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FacultyDepartment of Genetic Engineering / Graduate School of Biology-Oriented Science and Technology
PositionAssociate Professor
Commentator Guide
Last Updated :2020/09/30

Education and Career


  •  - 2000 , University of Tsukuba
  •  - 2000 , University of Tsukuba, Graduate School, Division of Agriculture
  •  - 1996 , University of Tsukuba
  •  - 1996 , University of Tsukuba, Second Cluster of College

Academic & Professional Experience

  •   2015 04 ,  - 現在, Faculty of Biology-Oriented Science and Technology, Kindai University
  •   2017 08 ,  - 2019 07 , Ministry of Education,Culture,Sports,Science and Technology
  •   2010 12 ,  - 2015 03 , Research Institute for Microbial Diseases, Osaka University
  •   2007 04 ,  - 2010 11 , Research fellow, CDB, RIKEN
  •   2003 01 ,  - 2007 03 , Graduate School of Life and Environmental Sciences, University of Tsukuba
  •   2001 04 ,  - 2002 12 , Osaka University
  •   2000 04 ,  - 2001 03 , Institute of Applied Biochemistry, University of Tsukuba
  •   1999 01 ,  - 2000 03 , Institute of Applied Biochemistry, University of Tsukuba

Research Activities

Research Areas

  • Life sciences, Applied molecular and cellular biology
  • Life sciences, Laboratory animal science
  • Life sciences, Laboratory animal science
  • Life sciences, Animals: biochemistry, physiology, behavioral science

Published Papers

  • Zygotic Nuclear F-Actin Safeguards Embryonic Development., Tomomi Okuno, Wayne Yang Li, Yu Hatano, Atsushi Takasu, Yuko Sakamoto, Mari Yamamoto, Zenki Ikeda, Taiki Shindo, Matthias Plessner, Kohtaro Morita, Kazuya Matsumoto, Kazuo Yamagata, Robert Grosse, Kei Miyamoto, Cell reports, Cell reports, 31(13), 107824 - 107824, Jun. 30 2020 , Refereed
  • Normal B cell development and Pax5 expression in Thy28/ThyN1-deficient mice, Kitaura F, Yuno M, Fujita T, Wakana S, Ueda J, Yamagata K, Fujii H, 14(7), e0220199, Jul. 2019 , Refereed
  • Histone H3K9 Methyltransferase G9a in Oocytes Is Essential for Preimplantation Development but Dispensable for CG Methylation Protection, Au Yeung WK, Brind'Amour J, Hatano Y, Yamagata K, Feil R, Lorincz MC, Tachibana M, Shinkai Y, Sasaki H, 27(1), 282 - 293, Apr. 2019 , Refereed
  • Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging., Yamagata K, Nagai K, Miyamoto H, Anzai M, Kato H, Miyamoto K, Kurosaka S, Azuma R, Kolodeznikov II, Protopopov AV, Plotnikov VV, Kobayashi H, Kawahara-Miki R, Kono T, Uchida M, Shibata Y, Handa T, Kimura H, Hosoi Y, Mitani T, Matsumoto K, Iritani A, Sci Rep, Sci Rep, 9(1), 4050, Mar. 2019 , Refereed
  • A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions., Takahashi T, Okeyo KO, Ueda J, Yamagata K, Washizu M, Oana H, Sci Rep, Sci Rep, 8(1), 13684, Sep. 2018 , Refereed
  • Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos, Yao T, Suzuki R, Furuta N, Suzuki Y, Kabe K, Tokoro M, Sugawara A, Yajima A, Nagasawa T, Matoba S, Yamagata K, Sugimura S, 8(1), 7460, May 2018 , Refereed
  • Peroxiredoxin as a functional endogenous antioxidant enzyme in pronuclei of mouse zygotes, Morita K, Tokoro M, Hatanaka Y, Higuchi C, Ikegami H, Nagai K, Anzai M, Kato H, Mitani T, Taguchi Y, Yamagata K, Hosoi Y, Miyamoto K, Matsumoto K, J Reprod Dev, J Reprod Dev, 64(2), 161 - 171, Apr. 2018 , Refereed
  • Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development, Higuchi C, Shimizu N, Shin SW, Morita K, Nagai K, Anzai M, Kato H, Mitani T, Yamagata K, Hosoi Y, Miyamoto K, Matsumoto K, 64(1), 65 - 74, Feb. 2018 , Refereed
  • Scaling relationship between intra-nuclear DNA density and chromosomal condensation in metazoan and plant, Hara Y, Adachi K, Kagohashi S, Yamagata K, Tanabe H, Kikuchi A, Okumura S-I, Kimura A, Chromosome Science, Chromosome Science, 19, 43 - 49, Sep. 2016 , Refereed
  • Generation of Hprt-disrupted rat through mouse <- rat ES chimeras, Ayako Isotani, Kazuo Yamagata, Masaru Okabe, Masahito Ikawa, SCIENTIFIC REPORTS, SCIENTIFIC REPORTS, 6, 24215, Apr. 2016 , Refereed
    Summary:We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse <- rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse <- rat ES chimera system. The mouse <- rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.
  • Behavior of Mouse Spermatozoa in the Female Reproductive Tract from Soon after Mating to the Beginning of Fertilization, Yuko Muro, Hidetoshi Hasuwa, Ayako Isotani, Haruhiko Miyata, Kazuo Yamagata, Masahito Ikawa, Ryuzo Yanagimachi, Masaru Okabe, BIOLOGY OF REPRODUCTION, BIOLOGY OF REPRODUCTION, 94(4), 1 - 7, Apr. 2016 , Refereed
    Summary:Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.
  • Micronucleus formation causes perpetual unilateral chromosome inheritance in mouse embryos, Cayetana Vazquez-Diez, Kazuo Yamagata, Shardul Trivedi, Jenna Haverfield, Greg FitzHarris, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 113(3), 626 - 631, Jan. 2016 , Refereed
    Summary:Chromosome segregation defects in cancer cells lead to encapsulation of chromosomes in micronuclei (MN), small nucleus-like structures within which dangerous DNA rearrangements termed chromothripsis can occur. Here we uncover a strikingly different consequence of MN formation in preimplantation development. We find that chromosomes from within MN become damaged and fail to support a functional kinetochore. MN are therefore not segregated, but are instead inherited by one of the two daughter cells. We find that the same MN can be inherited several times without rejoining the principal nucleus and without altering the kinetics of cell divisions. MN motion is passive, resulting in an even distribution of MN across the first two cell lineages. We propose that perpetual unilateral MN inheritance constitutes an unexpected mode of chromosome missegregation, which could contribute to the high frequency of aneuploid cells in mammalian embryos, but simultaneously may serve to insulate the early embryonic genome from chromothripsis.
  • Quantitative Assessment of Embryo Quality Based on a Live-Cell Imaging Technique, Yao T, Ueda J, Kobayashi T, Hori M, Yamagata K, Journal of Mammalian Ova Research, Journal of Mammalian Ova Research, 32(4), 149 - 157, Oct. 2015 , Refereed
  • Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced gamma H2AX accumulation, Tsunetoshi Nakatani, Kazuo Yamagata, Tohru Kimura, Masaaki Oda, Hiroyuki Nakashima, Mayuko Hori, Yoichi Sekita, Tatsuhiko Arakawa, Toshinobu Nakamura, Toru Nakano, EMBO REPORTS, EMBO REPORTS, 16(5), 582 - 589, May 2015 , Refereed
    Summary:In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (H2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of H2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.
  • Early Development of Cloned Bovine Embryos Produced from Oocytes Enucleated by Fluorescence Metaphase II Imaging Using a Conventional Halogen-Lamp Microscope, Daisaku Iwamoto, Kazuo Yamagata, Masao Kishi, Yoko Hayashi-Takanaka, Hiroshi Kimura, Teruhiko Wakayama, Kazuhiro Saeki, CELLULAR REPROGRAMMING, CELLULAR REPROGRAMMING, 17(2), 106 - 114, Apr. 2015 , Refereed
    Summary:Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals.
  • Visualization of epigenetic modifications in preimplantation embryos, Kimura H, Yamagata K, Methods Mol Biol, Methods Mol Biol, 1222, 127 - 147, 2015 , Refereed
  • Heterochromatin Dynamics during the Differentiation Process Revealed by the DNA Methylation Reporter Mouse, MethylRO, Jun Ueda, Kazumitsu Maehara, Daisuke Mashiko, Takako Ichinose, Tatsuma Yao, Mayuko Hori, Yuko Sato, Hiroshi Kimura, Yasuyuki Ohkawa, Kazuo Yamagata, STEM CELL REPORTS, STEM CELL REPORTS, 2(6), 910 - 924, Jun. 2014 , Refereed
    Summary:In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.
  • Improved and robust detection of cell nuclei from four dimensional fluorescence images, Sato Y, Mukai M, Ueda J, Muraki M, Stasevich T J, Horikoshi N, Kujirai T, Kita H, Kimura T, Hira S, Okada Y, Hayashi-Takanaka Y, Obuse C, Kurumizaka H, Kawahara A, Yamagata K, Nozaki N, Kimura H, Sci Rep, Sci Rep, (3), 2436, Aug. 2013 , Refereed
  • Long-term live-cell imaging of mammalian preimplantation development and derivation process of pluripotent stem cells from the embryos, Kazuo Yamagata, Jun Ueda, DEVELOPMENT GROWTH & DIFFERENTIATION, DEVELOPMENT GROWTH & DIFFERENTIATION, 55(4), 378 - 389, May 2013 , Refereed
    Summary:Mammalian fertilization is a process in which two highly specialized haploid gametes unite and endow totipotency to the resulting diploid zygote. This is followed by cell proliferation and the onset of differentiation during the brief period leading up to implantation. In these processes, a number of cellular components and structures are regulated spatially and temporally, as seen in repeated cell division, cell cycle progression, and epigenetic reprogramming. In mammals, the numbers of oocytes and embryos that can be collected are very limited. Therefore, analyses of molecular mechanisms are hampered because of difficulties in conducting biochemical analyses on such limited material. Furthermore, immunostaining methods require cell fixation and are insufficient for understanding ontogeny, because the processes observed in fertilization and early embryonic development progress in time-dependent manners and each phenomenon is connected with others by cause-and-effect relationships. Consequently, it is important to develop an experimental system that enables molecular imaging without affecting embryonic development. To achieve the above advantages, especially retrospective and prospective analyses, we have established a live-cell imaging system that enables observations under minimally invasive conditions. Using this approach, we have succeeded in visualizing and predicting the developmental potential of embryos after various perturbations. We also succeeded in imaging embryonic stem (ES) cell derivation in natural conditions. In this review, we describe a brief history of embryonic imaging and detailed protocols. We also discuss promising aspects of imaging in the fields of developmental and stem cell biology.
  • Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging, Togo Shimozawa, Kazuo Yamagata, Takefumi Kondo, Shigeo Hayashi, Atsunori Shitamukai, Daijiro Konno, Fumio Matsuzaki, Jun Takayama, Shuichi Onami, Hiroshi Nakayama, Yasuhito Kosugi, Tomonobu M. Watanabe, Katsumasa Fujita, Yuko Mimori-Kiyosue, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110(9), 3399 - 3404, Feb. 2013 , Refereed
    Summary:A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.
  • 4D imaging reveals a shift in chromosome segregation dynamics during mouse pre-implantation development, Kazuo Yamagata, Greg FitzHarris, CELL CYCLE, CELL CYCLE, 12(1), 157 - 165, Jan. 2013 , Refereed
    Summary:Cells of the early developing mammalian embryo frequently mis-segregate chromosomes during cell division, causing daughter cells to inherit an erroneous numbers of chromosomes. Why the embryo is so susceptible to errors is unknown, and the mechanisms that embryos employ to accomplish chromosome segregation are poorly understood. Chromosome segregation is performed by the spindle, a fusiform-shaped microtubule-based transient organelle. Here we present a detailed analysis of 4D fluorescence-confocal data sets of live embryos progressing from the one-cell embryo stage through to blastocyst in vitro, providing some of the first mechanistic insights into chromosome segregation in the mammalian embryo. We show that chromosome segregation occurs as a combined result of poleward chromosome motion (anaphase-A) and spindle elongation (anaphase-B), which occur simultaneously at the time of cell division. Unexpectedly, however, regulation of the two anaphase mechanisms changes significantly between the first and second embryonic mitoses. In one-cell embryos, the velocity of anaphase-A chromosome motion and the velocity and overall extent of anaphase-B spindle elongation are significantly constrained compared with later stages. As a result chromosomes are delivered close to the center of the forming two-cell stage blastomeres at the end of the first mitosis. In subsequent divisions, anaphase-B spindle elongation is faster and more extensive, resulting in the delivery of chromosomes to the distal plasma membrane of the newly forming blastomeres. Metaphase spindle length scales with cell size from the two-cell stage onwards, but is substantially shorter in the first mitosis than in the second mitosis, and the duration of mitosis-1 is substantially greater than subsequent divisions. Thus, there is a striking and unexpected shift in the approach to cell division between the first and second mitotic divisions, which likely reflects adaptations to the unique environment within the developing embryo.
  • Latrunculin A Can Improve the Birth Rate of Cloned Mice and Simplify the Nuclear Transfer Protocol by Gently Inhibiting Actin Polymerization, Yukari Terashita, Sayaka Wakayama, Kazuo Yamagata, Chong Li, Eimei Sato, Teruhiko Wakayama, BIOLOGY OF REPRODUCTION, BIOLOGY OF REPRODUCTION, 86(6), 180, Jun. 2012 , Refereed
    Summary:Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.
  • Abnormal chromosome segregation at early cleavage is a major cause of the full-term developmental failure of mouse clones, Eiji Mizutani, Kazuo Yamagata, Tetsuo Ono, Satoshi Akagi, Masaya Geshi, Teruhiko Wakayama, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 364(1), 56 - 65, Apr. 2012 , Refereed
    Summary:To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with alpha-tubulin (EGFP-alpha-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70 h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously. (C) 2012 Elsevier Inc. All rights reserved.
  • Effect of Fluorescent Mercury Light Irradiation on In Vitro and In Vivo Development of Mouse Oocytes after Parthenogenetic Activation or Sperm Microinjection, Yukari Terashita, Chong Li, Kazuo Yamagata, Eimei Sato, Teruhiko Wakayama, JOURNAL OF REPRODUCTION AND DEVELOPMENT, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 57(5), 564 - 571, Oct. 2011 , Refereed
    Summary:The detection of specific cellular components using fluorescent agents such as green fluorescent protein (GFP), red fluorescent protein or Hoechst dyes provides a powerful tool for studying cell biology. However, specimens must be exposed to high-intensity light, which might cause cellular damage. Here, we exposed mouse metaphase stage (M) II oocytes to fluorescent mercury vapor light at three wavelengths (539 nm, 488 nm and 341 nm) to determine the maximum exposure time that would avoid damage. When oocytes were activated parthenogenetically after exposure to these wavelengths for more than 20 min, 5 mm or 4 sec, respectively, the percentages of dead oocytes after activation increased, and none of the surviving embryos developed to blastocysts. However, embryos fertilized by intracytoplasmic sperm injection (ICSI) were more tolerant to light damage, even though the quality of blastocysts, judged by cell number and cell allocation to the inner cell mass and trophectoderm measured by immunostaining for Oct4 and Cdx2, was reduced as exposure times increased. Live, healthy offspring were obtained when these exposed embryos were transferred into recipient pseudopregnant females at the 2-cell stage. In addition, MII oocytes collected from GFP-expressing transgenic mice after 5 min of irradiation with 488-nm light were also able to develop to full term following ICSI. Thus, we determined the safe period of exposure to several wavelengths for oocyte manipulation or observation that would permit subsequent development.
  • Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling, Yoko Hayashi-Takanaka, Kazuo Yamagata, Teruhiko Wakayama, Timothy J. Stasevich, Takashi Kainuma, Toshiki Tsurimoto, Makoto Tachibana, Yoichi Shinkai, Hitoshi Kurumizaka, Naohito Nozaki, Hiroshi Kimura, NUCLEIC ACIDS RESEARCH, NUCLEIC ACIDS RESEARCH, 39(15), 6475 - 6488, Aug. 2011 , Refereed
    Summary:Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.
  • Offspring From Intracytoplasmic Sperm Injection of Aged Mouse Oocytes Treated With Caffeine or MG132, Tetsuo Ono, Eiji Mizutani, Chong Li, Kazuo Yamagata, Teruhiko Wakayama, GENESIS, GENESIS, 49(6), 460 - 471, Jun. 2011 , Refereed
    Summary:Postovulatory mammalian oocytes age significantly in culture. B6D2F1 or ICR strain mouse oocytes were collected 16 h after hCG injection and then cultured for up to 40 h post hCG at 37 degrees C under 5% CO2 in air. After intracytoplasmic sperm injection (ICSI), B6D2F1 and ICR oocytes lost full-term developmental potential by 30 h and 26 h after hCG administration, respectively. However, using supplementation with 10 mM caffeine or 1-5 mu M of MG132, we could obtain live offspring from oocytes at 34 h (BDF1, 5%-21%) or 28 h (ICR, 5%-18%), whereas none were obtained from untreated aged oocytes. Caffeine maintained normal meiotic spindle morphology, whereas MG132 maintained maturation-promoting factor activity. These treatments did not affect the potential of fresh oocytes for fertilization and subsequent development. Thus, it should be safe to use these chemicals in routine in vitro fertilization and offspring could be generated by ICSI of aged fertilization failed oocytes. genesis 49:460-471, 2011. (C) 2011 Wiley-Liss, Inc.
  • Understanding paternal genome demethylation through live-cell imaging and siRNA, Kazuo Yamagata, Yuki Okada, CELLULAR AND MOLECULAR LIFE SCIENCES, CELLULAR AND MOLECULAR LIFE SCIENCES, 68(10), 1669 - 1679, May 2011 , Refereed
    Summary:Identification of a DNA demethylase responsible for zygotic paternal DNA demethylation has been one of the most challenging goals in the field of epigenetics. Several candidate molecules have been proposed, but their involvement in the demethylation remains controversial, partly due to the difficulty of preparing a sufficient quantity of materials for biochemical analysis. In this review, we utilize a recently developed method for live-cell imaging of mouse zygotes combined with RNA interference (RNAi) to search for factors that affect zygotic paternal DNA demethylation. The combined use of various fluorescent probes and RNAi is a useful approach for the study of not only DNA demethylation but also the spatiotemporal dynamics of histone depositions in zygotes, although it is not appropriate for large-scale screening or knockdown of genes that are abundantly expressed before fertilization. This new technique enables us to understand the epigenetic hierarchy during cellular response and differentiation in preimplantation embryos.
  • DNA methylation profiling using live-cell imaging, Yamagata K, Methods, Methods, 52(3), 259 - 66, Nov. 2010 , Refereed
  • Long-term, six-dimensional live-cell imaging for the mouse preimplantation embryo that does not affect full-term development, Yamagata K, Suetsugu R, Wakayama T, J Reprod Dev, J Reprod Dev, 55(3), 343 - 350, Mar. 2009 , Refereed
  • Synthesis, processing, and subcellular localization of mouse ADAM3 during spermatogenesis and epididymal sperm transport, Kim E, Nishimura H, Iwase S, Yamagata K, Kashiwabara S, Baba T, J Reprod Dev, J Reprod Dev, 50(5), 571 - 578, Oct. 2004 , Refereed
  • Occurrence of small,round vesicles in the acrosome of elongating spermatids from a mouse mutant line with a partial deletion of the Y chromosome, Siruntawineti J, Yamagata K, Nakanishi T, Baba T, J Reprod Dev, J Reprod Dev, 48, 513 - 521, Apr. 2002 , Refereed
  • Difference of acrosomal serine protease system between mouse and other rodent sperm, Yamagata K, Honda A, Kashiwabara S, Baba T, Genesis, Genesis, 25(2), 115 - 122, Sep. 1999 , Refereed
  • A homologue of pancreatic trypsin is localized in the acrosome of mammalian sperm and is released during acrosome reaction, Ohmura K, Kohno N, Kobayashi Y, Yamagata K, Sato S, Kashiwabara S, Baba T, J Biol Chem, J Biol Chem, 274(41), 29426 - 29432, Apr. 1999 , Refereed
  • Improved and robust detection of cell nuclei from four dimensional fluorescence images, Yamagata K, Murayama K, Okabe M, Toshimori K, Nakanishi T, Kashiwabara S, Baba T, J Biol Chem, J Biol Chem, 273(17), 10470 - 10474, Apr. 1998 , Refereed
  • Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage, Mashiko D, Ikeda Z, Yao T, Tokoro M, Fukunaga N, Asada Y, Yamagata K, Sci Rep, Sci Rep, 10(1), 854, Jan. 2020 , Refereed
  • Editing DNA Methylation in Mammalian Embryos, Yamazaki T, Hatano Y, Taniguchi R, Kobayashi N, Yamagata K, Int. J. Mol. Sci, Int. J. Mol. Sci, 21(2), 637, Jan. 2020 , Refereed
  • Nuclear formation induced by DNA-conjugated beads in living fertilised mouse egg., Yuka Suzuki, Şükriye Bilir, Yu Hatano, Tatsuhito Fukuda, Daisuke Mashiko, Shouhei Kobayashi, Yasushi Hiraoka, Tokuko Haraguchi, Kazuo Yamagata, Sci Rep, Sci Rep, 9(1), 8461 - 8461, Jun. 11 2019 , Refereed
    Summary:Reformation of a functional nucleus at the end of mitosis is crucial for normal cellular activity. Reconstitution approaches using artificial beads in frog egg extracts have clarified the molecules required for nuclear formation in vitro. However, the spatiotemporal regulation of these components, which is required for the formation of a functional nucleus in living embryos, remains unknown. Here we demonstrate that exogenous DNA introduced in the form of DNA-conjugated beads induces the assembly of an artificial nucleus in living mouse cleavage-stage embryos. Live-cell imaging and immunofluorescence studies revealed that core histones and regulator of chromosome condensation 1 (RCC1) assembled on the DNA, suggesting that nucleosomes were formed. Electron microscopy showed that double-membrane structures, partly extended from annulate lamellae, formed around the beads. Nuclear pore complex-like structures indistinguishable from those of native nuclei were also formed, suggesting that this membranous structure resembled the normal nuclear envelope (NE). However, the reconstituted NE had no nuclear import activity, probably because of the absence of Ras-related nuclear protein (Ran). Thus, DNA is necessary for NE reassembly in mouse embryos but is insufficient to form a functional nucleus. This approach provides a new tool to examine factors of interest and their spatiotemporal regulation in nuclear formation.
  • Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells, Yuichiro Semba, Akihito Harada, Kazumitsu Maehara, Shinya Oki, Chikara Meno, Jun Ueda, Kazuo Yamagata, Atsushi Suzuki, Mitsuho Onimaru, Jumpei Nogami, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa, Nucleic Acids Res, Nucleic Acids Res, 45(15), 8758 - 8772, Sep. 2017 , Refereed
    Summary:Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.
  • Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2, Sugako Ogushi, Kazuo Yamagata, Chikashi Obuse, Keiko Furuta, Teruhiko Wakayama, Martin M. Matzuk, Mitinori Saitou, J Cell Sci, J Cell Sci, 130(14), 2416 - 2429, Jul. 2017 , Refereed
    Summary:The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental pericentromeric DNAs, which led to a significant mitotic delay and frequent chromosome mis-segregation upon the first mitotic division. A proteomic analysis identified nucleoplasmin 2 (NPM2) as a dominant component of the oocyte nucleolus. Consistently, Npm2-deficient oocytes, which lack a normal nucleolar structure, showed chromosome segregation defects similar to those in enucleolated oocytes, suggesting that nucleolar loss, rather than micromanipulation-related damage to the genome, leads to a disorganization of higher-order chromatin structure in pronuclei and frequent chromosome mis-segregation during the first mitosis. Strikingly, expression of NPM2 alone sufficed to reconstitute the nucleolar structure in enucleolated embryos, and rescued their first mitotic division and full-term development. The nucleolus rescue through NPM2 required the pentamer formation and both the N-and C-terminal domains. Our findings demonstrate that the NPM2-based oocyte nucleolus is an essential platform for parental chromatin organization in early embryonic development.
  • A delayed sperm penetration of cumulus layers by disruption of acrosin gene in rats, Ayako Isotani, Takafumi Matsumura, Masaki Ogawa, Takahiro Tanaka, Kazuo Yamagata, Masahito Ikawa, Masaru Okabe, Biol Reprod, Biol Reprod, 97(1), 61 - 68, Jul. 2017 , Refereed
    Summary:Acrosin, the trypsin-like serine protease in the sperm acrosome, was long viewed as a key enzyme required for zona pellucida penetration to fertilize eggs. However, gene disruption experiments in mice surprisingly showed that acrosin-disrupted males were fertile. Thus, the acrosin was considered to be not an essential enzyme for fertilization in mice. However, the involvement of acrosin in fertilization has been suggested in various species such as rat, bull, and pig. Moreover, it has been reported that serine protease (including acrosin) activity in mice is significantly weaker compared to other species, including rats. We analyzed the role of acrosin by disrupting the rat acrosin gene. It was found that, unlike in mice, acrosin was almost the sole source of serine protease in rat spermatozoa. Nevertheless, the acrosin-disrupted males were not infertile. However, the litter size from acrosin-disrupted males was decreased compared to heterozygous mutant rats. Further investigation using an in vitro fertilization system revealed that the acrosin-disrupted spermatozoa possessed an equal ability to penetrate the zona pellucida with wild-type spermatozoa, but the cumulus cell dispersal was slower compared to wild-type and heterozygous spermatozoa. This delay was presumed to be the cause of the small litter size of acrosin-disrupted male rats. Summary Sentence Acrosin was practically the sole source of serine protease in rat spermatozoa, but its disruption did not produce infertile males.
  • Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase, Taiga Yamazaki, Yu Hatano, Tetsuya Handa, Sakiko Kato, Kensuke Hoida, Rui Yamamura, Takashi Fukuyama, Takayuki Uematsu, Noritada Kobayashi, Hiroshi Kimura, Kazuo Yamagata, PLOS ONE, PLOS ONE, 12(5), e0177764, May 2017 , Refereed
    Summary:To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
  • Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules, Kei Miyamoto, Yosuke Tajima, Koki Yoshida, Mami Oikawa, Rika Azuma, George E. Allen, Tomomi Tsujikawa, Tomomasa Tsukaguchi, Charles R. Bradshaw, Jerome Jullien, Kazuo Yamagata, Kazuya Matsumoto, Masayuki Anzai, Hiroshi Imai, John B. Gurdon, Masayasu Yamada, Biol Open, Biol Open, 6(4), 415 - 424, Apr. 2017 , Refereed
    Summary:Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.
  • Viable offspring after imaging of Ca2+ oscillations and visualization of the cortical reaction in mouse eggs, Yuhkoh Satouh, Kaori Nozawa, Kazuo Yamagata, Takao Fujimoto, Masahito Ikawa, Biol Reprod, Biol Reprod, 96(3), 563 - 575, Mar. 2017 , Refereed
    Summary:During mammalian fertilization, egg Ca2+ oscillations are known to play pivotal roles in triggering downstream events such as resumption of the cell cycle and the establishment of blocks to polyspermy. However, viable offspring have not been obtained after monitoring Ca2+ oscillations, and their spatiotemporal links to subsequent events are still to be examined. Therefore, the development of imaging methods to avoid phototoxic damage while labeling these events is required. Here, we examined the usefulness of genetically encoded Ca2+ indicators for optical imaging (GECOs), in combination with spinning-disk confocal imaging. The Ca2+ imaging of fertilized mouse eggs with GEM-, G-, or R-GECO recorded successful oscillations (8.19 +/- 0.31, 7.56 +/- 0.23, or 7.53 +/- 0.27 spikes in the first 2 h, respectively), similar to those obtained with chemical indicators. Then, in vitro viability tests revealed that imaging with G-or R-GECO did not interfere with the rate of development to the blastocyst stage (61.8 or 70.0%, respectively, vs 75.0% in control). Furthermore, two-cell transfer to recipient female mice after imaging with G-or R-GECO resulted in a similar birthrate (53.3 or 52.0%, respectively) to that of controls (48.7%). Next, we assessed the quality of the cortical reaction (CR) in artificially activated or fertilized eggs using fluorescently labeled Lens culinaris agglutinin-fluorescein isothiocyanate. Multicolor imaging demonstrated that the first few Ca2+ spikes are sufficient for the completion of the CR and subsequent hardening of the zona pellucida in mouse eggs. These methods provide a framework for studying Ca2+ dynamics in mammalian fertilization.
  • Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis, Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata, Cell Rep, Cell Rep, 18(3), 593 - 600, Jan. 2017 , Refereed
    Summary:Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-typespecific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.
  • A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation, Yuko Sato, Tomoya Kujirai, Ritsuko Arai, Haruhiko Asakawa, Chizuru Ohtsuki, Naoki Horikoshi, Kazuo Yamagata, Jun Ueda, Takahiro Nagase, Tokuko Haraguchi, Yasushi Hiraoka, Akatsuki Kimura, Hitoshi Kurumizaka, Hiroshi Kimura, J Mol Biol, J Mol Biol, 428(20), 3885 - 3902, Oct. 2016 , Refereed
    Summary:Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K2Ome1). The specificity of the H4K2Ome1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K2Ome1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K2Ome1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (
  • Live imaging of X chromosome reactivation dynamics in early mouse development can discriminate naive from primed pluripotent stem cells, Shin Kobayashi, Yusuke Hosoi, Hirosuke Shiura, Kazuo Yamagata, Saori Takahashi, Yoshitaka Fujihara, Takashi Kohda, Masaru Okabe, Fumitoshi Ishino, Development, Development, 143(16), 2958 - 2964, Aug. 2016 , Refereed
    Summary:Pluripotent stem cells can be classified into two distinct states, naive and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naive state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.
  • Improved and Robust Detection of Cell Nuclei from Four Dimensional Fluorescence Images, Md Khayrul Bashar, Kazuo Yamagata, Tetsuya J. Kobayashi, PLOS ONE, PLOS ONE, 9(7), e101891, Jul. 2014 , Refereed
    Summary:Segmentation-free direct methods are quite efficient for automated nuclei extraction from high dimensional images. A few such methods do exist but most of them do not ensure algorithmic robustness to parameter and noise variations. In this research, we propose a method based on multiscale adaptive filtering for efficient and robust detection of nuclei centroids from four dimensional (4D) fluorescence images. A temporal feedback mechanism is employed between the enhancement and the initial detection steps of a typical direct method. We estimate the minimum and maximum nuclei diameters from the previous frame and feed back them as filter lengths for multiscale enhancement of the current frame. A radial intensity-gradient function is optimized at positions of initial centroids to estimate all nuclei diameters. This procedure continues for processing subsequent images in the sequence. Above mechanism thus ensures proper enhancement by automated estimation of major parameters. This brings robustness and safeguards the system against additive noises and effects from wrong parameters. Later, the method and its single-scale variant are simplified for further reduction of parameters. The proposed method is then extended for nuclei volume segmentation. The same optimization technique is applied to final centroid positions of the enhanced image and the estimated diameters are projected onto the binary candidate regions to segment nuclei volumes. Our method is finally integrated with a simple sequential tracking approach to establish nuclear trajectories in the 4D space. Experimental evaluations with five image-sequences (each having 271 3D sequential images) corresponding to five different mouse embryos show promising performances of our methods in terms of nuclear detection, segmentation, and tracking. A detail analysis with a sub-sequence of 101 3D images from an embryo reveals that the proposed method can improve the nuclei detection accuracy by 9 % over the previous methods, which used inappropriate large valued parameters. Results also confirm that the proposed method and its variants achieve high detection accuracies (> 98% mean F-measure) irrespective of the large variations of filter parameters and noise levels.
  • Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos, Yukari Terashita, Kazuo Yamagata, Mikiko Tokoro, Fumiaki Itoi, Sayaka Wakayama, Chong Li, Eimei Sato, Kentaro Tanemura, Teruhiko Wakayama, PLOS ONE, PLOS ONE, 8(10), e78380, Oct. 2013 , Refereed
    Summary:Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.
  • Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent, Fumiaki Itoi, Mikiko Tokoro, Yukari Terashita, Kazuo Yamagata, Noritaka Fukunaga, Yoshimasa Asada, Teruhiko Wakayama, PLOS ONE, PLOS ONE, 7(10), e47512, Oct. 2012 , Refereed
    Summary:To culture preimplantation embryos in vitro, water-jacketed CO2 incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37 degrees C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37 degrees C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O-2 was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.
  • Fluorescence Cell Imaging and Manipulation Using Conventional Halogen Lamp Microscopy, Kazuo Yamagata, Daisaku Iwamoto, Yukari Terashita, Chong Li, Sayaka Wakayama, Yoko Hayashi-Takanaka, Hiroshi Kimura, Kazuhiro Saeki, Teruhiko Wakayama, PLOS ONE, PLOS ONE, 7(2), e31638, Feb. 2012 , Refereed
    Summary:Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.
  • Inhibition of class IIb histone deacetylase significantly improves cloning efficiency in mice., YAMAGATA Kazuo, Biology of Reproduction, Biology of Reproduction, 83, 90 - 101, Oct. 2010 , Refereed
  • Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system, Kazuo Yamagata, Jun Ueda, Eiji Mizutani, Mitinori Saitou, Teruhiko Wakayama, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 346(1), 90 - 101, Oct. 2010 , Refereed
    Summary:Despite the broad literature on embryonic stem cells (ESCs), their derivation process remains enigmatic. This may be because of the lack of experimental systems that can monitor this prolonged cellular process. Here we applied a live-cell imaging technique to monitor the process of ESC derivation over 10 days from morula to outgrowth phase using an Oct4/eGFP reporter system. Our imaging reflects the 'natural' state of ESC derivation, as the ESCs established after the imaging were both competent in chimeric mice formation and germ-line transmission. Using this technique, ESC derivation in conventional conditions was imaged. After the blastocoel was formed, the intensity of Oct4 signals attenuated in the trophoblast cells but was maintained in the inner cell mass (ICM). Thereafter, the Oct4-positive cells scattered and their number decreased along with apoptosis of the other Oct4-nagative cells likely corresponds to trophoblast and hypoblast cells, and then only the surviving Oct4-positive cells proliferated and formed the colony. All embryos without exception passed through this cell death phase. Importantly, the addition of caspase inhibitor Z-VAD-FMK to the medium dramatically suppressed the loss of Oct4-positive cells and also other embryo-derived cells, suggesting that the cell deaths was induced by a caspase-dependent apoptotic pathway. Next we imaged the ESC derivation in 3i medium, which consists of chemical compounds that can suppress differentiation. The most significant difference between the conventional and 3i methods was that there was no obvious cell death in 3i, so that the colony formation was rapid and all of the Oct4-positive cells contributed to the formation of the outgrown colony. These data indicate that the prevention of cell death in epiblast cells is one of the important events for the successful establishment of ESCs. Thus, our imaging technique can advance the understanding of the time-dependent cellular changes during ESC derivation. (C) 2010 Elsevier Inc. All rights reserved.
  • Visualization of DNA methylation and histone modifications in living cells, Hiroshi Kimura, Yoko Hayashi-Takanaka, Kazuo Yamagata, CURRENT OPINION IN CELL BIOLOGY, CURRENT OPINION IN CELL BIOLOGY, 22(3), 412 - 418, Jun. 2010 , Refereed
    Summary:DNA methylation and histone modifications play important roles in genome function, including epigenetic gene regulation. These modifications undergo drastic changes when nuclei are reprogrammed during development and differentiation. Recent studies have enabled the detection of the dynamics of modifications in living cultured cells and mouse preimplantation embryos. DNA methylation was visualized using the methyl-CpG-binding domain of the human MBD1 protein. The level and distribution of histone modifications can be monitored by two different methods. One approach uses fluorescence/Forster resonance energy transfer (FRET)-based sensors and another uses fluorescently labeled antigen binding fragments of specific antibodies. These visualization techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease.
  • A role for the elongator complex in zygotic paternal genome demethylation, Yuki Okada, Kazuo Yamagata, Kwonho Hong, Teruhiko Wakayama, Yi Zhang, NATURE, NATURE, 463(7280), 554 - U177, Jan. 2010 , Refereed
    Summary:The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs(1). One of the molecular events that takes place during this transition is the demethylation of the paternal genome(2,3). Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified(4). To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through short-interfering-RNA-mediated knockdown in mouse zygotes, we identified Elp3 (also called KAT9), a component of the elongator complex(5), to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulphite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of messenger RNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation, indicating that the SAM radical domain is involved in the demethylation process. Our study not only establishes a critical role for the elongator complex in zygotic paternal genome demethylation, but also indicates that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.
  • Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase, Yoko Hayashi-Takanaka, Kazuo Yamagata, Naohito Nozaki, Hiroshi Kimura, JOURNAL OF CELL BIOLOGY, JOURNAL OF CELL BIOLOGY, 187(6), 781 - 790, Dec. 2009 , Refereed
    Summary:Posttranslational histone modifications regulate both gene expression and genome integrity. Despite the dynamic nature of these modifications, appropriate real-time monitoring systems are lacking. In this study, we developed a method to visualize histone modifications in living somatic cells and preimplantation embryos by loading fluorescently labeled specific Fab antibody fragments. The technique was used to study histone H3 Ser10 (H3S10) phosphorylation, which occurs during chromosome condensation in mitosis mediated by the aurora B kinase. In aneuploid cancer cells that frequently missegregate chromosomes, H3S10 is phosphorylated just before the chromosomes condense, whereas aurora B already accumulates in nuclei during S phase. In contrast, in nontransformed cells, phosphorylated H3S10 foci appear for a few hours during interphase, and transient exposure to an aurora B-selective inhibitor during this period induces chromosome missegregation. These results suggest that, during interphase, moderate aurora B activity or H3S10 phosphorylation is required for accurate chromosome segregation. Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies.
  • Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection, Kazuo Yamagata, Rinako Suetsugu, Teruhiko Wakayama, HUMAN REPRODUCTION, HUMAN REPRODUCTION, 24(10), 2490 - 2499, Oct. 2009 , Refereed
    Summary:Intracytoplasmic sperm injection (ICSI) is a technique in which sperm are injected directly into unfertilized oocytes, whereby offspring can be obtained even with dysfunctional sperm. Despite its advantages in human and animal reproductive technology, the low rate of resultant live offspring is perturbing. One major cause is thought to be embryonic chromosomal abnormalities. However, there is no direct evidence of how these occur or how they affect pregnancy outcomes. Chromosomal dynamics during the first mitotic division of mouse embryos were analyzed using a new live-cell imaging technology. After imaging, the embryos' developmental capacities were determined. When ICSI-generated embryos were monitored for their chromosome integrity, some embryos with apparent normal morphology seen by conventional light microscopy had abnormal chromosome segregation (ACS) at the first mitotic division. Chromosomal fragments were misaligned during the first metaphase and formed micronuclear-like structures at the interphase of the 2-cell stage. Similar ACS was also found in mouse embryos produced by microinjecting round spermatids, with even higher frequency. Giemsa staining and immunostaining revealed that these fragments were derived from double-strand DNA breaks in the paternal genome. About half of the embryos with ACS developed into normal-looking morulae or blastocysts and implanted, but almost all of them aborted spontaneously before embryonic day 7.5. ACS during first mitosis appears to be a major cause of early pregnancy losses in ICSI-generated mouse embryos. Moreover, this novel imaging technology could be applicable as a method for the assessment of embryo quality.
  • Detrimental Effects of Microgravity on Mouse Preimplantation Development In Vitro, Sayaka Wakayama, Yumi Kawahara, Chong Li, Kazuo Yamagata, Louis Yuge, Teruhiko Wakayama, PLOS ONE, PLOS ONE, 4(8), e6753, Aug. 2009 , Refereed
    Summary:Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (mu G) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under mG. However, although we obtained 75 healthy offspring from mu G-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under mG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under mG environment in a mammal, but normal preimplantation embryo development might require 1G.
  • Increasing the cell number of host tetraploid embryos can improve the production of mice derived from embryonic stem cells, Hiroshi Ohta, Yuko Sakaide, Kazuo Yamagata, Teruhiko Wakayama, BIOLOGY OF REPRODUCTION, BIOLOGY OF REPRODUCTION, 79(3), 486 - 492, Sep. 2008 , Refereed
    Summary:Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1X) to triple (3X) host 4n embryos. We found that birth rate of ES mice using 1X 4n embryos was quite low (0-2%) regardless of cell line, whereas except for one cell line, approximately 6-14% of embryos developed to full term in the case of 3X 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3X 4n host embryos. These results demonstrate that the use of 3X 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.
  • Nuclear localization of profilin III-ArpM1 complex in mouse spermiogenesis, Yuki Hara, Kazuo Yamagata, Kei Oguchi, Tadashi Baba, FEBS LETTERS, FEBS LETTERS, 582(20), 2998 - 3004, Sep. 2008 , Refereed
    Summary:Actin-related proteins (Arps) have been reported to be localized in the cell nucleus, and implicated in the regulation of chromatin and nuclear structure, as well as being involved in cytoplasmic functions. We demonstrate here that mouse ArpM1, which closely resembles the conventional actin, is expressed exclusively in the testis, particularly in haploid germ cells. ArpM1 protein first appears in the round spermatid and changes its localization dynamically in the nucleus during spermiogenesis. By co-immunoprecipitation analysis, profilin III was identified as ArpM1-interacting protein. Our findings suggest that the testis-specific profilin III-ArpM1 complex may be involved in conformational changes in the organization of the sperm-specific nucleus.
  • The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice, Kenji Miyado, Keiichi Yoshida, Kazuo Yamagata, Keiichi Sakakibara, Masaru Okabe, Xiaobiao Wang, Kiyoko Miyamoto, Hidenori Akutsu, Takahiko Kondo, Yuji Takahashi, Tadanobu Ban, Chizuru Ito, Kiyotaka Toshimori, Akihiro Nakamura, Masahiko Ito, Mami Miyado, Eisuke Mekada, Akihiro Umezawa, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(35), 12921 - 12926, Sep. 2008 , Refereed
    Summary:Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.
  • Cd52, known as a major maturation-associated sperm membrane antigen secreted from the epididymis, is not required for fertilization in the mouse, Ryo Yamaguchi, Kazuo Yamagata, Hidetoshi Hasuwa, Emiko Inano, Masahito Ikawa, Masaru Okabe, GENES TO CELLS, GENES TO CELLS, 13(8), 851 - 861, Aug. 2008 , Refereed
    Summary:CD52 is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes and in epididymal epithelial cells. CD52 is also known as "maturation-associated sperm antigen" but its function is unknown. We therefore generated Cd52 disrupted mice. The resulting Cd52 null mice were healthy, even though Cd52 is expressed on cells of the immune system. We then examined a possible role for CD52 in reproduction. Sperm from Cd52-deficient males were investigated and the viability, motility, morphology, and incidence of spontaneous acrosome reactions were found to be all similar to values for wild-type sperm. In in vitro fertilization system, the sperm showed normal fertilizing ability. As CD52 was found to be transferred onto sperm only after they had migrated into the vas deferens, we examined the behavior of sperm from Cd52-deficient mice in vivo. The mice mated naturally and we observed that a normal number of sperm passed through the uterotubal junction, known to the crucial hurdle for various gene knockouts resulting in infertile sperm. As a consequence, there was no difference in the litter size from the wild-type and Cd52-null males. Our results therefore indicate CD52 is not required for fertilization in the mouse either in vivo or in vitro.
  • Capturing epigenetic dynamics during pre-implantation development using live cell imaging, Kazuo Yamagata, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 143(3), 279 - 286, Mar. 2008 , Refereed
    Summary:During mammalian fertilization and pre-implantation development, the highly differentiated gametes revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in male and female gamete chromatin structure, in global epigenetic modifications and in nuclear architecture. We have developed a live cell imaging technique for oocytes and early embryos to understand these series of phenomena. Using this technique, we were able to observe dynamic changes in DNA methylation status in living embryos. Furthermore, epigenetic abnormalities were detected in reconstructed embryos generated by round spermatid injection or by somatic cell nuclear transfer. In this review, I will discuss the usefulness and possibilities of this imaging technique in studies on nuclear dynamics during fertilization and preimplantation development.
  • Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages, Kazuo Yamagata, Taiga Yamazaki, Hiromi Miki, Narumi Ogonuki, Kimiko Inoue, Atsuo Ogura, Tadashi Baba, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 312(1), 419 - 426, Dec. 2007 , Refereed
    Summary:Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature. (c) 2007 Elsevier Inc. All rights reserved.
  • Molecular dynamics of heterochromatin protein 1 beta, HP1 beta, during mouse preimplantation development, Taiga Yamazaki, Satoru Kobayakawa, Kazuo Yamagata, Kuniya Abe, Tadashi Baba, J Reprod Dev., J Reprod Dev., 53(5), 1035 - 1041, Oct. 2007 , Refereed
    Summary:To elucidate the molecular dynamics of HP1 beta in mouse preimplantation embryos, we examined the localization, dynamics, and mobility of HP1 beta in the (pro)nucleus by live cell imaging. Time-lapse observation revealed that the chromatin association of HP1 beta is regulated in a cell cycle-dependent manner. HP1 beta was localized in the interphase nucleus and was dynamically dissociated from the nucleus during the metaphase stage. The HP1 beta assembly and clustered heterochromatin structure were both found in the nuclei of 2-cell and later-stage embryos. Moreover, fluorescent recovery after photobleaching analysis implied that HP1 beta is more freely mobile in the pronucleus of the 1-cell embryo than in the 4-cell nucleus. These results suggest that the chromatin configuration may be regulated by the stability and mobility of chromatin-associated proteins including HP1 beta during early embryonic stages.
  • Acrosome reaction of mouse epididymal sperm on oocyte zona pellucida, Misuzu Yamashita, Kazuo Yamagata, Keiko Tsumura, Tomoko Nakanishi, Tadashi Baba, J Reprod Dev., J Reprod Dev., 53(2), 255 - 262, Apr. 2007 , Refereed
    Summary:To improve assessment of the acrosome reaction of mouse epididymal sperm, we employed anti-Izumo1 antibody instead of antibodies against acrosomal proteins. The acrosomal states among acrosome-intact, spontaneously acrosome-reacted, truly acrosome-reacted, and probably dead and/or membrane-damaged sperm were clearly distinguished by combined application of anti-Izumo1 antibody, DNA dye Hoechst 33342, and monoclonal antibody MN7 to paraformaldehyde-fixed sperm. When the acrosome reaction of capacitated epididymal sperm on the oocyte zona pellucida was examined using anti-Izumo1 antibody, approximately 20% of sperm bound onto the zona pellucida were acrosome-reacted 30 min after insemination. We also observed the moment of the acrosome reaction of live sperm on the zona pellucida by time-lapse monitoring using fluorescein isothiocyanate-conjugated anti-Izumo1 antibody.
  • Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging, Taiga Yamazaki, Kazuo Yamagata, Tadashi Baba, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 304(1), 409 - 419, Apr. 2007 , Refereed
    Summary:Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin. (c) 2006 Elsevier Inc. All rights reserved.
  • Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice, Ryo Yamaguchi, Kazuo Yamagata, Masahito Ikawa, Stuart B. Moss, Masaru Okabe, BIOLOGY OF REPRODUCTION, BIOLOGY OF REPRODUCTION, 75(5), 760 - 766, Nov. 2006 , Refereed
    Summary:Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from C/gn(-/-) mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn(-/-) and Ace(-/-) sperm. ADAM3 was absent from Clgn(-/-) sperm. In the Ace-/- mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergentenriched phase may explain the inability of Clgn(-/-) and Ace sperm to bind to the zona pellucida.
  • Noninvasive visualization of molecular events in the mammalian zygote, K Yamagata, T Yamazaki, M Yamashita, Y Hara, N Ogonuki, A Ogura, GENESIS, GENESIS, 43(2), 71 - 79, Oct. 2005 , Refereed
    Summary:Following fertilization, a number of molecular events are triggered in the mammalian zygote. As biochemical studies using mammalian gametes and zygotes have inherent difficulties, the molecular nature of these processes is currently unclear. We have developed a method to visualize these events. In vitro transcribed mRNAs encoding for proteins fused with green fluorescent protein were microinjected into oocytes or embryos and fluorescence signals were observed. Using this technique we succeeded in obtaining images of the DNA methylation status in living mouse and rabbit embryos. Moreover, time-lapse images were acquired of spindle and nuclear formation during second meiosis and first mitosis. Importantly, the microinjected embryos developed to the normal offspring even after observation, suggesting that the technique is relatively noninvasive. Thus, our method may help elucidate the molecular aspects of fertilization and preimplantation development and, based on the real-time genetic and epigenetic status, could become a tool to select "good quality" embryos before implantation. genesis 43:71-79, 2005. (c) 2005 Wiley-Liss, Inc.
  • Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization, G Kondoh, H Tojo, Y Nakatani, N Komazawa, C Murata, K Yamagata, Y Maeda, T Kinoshita, M Okabe, R Taguchi, J Takeda, NATURE MEDICINE, NATURE MEDICINE, 11(2), 160 - 166, Feb. 2005 , Refereed
    Summary:The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.
  • Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane, K Yamagata, T Nakanishi, M Ikawa, R Yamaguchi, SB Moss, M Okabe, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 250(2), 348 - 357, Oct. 2002 , Refereed
    Summary:Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin alpha/beta and the appearance of fertilin beta on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin beta-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin(-/-) animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin(+/-) mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm-EPM binding but rather may involve either sperm-ZP binding and/or sperm transit to the oviduct. Because fertilin beta is absent from calmegin-deficient mice, these results also suggest that the role of fertilin beta in sperm-EPM interaction needs to be reevaluated. (C) 2002 Elsevier Science (USA).
  • Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg, D Baba, SI Kashiwabara, A Honda, K Yamagata, Q Wu, M Ikawa, M Okabe, T Baba, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 277(33), 30310 - 30314, Aug. 2002 , Refereed
    Summary:The function of glycosylphosphatidylinositol-anchored sperm hyaluronidase PH-20 in fertilization has long been believed to enable acrosome-intact sperm to pass through the layer of cumulus cells and reach the egg zona pellucida. In this study, we have produced mice carrying a null mutation in the PH-20 gene using homologous recombination. Despite the absence of sperm PH-20, the mutant male mice were still fertile. In vitro fertilization assays showed that mouse sperm lacking PH-20 possess a reduced ability to disperse cumulus cells from the cumulus mass, resulting in delayed fertilization solely at the early stages after insemination. Moreover, SDS-PAGE of sperm extracts and subsequent Western blot analysis revealed the presence of other hyaluronidase(s), except PH-20, presumably within the acrosome of mouse sperm. These data provide evidence that PH-20 is not essential for fertilization, at least in the mouse, suggesting that the other hyaluronidase(s) may play an important role in sperm penetration through the cumulus cell layer and/or the egg zona pellucida, possibly in cooperation with PH-20, although the importance of sperm motility cannot be neglected.
  • A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein, A Honda, K Yamagata, S Sugiura, K Watanabe, T Baba, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 277(19), 16976 - 16984, May 2002 , Refereed
    Summary:We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.
  • Identification of a novel isoform of poly(A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells, S Kashiwabara, TG Zhuang, K Yamagata, J Noguchi, A Fukamizu, T Baba, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 228(1), 106 - 115, Dec. 2000 , Refereed
    Summary:We have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TRAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)(12) RNA primer in the presence of MnCl2. Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs. (C) 2000 Academic Press.
  • p-aminobenzamidine-sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the egg zona pellucida in mouse, K Yamagata, K Murayama, N Kohno, S Kashiwabara, T Baba, ZYGOTE, ZYGOTE, 6(4), 311 - 319, Nov. 1998 , Refereed
    Summary:It has been reported that a significant delay in protein dispersal from the acrosomal matrix is observed in wild-type sperm by adding p-aminobenzamidine, a trypsinlacrosin inhibitor, to the incubation medium. The pattern of this delayed release was similar to that of the acrosin-deficient mutant mouse sperm (Yamagata et al., J. Biol. Chem., 273, 10470-4, 1998). In the present study, no further delay in protein dispersal was found when the acrosin-deficient sperm were treated with p-aminobenzamidine, indicating that among the p-aminobenzamidine-sensitive protease(s) only acrosin may function to accelerate this process. Although the acrosin-deficient sperm penetrated the zona pellucida (Baba et al., J. Biol. Chem., 269, 31845-9, 1994), the addition of p-aminobenzamidine to the fertilisation medium caused a significant inhibition of fertilisation in vitro. This indicates that there is a p-aminobenzamidine-sensitive protease(s) other than acrosin participating in the zona penetration step. Indeed, we demonstrated that a non-acrosin protease with a size of 42 kDa was present in the supernatant of the acrosome-reacted sperm suspension. The enzyme was inhibited by p-amimobenzamidine, diisopropyl fluorophosphate and NU-tosyl-L-lysine chloromethyl ketone, and was apparently activated by acrosin.
  • Two novel testicular serine proteases, TESP1 and TESP2, are present in the mouse sperm acrosome, N Kohno, K Yamagata, S Yamada, S Kashiwabara, Y Sakai, T Baba, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 245(3), 658 - 665, Apr. 1998 , Refereed
    Summary:To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of two similar but different serine proteases, TESP1 and TESP2, have been identified. The nucleotide sequences of these clones indicate that mouse TESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366 amino acids, respectively. Comparison of the two TESP sequences with those of typical serine proteases suggests that each TESP zymogen is probably converted into a two-chain mature enzyme consisting of light and heavy chains covalently linked by a single pre-existing disulfide bond. The conversion may be accomplished by another protease(s) with a trypsin-like cleavage specificity, since it is unlikely that the mature TESP1 and TESP2 are capable of splitting the Lys-Ile bond between the light and heavy chains. Northern blot analysis of total cellular RNA demonstrates that the TESP1 and TESP2 genes are expressed only in the testis, and the transcripts are abundantly present in the haploid round spermatids. Moreover, immunocytochemical analysis of mouse cauda epididymal sperm using affinity-purified antibodies reveals that these two TESPs are both localized in the sperm acrosome and are released during the acrosome reaction induced by calcium ionophore A23187. These findings provide additional clues for elucidating the mechanisms involved in the sperm/egg interactions, including penetration of the zona pellucida by sperm. (C) 1998 Academic Press.

Books etc

  • Analysis of rabbit haploid embryos toward to derivation of haploid embryonic stem cells, Joint author, Memoirs of Institute of Advanced Technology, Kindai University,   2017 03
  • Generation of MethyIRO mouse to study DNA methylation dynamics in normal and pathological states, Jun Ueda, Kazuo Yamagata, Joint author,   2015 05
  • DNA methylation dynamics studies using DNA methylation reporter mouse, MethyIRO, Jun Ueda, Kazumitsu Maehara, Yasuyuki Ohkawa, Kazuo Yamagata, Joint author,   2015 01

Conference Activities & Talks

  • Manipulate & Reconstitute the chromatin, Yamagata Kazuo, RIKEN SEMINAR 7th Epigenetics Seminar Series 2019,   2019 10 15 , 招待有り
  • ゲノム編集を応用したマウス受精卵のエピゲノム編集, YAMAGATA Kazuo, 第3回日本ゲノム編集学会,   2018 06 19 , 招待有り
  • Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase., Yamagata Kazuo, HMGU-Japan Epigenetics and Chromatin Symposium,   2017 09 04 , 招待有り
  • 精巣特異的ヒストンバリアントH3tは精子幹細胞の精子形成過程への移行に必須である, Yamagata Kazuo, 第35回日本受精着床学会,   2017 07 21 , 招待有り
  • Understanding of nuclear function by live imaging of chromatin dynamics, Yamagata Kazuo, 39th Annual Meeting of the Molecular Society of Japan,   2016 11 , 招待有り
  • Quantification of Embryo Quality by Live-Cell Imaging, Yamagata Kazuo, Society for the Study of Reproduction,   2016 07 , 招待有り
  • Live-cell Imaging of Chromatin and DNA-methylation Dynamics Using MethylRO Mouse, Yamagata Kazuo, 6th Hiroshima Conference on Education and Science in Dentistry,   2015 10
  • Visualization of Dynamics of Methylated DNA in Living Cell and Animal, Yamagata Kazuo, The International Symposium on Chromatin Structure, Dynamics, and Function,   2015 08
  • Visualization of Dynamics of Methylated DNA in living cell and animal., Yamagata Kazuo, International Symposium on Bio-imaging and,   2015 02
  • Quantification of embryo quality by live-cell imaging, Yamagata Kazuo, 47th Annual Meering of the Japanese Society of Developmental Biologists,   2014 05
  • Quantification of embryo quality, YAMAGATA Kazuo,   2012 05
  • Genetic and Epigenetic Landscape of Mouse Preimplantation Development Revealed by Live-cell Imaging, Yamagata Kazuo, 2nd World Congress of Reproductive Biology,   2011 10
  • Genetic and Epigenetic Landscape of Mouse Preimplantation Development Revealed by Live-cell Imaging, Yamagata Kazuo, CDB symposium 2011,   2011 03
    Summary:“Epigenetic Landscape in Development and Disease” RIKEN, Center for Developmental Biology
  • Nuclear and Chromosomal Dynamics during Preimplantation Development Revealed by Multi-dimensional, Long-term Live-cell imaging, Yamagata Kazuo, International Symposium on the Physicochemical Field for Genetic Activities,   2011 01
  • A novel approach based on the live-cell imaging which allows the identification of defective embryos in mouse ART, Yamagata Kazuo, International Symposium for Immunology of Reproduction,   2010 08
  • Assessment of Embryo Quality by Live-cell Imaging, Yamagata Kazuo, ESHRE Campus course at the 2nd World Congress of Reproductive Biology,   2010 07
  • Long-term, six-dimensional, and safe live-cell imaging for mouse preimplantation development and its application for the embryo assessment, Yamagata Kazuo, 42nd Annual Meeting for the Japanese Society of Developmental Biologists,   2009 05
  • Live-cell imaging technique for the preimplantation embryos and its application, Yamagata Kazuo, The 4th Annual Conference of the Asian Reproductive Biotechnology Society,   2007 11
    Summary:Taiga Yamazaki, Hiromi Miki, Narumi Ogonuki, Kimiko Inoue, Atsuo Ogura, and Tadashi Baba
  • Roles of ADAMs in Sperm/egg Interactions, Yamagata Kazuo, University of Pennsylvania Medical Center,   2003 08


  • 初期胚発生におけるイベントのタイミングは細胞数・時間のどちらに依存しているのか~三次元画像解析による時間定量~, 池田善喜, 小林徹也, 八尾竜馬, 野老美紀子, 増子大輔, 細井美彦, 山縣一夫, 定量生物学の会 第9回年会,   2019 01
  • レトロトランスポゾンMERVLの活性化機構の解析, 奥野智美, 山口壮輝, 樋口智香, 神谷拓磨, 山本真理, 越智浩介, 西野亜理紗, 井橋俊哉, 辻本佳加理, 坂本裕子, 松橋珠子, 安齋政幸, 黒坂哲, 三谷匡, 山縣一夫, 細井美彦, 松本和也, 宮本圭, 第41回日本分子生物学会年会,   2018 11 ,
  • DNAビーズを用いたマウス受精卵での核構築, 鈴木由華, SukriyeBilir, 小坂田裕子, 小林昇平, 平岡泰, 原口徳子, 山縣一夫, 第41回日本分子生物学会年会,   2018 11
  • ライブセルイメージングを用いたマウス受精卵におけるDNA損傷の可視化及び定量化, 小栗未生奈, 半田哲也, 鈴木由華, 波多野裕, 野老美紀子, 八尾竜馬, 小林昇平, 細井美彦, 野崎直仁, 原口徳子, 木村宏, 山縣一夫, 第41回日本分子生物学会年会,   2018 11
  • DNAビーズを用いた未受精卵内でのキネトコアの再構成, 田中菜穂子, 福田龍人, 小林昇平, 細井美彦, 原口徳子, 山縣一夫, 第41回日本分子生物学会年会,   2018 11
  • マウス初期胚発生におけるセントロメア/ペリセントロメアのDNAメチル化機能, 波多野裕, 山﨑大賀, 谷口稜弥, 増子大輔, 野老美紀子, 八尾竜馬, 小林憲忠, 山縣一夫, 第41回日本分子生物学会年会,   2018 11
  • 再構成的アプローチによる受精卵核構築の理解, 鈴木由華, 山縣一夫,   2018 10
  • セントロメア/ペリセントロメア領域における人為的なDNAメチル化導入が発生に与える影響(奨励賞受賞), 波多野裕, 山崎大賀, 加藤佐樹子, 野老美紀子, 細井美彦, 山縣一夫, 第13回日本生殖再生医学会・学術集会,   2018 03
  • 受精直後の精子核DNA二本鎖切断を生きたまま可視化・定量化する方法の開発, 小栗未生奈, 半田哲也, 杉本瑞紀, 鈴木由華, 波多野裕, 野老美紀子, 八尾竜馬, 細井美彦, 野崎直仁, 木村宏, 山縣一夫,   2017 12
  • Understanding of nuclear assembly in mouse preimplantation embryo by reconstitution approach., HMGU-Japan Epigenetics and Chromatin Symposium,   2017 09
  • 受精後のユビキチン・プロテアソーム系による母性タンパク質の分解はマウス初期胚発生に重要である, 樋口智香, 守田昂太郎, 山口壮輝, 松橋珠子, 永井宏平, 安齋政幸, 安齋政幸, 山縣一夫, 細井美彦, 宮本圭, 松本和也,   2017 04 ,
  • マウス初期胚発生過程におけるH3R2me2sの役割, 守田昂太郎, 樋口智香, 山口壮輝, 松橋珠子, 松橋珠子, 永井宏平, 安齋政幸, 安齋政幸, 加藤博巳, 加藤博巳, 山縣一夫, 細井美彦, 宮本圭, 松本和也,   2017 04 ,
  • マウス卵胞培養過程で生じる細胞死を指標とした評価基準の作成, 藤村雪乃, 野老美紀子, 八尾竜馬, 山縣一夫, 細井美彦, 日本生殖再生医学会 第12回学術集会,   2017 03
  • ペリセントロメア領域における人為的なDNAメチル化導入が発生に与える影響, 波多野裕, 山﨑大賀, 加藤佐樹子, 野老美紀子, 穂井田謙介, 細井美彦, 山縣一夫, 第39回 日本分子生物学会年会,   2016 12
  • ライブセルイメージングを用いたマウス着床前初期胚発生におけるATP動態の可視化と定量化, 鈴木由華, 八尾竜馬, 山本正道, 野老美紀子, 福永憲隆, 浅田義正, 細井美彦, 山縣一夫, 第39回 日本分子生物学会年会,   2016 12
  • メチローマウス由来単一ES細胞におけるメチル化DNA動態の動的観察法の開発, 穂井田謙介, 上田潤, 山田健, 野老美紀子, 細井美彦, 山縣一夫, 第39回 日本分子生物学会年会,   2016 12
  • マウス卵胞培養過程で生じる細胞死を利用した評価基準の作成, 藤村雪乃, 玉置幸嵩, 大西佐知, 山縣一夫, 細井美彦, 第19回 日本IVF学会学術集会,   2016 10
  • Evaluation of bovine embryo competence using a live-cell imaging technique., 鈴木理恵, 八尾竜馬, 鈴木由華, 古川晋也, 杉村智史, 細井美彦, 山縣一夫, 国際核移植学会シンポジウム,   2016 03
  • Visualization and quantification of ATP dynamics in mouse preimplantation embryos using live-cell imaging., 鈴木由華, 八尾竜馬, 鈴木理恵, 古川晋也, 山本正道, 細井美彦, 山縣一夫, 国際核移植学会シンポジウム,   2016 03
  • Dynamics of methylated DNA in ES cell replication and differentiation revealed by live-cell imaging., 穂井田謙介, 上田潤, 大日向康秀, 財田大地, 野老美紀子, 細井美彦, 山縣一夫, International Symposium on Epigenome Dynamics and Regulation in Germ Cell,   2016 02
  • メチローマウスを用いた各種幹細胞の分化過程におけるDNAメチル化状態の動的観察, Hoida Kensuke, 先端技術総合研究所公開シンポジウム,   2015 10
  • Generation of MethylRO mouse to study DNA methylation dynamics of normal and pathological states, Jun Ueda, Kazuo Yamagata, Journal of Clinical and Experimental Medicine, 253, 6, 523, 524,   2015 05 , 招待有り
  • Reconstitution of Developmental Dynamics of Mammalian Embryogenesis from 3D Confocal Microscope Images, KOBAYASHI Tetsuya, OZEKI Mitsunori, YAMAGATA Kazuo, Funahashi Akira, IEICE technical report., 114, 482, 1, 6,   2015 03 02 , Refereed,
    Summary:Reconstruction of developmental dynamics of mammalian preimplantation embryos from bioimaging data by using image analysis is now becoming an important challenge in the fields of basic science and assisted reproductive technology. In this work, we firstly review the recent progress on the image analysis techniques to reconstruct cell lineage from bioimaging data, and then introduce our recent result on the tracking of developmental dynamics of mammalian embryo by using integer programing.
  • DNA methylation dynamics studies using DNA methylation reporter mouse, MethylRO, Ueda, J, Maehara, K, Ohkawa, Y, Yamagata, K, Experimental Medicine (Jikkenigaku, Japanese Review), 33, 3, 481, 489,   2015 02 , 招待有り
  • A transient inhibition of proteasome pathway entails a delay in the onset of ZGA and impairs full term development of mice, HIGUCHI Chika, SHIMIZU Natsumi, MORITA Kohtaro, UCHIBORI Sho, TSUKAGUCHI Tomomasa, NAGAI Kohei, ANZAI Masayuki, YAMAGATA Kazuo, HOSOI Yoshihiko, MIYAMOTO Kei, MATSUMOTO Kazuya, The Journal of Reproduction and Development Supplement, 108, 0, P, 12-P-12,   2015 ,
  • Involvement of Peroxiredoxin (Prdx) in reducing hydrogen peroxide in pronuclei of mouse zygotes., MORITA Kohtaro, TOKORO Mikiko, HIGUCHI Chika, UCHIBORI Sho, TSUKAGUCHI Tomomasa, NAGAI Kohei, ANZAI Masayuki, YAMAGATA Kazuo, HOSOI Yoshihiko, MIYAMOTO Kei, MATSUMOTO Kazuya, The Journal of Reproduction and Development Supplement, 108, 0, P, 15-P-15,   2015 ,
  • The effect of Coenzyme Q10 on in vitro fertilization and subsequent preimplantation development of post-ovulatory aged oocytes in mice, UCHIBORI Sho, HIGUCHI Chika, MORITA Kohtaro, TSUKAGUCHI Tomomasa, ANZAI Masayuki, YAMAGATA Kazuo, HOSOI Yoshihiko, MIYAMOTO Kei, MATSUMOTO Kazuya, The Journal of Reproduction and Development Supplement, 108, 0, P, 10-P-10,   2015 ,
  • マウスクローン胚のライブセルイメージングと単一胚移植による解析, 水谷英二, 山縣一夫, 若山照彦, J Mamm Ova Res, 30, 2, S61,   2013 04 01 ,
  • ES細胞のできるまで, 山縣一夫, 上田潤, 水谷英二, 斎藤通紀, 若山照彦, J Reprod Dev, 58, Suppl Japanese Issue, J101,   2012 08 20 ,
  • ウシ体細胞クローン技術における新規の除核方法の検討, IWAMOTO DAISAKU, YAMAGATA KAZUO, KISHI MASAO, KIMURA HIROSHI, HAYASHI YOKO, WAKAYAMA TERUHIKO, SAEKI KAZUHIRO, 日本畜産学会大会講演要旨, 115th, 122, 122,   2012 03 28 ,
  • 単一胚ライブセルイメージングによる初期胚発生核ダイナミクスの可視化, 山縣一夫, 水谷英二, 林(高中)陽子, 木村宏, 若山照彦, 日本蛋白質科学会年会プログラム・要旨集, 11th, 25,   2011 05 27 ,
  • 種々のヒストン脱アセチル化酵素の阻害剤を用いたマウス体細胞核移植に影響を与えているHDACの特定の試み, 小野哲男, 李羽中, 水谷英二, 山縣一夫, 若山照彦, J Reprod Dev, 56, Suppl Japanese Issue, J131,   2010 08 20 , 10.14882/jrds.,
  • 6次元長期間ライブセルイメージングによるマウスクローン胚発生過程の解析, 水谷英二, 山縣一夫, 若山照彦, J Mamm Ova Res, 26, 2, S67,   2009 04 01 ,
  • 長時間ライブセルイメージングが明らかにするマウスクローン初期胚の遺伝情報場異常, 山縣一夫, 水谷英二, 若山照彦, 日本分子生物学会年会講演要旨集, 32nd, Vol.2, 16,   2009 ,
  • ライブセルイメージングによるマウス体細胞クローン胚の解析およびクローン産仔の作出, 水谷英二, 山縣一夫, 若山照彦, J Reprod Dev, 54, Supplement, J90,   2008 08 25 , 10.14882/jrds.,
  • Live-cell imaging of epigenetic dynamics during preimplantation development, 山縣, 一夫, 山崎, 大賀, 馬場, 忠, Protein, nucleic acid and enzyme, 52, 16, 2216, 2222,   2007 12
  • Identification and characterization of novel serine proteases in mouse sperm, HONDA,Arata, YAMAGATA,Kazuo, BABA,Tadashi, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 617,   1998 12
  • p-Aminobenzamidine-sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the eggzona pellucida in mouse, YAMAGATA,Kazuo, BABA,Tadashi, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 617,   1998 12
  • げっ歯動物雄性生殖細胞特異的セリンプロテアーゼSSSP染色体遺伝子に存在する第三イントロンの解析, 山縣, 一夫, 河野, 展久, 馬場, 忠, 日本分子生物学会年会プログラム・講演要旨集, 19, 0, 576,   1996 08

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(挑戦的萌芽研究), Visualization of chromosome aneuploidy in living embryo, Chromosomal aneuploidy in oocyte and early embryo is major cause of early pregnancy loss and evokes severe chromosome disorder such as Down syndrome after birth. However, the mechanisms for the chromosome abnormality were still enigmatic due to the experimental limitations inherent to the early embryo. In this study, we established a new protocol to visualize the chromosomal aneuploidy in living embryo by developing the fluorescently probe that binds to the specific chromosomes.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(若手研究(B)), Novel method for the embryo sexing using live-cell imaging
  • The Other Research Programs, -