetics, Culture Media, Serum-Free, Diethylstilbestrol pharmacology, Estrogens, Non-Steroidal pharmacology, Female, Genes, Reporter, Luminescence, Ovary cytology, Ovary growth & development, Period Circadian Proteins biosynthesis, Period Circadian Proteins genetics, RNA biosynthesis, RNA genetics, Rats, Real-Time Polymerase Chain Reaction, Transcription, Genetic physiology, Circadian Rhythm physiology, Follicle Stimulating Hormone physiology, Granulosa Cells physiology, Triiodothyronine physiology
- Abstract
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.
- Published
- 2012
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112. Effects of tumor necrosis factor-alpha on cell proliferation, prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro.
- Author
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Gamo T, Yamauchi N, Nishimura K, Watanabe R, Matsumoto K, Oozono S, Kubota K, He PJ, Soh T, and Hattori MA
- Subjects
- Animals, Cells, Cultured, Dose-Response Relationship, Drug, Embryo, Mammalian cytology, Endometrium metabolism, Endometrium pathology, Epidermal Growth Factor pharmacology, Female, Fibroblast Growth Factor 2 pharmacology, Male, Pregnancy, Rats, Rats, Wistar, Stromal Cells drug effects, Stromal Cells metabolism, Stromal Cells pathology, Cell Proliferation drug effects, Dinoprost metabolism, Dinoprostone metabolism, Endometrium drug effects, Matrix Metalloproteinases metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Tumor necrosis factor-alpha (TNF-alpha) is known as a pluripotent cell mediator, and it is implicated in the control of uterine cell growth, differentiation and function during estrous cycle and pregnancy. In this study, we investigated the effect of TNF-alpha on endometrial stromal cells derived from rat uterus (rat endometrial stromal cells, RES). RES were isolated from rat endometrium at day 5 of pregnancy. Proliferation activities of RES were measured by using bromodeoxyuridine (BrdU) labeling kit, the productions of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) were measured by enzyme immunoassay kits and the production of matrix metalloproteinases (MMPs) was analyzed by gelatin-zymography. TNF-alpha, as well as epidermal growth factor and fibroblast growth factor-2, significantly increased the proliferation activity of RES (P<0.05). TNF-alpha selectively stimulated the production of PGE2 in RES (P<0.05), but not the production of PGF2alpha. Additionally, TNF-alpha did not stimulate the production of MMPs in RES at the concentration of 5 ng/mL, compared with the control groups (P>0.05). In conclusion, this study demonstrates several regulational functions of TNF-alpha on RES using in vitro culture system. The effects of TNF-alpha on proliferation and MMP production of RES have been shown for the first time. We believe that these results demonstrate part of the functions of TNF-alpha in endometrium and contribute to the better understanding of endometrial functions.
- Published
- 2007
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113. Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA.
- Author
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Sato F, Kurokawa M, Yamauchi N, and Hattori MA
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- Animals, Cell Culture Techniques, Cell Shape, Chick Embryo, Culture Media, Serum-Free, Cyclin-Dependent Kinase Inhibitor p21 genetics, Muscle Fibers, Skeletal cytology, MyoD Protein genetics, Myogenin genetics, Myostatin, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Cell Differentiation, Myoblasts cytology, RNA Interference, RNA, Small Interfering genetics, Transforming Growth Factor beta physiology
- Abstract
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)(10)-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.
- Published
- 2006
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114. Temporal and spatial expression of TGF-beta2 in chicken somites during early embryonic development.
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Aramaki S, Sato F, Soh T, Yamauchi N, Sakai T, and Hattori MA
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- Age Factors, Animals, DNA Primers, DNA, Complementary genetics, Histological Techniques, In Situ Hybridization, Reverse Transcriptase Polymerase Chain Reaction, Transforming Growth Factor beta2, Chick Embryo embryology, Chick Embryo metabolism, Gene Expression Regulation, Developmental physiology, Somites metabolism, Transforming Growth Factor beta metabolism
- Abstract
A multifunctional growth and differentiation factor TGF-beta is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF-beta2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF-betas were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole-mount in situ hybridization. TGF-beta2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF-beta2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29-31 (6-7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF-beta2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF-beta2 were clearly observed in the myotomal somite tips as well as the neural tube. RT-PCR analysis showed that TGF-beta2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF-beta2 may be a regulatory factor participating in the somitogenesis of chicken embryos., (Copyright 2005 Wiley-Liss, Inc.)
- Published
- 2005
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115. Function of TGF-beta2 in the growth of chicken primordial germ cells and germinal ridge stroma cells during embryonic development.
- Author
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Fujioka T, Soh T, Fujihara N, and Hattori MA
- Subjects
- Analysis of Variance, Animals, Cells, Cultured, Chick Embryo metabolism, Growth Substances metabolism, Immunohistochemistry, Periodic Acid-Schiff Reaction, Reverse Transcriptase Polymerase Chain Reaction, Transforming Growth Factor beta metabolism, Transforming Growth Factor beta2, Chick Embryo embryology, Gene Expression Regulation, Developmental, Germ Cells growth & development, Gonads embryology, Transforming Growth Factor beta physiology
- Abstract
The development of chicken embryonic gonads is locally regulated by the systematic action of growth factors. Recently, we used suppressive subtraction cloning to identify transforming growth factor beta2 (TGF-beta2) as a growth factor gene preferentially expressed in chicken embryonic ovaries and testes during the early periods of development (Hattori et al. 2002a. Prominent expression of transforming growth factor beta2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning. Gen Comp Endocrinol 125:311-316). In the present study, the function of TGF-beta2 in chicken embryonic gonads was investigated using a serum-free culture system in the presence of several growth factors, which may behave as mitogenic or survival factors of primordial germ cells (PGCs). Chicken germinal ridges containing PGCs and germinal ridge stroma cells (GRSCs) were collected from six-day embryos. Addition of TGF-beta2 caused a dose-dependent inhibition of the number of co-cultured PGCs and GRSCs in the presence of these growth factors. However, there was no obvious difference between embryonic ovaries and testes in the effects of TGF-beta2. Immunocytochemical analysis using anti-SSEA-1 antibody revealed that TGF-beta2 induced fragmentation of PGCs. Expression of the TGF-beta2 gene was estimated in the co-cultured PGCs and GRSCs by semi-quantitative RT-PCR. The mRNA level of TGF-beta2 was significantly suppressed in the presence of the growth factors. These results suggest that TGF-beta2 is a gonadal regulator preferentially expressed at the early stages of chicken embryonic development and reduces the growth of PGCs and GRSCs by suppressing proliferation. However, expression of TGF-beta2 may be controlled by mitogenic or survival factors of PGCs., (Copyright 2004 Wiley-Liss, Inc.)
- Published
- 2004
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116. Evaluation of deoxyribonuclease activity in seminal plasma of ejaculated chicken semen.
- Author
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Sato F, Soh T, Hattori MA, and Fujihara N
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- Animals, Cations, Divalent pharmacology, DNA analysis, DNA Primers, Edetic Acid pharmacology, Hot Temperature, Indicators and Reagents, Male, Reverse Transcriptase Polymerase Chain Reaction, Chickens physiology, Deoxyribonucleases analysis, Semen enzymology
- Abstract
Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents., Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light., Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents., Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.
- Published
- 2003
117. Utilization of central disk of blastoderm and germinal crescent region for production of interspecific germline chimera between chicken and quail.
- Author
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Soh T, Inoue Y, Xi YM, Kato Y, and Hattori MA
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- Animals, Base Sequence, Blastoderm ultrastructure, Brain embryology, Brain Chemistry, Chickens, Chimera, DNA Primers, DNA, Complementary genetics, Female, Male, Ovary embryology, Polymerase Chain Reaction, Quail, Testis embryology, Blastoderm physiology, Chick Embryo physiology, Embryo, Nonmammalian physiology, Germ-Line Mutation physiology, Ovalbumin genetics
- Abstract
Aim: The production of interspecific germline chimeras between chicken and quail were attempted employing the dissociated cells derived from the blastodermal central disk (stage X) and the germinal crescent region of embryo (stage 7-8)., Methods: The central disk (CD) of the area pellucida in chicken blastoderm (stage X) and the germinal crescent region (GCR) of embryo (stage 7-8) were dispersed and injected into the subgerminal cavity of quail blastoderm (stage X). Injected eggs were incubated for 7 days or to hatching. The donor chicken DNA was detected by the polymerase chain reaction., Results: In day-7 embryos, chicken DNA was detected in 5 gonads and 9 brains from 53 survived embryos received chicken CD cells, and 1 gonads and 6 brains from 27 survived embryos received chicken GCR. Chicken DNA was also detected from the semen of one adult male hatched from eggs received chicken GCR cells., Conclusion: CD and GCR cells as the donors showed the possibility to produce the interspecific germline chimera, but further studies are needed to make necessary improvement.
- Published
- 2002
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