Y Obata

Tokyo University of Agriculture, Edo, Tōkyō, Japan

Are you Y Obata?

Claim your profile

Publications (116)673.39 Total impact

  • Yayoi Obata
    [Show abstract] [Hide abstract]
    ABSTRACT: Epigenetic modification is the main mechanism of transcriptional regulation that does not involve changes in DNA sequences, such as DNA methylation, acetylation and methylation of the N-terminal tail of histone. Recently, next-generation sequencing technology has provided detailed information about the DNA methylation status of the whole mouse genome in full-grown oocytes. However, it is still very hard to read histone codes in oocytes because a large number of cells (1 × 106 cells or more) are needed for such analyses. In addition, information that can be obtained from immunostaining analysis is limited to a global image of histone modification in oocytes. Consequently, a complete picture of individual epigenetic modifications in mouse oocytes has not yet been understood. In this paper, the DNA methylation required for functional oocytes is reviewed. The differences in DNA methylation between oocytes grown in vivo and in vitro, and the potential for manipulating epigenetic modifications in oocytes are also discussed.
    No preview · Article · Oct 2014 · Journal of Mammalian Ova Research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.
    Preview · Article · Mar 2014 · Journal of Reproduction and Development
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analysed DNA methylation imprints. The results showed that two- to four-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing oocytes versus fully grown oocytes derived from wild-type mice, but the analysed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in non-growing oocytes is insufficient for methylation imprints and imprinted regions are resistant to DNMT3A/DNMT3L in non-growing oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1, and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn, and Impact was observed in non-growing oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.
    Preview · Article · Mar 2014 · Human Molecular Genetics
  • Source
    Yayoi Obata · Takuya Wakai · Satoshi Hara · Tomohiro Kono
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However, the mechanisms underlying gamete-specific imprinting remain unclear. Here, we investigated whether male and female gametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the niche surrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fused with enucleated fully grown oocytes and exposed to the ooplasm for 5-6 days, the DNA methylation status of the reconstituted oocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest that the imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. In contrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grown oocytes and the reconstituted oocytes were then cultured for 5-6 days, the Igf2r, Kcnq1ot1, and unexpectedly H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from 5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes, the chromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs, and that mechanisms for maintaining nonmethylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.
    Preview · Article · Oct 2013 · Reproduction
  • Source
    Baozeng Xu · Yayoi Obata · Feng Cao · Teruko Taketo
    [Show abstract] [Hide abstract]
    ABSTRACT: The oocytes of B6.Y(TIR) sex-reversed female mouse mature in culture but fail to develop after fertilization because of their cytoplasmic defects. To identify the defective components, we compared the gene expression profiles between the fully-grown oocytes of B6.Y(TIR) (XY) females and those of their XX littermates by cDNA microarray. 173 genes were found to be higher and 485 genes were lower in XY oocytes than in XX oocytes by at least 2-fold. We compared the transcript levels of selected genes by RT-PCR in XY and XX oocytes, as well as in XO oocytes missing paternal X-chromosomes. All genes tested showed comparable transcript levels between XX and XO oocytes, indicating that mRNA accumulation is well adjusted in XO oocytes. By contrast, in addition to Y-encoded genes, many genes showed significantly different transcript levels in XY oocytes. We speculate that the presence of the Y-chromosome, rather than the absence of the second X-chromosome, caused dramatic changes in the gene expression profile in the XY fully-grown oocyte.
    Preview · Article · Jul 2012 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes.
    Preview · Article · Aug 2011 · Oncogene
  • [Show abstract] [Hide abstract]
    ABSTRACT: Loss of imprinting (LOI) is occasionally observed in human imprinting disorders. However, the process behind the LOI is not fully understood. To gain a better understanding, we produced embryos and pups from mouse oocytes that lacked a complete methylation imprint using a method that involved transferring the nuclei of growing oocytes into the cytoplasm of enucleated fully grown oocytes following in vitro fertilization (IVF). We then analyzed the imprinting statuses. Our findings show that the incomplete methylation imprint derived from growing oocytes results in epigenetic mosaicism or a loss of methylation imprint (LOM) at maternal alleles in embryos. In some embryos, both hypo- and hypermethylated maternal Kcnq1ot1 alleles were detected, whereas either hypo- or hypermethylated maternal Kcnq1ot1 alleles were detected in others. Such tendencies were also observed at the Igf2r and Mest loci. Gene expression levels of imprinted genes were linked with their methylation statuses in some but not all embryos. Possible explanations of the inconsistency between the data from DNA methylation and gene expression include epigenetic mosaicism in embryos. Pups were successfully produced from growing oocytes at a quite low frequency. They exhibited an obese phenotype and LOI with respect to Igf2r, Snrpn and Mest. Our finding suggests the possibility that LOI/LOM at maternal alleles in human concepti could be derived from epigenetically immature/mutated oocytes.
    No preview · Article · Jun 2011 · Journal of Reproduction and Development
  • Yayoi Obata
    [Show abstract] [Hide abstract]
    ABSTRACT: In mammals, both parental genomes are essential for normal ontogeny because epigenetic modifications imposed in the parents' gametes lead to parent-of-origin specific gene expression in their offspring. These phenomena are referred to as genomic imprinting. It has been shown that maternal imprinting is established during oocyte growth, lack of maternal imprinting in zygotes leads to early embryonic death, and in vitro system that allows establishment of maternal imprinting is developed. In this review, I describe the history of the discovery of genomic imprinting, the regulatory mechanisms of mammalian development by maternal imprinting, and the molecular mechanisms of genomic imprinting.
    No preview · Article · Feb 2011 · Journal of Reproduction and Development
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2-knockout (Jdp2KO) mice by targeting exon-1 to disrupt the site of initiation of transcription. The epidermal thickening of skin from the Jdp2KO mice after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) proceeded more rapidly than that of control mice, and more proliferating cells were found at the epidermis. Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation.
    Full-text · Article · Nov 2010 · Oncogene
  • [Show abstract] [Hide abstract]
    ABSTRACT: To analyze an abnormal gait pattern in mutant mice (Hugger), we conducted coarse-grained motion capture. Using a simple retroreflective marker-based approach, we could detect high-resolution mutant-specific gait patterns. The phenotypic gait patterns are caused by extreme vertical motion of limbs, revealing inefficient motor functions. To elucidate the inefficiency, we developed a musculoskeletal computer model of the mouse hindlimb based on X-ray CT data. By integrating motion data with the model, we determined mutant-specific musculotendon lengths, suggesting that three major muscles were involved in the abnormal gait. This approach worked well on laboratory mice, which were putatively too small to be motion capture subjects. Motion capture technology was originally developed for human study, and our approach may help fill neuroscience gaps between mouse and human behavioral phenotypes.
    No preview · Article · Sep 2009 · Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cluster of imprinted genes located in the Dlk1–Dio3 domain spanning 1 Mb plays an essential role in controlling pre- and postnatal growth and differentiation in mice and humans. The failure of parent-of-origin-dependent gene expression in this domain results in grave disorders, leading to death in some cases. However, little is known about the role of maternally expressed non-coding RNAs (ncRNAs) including many miRNAs and snoRNAs in this domain. In order to further understand the role of these ncRNAs, we created Gtl2-mutant mice harboring a 10 kb deletion in exons 1–5. The mutant mice exhibited a very unique inheritance mode: when the deletion was inherited from the mother (Mat-KO), the pups were born with normal phenotypes; however, all of them died within 4 weeks after birth, probably due to severely hypoplastic pulmonary alveoli and hepatocellular necrosis. Mice carrying the paternal deletion (Pat-KO) showed severe growth retardation and perinatal lethality. Interestingly, the homozygous mutants (Homo-KO) survived and developed into fertile adults. Our results show that these phenotypes occur due to altered expression of the Dlk1–Dio3 cluster genes including miRNAs and snoRNAs via the cis and trans effects.
    Preview · Article · Apr 2009 · Human Molecular Genetics
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The B6.Y(TIR) sex-reversed female mouse is anatomically normal at young ages but fails to produce offspring. We have previously shown that its oocytes go through the meiotic cell cycle up to the second metaphase; however, the meiotic spindle is not properly organized, the second meiotic division goes awry after activation or fertilization, and none of the oocytes initiate embryonic development. In the present study, we transferred the nuclei of GV-stage oocytes from XY females into the enucleated GV-stage oocytes from (B6.DBA)F1.XX females. The resultant reconstructed oocytes properly assembled second meiotic spindles after in vitro maturation and produced healthy offspring after in vitro fertilization. Some male pups inherited maternal Y chromosomes. We conclude that the cytoplasm of the XY oocyte is insufficient to support spindle formation at the second metaphase whereas its replacement with the cytoplasmic material from an XX oocyte allows normal development.
    Preview · Article · Oct 2008 · Proceedings of the National Academy of Sciences
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.
    Full-text · Article · Jun 2008 · Nature
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.
    Preview · Article · May 2008 · Biology of Reproduction
  • [Show abstract] [Hide abstract]
    ABSTRACT: A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.
    No preview · Article · Feb 2008 · Nature Protocol

  • No preview · Article · Dec 2007 · Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme
  • Yayoi Obata · Yuhei Maeda · Izuho Hatada · Tomohiro Kono
    [Show abstract] [Hide abstract]
    ABSTRACT: Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.
    No preview · Article · Dec 2007 · Journal of Reproduction and Development
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.
    Full-text · Article · May 2007 · FEBS Letters
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Among the events that control cellular differentiation, the acetylation of histones plays a critical role in the regulation of transcription and the modification of chromatin. Jun dimerization protein 2 (JDP2), a member of the AP-1 family, is an inhibitor of such acetylation and contributes to the maintenance of chromatin structure. In an examination of Jdp2 'knock-out' (KO) mice, we observed elevated numbers of white adipocytes and significant accumulation of lipid in the adipose tissue in sections of scapulae. In addition, mouse embryo fibroblasts (MEFs) from Jdp2 KO mice were more susceptible to adipocyte differentiation in response to hormonal induction and members of the CCAAT/enhancer-binding proteins (C/EBP) gene family were expressed at levels higher than MEFs from wild-type mice. Furthermore, JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.
    Full-text · Article · Apr 2007 · Cell Death and Differentiation
  • Y. Obata · T. Kono
    [Show abstract] [Hide abstract]
    ABSTRACT: Long-term effects of in vitro maturation of oocytes and in vitro culture of fertilized eggs have been reported in ruminants, mice, and humans. However, effects of in vitro oocyte growth are unknown. Although a large number of non-growing oocytes can be a gamete resource, very few oocytes ever acquire competence to support full-term development after in vitro growth. The objective of the study was to evaluate different culture conditions and the long-term effects of in vitro oocyte growth on the production of offspring. Oocytes of newborn, 10-day-old, and adult BDF1 (C57BL/6N × DBA2) mice were cultured for 22, 11, and 1 day(s), respectively. The results showed that alpha-MEM medium was superior to Waymouth medium in oocyte growth (68.6 ± 3.87 µm vs. 61.7 ± 3.26 µm, respectively; P < 0.001), and in maintenance of follicular integrity (69% vs. 30%; P < 0.001) when non-growing oocytes from newborn mice were cultured. However, oocytes grown in vitro were incompetent to support meiotic maturation by themselves in the case of either the 22-day culture of oocytes from newborn mice (1/59 in alpha-MEM vs. 1/65 in Waymouth) or the 11-day culture of oocytes from 10-day-old mice (51/140 in alpha-MEM vs. 2/157 in Waymouth), and none of them developed to the blastocyst stage. Subsequently, to examine the nucleic competence of oocytes grown in vitro, serial nuclear transfers were carried out. Karyoplasts from oocytes grown in vitro using alpha-MEM were fused with the GV oocytes grown in vivo after enucleation. The reconstituted oocytes were cultured in alpha-MEM. After 14 h, MII chromosomes of the reconstituted oocytes were transferred into the enucleated and ovulated MII oocytes in order to provide cytoplasmic competency. The results showed that when the donor oocytes attained a diameter of e60 µm, the reconstituted oocytes could develop into pups at extremely high rates (30-41%) after in vitro fertilization (IVF) and embryo transfer in the case of either the 22-day culture of oocytes from newborn mice (7/17) or the 11-day culture of oocytes from 10-day-old mice (25/77). A significant difference was not observed in the competence to develop to term of the reconstituted oocytes when compared with that of the oocytes reconstituted from the control GV (25/52; P > 0.05). When the donor oocytes attained a diameter of 50–60 µm, the reconstituted oocytes also could develop into pups (7/33); however, their efficiency was significantly reduced when compared with that of the reconstituted oocytes from the control GV (P < 0.05). On the other hand, the weight of the offspring depended on the duration of culture, and offspring from non-growing oocytes (1.48 ± 0.17 g) were heavier than those of the IVF control (1.25 ± 0.14 g; P < 0.05). In conclusion, we have demonstrated that using a nuclear transfer technique combined with in vitro growth of oocytes was sufficient to produce functional oocytes, and long-term culture for oocyte growth did not affect the nucleic ability of oocytes to develop to term; however, fetal growth may be susceptible to the duration of culture.
    No preview · Article · Jan 2007 · Reproduction Fertility and Development

Publication Stats

4k Citations
673.39 Total Impact Points


  • 1996-2014
    • Tokyo University of Agriculture
      • • Department of Bioscience
      • • Department of Animal Science
      Edo, Tōkyō, Japan
  • 2007
    • RIKEN
      Вако, Saitama, Japan
  • 2002
    • National Institute of Genetics
      • Division of Human Genetics
      Мисима, Shizuoka, Japan
  • 2000-2002
    • Gunma University
      • Laboratory of Genome Science
      Maebashi, Gunma Prefecture, Japan
  • 2001
    • City of Hope National Medical Center
      Duarte, California, United States
  • 1986-2001
    • Aichi Cancer Center
      Nagoya, Aichi, Japan
  • 1991
    • Nagasaki University Hospital
      Nagasaki, Nagasaki, Japan
  • 1975-1989
    • Memorial Sloan-Kettering Cancer Center
      New York City, New York, United States
  • 1985
    • Kyoto University
      Kioto, Kyōto, Japan