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Seung-Wook Shin,
Natsumi Shimizu,
Mikiko Tokoro,
Satoshi Nishikawa,
Yuki Hatanaka,
Masayuki Anzai,
Jun Hamazaki,
Satoshi Kishigami, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Shigeo Murata,
Kazuya Matsumoto
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ABSTRACT: During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.
Biology open. 02/2013; 2(2):170-82.
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Takayuki Yamochi,
Yuta Kida,
Noriyoshi Oh,
Sei Ohta,
Tomoko Amano,
Masayuki Anzai,
Hiromi Kato,
Satoshi Kishigami,
Tasuku Mitani,
Kazuya Matsumoto, Kazuhiro Saeki,
Makoto Takenoshita,
Akira Iritani,
Yoshihiko Hosoi
[show abstract]
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ABSTRACT: Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.
Zygote 04/2012; · 1.17 Impact Factor
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ABSTRACT: To assess the developmental kinetics of human embryos and their ability to develop to morphologically normal blastocysts.
Experimental study on human embryos donated for research using a time-lapse imaging system based on individual embryo culture in poly(dimethylsiloxane) microwells and monitored using a microscope inside the incubator.
Private fertility clinic.
Surplus embryos donated by couples after undergoing fertility treatment.
None.
Blastocyst score and times required from beginning to completion of the second and third mitotic divisions.
The time required for completion of the second division (the three- to four-cell stage) was shorter in embryos that developed to high-scoring blastocysts (0.7 hours, n = 17) than in those forming low-scoring blastocysts (3.7 hours, n = 24). Similarly, the mean time required to completion of the third division (five- to eight-cell stage) was also significantly shorter in embryos forming high-scoring blastocysts (5.7 hours) than among those forming low-scoring blastocysts (16.9 hours).
Individual embryos with the potential to develop to high-scoring blastocysts could be selected at 2-3 days of culture using this system by examining the times required to complete the second and third mitotic divisions.
Fertility and sterility 01/2012; 97(2):332-7. · 3.97 Impact Factor
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ABSTRACT: The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the β-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.
Cellular reprogramming. 12/2011; 14(1):20-8.
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Seung-Wook Shin,
Mikiko Tokoro,
Satoshi Nishikawa,
Hyang-Heun Lee,
Yuki Hatanaka,
Takuji Nishihara,
Tomoko Amano,
Masayuki Anzai,
Hiromi Kato,
Tasuku Mitani,
Satoshi Kishigami, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.
Journal of Reproduction and Development 12/2010; 56(6):655-63. · 1.46 Impact Factor
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Mikiko Tokoro,
Seung-Wook Shin,
Satoshi Nishikawa,
Hyang-Heun Lee,
Yuki Hatanaka,
Tomoko Amano,
Tasuku Mitani,
Hiromi Kato,
Masayuki Anzai,
Satoshi Kishigami, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pβ-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken β-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.
Journal of Reproduction and Development 12/2010; 56(6):607-15. · 1.46 Impact Factor
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Tomoko Amano,
Kaori Tokunaga,
Reiko Kakegawa,
Ayaka Yanagisawa,
Atsushi Takemoto,
Atsuhiro Tatemizo,
Tatsuya Watanabe,
Yuki Hatanaka,
Akinori Matsushita,
Masao Kishi,
Masayuki Anzai,
Hiromi Kato,
Tasuku Mitani,
Satoshi Kishigami, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85-100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA.
Animal reproduction science 09/2010; 121(3-4):225-35. · 1.56 Impact Factor
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ABSTRACT: In bovine somatic cell nuclear transfer (NT), embryos are more likely to develop to full term when they are derived from fibroblasts at the G1 phase instead of cells at the G0/G1 phase. To better understand the reason for this difference, we examined morphological development in the early pregnancy of NT embryos using G1 phase cells (G1-NT embryos) and G0/G1 phase cells (G0/G1-NT embryos). Blastocysts derived from G1 and G0/G1-NT embryos were transferred to recipient heifers, and the conceptuses at day 50 of gestation were retrieved non-surgically using prostaglandin F(2alpha) and oxytocin. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The percentages of embryos that developed to the blastocyst stage did not differ between G1 and G0/G1-NT embryos. Pregnancy rates at day 30 of recipient heifers carrying G1-NT, G0/G1-NT, IVF, parthenogenetic and AI embryos were similar (57-100%). Two recipient heifers carrying parthenogenetic embryos returned to estrus between days 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at day 50 of gestation of all experimental groups (83%) were recovered non-surgically by several PGF(2alpha) and oxytocin treatments. Recovery rates of normal fetuses derived from G1-NT embryos (83%), IVF embryos (80%) and AI embryos (88%) were greater than those of G0/G1-NT embryos (33%) and parthenogenetic embryos (0%). Our results suggest that NT embryos reconstructed with cells at the G1 phase have a high developmental competence from the time of embryo transfer to day 50 of gestation.
Animal reproduction science 02/2010; 119(3-4):191-7. · 1.56 Impact Factor
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ABSTRACT: Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.
Stem cells and development 08/2009; 18(10):1433-40. · 4.15 Impact Factor
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ABSTRACT: Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.
Zygote 05/2009; 17(3):209-15. · 1.17 Impact Factor
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Takeshi Teramura,
Yuta Onodera,
Hideki Murakami,
Syunsuke Ito,
Toshihiro Mihara,
Toshiyuki Takehara,
Hiromi Kato,
Tasuku Mitani,
Masayuki Anzai,
Kazuya Matsumoto, Kazuhiro Saeki,
Kanji Fukuda,
Norimasa Sagawa,
Yoshihiko Osoi
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ABSTRACT: The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.
Journal of Reproduction and Development 04/2009; 55(3):283-92. · 1.46 Impact Factor
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Manabu Satoh,
Mikiko Tokoro,
Haruka Ikegami,
Kouhei Nagai,
Youhei Sono,
Seung-Wook Shin,
Satoshi Nishikawa, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Aisaku Fukuda,
Yoshiharu Morimoto,
Kazuya Matsumoto
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ABSTRACT: Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function.
Journal of Reproduction and Development 04/2009; 55(3):316-26. · 1.46 Impact Factor
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Yoriko Indo,
Atsuhiro Tatemizo,
Yuki Abe,
Iwane Suzuki,
Kazuya Matsumoto,
Yoshihiko Hosoi,
Mikio Kinoshita,
Koji Mikami,
Norio Murata,
Akira Iritani, Kazuhiro Saeki
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ABSTRACT: Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.
Biochimica et Biophysica Acta 03/2009; 1791(3):183-90. · 4.66 Impact Factor
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ABSTRACT: Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.
PLoS ONE 02/2009; 4(1):e4142. · 4.09 Impact Factor
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ABSTRACT: In the mouse, maturing oocytes and zygotes until the late 1-cell stage are transcriptionally inert. The development of early preimplantation embryos, including reprogramming of differentiated germ cells into totipotent embryos, is regulated by the translation of mRNAs in oocytes preliminarily stored during oocytogenesis (maternal mRNAs). In the period of oocyte to zygote transition in mammals, the translational promotion or repression of maternal mRNAs does not start in unison. For example, a set of maternal mRNAs that are translationally quiescent in the growing stage become translated after the start of maturation and/or fertilization, but another group of maternal mRNAs that are actively translated in the growing stage become inactivated during maturation. This selective and temporal translational profile of maternal mRNAs seems to be regulated by RNA-binding proteins that bind to maternal mRNAs and decide the timing of their entry into the ribosome and by special short sequences in maternal mRNAs that recognize specific RNA-binding proteins. In this review, we focus on the mechanisms that modulate the profile of post-transcriptional regulation in the period of oocyte to zygote transition in mammals.
Journal of Mammalian Ova Research 01/2009;
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Tomoko Amano,
Akinori Matsushita,
Yuki Hatanaka,
Tatsuya Watanabe,
Katsutaka Oishi,
Norio Ishida,
Masayuki Anzai,
Tasuku Mitani,
Hiromi Kato,
Satoshi Kishigami, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.
Biology of Reproduction 12/2008; 80(3):473-83. · 4.01 Impact Factor
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Miyuri Kawasumi,
Yuichi Unno,
Toshiki Matsuoka,
Megumi Nishiwaki,
Masayuki Anzai,
Tomoko Amano,
Tasuku Mitani,
Hiromi Kato, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Satoshi Kishigami,
Kazuya Matsumoto
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ABSTRACT: Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.
Molecular Reproduction and Development 11/2008; 76(4):342-50. · 2.53 Impact Factor
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ABSTRACT: Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.
Journal of Reproduction and Development 10/2008; 54(5):352-7. · 1.46 Impact Factor
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Kazunobu Tsunemoto,
Masayuki Anzai,
Toshiki Matsuoka,
Mikiko Tokoro,
Seung-Wook Shin,
Tomoko Amano,
Tasuku Mitani,
Hiromi Kato,
Yoshihiko Hosoi, Kazuhiro Saeki,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.
Molecular Reproduction and Development 08/2008; 75(7):1104-8. · 2.53 Impact Factor
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Toshiki Matsuoka,
Manabu Sato,
Mikiko Tokoro,
Seung-Wook Shin,
Atsuto Uenoyama,
Kazunari Ito,
Syuji Hitomi,
Tomoko Amano,
Masayuki Anzai,
Hiromi Kato,
Tasuku Mitani, Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.
Journal of Reproduction and Development 07/2008; 54(3):192-7. · 1.46 Impact Factor
