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KATO Yoko

    Department of Advanced Bioscience　Professor
Last Updated :2024/04/25

Researcher Information

Degree

  • Ph.D

J-Global ID

Research Areas

  • Life sciences / Laboratory animal science
  • Life sciences / Animal production science

Academic & Professional Experience

  • 2009 - Today  KINDAI UniversityAgricultureProfessor

Published Papers

  • Nagi Fujii; Yuta Nakata; Yoko Kato
    Experimental animals 2022/08 [Refereed]
     
    It is well known that the survivability of gametes of postmortem carcass was decreased as time passes after death. In this study, it was examined whether cytoplasmic replacement rescues the survivability of germinal vesicle stage (GV) oocytes of postmortem carcass in the mouse. Reactive oxygen species (ROS) levels and mitochondria numbers in GV oocytes of the dead mice stored at 4 degrees were significantly impaired after 44 h postmortem compared to the control (0h). However, when kayoplasts of GV oocytes of postmortem carcass was transferred to recipient ooplasts (GV transfer), proportion of in vitro maturation (IVM), normal spindle morphology, in vitro and in vivo developmental ability after IVF of reconstituted oocytes was improved. Moreover, secondary follicle oocytes of postmortem carcass were developed, matured and fertilized in vitro and developed to go to term, when GV transfer was conducted at the GV phase. Thus, transfer of GV karyoplast recovered from postmortem carcass, which viability was decreased, into fresh GV recipient ooplasm, rescues survivability of reconstituted oocytes. It suggested the effective use of oocytes of dead animals in the mouse and this achievement must apply to other rare animal species, especially animals under control by human.
  • Yoko Kato
    Encyclopedia of Dairy Sciences (Third edition) Elsevier 868 - 873 2022 [Refereed][Invited]
  • Effect of phytohemagglutinin (PHA) on nuclear transfer pig embryos in vitro
    Ndubuisi Machebe; Soichiro Shimizu; Masaki Hata; Kazuki Ohata; Tetsuya Tani; Yoko Kato
    Journal of Mammalian Ova Research 36 33 - 43 1347-5878 2019/04 [Refereed]
  • Elhameh Jahanbakhsh-Asl; Mohammad Salehi; Marefat Gha ari-Novin; Yoko Kato
    International Journal of Women's health and Reproduction Science 6 (2) 1 - 8 2018 [Refereed]
  • Tetsuya Tani; Yoko Kato
    CELLULAR REPROGRAMMING MARY ANN LIEBERT, INC 19 (2) 95 - 106 2152-4971 2017/04 [Refereed]
     
    For reprogramming a somatic nucleus during mammalian cloning, metaphase of the second meiotic division (MII) oocytes has been widely used as recipient cytoplasm. High activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) is believed to accelerate the remodeling and/or reprogramming of a somatic nucleus introduced into the ooplasm by somatic cell nuclear transfer. We demonstrated previously that the first step in nuclear reprogramming is not directly regulated by MPF and MAPK because activated oocytes in which MPF activity is diminished and MAPK activity is maintained can develop to the blastocyst stage after receiving an M phase somatic nucleus in bovine cloning. In this study, our aim was to test whether MAPK activity is necessary for the first step in nuclear reprogramming and/or chromatin remodeling (phosphorylation of histone H3 at Ser3, trimethylation of histone H3 at Lys 9, and acetylation of histone H3 at Lys14) in bovine somatic cloning. We found that it was not necessary, and neither was MPF activity.
  • Kazuki Ohata; Yoko Kato
    Journal of Mammalian Ova Research Japanese Society of Mammalian Ova Research 33 (1) 55 - 61 1347-5878 2016/04 [Refereed]
     
    The survival probability of mouse blastocysts developed from nuclear transfer (NT) was estimated prior to embryo transfer using a novel biopsy method. NT embryos were separated into two at the 2-cell stage to produce monozygotic twin blastocysts from a single NT oocyte. We first examined efficient culture methods to produce monozygotic twin blastocysts. Then one of the two blastocysts was used for gene expression analysis, and the other was transferred to a recipient mouse. Co-culture of monozygotic twin blastomeres with fertilized embryos or culture in a small well in vitro improved the developmental potential to the blastocyst stage and increased the blastocyst cell number. Although 21.9% of twin somatic cell NT embryos had the same expression levels of Oct4 and Sox2 genes as fertilized embryos, no fetuses were obtained after transfer to recipients.
  • Kenji Ezoe; Akiko Yabuuchi; Tetsuya Tani; Chiemi Mori; Tetsuya Miki; Yuko Takayama; Zeki Beyhan; Yoko Kato; Takashi Okuno; Tamotsu Kobayashi; Keiichi Kato
    PLOS ONE PUBLIC LIBRARY SCIENCE 10 (5) e0126801  1932-6203 2015/05 [Refereed]
     
    Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.
  • Mohammad Salehi; Yoko Kato; Yukio Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 22 (2) 213 - 217 0967-1994 2014/05 [Refereed]
     
    The beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10(-6) to 10(-12) M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10(-12) M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.
  • Yoko Kato; Yukio Tsunoda
    Principles of Cloning: Second Edition Elsevier Inc. 127 - 135 2013/10 
    After the first successful nuclear transfer in mammals in 1983 with pronuclear exchanges, this technique was applied to produce cloned animals using unfertilized oocytes. In 1997 first somatic cell cloning was succeeded in sheep, and after that in more than 20 species, somatic cell nuclear transfer has been succeeded. But the successful rate to produce healthy cloned animals is still low. In this chapter, whether donor cell types affect on the cloning efficiency is discussed, with two categories such as germ line cell types and somatic cell types as donor. © 2014 Elsevier Inc. All rights reserved.
  • KATO Yoko
    TRENDS IN THE SCIENCES 公益財団法人 日本学術協力財団 18 (4) 4_62 - 4_67 1342-3363 2013
  • Yukio Tsunoda; Yoko Kato
    Transgenic Animal Technology: A Laboratory Handbook: Second Edition Elsevier Inc. 195 - 231 2012/12
  • Yuta Tsuji; Yoko Kato; Yukio Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 20 (3) 261 - 267 0967-1994 2012/08 [Refereed]
     
    Somatic cell nuclear-transferred (SCNT) oocytes have a high potential for development in vitro, but a large proportion of embryos that are transferred to recipients is aborted before parturition. The precise mechanism for the high abortion rate is unknown, but abnormal placenta formation is frequently observed in SCNT-cloned pregnancies. The present study examined the effects of treating the recipients with cyclosporin A (CsA), an immunoprotectant, on the proportion of fetuses resulting from SCNT-cloned pregnancies. Cloned embryos developed from enucleated oocytes and receiving cumulus cells from F1 (C57BL/6 x DBA, H-2(b/d)) females were transferred to outbred ICR (in which the H-2 complex was not fixed) recipient females. Each recipient received an intraperitoneal injection of CsA or vehicle. Compared with vehicle, administration of CsA to recipients on day 4.5 of pregnancy significantly increased the proportion of fetuses observed on day 10.5. The proportion of fetuses at day 18.5 of pregnancy in recipients receiving CsA treatment was slightly higher than that in controls. This study is the first to report that CsA administration increases the proportion of fetuses resulting from SCNT-cloned pregnancies.
  • Mayu Nakano; Yoko Kato; Yukio Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 20 (2) 199 - 207 0967-1994 2012/05 [Refereed]
     
    Melatonin secreted from the mammalian pineal gland is a free-radical scavenger that protects tissues from cell damage. The present study examined the effects of addition of melatonin to the culture medium on the developmental potential of parthenogenetic and somatic cell nuclear-transferred (SCNT) porcine oocytes. Supplementation of the maturation medium with melatonin did not increase the maturation rate, the proportion of oocytes that cleaved and developed into blastocysts after parthenogenetic activation, or the blastocyst cell number compared to controls. When 10(-7) M melatonin was added to the culture medium, the proportion of parthenogenetic oocytes that developed to the 2-cell and 4-cell stages was significantly higher than that of controls. The potential of melatonin-treated oocytes to develop into blastocysts was high but not significantly different from that of controls. The addition of 10(-7) M melatonin to the culture medium did not increase the preimplantation development of SCNT oocytes. Melatonin treatment significantly reduced the levels of reactive oxygen species in 4-cell parthenogenetic and SCNT embryos, but did not reduce the proportion of apoptotic cells in parthenogenetic and SCNT blastocysts. Although the results indicated that parthenogenetic and SCNT melatonin -treated embryos had significantly lower levels of reactive oxygen species than controls, the potential of melatonin-treated embryos to develop into blastocysts was not significantly higher than that of controls, in contrast to previous reports. The beneficial effects of melatonin on the developmental potential of oocytes might depend on the culture conditions.
  • Yuta Tsuji; Yoko Kato; Yukio Tsunoda
    CELLULAR REPROGRAMMING MARY ANN LIEBERT INC 14 (1) 38 - 44 2152-4971 2012/02 [Refereed]
     
    In our previous study (Tsuji et al., 2010), administration of hCG to recipients around the timing of implantation significantly increased the in vivo development of mouse embryos after somatic cell nuclear transfer (SCNT) until day 10.5, but did not increase the development to full term. The present study was undertaken to examine whether cotransfer of fertilized embryos or parthenogenetic embryos prevents the embryonic loss of SCNT embryos after day 10.5, allowing them to develop to full term. We found that compared with SCNT embryo transfer alone, full-term development of SCNT embryos slightly, but not significantly, increased by cotransfer of mouse hybrid blastocysts derived from BDF1 (C57BL/6 x DBA) female x ICR male into the oviducts of recipients administered hCG (2.0% vs. 5.5%). This was not the case with the cotransfer of blastocysts from an ICR female x ICR male (2.5% vs. 2.2%) or parthenogenetic blastocysts from BDF1 female (3.0% vs. 2.0%). Furthernore, when SCNT blastocysts were transferred into the uteri of recipients, full-term development did not increase even with the cotransfer of hybrid blastocysts. The mechanisms of the effect of cotransfer of fertilized and parthenogenetic embryos on the full-term development of SCNT mouse embryos are discussed.
  • Takaaki Sugimoto; Yoko Kato; Yukio Tsunoda
    Journal of Mammalian Ova Research 29 (1) 75 - 81 1341-7738 2012 
    As reported previously, berberine, the main component extracted from Coptis rhizome and Phellodendron, has potential as a contraceptive for animals since berberine has a strong inhibitory effect on the in vitro development of mouse zygotes and on fetal development in vivo. The present study was undertaken to examine the effect of berberine treatment on the reversibility of the development of zygotes and gametes, and on the fertilization and subsequent development in the mouse. The reversibility of the berberine-induced inhibition of the development of mouse zygotes was dependent on the concentration used and treatment period. Berberine treatment did not inhibit the fertilizing capacity of epididymal spermatozoa and the fertilizability of oocytes at the second metaphase stage. The present study demonstrated that in vitro development of mouse zygotes is about 100 times more sensitive to berberine than the in vitro growth of mouse fetal fibroblast cells. The high stability of berberine at low temperatures for at least for 12 months and the high sensitivity of preimplantation embryos to berberine is useful information when considering the administration of berberine to females as a contraceptive. © 2012 Japanese Society of Mammalian Ova Research.
  • Takaaki Sugimoto; Yuta Tsuji; Yoko Kato; Yukio Tsunoda
    Journal of Mammalian Ova Research 29 (3) 128 - 134 1341-7738 2012 
    After screening 408 crude drugs, we found that Rizoma Arisaematis increased the cell numbers of mouse blastocysts developed from in vivo zygotes. We examined the effects of Rizoma Arisaematis on the in vitro development of mouse zygotes and embryos produced by ICSI and SCNT, as well as on fetal development. Mouse zygotes were cultured in media containing a water-soluble extract of Rizoma Arisaematis at various concentrations, and the potential of zygotes to develop into blastocysts and the cell numbers of blastocysts were examined. In addition, the effects of Rizoma Arisaematis on the in vitro and in vivo developmental potential of embryos produced by ICSI and nuclear transfer were examined. In vitro treatment of zygotes with Rizoma Arisaematis increased the cell numbers of blastocysts. The proportions of the blastocysts that implanted and developed into fetuses were slightly higher in the blastocysts which were developed from zygotes treated with Rizoma Arisaematis than those of the control. The Rizoma Arisaematis treatment of mouse fetal fibroblast cells or embryos produced by ICSI and SCNT did not affect the growth of the cells, or the in vitro development of the zygotes. The present study demonstrated that Rizoma Arisaematis improved the quality of embryos developed from in-vivo zygotes, but not that of embryos produced by ICSI and nuclear transfer. © 2012 Japanese Society of Mammalian Ova Research.
  • Y. Kato; Y. Tsunoda
    Encyclopedia of Dairy Sciences: Second Edition Elsevier Inc. 610 - 615 2011/01 [Refereed]
     
    The word 'clone', defined as 'the descendant of a single plant or animal nonsexually produced from any one cell and with exactly the same form as the parent', was first used by Webber in 1903. Now, the term is broadly used also to include cell or gene replication (e.g., cell cloning or gene cloning). Animal clones are produced by embryo separation, splitting, and nuclear transfer (NT). NT includes two types: using blastomeres as donor cells and using somatic cells as donors. Since the first sensational report of somatic cell nuclear transfer (SCNT) in 1997, it has been successfully used in many kinds of species during the past two decades, and examination of the reprogramming factors or mechanisms occurring in the ooplasm after NT has started and these mechanisms are getting clarified slowly. This article provides an overview of nuclear cloning, history of breed improvement in bovines, the individual procedures of nuclear cloning such as separation of blastomeres, splitting the morula to blastocyst stage embryos, NT of blastomeres, and SCNT, and provides also conclusions and speculation on animal cloning.
  • Yoko Kato; Yukio Tsunoda
    THAI JOURNAL OF VETERINARY MEDICINE CHULALONGKORN UNIV 41 39 - 42 0125-6491 2011 [Refereed]
     
    After the first success of somatic cell nuclear transfer (SCNT) in 1997 (Wilmut at al., 2008), a great deal of researches have examined whether any somatic cell types could be reprogrammed in ooplasm and any mammalian species could be adopted for cloning such as endangered animals. It is clear that the successful rate of animal cloning is not dramatically improved after the first success in 1997. One of the possible reasons for the low success is the failure of reprogramming of somatic cells in ooplasm after nuclear transfer. The failure of the reprogramming causes varied abnormalities to SCNT embryos and results in low successful rate. The gradual loss of SCNT embryos at any developmental stages from nuclear transfer to parturition remains unclear. We examined whether any donor cell types could be reprogrammed in ooplasm, and how to improve the developmental potential of SCNT embryos in vitro and in vivo.
  • Yukio Tsunoda; Yoko Kato
    Journal of Mammalian Ova Research 28 (1) 40 - 46 1341-7738 2011 
    After screening 269 crude drugs for their ability to inhibit the development of mouse zygotes, we found Coptis rhizome and Phellodendron bark to have inhibitory effects. We examined the effects of both extracts and of berberine, a major component of these plants, on in vitro development of zygotes and on fullterm fetal development in the mouse. Mouse zygotes were cultured in medium containing water-soluble extracts of Coptis rhizome or Phellodendron bark, or berberine at various concentrations for 5 days and the potential of zygotes to develop to blastocysts was examined. In addition, superovulated mice were intramuscularly injected with berberine and mated, and examined for the in vivo development of fertilized eggs to blastocysts and full-term fetuses. In vitro development of zygotes to blastocysts was almost completely inhibited when they were cultured in medium containing more than 0.1 μg/ml Coptis rhizome, 10 μg/ml Phellodendron bark, or 0.01 μg/ml berberine chloride or berberine sulfate. When superovulated and mated females received 100 μg berberine chloride once a day for 2 to 14 days, the proportions of recovered blastocysts and full-term fetuses were significantly decreased. The present study indicates the potential use of berberine as a contraceptive for animals.
  • Yuta Tsuji; Yoko Kato; Yukio Tsunoda
    CELLULAR REPROGRAMMING MARY ANN LIEBERT INC 12 (2) 183 - 189 2152-4971 2010/04 [Refereed]
     
    Somatic cell nuclear-transferred (SCNT) oocytes have a relatively high potential to develop into blastocysts in vitro, but a large proportion embryos die at various pre- and postimplantation stages after transfer to recipients. Although the reason for the high mortality of SCNT embryos at the peri- and postimplantation stages is not clear, epigenetic abnormalities of SCNT embryos are considered to be the main cause. Such abnormalities of SCNT embryos may decrease their ability to maintain the corpora lutea, which is necessary for initiating implantation and maintaining fetal development. To examine this hypothesis, human chorionic gonadotropin (hCG) and progesterone were administered at different times to recipients that received SCNT embryos. When hCG was administered daily from day 3.5 to day 6.5 of pregnancy, the implantation and fetal development rates increased significantly compared to those of controls. The potential of SCNT embryos to develop to full term, however, was not greater than that of controls, even if hCG administration was continued to day 11.5 or day 17.5 and progesterone was administered from day 7.5 to day 17.5 after hCG injection. These findings demonstrated that administering hCG to recipients protects the in vivo development of SCNT embryos until day 10.5, but other treatment is necessary to support the progression of the embryos to full-term development.
  • Shigetoshi Mizumoto; Yoko Kato; Yukio Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 18 (1) 9 - 15 0967-1994 2010/02 [Refereed]
     
    We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes, produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes; continued to elongate with an increase in the Culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes; that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.
  • Yoko Kato; Yukio Tsunoda
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY U B C PRESS 54 (11-12) 1623 - 1629 0214-6282 2010 [Refereed]
     
    Cloning efficiency has not been dramatically improved after the first success of somatic cell nuclear transfer (SCNT) in sheep in 1997. The reasons for the low efficiency of SCNT embryos must be attributed to the insufficient reprogramming of the donor nucleus in ooplasm. It has been clarified that the methylation and acetylation status are disordered in SCNT embryos and the gene expression pattern is different and widely varied in SCNT embryos, compared with fertilized embryos. In this paper, we focused on the role of the donor nuclei in cloning efficiency, and discuss whether ooplasm can reprogram any nucleus.
  • Yuta Tsuji; Yoko Kato; Yukio Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 17 (2) 109 - 115 0967-1994 2009/05 [Refereed]
     
    To facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.
  • Shigetoshi Mizumoto; Yoko Kato; Yukio Tsunoda
    CLONING AND STEM CELLS MARY ANN LIEBERT INC 10 (4) 453 - 459 1536-2302 2008/12 [Refereed]
     
    We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in rat oocytes. First, we compared the effects of two types of inhibitors of spontaneous activation, MG132 and demecolcine, on the developmental potential of parthenogenetic oocytes. The potential of activated oocytes to develop into blastocysts significantly decreased 2 h after oocyte recovery (77% vs. 7%). The developmental potential of oocytes preserved in MG132-supplemented medium for 1 to 4 h was high (62% to 77%), but the potential of those preserved in demecolcine-supplemented medium for 3 and 4 h was low (77% vs. 41% and 37%, respectively). Second, the effect of the duration of parthenogenetic activation on the developmental potential was examined. When oocytes preserved in MG132 for 4 h were treated with 10 mM. strontium for 5 or 6 h, the potential of activated oocytes to develop into blastocysts was high (78% and 70%, respectively). Using the optimal conditions for parthenogenetic activation, we examined the potential of rat enucleated oocytes receiving cumulus cells to develop into blastocysts. In contrast to parthenogenotes, the potential of SCNT rat oocytes to develop into blastocysts was low (2%) even if then oocytes were treated with the histone deacetylation inhibitor trichostatin A. The reason for the low developmental potential of rat SCNT oocytes is discussed.
  • M. Kawakami; Y. Kato; Y. Tsunoda
    ANIMAL REPRODUCTION SCIENCE ELSEVIER SCIENCE BV 106 (3-4) 402 - 411 0378-4320 2008/07 
    The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 mu l). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20-24 h after activation, 25%) was slightly or significantly (P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13 %) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed. (C) 2007 Published by Elsevier B.V.
  • Xiangping Li; Yoko Kato; Yuta Tsuji; Yukio Tsunoda
    CLONING AND STEM CELLS MARY ANN LIEBERT INC 10 (1) 133 - 142 1536-2302 2008/03 [Refereed]
     
    Trichostatin A (TSA) is the most potent histone deacetylase (HDAC) inhibitor known. We previously reported that treatment of mouse somatic cell nuclear-transferred (SCNT) oocytes with TSA significantly increased the blastocyst rate, blastocyst cell number, and full-term development. How TSA enhances the epigenetic remodeling ability of somatic nuclei and the expression of development-related genes, however, is not known. In the present study, we compared the expression patterns of nine genes involved in chromatin structure and DNA methylation, and seven development-related genes in blastocysts developed from SCNT oocytes treated with and without TSA, and in blastocysts, developed in vivo and in vitro using real-time reverse transcription-polymerase chain reaction. In vivo-recovered blastocysts and blastocysts developed from TSA-treated SCNT oocytes exhibited similar expression patterns for Hdac1, 2, and 3, CBP, PCAF, and Dnmt3b genes compared with in vitro-developed blast ocysts and blastocysts developed from SCNT oocytes without TSA treatment. There were significantly lower expression levels of Hdac1 and Hdac2 transcripts in TSA-treated and in vivo-recovered blastocysts than in TSA-untreated and in vitro-developed blastocysts. The finding that TSA treatment of SCNT oocytes significantly upregulated Sox2 and cMyc transcripts in blastocysts indicated that both transcripts are TSA-responsive genes. Thus, TSA treatment of mouse SCNT oocytes decreased the expression of chromatin structure- and DNA methylation-related genes, and increased the expression of Sox2 and cMyc genes in blastocysts. Such modifications might be a reason for the high developmental potential of mouse SCNT oocytes treated with TSA.
  • Dasari Amarnath; Xiangping Li; Yoko Kato; Yukio Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 53 (6) 1247 - 1263 0916-8818 2007/12 [Refereed]
     
    Aberrant gene expression in somatic cell nuclear-transferred (NT) embryos due to abnormal epigenetic modifications of the donor nucleus likely accounts for much of the observed diminished viability and developmental abnormalities. We compared the expression of 13 developmentally important genes in individual 8-cell and blastocyst stage NT embryos produced from adults female cumulus cells and adult male skin fibroblast cells with low and high incidences of neonatal abnormalities [1, 2]. In vitro-fertilized (IVF) embryos were used as control embryos. Among the genes tested, the relative abundance of Glut-1, IGF-IR, E-cad, and Cx43 transcripts varied significantly between the two types of NT embryos at the 8-cell stage. The relative abundance of manganese super oxide dismutase (MnSOD) and Stat3 transcripts was significantly higher in IVF embryos compared with both types of NT embryos. At the blastocyst stage, there was a significant difference in the relative expression of only one gene, Bcl-2, between the two types of NT embryos. Although the level of Glut-1 expression did not vary between the two types of NT blastocysts, its expression in both types of NT blastocysts was significantly lower than that in IVF blastocysts. The MnSOD expression level tended to be higher in NT blastocysts. The gene expression profile for any single gene, however, was highly variable among individual embryos and was independent of embryo morphology. The present study demonstrated that the expression profiles of the 13 genes examined in Day 9 NT blastocysts produced from two different types of donor cells with different incidences of neonatal abnormalities are largely indistinguishable.
  • Guohui Liu; Yoko Kato; Yukio Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 53 (4) 785 - 790 0916-8818 2007/08 [Refereed]
     
    Parthenogenetic activation is an important factor in successful production of cloned mammals. Because it has been reported that aged oocytes are more sensitive to parthenogenetic activation than young oocytes, the present study examined the effects of oocyte aging on the in vitro and in vivo developmental potential of nuclear-transferred (NT) mouse oocytes receiving cumulus cells. The potentials of young NT oocytes (14 h after human chorionic gonadotrophin [hCG] injection) to develop into blastocysts was, however, significantly higher than that of aged oocytes (20 h after hCG injection; 16% vs 6%). When the nuclei of NT oocytes at the 2-cell stage were fused with enucleated fertilized 2-cell embryos, the potentials of the serial NT embryos to develop into blastocysts were no different for both young and aged oocytes (74% vs 74%). Live young, however, were obtained only after transfer of serial NT blastocysts developed from young NT oocytes (2%). In contrast to a report using embryonic nuclei as the nuclear donors, the results of the present study indicate that young oocytes are superior to aged oocytes as a source of recipient cytoplasm for mouse somatic cell cloning.
  • Dasari Amarnath; Yoko Kato; Yukio Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 53 (3) 491 - 497 0916-8818 2007/06 [Refereed]
     
    The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.
  • Tetsuya Tani; Yoko Kato; Yukio Tsunoda
    FRONTIERS IN BIOSCIENCE FRONTIERS IN BIOSCIENCE INC 12 2693 - 2705 1093-9946 2007/01 [Refereed]
     
    Nuclear, microtubular dynamics and spindle assembly checkpoint ( SAC) in bovine somatic cell nuclear transfer ( SCNT) oocytes receiving G1/0 or M phase somatic cell nuclei were studied. SCNT oocytes assembled microtubules, however, the spindles were structurally abnormal, including bi- ,tri-polar or elongated spindles with scattered premature chromosome condensation ( PCC) in G1/0 phase nuclei, and some miniature spindles with unaligned chromosomes in M phase nuclei. In contrast, demecolcine-treated SCNT oocytes formed chromosome clusters with membrane protrusion and significantly induced maturation-promoting factor ( MPF) activity elevation ( up to 177%) for 3 hours, indicating that first SAC at second metaphase ( MII) is established upon spindle disruption in SCNT oocytes. After parthenogenetic stimuli, unlike MII oocytes which prevent exit from MII arrest with high MPF activity upon spindle disruption by second SAC, demecolcine-treated SCNT oocytes could not prevent exit from MII arrest with inactivation of MPF activities, whereas MG132-treated SCNT oocytes could persist at MII arrest, indicating that SCNT oocytes lack the ability for second SAC establishment, however, two G1/0 phase nuclei in an ooplasm restored second SAC establishment upon spindle disruption. Furthermore, the developmental potential of demecolcine-treated SCNT oocytes receiving G1/0 phase nuclei to blastocyst stage was not significantly different than untreated SCNT oocytes ( 29% vs 31%). These results indicate that unlike MII oocytes, SCNT oocytes have aberrant spindle morphology and SAC at MII due to insufficient SAC signals from somatic cell nuclei, thus aberrant remodeling has started immediately after somatic cell nuclear transfer and may be responsible for chromosome instability in SCNT embryos as well as the low successful efficiency of cloning.
  • Tetsuya Tani; Hiroaki Shimada; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 9 (2) 267 - 280 1536-2302 2007 [Refereed]
     
    Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary. © Mary Ann Liebert, Inc.
  • Yoko Kato; Xiangping Li; Dasari Amarnath; Koichi Ushizawa; Kazuyoshi Hashizume; Tomoyuki Tokunaga; Masanori Taniguchi; Yukio Tsunoda
    Cloning and Stem Cells 9 (4) 495 - 511 1536-2302 2007 [Refereed]
     
    Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-τ, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos few or none of the IVF or CNT eight-cell embryos expressed IFN-τ mRNA, but all eight-cell FNT embryos expressed IFN-τ. IFN-τ mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-τ mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-τ mRNA and inferior developmental potential in cloned embryos should be examined further. © 2007 Mary Ann Liebert, Inc.
  • Andrei Rybouchkin; Yoko Kato; Yukio Tsunodaz
    BIOLOGY OF REPRODUCTION OXFORD UNIV PRESS INC 74 (6) 1083 - 1089 0006-3363 2006/06 [Refereed]
     
    Before fertilization, chromatins of both mouse oocytes and spermatozoa contain very few acetylated histones. Soon after fertilization, chromatins of both gametes become highly acetylated. The same deacetylation-reacetylation changes occur with histones of somatic nuclei transferred into enucleated oocytes. The significance of these events in somatic chromatin reprogramming to the totipotent state is not known. To investigate their importance in reprogramming, we injected cumulus cell nuclei into enucleated mouse oocytes and estimated the histone deacetylation dynamics with immunocytochemistry. Other reconstructed oocytes were cultured before and/or after activation in the presence of the highly potent histone deacetylase inhibitor trychostatin A (TSA) for up to 9 h postactivation. The potential of TSA-treated and untreated oocytes to develop to the blastocyst stage and to full term was compared. Global deacetylation of histones in the cumulus nuclei occurred between 1 and 3 h after injection. TSA inhibition of histone deacetylation did not affect the blastocyst rate (37% with and 34% without TSA treatment), whereas extension of the TSA treatment beyond the activation point significantly increased the blastocyst rate (up to 81% versus 40% without TSA treatment) and quality (on average, 59 versus 45 cells in day 4 blastocysts with and without TSA treatment, respectively). TSA treatment also slightly increased full-term development (from 0.8% to 2.8%). Thus, deacetylation of somatic histones is not important for reprogramming, and hyperacetylation might actually improve reprogramming.
  • Tetsuya Tani; Hiroaki Shimada; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 8 (1) 61 - 66 1536-2302 2006 [Refereed]
     
    The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning. © Mary Ann Liebert, Inc.
  • Xiangping Li; Dasari Amarnath; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 8 (1) 41 - 50 1536-2302 2006 [Refereed]
     
    The high incidence of abnormalities in cloned calves is a most serious problem for bovine somatic cell nuclear transfer (NT) technology. Because there is little information on the differences in mRNA expression in cloned blastocysts with donor cells of different sex and origin, we compared development-related gene expression in two types of cloned bovine blastocysts with different potentials to develop into normal calves, a female adult cumulus cell line (high potential to develop into live calves) and a male fibroblast cell line (low potential to develop into live calves) to examine the correlation between the normality of cloned calves and blastocyst mRNA expression patterns. We analyzed 12 genes involved in apoptosis, growth factor signaling, metabolism, and DNA methylation in blastocysts originating from two types of donor cells and in vitro-fertilized blastocysts using quantitative real-time polymerase chain reaction. Expression of the pro-apoptotic Bax gene and anti-apoptotic Bcl-2 and Glut-1 genes in fibroblast-derived blastocysts was significantly higher than in cumulus cell-derived and in vitro-fertilized blastocysts. The high Bcl-2 and Glut-1 gene expression suggests that some embryonic cells with damaged DNA in fibroblast-derived blastocysts are not removed, and their descendants later manifest abnormal placenta or fetus formation. Transfer of pre-selected cloned blastocysts into recipients is required, however, to determine whether the expression pattern of these apoptosis-related genes reflects differences in the potential to develop into normal calves. © Mary Ann Liebert, Inc.
  • Xiangping Li; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 8 (3) 214 - 224 1536-2302 2006 [Refereed]
     
    The evaluation of embryo morphology, widely used for selecting mammalian embryos before transfer, is not an adequate standard for selecting nuclear-transferred (NT) embryos. To search for markers useful for predicting the potential of NT embryos to develop into young, we examined the relation between the morphology of embryos with different developmental potential and gene expression of Oct 4, Nanog, Stat3, FGF4, Stella, and Sox2. In the present study, we examined pronuclear-exchanged blastocysts and morula blastomere, embryonic stem (ES) cell, and cumulus cell NT blastocysts, and in vivo-developed and in vitro-developed blastocysts. Based on the small variations in the gene expression levels among the in vivo-developed blastocysts, and the significant differences in gene expression between in vivo-developed (high developmental potential), and ES cell and cumulus cell NT blastocysts (low developmental potential), down-regulation of Sox2 and Oct4 genes is considered to be a candidate marker for the low potential of NT embryos to develop into young. © Mary Ann Liebert, Inc.
  • XP Li; Y Kato; Y Tsunoda
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 72 (2) 152 - 160 1040-452X 2005/10 [Refereed]
     
    The potential of embryonic and somatic cell nuclear-transferred (NT) mouse oocytes to develop into young is low compared with bovine NT oocytes. To examine the reasons for the low developmental potential of NT mouse oocytes, we analyzed the gene expression patterns of six development-related genes (Oct4, Nanog, Stat3, stella, FGF4, and Sox2) during preimplantation development in manipulated oocytes with different potentials to develop into young using real-time polymerase chain reaction (PCR) methods. The manipulated oocytes were parthenogenetically activated oocytes and embryonic stem cell, cumulus cell, morula blastomere NT oocytes, and in vitro-cultured and in vivo-recovered embryos. The mRNA expression patterns in mouse NT-derived embryos markedly differed from in vivo and in vitro counterparts. Some transcript expression patterns in embryonic stem-cell NT oocytes resembled those of parthenogenetic oocytes. Of the six developmentally important transcripts examined in NT embryos, four had a downregulated expression pattern at the blastocyst stage. Our findings indicate that abnormal expression patterns of development-related genes during preimplantation development correlate with the low potential of NT oocytes to develop into young. Although more detailed information is required, Sox2 mRNA expression pattern in blastocysts seems to closely correlate with the developmental potential of NT embryos.
  • クローン家畜の作成と利用
    角田幸雄; 加藤容子
    総合臨床 54 56 - 61 2005 [Refereed]
  • Masahiro Kawakami; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 7 (3) 167 - 177 1536-2302 2005 [Refereed]
     
    Several studies report that meiotic maturation of porcine oocytes can be reversibly preserved. The present study examined how long meiotic maturation can be suppressed. The first experiment determined the preservation medium suitable for reversibly suppressing meiotic maturation of porcine oocytes. The second experiment examined the in vitro developmental potential of oocytes maintained in meiotic arrest after parthenogenetic activation and nuclear transfer of somatic cells. Preservation of cumulus-oocyte complexes with NCSU-37 medium containing 10% follicular fluid, 1 mM dibutyryl cyclic AMP, and follicular shell pieces for 24-96 h at 39°C did not affect oocyte maturation compared with controls (94-98% vs. 98%). The potential of parthenogenetically activated and nuclear-transferred oocytes maintained in meiotic arrest for 24-48 h to develop into blastocysts was not significantly different from that of controls (20-25% vs. 18% and 8-11% vs. 9%, respectively). The present study demonstrated that meiotic maturation of porcine oocytes can be suppressed after preservation for 48 h at 39°C without decreasing oocyte maturation competence or the ability of oocytes to develop to at least the blastocyst stage. © Mary Ann Liebert, Inc.
  • D Amarnath; Y Kato; Y Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 50 (5) 593 - 598 0916-8818 2004/10 [Refereed]
     
    The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.
  • S Matsushita; T Tani; Y Kato; Y Tsunoda
    ANIMAL REPRODUCTION SCIENCE ELSEVIER SCIENCE BV 84 (3-4) 293 - 301 0378-4320 2004/09 
    The present study examined the competence of oocytes from bovine ovaries stored at low temperatures for at least 1 day, which is the necessary time period to complete inspection for bovine spongiform encephalopathy. Storage of ovaries at 10degreesC for 24h did not affect oocyte maturation (68% versus 68%) or the potential of oocytes to develop into day 8 blastocysts after in vitro fertilization (25% versus 27%), parthenogenetic activation (19% versus 25%), or somatic cell nucleus transfer (27% versus 32%) compared with controls. In vitro-fertilized and parthenogenetic oocytes from ovaries stored at 10degreesC for 48h had a significantly decreased maturation rate and developmental potential, but nucleus-transferred oocytes that received cultured cumulus cells did not (27% versus 32%). Thus, bovine ovaries can be stored at 10degreesC for at least 24h without decreasing oocyte maturation competence or the developmental potential of in vitro-fertilized, parthenogenetically activated, and somatic cell nucleus-transferred oocytes, at least to the blastocyst stage. The present study provides valuable information with regard to removing bovine ovaries from abattoirs after testing for bovine spongiforn encephalopathy. (C) 2004 Elsevier B.V. All rights reserved.
  • T Kobayashi; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 62 (5) 854 - 860 0093-691X 2004/09 
    The present study examined whether the timing of the first cleavage has an effect on the in vitro and in vivo developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells. First; the timing of the first cleavage and the developmental potential of nuclear-transferred oocytes were examined every hour from 12 to 24 h after the start of culture and compared with in vitro-fertilized oocytes. The developmental potential of in vitro-fertilized oocytes decreased gradually according to the time required for cleavage (84% (32/38) for 15 h to 50% (1/2) for 20 h), but intermediate-cleaved (15-16 h) nuclear-transferred oocytes had a higher potential to develop into blastocysts (55% (17/31) to 67% (45/67) versus 0-43% (6/14)). Second the nuclear-transferred oocytes were divided into three groups according to the timing of the first cleavage; each group was cultured to blastocysts in vitro, and then transferred to recipients. The potential of intermediate-cleaved oocytes (15-16 h) to develop into blastocysts was significantly higher than fast-cleaved (before 15 h) and slow-cleaved (after 16 h) oocytes (65, 46, and 37%). The proportion of fetuses on Day 10.5 of pregnancy was highest in the intermediate-cleaved group (4 versus 2 and 1%, respectively) and a full-term fetus was obtained from this group. The present study demonstrated that the timing of the first cleavage could be used to determine the potential of nuclear-transferred oocytes with embryonic stem cells to develop to the blastocyst stage in vitro, but not to determine post-implantation viability after transfer to recipients. (c) 2004 Elsevier Inc. All rights reserved.
  • A Yabuuchi; Y Yasuda; Y Kato; Y Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 50 (2) 263 - 268 0916-8818 2004/04 
    Enucleated oocytes receiving mouse embryonic stem (ES) cells develop into fertile young. The developmental potential to young is low, however, and the rate of postnatal death is high. We examined the effect of various nuclear transfer procedures on the in vitro and in vivo developmental potential of nuclear-transferred oocytes. The potential of oocytes receiving ES cells at M phase to develop into blastocysts after fusion by Sendai virus was high compared with that after direct injection (67% vs. 30%). The developmental potential of oocytes receiving ES cells at the M phase is higher than that of oocytes receiving ES cells at the G(1) phase (30-67% vs. 2-5%). Developmental ability to live young was low in all groups (0-4%). Different activation protocols affected the potential to develop into blastocysts to a different extent (27-62%), but did not affect the potential to develop into live young (0-3%). The present study demonstrated that the various conditions examined did not affect the potential of nuclear-transferred oocytes receiving ES cells to develop into live young or the incidence of postnatal death.
  • Y Kato; H Imabayashi; T Mori; T Tani; M Taniguchi; M Higashi; M Matsumoto; A Umezawa; Y Tsunoda
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 70 (2) 415 - 418 0006-3363 2004/02 [Refereed]
     
    Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111: 589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.
  • 角田幸雄; 加藤容子
    遺伝 裳華房 58 (1) 64 - 68 0387-0022 2004 [Refereed]
  • Tsunoda Y; Kato Y
    Methods in molecular biology (Clifton, N.J.) 254 195 - 212 1064-3745 2004 [Refereed]
  • Kojiro Kawano; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells 6 (2) 67 - 72 1536-2302 2004 [Refereed]
     
    The present study compared the potential of nuclear-transferred porcine oocytes receiving fetal somatic cells by direct injection and cell fusion procedures to develop into blastocysts. After brief treatment of in vitro matured oocytes with demecolcine and sucrose, the protrusion containing the condensed chromosome mass was mechanically removed. Single donor cells were fused with enucleated oocytes following electric pulses or injected into oocytes by piezoactuated microinjection. The reconstruction rate by direct injection was significantly higher than that following cell fusion (89 vs. 48%). The potential of nuclear-transferred oocytes to develop into blastocysts, however, was not different between injection and fusion methods (13% vs. 18%). Total cell number, inner cell mass, and trophectoderm cell numbers of cloned blastocysts were also not different between the two groups.
  • T Tani; Y Kato; Y Tsunoda
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 69 (6) 1890 - 1894 0006-3363 2003/12 [Refereed]
     
    Cloned mammals with normal fertility have been produced by nuclear transfer. Thus, oocyte cytoplasm has the ability to convert differentiated somatic cell nuclei into a state that resembles the conditions that occur at fertilization (nuclear reprogramming). Despite the long-held assumption that reprogramming factors are present in mammalian oocytes, the molecular nature of these factors is not known. The present study demonstrates that the process of nuclear reprogramming is not directly regulated by maturation promoting factor or mitogen-activated protein kinase activity. The potential for nuclear-transferred oocytes to develop to the blastocyst stage was not different when somatic cells at the M phase were fused with oocytes activated with ionomycin and cycloheximide 1-5 h before (12%-22%) but was significantly decreased when oocytes were activated 6 h before (1%). Further molecular studies on the differences between oocytes with and without reprogramming potential are required and will be useful for the identification of reprogramming factors.
  • Tsunoda Y; Kato Y
    Nihon rinsho. Japanese journal of clinical medicine 61 (3) 406 - 410 0047-1852 2003/03 [Refereed]
     
    体細胞を核移植した時に生じる核の初期化機構を中心に紹介した。
  • Masahiro Kawakami; Tetsuya Tani; Akiko Yabuuchi; Tatsuya Kobayashi; Hiroshi Murakami; Tatsuya Fujimura; Yoko Kato; Yukio Tsunoda
    Cloning and Stem Cells Mary Ann Liebert Inc. 5 (4) 379 - 387 1536-2302 2003 [Refereed]
     
    Brief treatment of metaphase II (MII) stage porcine oocytes with 0.4 μg/mL demecolcine in the presence of 0.05 M sucrose produces a membrane protrusion that contains a condensed chromosome mass. The present study examined the optimal conditions for demecolcine and nocodazole treatment in chemically assisted removal of chromosomes. When matured oocytes were treated with 0.1-0.4 μg/mL demecolcine for 60 min or with 0.4 μg/mL demecolcine for 30 min or 3 μrg/mL nocodazole for 30 or 60 min, more than 70% of oocytes had a membrane protrusion containing condensed chromosomes were located. There was no difference in the in vitro developmental potential of enucleated oocytes assisted by 0.1 and 0.4 μg/mL demecolcine or 3 μrg/mL nocodazole that received porcine somatic cells. After transfer to 10 recipients, however, two of six recipients that received demecolcine-treated enucleated eggs produced four healthy cloned piglets, but none of the four recipients of nocodazole-treated enucleated eggs produced piglets. Further studies are required to increase the successful development to term because the proportion of live piglets was low (4/2, 672, 0.15%).
  • Akiko Yabuuchi; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 48 (4) 393 - 397 0916-8818 2002/12 
    Nuclear-transferred mouse eggs receiving embryonic stem cells develop into blastocysts at a high rate, but their potential to develop into fetuses is very low. Therefore, the present study examined the effects of aggregating of two nuclear-transferred eggs at the 8-cell stage to increase the cell number of nuclear-transferred embryos. The proportion of implantation sites on gestational day 10.5 after transfer of aggregated nuclear-transferred embryos (53%) was significantly higher than after single nuclear transfer (29%) or serial nuclear-transfer (37%). The proportion of implantation sites with fetuses did not differ significantly among the three groups (7 to 17%). These results demonstrate that the low potential of nuclear-transferred embryos with embryonic stem cells to develop into fetuses is not due to the lower cell number of nuclear-transferred embryos.
  • XJ Yin; Y Kato; Y Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 10 (3) 217 - 222 0967-1994 2002/08 [Refereed]
     
    To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.
  • M Kawakami; T Tani; XJ Yin; Y Kato; Y Tsunoda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT SOCIETY REPRODUCTION & DEVELOPMENT-SRD 48 (4) 409 - 414 0916-8818 2002/08 
    The effects of oxygen tension (5% and 20%) during in vitro culture of oocytes and fetal fibroblast cells on the developmental potential of parthenogenetic and nuclear-transferred porcine oocytes were examined. The potential of parthenogenetic oocytes matured and cultured under different oxygen tensions to develop into blastocysts was not significantly different (5 to 16%). The proportions of enucleated oocytes receiving fetal fibroblast cells that developed into blastocysts were significantly lower when somatic cells were cultured under low oxygen tension (2 and 3%) than under high oxygen tension (7%). The effects of oxygen tension during culture of oocytes and somatic cells on the developmental potential of parthenogenetic and nuclear-transferred oocytes is discussed.
  • XJ Yin; T Tani; Yonemura, I; M Kawakami; K Miyamoto; R Hasegawa; Y Kato; Y Tsunoda
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 67 (2) 442 - 446 0006-3363 2002/08 [Refereed]
     
    The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.
  • XJ Yin; Y Kato; Y Tsunoda
    REPRODUCTION SOC REPRODUCTION FERTILITY 124 (1) 41 - 47 1470-1626 2002/07 
    Enucleated oocytes matured in vitro, from which chromosomes were removed by treatment with ionomycin and demecolcine, were used as recipient oocytes for nuclear transfer of fibroblast cells from a mature male rabbit. The enucleated oocytes with donor nuclei were electrically activated 2 h after fusion. The potential of nuclear-transferred oocytes matured in vitro and ovulated oocytes to develop into blastocysts was high (33-55%), except for oocytes cultured for 8.0 (19%) and 8.5 h (25%) in vitro. After transfer of nuclear-transferred oocytes to recipients, ten of 62 (16%) and one of eight (13%) recipients that received in vitro-matured and ovulated oocytes, respectively, had 19 (1%) and one (0.6%) implantation sites at the time of laparotomy on days 8-17 after transfer. Four fetuses, including two with beating hearts, were obtained on day 15 of gestation after transfer of nuclear-transferred oocytes matured in vitro. The reason for the low efficiency of fetus production was not clear. One possibility is chromosomal abnormalities of nuclear-transferred oocytes, as most (21 of 22) of the oocytes had chromosomes dispersed along the spindle fibre at the first cell cycle. This is the first report of successful production of fetuses after nuclear transfer of rabbit somatic cells.
  • T Amano; Y Kato; Y Tsunoda
    CELL AND TISSUE RESEARCH SPRINGER-VERLAG 307 (3) 367 - 370 0302-766X 2002/03 
    The present study examined the causes of the low developmental potential of enucleated oocytes that have received ES cells and consequent postnatal death of the young. The inner cell masses (ICM) of nuclear-transferred blastocysts or diploid blastocysts were injected into tetraploid blastocysts (group B) or nuclear-transferred tetraploid blastocysts (group C), respectively. The developmental potential of these groups was compared with tetraploid blastocysts injected with ICM of diploid blastocysts (group A), The potential of reconstituted blastocysts to develop into live young in group B increased slightly (5%) but was significantly lower than that in group A (45%). The rate of postnatal death of young in group B did not decrease. The implantation rate of reconstituted blastocysts in group C was very low and no live fetuses were obtained, The results of the present study indicate that the inferior potential of both ICM and trophectoderm cells of nuclear-transferred blastocysts underlies the low developmental rate of nuclear-transferred oocytes receiving ES cells and the higher rate of postnatal death of ES cell-derived young.
  • T Yamada; M Yoshikawa; S Kanda; Y Kato; Y Nakajima; S Ishizaka; Y Tsunoda
    STEM CELLS ALPHAMED PRESS 20 (2) 146 - 154 1066-5099 2002 
    Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found the emergence of cell clusters that show the cellular uptake of indocyanine green (ICG) in the culture of differentiated ES cells. ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. The aim of the present study was to investigate the hepatic characteristics of ICG-stained cells. Methods. Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to outgrow in the placed culture. Gene expression of hepatocyte markers was analyzed by reverse transcriptase-polymerase chain reaction, and albumin production was examined immunohistochemically. Morphology and cellular components were investigated by electron microscopy. ICG-stained cells were further transplanted into the portal vein of mice. Results. ICG-stained cells appeared around 14 days of the EB culture and formed distinct three-dimensional structures. They were inummoreactive to albumin and expressed mRNAs such as albumin, alpha-fetoprotein, transthyretin, hepatocyte nuclear factor 3 beta, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, urea cycle enzyme, gluconeogenic enzyme, and liver-specific organic anion transporter-1. An ultrastructural analysis revealed a well-developed system of organelles such as mitochondria, lysosomes, Golgi apparatus, and rough and smooth endoplasmic reticulum. The transplantation of ICG-positive cells into the portal vein resulted in the incorporation into mice livers, where they were morphologically indistinguishable from neighboring hepatocytes. Conclusions. ES cell-derived ICG-positive cells possess characteristics of hepatocytes, and ICG-staining is a useful marker to identify differentiated hepatocytes from EBs in vitro.
  • Takatsugu Yamada; Masahide Yoshikawa; Miyako Takaki; Shigeko Torihashi; Yoko Kato; Yoshiyuki Nakajima; Shigeaki Ishizaka; Yukio Tsunoda
    Stem Cells AlphaMed Press 20 (1) 41 - 49 1066-5099 2002 
    Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found that ES cells can give rise to a functional gut-like unit, which forms a three-dimensional dome-like structure with lumen and exhibits mechanical activity, such as spontaneous contraction and peristalsis. The aim of the present study was to investigate the electrophysiological and morphological properties of ES cell-derived contracting clusters. Methods. Electrical activity was examined by an extracellular recording. Morphology and cellular components were investigated by immunohistochemistry and electron microscopy. Results. Clusters with rhythmic contractions displayed electrical slow waves at a regular rhythm, and clusters with highly coordinated peristalsis showed regular slow waves and spontaneous spike action potentials. Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICC), was observed in dense network structures. Neuronal marker PGP9.5 immunoreactivity was observed only in clusters with peristalsis. The topographical structure of the wall was organized by an inner epithelial layer and outer smooth muscle layer. The smooth muscle layer was provided with an ICC network and innervated with enteric neurons. Conclusions. ES cells can differentiate into a functional gut-like organ in vitro that exhibits physiological and morphological properties characteristic of the gastrointestinal (GI) tract. This ES cell-derived gut provides a powerful tool for studying GI motility and gut development in vitro, and has potential for elucidating and treating a variety of motility disorders.
  • クローンウシにおける核の初期化と頻発異常
    角田幸雄; 加藤容子
    蛋白質核酸酵素 47 1797 - 1803 2002 [Refereed]
  • Y Tsunoda; Y Kato
    DIFFERENTIATION BLACKWELL VERLAG GMBH 69 (4-5) 158 - 161 0301-4681 2002/01 
    It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.
  • Yukio Tsunoda; Yoko Kato
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 47 1797 - 1803 0039-9450 2002/01
  • T Yamada; M Yoshikawa; S Kanda; Y Kato; Y Nakajima; S Ishizaka; Y Tsunoda
    STEM CELLS ALPHAMED PRESS 20 (2) 146 - 154 1066-5099 2002 
    Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found the emergence of cell clusters that show the cellular uptake of indocyanine green (ICG) in the culture of differentiated ES cells. ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. The aim of the present study was to investigate the hepatic characteristics of ICG-stained cells. Methods. Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to outgrow in the placed culture. Gene expression of hepatocyte markers was analyzed by reverse transcriptase-polymerase chain reaction, and albumin production was examined immunohistochemically. Morphology and cellular components were investigated by electron microscopy. ICG-stained cells were further transplanted into the portal vein of mice. Results. ICG-stained cells appeared around 14 days of the EB culture and formed distinct three-dimensional structures. They were inummoreactive to albumin and expressed mRNAs such as albumin, alpha-fetoprotein, transthyretin, hepatocyte nuclear factor 3 beta, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, urea cycle enzyme, gluconeogenic enzyme, and liver-specific organic anion transporter-1. An ultrastructural analysis revealed a well-developed system of organelles such as mitochondria, lysosomes, Golgi apparatus, and rough and smooth endoplasmic reticulum. The transplantation of ICG-positive cells into the portal vein resulted in the incorporation into mice livers, where they were morphologically indistinguishable from neighboring hepatocytes. Conclusions. ES cell-derived ICG-positive cells possess characteristics of hepatocytes, and ICG-staining is a useful marker to identify differentiated hepatocytes from EBs in vitro.
  • T Amano; Y Kato; Y Tsunoda
    ZYGOTE CAMBRIDGE UNIV PRESS 9 (2) 153 - 157 0967-1994 2001/05 
    The present study compared the production efficiency and incidence of postnatal death in mice derived by injecting embryonic stem (ES) cells into either heat-treated blastocysts or tetraploid blastocysts. The proportion of completely ES-cell-derived mice from the tetraploid blastocyst group (3.3%) was significantly higher than that obtained from the heat-treated blastocyst group (1.5%). The incidence of postnatal death was the same between the two groups: 10 of 15 young (67%) in the heat-treated group and 21 of 34 young (62%) in the tetraploid group died within 13 days of birth. The remaining young grew to adulthood, had normal fertility, and their germ cells were of ES cell origin. There was no clear correlation, however, between the postnatal lethality of ES-cell-derived mice and the genetic background of the ES cells. The causes of postnatal death are discussed.
  • T Amano; T Tani; Y Kato; Y Tsunoda
    JOURNAL OF EXPERIMENTAL ZOOLOGY WILEY-LISS 289 (2) 139 - 145 0022-104X 2001/02 [Refereed]
     
    Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001. (C) 2001 Wiley-Liss, Inc.
  • A Yabuuchi; T Tani; Y Kato; Y Tsunoda
    JOURNAL OF EXPERIMENTAL ZOOLOGY WILEY-LISS 289 (3) 208 - 212 0022-104X 2001/02 
    The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 mug/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. (C) 2001 Wiley-Liss, Inc.
  • クローンウシと再プログラム化
    角田幸雄; 加藤容子
    実験医学、 19 1494 - 1498 2001 [Refereed]
  • 体細胞クローンウシの作製
    谷哲弥; 加藤容子; 角田幸雄
    実験医学別冊 ポストゲノム時代の実験講座４， 217 - 222 2001 [Refereed]
  • 体細胞クローン技術の新展開，
    角田幸雄; 加藤容子
    Hormone Frontier in Gynecology 8 33 - 38 2001 [Refereed]
  • T Tani; Y Kato; Y Tsunoda
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 64 (1) 324 - 330 0006-3363 2001/01 
    We examined the in vitro developmental potential of nonactivated and activated enucleated ova receiving cumulus cells at various stages of the cell cycle. Eleven to 29% of activated ova receiving donor cells stopped developing at the 8-cell stage but 21% to 50% of nonactivated ova receiving donor cells at either the G(0), G(1), G(2), or M phase, or cycling cells developed into blastocysts. One normal calf was born after transferring five blastocysts that had developed from ova receiving donor cells at the M phase. The present study demonstrated that direct exposure of donor chromosomes to nonactivated ovum cytoplasm is effective for somatic cell nucleus reprogramming, and activated ovum cytoplasm does not reprogram the nucleus.
  • T. Amano; Y. Kato; Y. Tsunoda
    Reproduction Journals of Reproduction and Fertility Ltd 121 (5) 729 - 733 1470-1626 2001 
    The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.
  • Y Tsunoda; Y Kato
    ZOOLOGICAL SCIENCE ZOOLOGICAL SOC JAPAN 17 (9) 1177 - 1184 0289-0003 2000/12 
    Differentiated mammalian somatic cell nuclei and embryonic nuclei can now be reprogrammed to develop into young if they are introduced into enucleated oocytes. The success rates for cloning are generally low, however, and peri- and postnatal death rates of the young are high. Cloning technology will be useful for the genetic improvement of farm animals, therapeutic human protein production, and organ or tissue transplantation into humans. In addition, the information obtained on nuclear reprogramming will be helpful for understanding the fundamental mechanisms of differentiation and aging.
  • XJ Yin; T Tani; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 54 (9) 1469 - 1476 0093-691X 2000/12 
    The present study determined a suitable parthenogenetic activation procedure for rabbit oocytes and examined the developmental potential of enucleated oocytes receiving cultured cumulus cells. Unfertilized oocytes recovered from superovulated rabbits were activated with one or two sets of electrical pulses, with or without subsequent administration of 6-dimethylaminopurine (6-DMAP). The proportion of oocytes treated with one or two sets of electrical pulses and 6-DMAP that cleaved (87% and 98%, respectively) and developed into blastocysts (77% and 85%, respectively) was significantly higher (P < 0.05) than those activated with electrical pulses alone (30% and 42% for cleavage, 7% and 17% for blastocysts). Cumulus cells separated from ovulated oocytes obtained from mature rabbits were cultured for three to five passages and then induced to quiescence by serum starvation before nuclear transfer. The enucleated oocytes receiving cumulus cells were activated with electrical pulses followed by the addition of 6-DMAP, and cultured in vitro far 5 to 6 d or transferred to pseudopregnant recipient females 1 d after activation. Of 186 nuclear-transferred oocytes, 123 (66%) cleaved and 42 (23%) developed into blastocysts. After transfer of 174 nuclear-transferred oocytes to 8 recipient females, a total of 3 implantation sites were observed in 3 recipient females but no fetuses were obtained. (C) 2000 by Elsevier Science Inc.
  • Y Kato; T Tani; Y Tsunoda
    JOURNAL OF REPRODUCTION AND FERTILITY JOURNALS OF REPRODUCTION FERTILITY LTD 120 (2) 231 - 237 0022-4251 2000/11 
    Twenty-four calves were cloned from six somatic cell types of female and male adult, newborn and fetal cows. The clones were derived from female cumulus (n = 3), oviduct (n = 2) and uterine (n = 2) cells, female and male skin cells (n = 10), and male ear (n = 5) and Liver (n = 2) cells. On the basis of the number of cloned embryos transferred (n = 172) to surrogate cows, the overall rate of success was 14%, but based on the number of surrogate mothers that became pregnant (n = 50), the success rate was 48%. Cell nuclei from uterus, ear and liver cells, which have not been tested previously, developed into newborn calves after nuclear transfer into enucleated oocytes. To date, seven female and six male calves have survived: six of the females were from adult cells (cumulus (n = 3), oviduct (n = 2) and skin (n = 1) cells) and one was from newborn skin cells, whereas the male calves were derived from adult ear cells (n = 3), newborn liver and skin cells (II = 2), and fetal cells (n = 1). Clones derived from adult cells frequently aborted in the later stages of pregnancy and calves developing to term showed a higher number of abnormalities than did those derived from newborn or fetal cells. The telomeric DNA lengths in the ear cells of three male calves cloned from the ear cells of a bull aged 10 years were shorter than those of the original bull. However, the telomeric DNA lengths from the white blood cells of the clones were similar to those of an age-matched control and the original bull, which indicates that telomeric shortening varies among tissues
  • T Tani; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 53 (8) 1623 - 1629 0093-691X 2000/05 
    An efficient method for freezing donor cells is necessary when using nucleus transfer of somatic cells for large-scale cloning. In the present study, we developed a method for freezing and thawing bovine cumulus cell-derived cultured cells to be used as nucleus donors. Cumulus cells were obtained from ovaries of living and slaughtered bovine and cultured in vitro. Cumulus cell-derived cultured cells were serum-starved for several days to induce a quiescent state and then frozen at -70 degrees C for at least 2 d. Immediately thereafter or 2 h after thawing, the cells were used as donor cells for nuclear transfer without additional in vitro culture. The fusion rate with recipient cytoplasts was not affected by the cumulus cell source (slaughtered or living) or time after thawing (0 and 2 h). The cleavage rate of frozen-thawed cumulus cell-derived cultured cells from slaughtered cows immediately after thawing (0 h) was highest (97%) and was significantly higher than that of controls (85%) or cells transferred 2 h after thawing (85%). There were no significant differences among any of the groups in the potential of the nuclear transfer embryos to develop into blastocysts (34 vs 44 and 44%, 39 vs 45 and 46%). Thus, storage of bovine cumulus cell-derived cultured cells in the quiescent state at -70 degrees C is effective and might be useful and convenient for large-scale cloning. The maximum storage periods and developmental potential of embryos after such nucleus transfers requires further examination. (C) 2000 by Elsevier Science Inc.
  • T Amano; K Nakamura; T Tani; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 53 (7) 1449 - 1458 0093-691X 2000/04 
    The sensitivity of the inner cell mass (ICM) and trophectoderm (TE) of mouse blastocysts to high temperatures was examined. When blastocysts with a diameter of 100 to 120 mu m treated for 15 to 20 min at 45 degrees C were cultured in vitro, the cell number in the ICM did not increase, although that in the TE did increase. After transfer of treated blastocysts to recipients, implantation was not drastically inhibited but no live fetuses were obtained. These results demonstrated that the ICM at the blastocyst stage was more sensitive to high temperature than the TE. ICM clumps or ES cells were injected into blastocysts treated for 20 min at 45 degrees C. After transfer of injected blastocysts to recipients, we obtained mice derived completely from ICM or ES cells as judged by GPI analysis. Since 4 of 7 ES-cell derived mice, but none of the 6 mice derived from the ICM died after birth, an as yet unidentified epigenetic alteration might have occurred during the establishment and/or culture of ES cells. (C) 2000 by Elsevier Science Inc.
  • BX Nguyen; Y Sotomaru; T Tani; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 53 (7) 1439 - 1448 0093-691X 2000/04 
    Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment I, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification. (C) 2000 by Elsevier Science Inc.
  • 21世紀の医学を展望する 核移植と再生医学
    角田幸雄; 加藤容子
    最新医学別冊，再生医学 23 - 30 2000 [Refereed]
  • 角田幸雄; 加藤容子
    現代化学 東京化学同人 (11月) 45 - 49 0386-961X 2000 [Refereed]
  • 家畜における核移植
    加藤容子; 角田幸雄
    遺伝子医学 12 49 - 53 2000 [Refereed]
  • 体細胞核移植による動物クローニング
    角田幸雄; 加藤容子
    蛋白質核酸酵素 45 2015 - 2020 2000 [Refereed]
  • Y. Tsunoda; Y. Kato
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 45 2015 - 2020 0039-9450 2000/01
  • Y Kato; A Yabuuchi; N Motosugi; J Kato; Y Tsunoda
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 61 (4) 1110 - 1114 0006-3363 1999/10 
    The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 mu m; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.
  • Y Sotomaru; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 52 (2) 213 - 220 0093-691X 1999/07 
    Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to the blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (IPN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with IPN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 mates and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique. (C) 1999 by Elsevier Science Inc.
  • Y Kato; WM Rideout; K Hilton; SC Barton; Y Tsunoda; MA Surani
    DEVELOPMENT COMPANY OF BIOLOGISTS LTD 126 (9) 1823 - 1832 0950-1991 1999/05 
    There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes, However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes, Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was 'rescued' from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.
  • 角田幸雄; 加藤容子
    日経サイエンス 日経サイエンス 29 (3) 44 - 47 0917-009X 1999 [Refereed]
  • 体細胞クローン動物
    角田幸雄; 谷哲弥; 加藤容子
    J.Reprod.Dev1999年度日本繁殖生物学会シンポジウム 45 61 - 64 1999
  • Y Kato; T Tani; Y Sotomaru; K Kurokawa; JY Kato; H Doguchi; H Yasue; Y Tsunoda
    SCIENCE AMER ASSOC ADVANCEMENT SCIENCE 282 (5396) 2095 - 2098 0036-8075 1998/12 
    Eight carves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). ALL carves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn carves.
  • TSUNODA Yukio; KATO Yoko
    化学と生物 学会出版センタ- 36 (9) 572 - 577 0453-073X 1998/09 [Refereed]
  • Y Sotomaru; Y Kato; Y Tsunoda
    CRYOBIOLOGY ACADEMIC PRESS INC 37 (2) 139 - 145 0011-2240 1998/09 
    We examined the viability of mouse bisected embryos after freezing and thawing and produced monozygotic twin mice from these embryos. Two-cell embryos were collected from superovulated mature agouti, Fl hybrid (C57BL/6 X CBA) female mice. For bisection, one blastomere of the embryo was aspirated with a micropipette and injected into an empty zona pellucida. After culture for 24 to 28 h to the compacted 4- to 8-cell stage or 48 to 52 h to the late morula to blastocyst stage, the embryos were slowly frozen (-0.5 to -1.0 degrees C/min), thawed (30 degrees C/min), cultured for 24 h, and then transferred to recipient females. The bisected embryos without zonae pellucidae had developmental ability in vitro similar to those with zonae pellucidae (88% vs 89%). However, after freezing and thawing at the compacted 4- to 8-cell stage, bisected embryos with zonae pellucidae had higher viability than those without (60% vs 15%). Zona enclosed, bisected embryos frozen at the compacted 4- to 8-cell stage were more resistnat to freezing and thawing than those at the late morula to blastocyte stage (60% vs 23%). After transfer to recipients 26% of the zona enclosed bisected embryos frozen-thawed at the 4- to 8-cell stage developed to living fetuses a day 17.5 to 18.0 of pregnancy, which was slightly but not significantly lower than that of fresh bisected embryos (48%). On the other hand, only 5% of bisected embryos frozen-thawed at the late morula to blastocyst stage developed to young. The transfer of 15 sets of twin blastocysts as pairs that had been frozen and thawed at the compacted 4- to 8-cell stage yielded 2 (13%) sets of monozygotic twins. (C) 1998 Academic Press.
  • Y Tsunoda; Y Kato
    JOURNAL OF REPRODUCTION AND FERTILITY JOURNALS OF REPRODUCTION FERTILITY LTD 113 (2) 181 - 184 0022-4251 1998/07 
    The nuclei of mouse trophectoderm cells were found to have developmental totipotency like inner cell mass cells after serial nuclear transfer. Single inner cell mass or trophectoderm cells from expanded blastocysts synchronized with the cell cycle by treatment with nocodazole and aphidicolin to the G1 stage were injected into the perivitelline space of enucleated metaphase II oocytes together with Sendai virus. All oocytes were given three electrical pulses to induce fusion and activation (first nuclear transfer). Aphidicolin was present in all media used until fusion. When reconstituted oocytes developed to the two-cell stage, the nuclei of the reconstituted eggs were fused with the enucleated blastomeres of fertilized two-cell embryos by inactivated Sendai virus (second nuclear transfer). The reconstituted embryos were cultured in vitro and transferred to recipients. After the second nuclear transfer, 23-64% (for inner cell mass cells) and 32-62% (for trophectoderm cells) developed to morula or blastocyst stage. Better development of second nuclear transfer embryos was observed when oocytes fused with trophectoderm nuclei did not extrude a polar body after the first nuclear transfer. After transfer of morulae and blastocysts to recipients, four males were obtained, two from inner cell mass and two from trophectoderm nuclei. These findings indicate that the nucleus of inner cell mass and trophectoderm cells of mouse blastocysts can be reprogrammed within the cytoplasm of unfertilized oocytes and then in fertilized embryos.
  • 日本のクローン技術，その現在と未来
    角田幸雄; 加藤容子
    科学 68 679 - 681 1998 [Refereed]
  • 加藤容子; 角田幸雄
    蛋白質核酸酵素(増刊), 共立出版 43 (4) 397 - 404 0039-9450 1998 [Refereed]
  • Y. Kato; Y. Tsunoda
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 43 397 - 404 0039-9450 1998/01
  • Yusuke Sotomaru; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development The Japanese Society of Animal Reproduction (JSAR) 44 (1) 1 - 6 1348-4400 1998 
    Trophectoderm (TE) and inner cell mass (ICM) cells from blastocytsts were compared for their ability to produce chimeric mice or conceptuses (pluripotency) using nuclear transplantation in which a single cell was transplanted into an enucleated blastomere of a 2-cell embryo. The developmental ability of the embryos reconstituted with TE and ICM cells was dependent on when the recipient 2-cell embryos were recovered after hCG administration. When 2-cell embryos obtained 38-42 and 43-46 h post hCG injection were used, cell division of the reconstituted embryos rarely progressed to the 4-cell stage. However, when embryos recovered 48-51 h post hCG were used, the proportion of cleaved embryos increased significantly, developing to blastocysts with several large blastomeres. When reconstituted embryos were treated with nocodazole after nuclear transplantation, the percentage of cleaved embryos significantly increased, with some developing to blastocysts. Such blastocysts were transferred to recipient females, and no donor nuclei were detected in the conceptuses at midgestation. The in vitro developmental ability of the chimeric 2cell embryos reconstituted with TE and ICM cells from blastocysts was quite limited and no chimeric conceptuses were obtained.
  • TSUNODA Yukio; KATO Yoko
    Journal of Reproduction and Development 日本繁殖生物学会 43 (6) j123 - j126 0916-8818 1997/12 
    1952年以降ヒョウガエルやツメガエルを用いて行われてきた実験から，オタマジャクシの小腸細胞のような分化した体細胞の核移植によって正常なカエルが得られることが示されている．しかしながら，成体のカエルの体細胞からはオタマジャクシは得られるが，正常なカエルは得られていない．今回Wilmutら[1]は，少なくとも一部の成体の体細胞核は個体への発生能力を維持していることを動物で初めて示した．本稿では，これまでの研究の歩みをふりかえりながら，哺乳動物における体細胞核移植の意義と将来の研究方向を展望してみたい．
  • KATO Yoko
    Journal of Reproduction and Development 日本繁殖生物学会 43 (6) j47 - j54 0916-8818 1997/12 
    In the present study, the pluripotency of embryonic cells on the germ line, especially fetal germ cells, was examined. The results obtained are as follows; when donor cells on the germ line were aggregated with precompacted stage embryos or injected into blastocysts, fertile chimeric mice or fetuses were obtained from 3.5, 4.5 and 5.5 days cells and ES-cells, respectively. However, further stage cells never contributed to the conceptuses even though analyzed at 10.5 days of pregnancy. When fetal germ cells on days 14.5-16.5 were reprogrammed by fusion with enucleated oocytes and then treated with fertilized embryos, they can form chimeric fetuses and extraembryonic tissues. It was suggested that differentiated cells on the germ line such as fetal germ cells can also develop to chimeric fetuses after reprogramming. However, it was also clear that they died at the midgestation probably due to the inappropriate imprinting.
  • 角田 幸男; 加藤 容子
    畜産の研究 養賢堂 51 (10) 1094 - 1098 0009-3874 1997/10
  • Y Sotomaru; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 48 (6) 977 - 984 0093-691X 1997/10 
    The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods. (C) 1997 by Elsevier Science Inc.
  • Y Tsunoda; Y Kato
    JOURNAL OF EXPERIMENTAL ZOOLOGY WILEY-LISS 278 (4) 250 - 254 0022-104X 1997/07 [Refereed]
     
    The developmental ability of enucleated mouse oocytes reconstituted between the nucleus from 4-cell mouse embryos at different cell stages and recipient cytoplasms at different conditions, and the developmental ability of oocytes receiving nuclei from compacted morulae were examined. The highest development was observed with the nucleus at the G(1) stage fused with cytoplasm at the M stage. Although the enucleated oocytes receiving a nucleus from 4-cell embryos did not develop after transfer to a recipient female, young were obtained after renuclear transfer to enucleated fertilized eggs. Live mice were also obtained from the nucleus of compacted morula. (C) 1997 Wiley-Liss, Inc.
  • TSUNODA Yukio; KATO Yoko
    Nihon Chikusan Gakkaiho 公益社団法人 日本畜産学会 68 (6) 596 - 602 1346-907X 1997/06 [Refereed]
  • H Takano; C Kozai; S Shimizu; Y Kato; Y Tsunoda
    THERIOGENOLOGY ELSEVIER SCIENCE INC 47 (7) 1365 - 1373 0093-691X 1997/05 [Refereed]
     
    The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2+/-4.3, twice: 19.8+/-9.2 and three times: 30.0+/-14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts, respectively. (C) 1997 by Elsevier Science Inc.
  • OHKOSHI Katsuhiro; HATA Masaki; KATO Yoko; TSUNODA Yukio
    Nihon Chikusan Gakkaiho 公益社団法人 日本畜産学会 68 (1) 7 - 12 1346-907X 1997/01 
    The in vitro developmental ability of frozen-thawed enucleated oocytes receiving a donor blastomere of embryos fertilized in vitro was studied. All recipient oocytes were enucleated before freezing and were activated 9 hours before fusion with a donor blastomere from 8-to 20-cell stage embryos. The enucleated oocytes were frozen in 1.0 or 1.5M glycerol by conventional slow cooling methods. The developmental ability of oocytes was slightly higher when they were frozen before rather than after activation (in experiment 1). The developmental ability to 2-cell and 8-cell stage was high (70% and 26%, respectively) when aged oocytes (32 hours of maturation at activation) were used, but the development to blastocyst was only observed in young oocytes (23.5 hours) receiving a donor blastomere (2% in experiment 2). Although the developmental ability to 8-cell stage of frozen-thawed nuclear transferred oocytes was low (15%), a small ratio of them developed to blastocysts (2%).
  • 角田幸雄; 加藤容子
    科学 岩波書店 67 (5) 344 - 347 0022-7625 1997 [Refereed]
  • 角田幸雄; 加藤容子
    J.Reprod.Dev Japanese Society of Animal Reproduction 43 (6) j123 - 126 0916-8818 1997 [Refereed]
  • 加藤容子
    J.Reprod.Dev 43 j47 - 54 1997 [Refereed]
  • Katsuhiro Ohkoshi; Masaki Hata; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development The Japanese Society of Animal Reproduction (JSAR) 43 (3) 261 - 265 1348-4400 1997 [Refereed]
     
    The effect of the age of a donor embryo on the developmental ability of bovine nuclear transferred eggs was examined. A blastomere of an in vitro fertilized embryo cultured for 3.5 to 6.5 days, or an inner cell mass cell (ICM) from a blastocyst cultured for 7.5 days was fused with an enucleated, preactivated oocyte matured in vitro by electrical stimulation. Fused eggs were cocultured with cumulus cells for 10 days in vitro. The fusion rate of blastomeres from day 3.5 to 6.5 embryos with enucleated oocytes (78 to 88%) and the developmental ability of nuclear transferred eggs to blastocysts (6 to 10%) were not significantly affected by the age of the donor embryo. However, the fusion rate of the ICM cell was significantly low (28%). Although nuclear transferred eggs receiving an ICM cell developed to 2-cell (73%) and 8-cell (22%) stages at the same rates as those obtained in other groups (68 to 81% for 2-cell, 30 to 34% for 8-cell), none of them developed to blastocysts.
  • Katsuhiro Ohkoshi; Masaki Hata; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development The Japanese Society of Animal Reproduction (JSAR) 43 (3) 261 - 265 1348-4400 1997 
    The effect of the age of a donor embryo on the developmental ability of bovine nuclear transferred eggs was examined. A blastomere of an in vitro fertilized embryo cultured for 3.5 to 6.5 days, or an inner cell mass cell (ICM) from a blastocyst cultured for 7.5 days was fused with an enucleated, preactivated oocyte matured in vitro by electrical stimulation. Fused eggs were cocultured with cumulus cells for 10 days in vitro. The fusion rate of blastomeres from day 3.5 to 6.5 embryos with enucleated oocytes (78 to 88%) and the developmental ability of nuclear transferred eggs to blastocysts (6 to 10%) were not significantly affected by the age of the donor embryo. However, the fusion rate of the ICM cell was significantly low (28%). Although nuclear transferred eggs receiving an ICM cell developed to 2-cell (73%) and 8-cell (22%) stages at the same rates as those obtained in other groups (68 to 81% for 2-cell, 30 to 34% for 8-cell), none of them developed to blastocysts.
  • Minkang Wang; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development The Japanese Society of Animal Reproduction (JSAR) 43 (1) 91 - 95 1348-4400 1997 [Refereed]
     
    Monozygotic mouse twins were produced by micromanipulation of 2-cell stage embryos and factors affecting the embryonic development were examined. In experiment 1, 2-cell embryos obtained from superovulated CD-1 strain or Fl (C57BLJ6 x CBA) females mated with CD-1 strain males were separated into a single blastomere. Pairs of twin blastomeres were cultured with or without supplementation of growth factors, a mixture of IGF-1, TGF-β and EGF for 3 days and twin blastocysts developed were transferred to recipient females. Of 174 pairs of Fl embryos, 88 to 100% developed to twin blastocysts. In contrast, development of CD-1 embryos (835 pairs) was significantly lower (25 to 36%). Of 63 Fl twin blastocysts transferred, 7 to 29% developed to twin fetuses, whereas only 0 to 8% of 62 pairs of CD-1 embryos developed to twins. In experiment 2, one nucleus of a 2-cell stage Fl embryo was electrically fused with an enucleated recipient 2-cell embryo. The 2-cell embryo from which a nucleus was removed was cultured together with the reconstituted embryo as a monozygotic twin pairs. Of 24 and 28 twin pairs cultured with or without growth factors, 92 and 86% developed to twin blastocysts, respectively. After transfer of 13 twin blastocysts to recipients, 3 and 2 twin fetuses were obtained. These findings indicated that monozygotic twin mice can be produced more efficiently from Fl embryos than from CD-1 embryos. Methods of removal of zona pellucida and addition of growth factors to the culture medium had no effect on the efficiency of production of monozygotic twins.
  • Yoko Kato; Miho Tanimura; Yukio Tsunoda
    Journal of Reproduction and Development The Japanese Society of Animal Reproduction (JSAR) 43 (3) 205 - 211 1348-4400 1997 [Refereed]
     
    Glucose phosphate isomerase (GPI)-IAA and GPI-1BB homozygotes were produced by natural mating of CD-1 strain mice and the several reproductive characteristics of both homozygotes were compared. The pregnancy rate, period required for copulation and litter size were essentially the same. The developmental ability of GPI-1AA zygotes and 2-cell embryos in vitro was significantly lower than that of GPI-1BB (60 vs 75% and 75 vs 98%, respectively). After preservation at 4C for 24 h, the proportion of the GPI-1 AA-2-cell embryos developed to blastocysts was significantly low (28 vs 57%). After transfer of GPI-1AA embryos to recipient mice, the developmental potential into fetus was similar or significantly superior to that of GPI-1BB (19 vs 24% and 47 vs 14%, respectively). When both embryos were aggregated at the 8-16-cell stage and transferred to recipients, 76% of the young (13/17) was chimeras and both isozymes types were observed in most tissues analyzed. In conclusion, both GPI-1AA and GPI-1BB embryos of the CD-1 strain mice were equally useful for researches in developmental biology and biotechnology, their features being basically the same.
  • TAKANO Hiroshi; KOZAI Chiaki; SHIMIZU Satoru; KATO Yoko; TSUNODA Yukio
    Nihon Chikusan Gakkaiho 公益社団法人 日本畜産学会 67 (11) 991 - 995 1346-907X 1996/11 [Refereed]
     
    Effect of aging of oocytes on the in vitro developmental ability of bovine parthenogenetic eggs were examined. The oocytes with a first polar body were activated at 24 (young) or 44 hours (aged) after starting of in vitro maturational culture by electric stimulation, then they were incubated in the medium supplemented with cytochalasin B and cycloheximide (CY) for 6 hours. After incubation, they were cultured with cumulus cells for 8 days in vitro. Karyoplast and cytoplast were separated from young and aged oocytes, respectively. Then, 4 combinations of reconstituted oocytes were produced, activated and cultured in vitro. A blastomere from in vitro fertilized 32 to 64 stage embryos was fused with enucleated young or aged cytoplast, and reconstituted oocytes were cultured in vitro. Results obtained were as follows. (1) The activation rate of young oocytes significantly increased after CY treatment (19% vs 97%). (2) The developmental ability of aged parthenogenetic eggs was significantly low compared with that of young oocytes. (3) Experiments on the exchang of karyoplast and cytoplast between young and aged oocytes, and nuclear transfer revealed that the low developmental ability of aged parthenogenetic oocytes was due to the developmental deficiency of chromosomes and cytoplasmic factor (s).
  • HATA Masaki; OHKOSHI Katsuhiro; KATO Yoko; TSUNODA Yukio
    日本不妊学会雑誌 = Japanese journal of fertility and sterility 日本不妊学会 41 (3) 77 - 82 0029-0629 1996/07
  • Y Kato; Y Tsunoda
    THERIOGENOLOGY BUTTERWORTH-HEINEMANN 45 (5) 1029 - 1035 0093-691X 1996/04 [Refereed]
     
    Mouse fetal germ cells (FGCs) isolated or within genital ridges from male fetuses at 15.5 d post coitum were preserved at 4 degrees C for 3 similar to 15 d with TCM-199 medium supplemented with 10, 50 or 100% fetal bovine serum (FCS) and 0.1, 0.6 or 1.0 M sucrose. The viability of FGCs was assessed by the dye exclusion test with trypan blue and nuclear transfer into enucleated oocytes and then into enucleated 2-cell embryos. Forty-one to 45% of FGCs survived for 5 d after preservation in the medium containing 50% FCS and 0.1 M sucrose according to the results of the dye exclusion test. But significant good effect of sucrose was not observed regardless of its concentration. When genital ridges were preserved, 15 similar to 22% of FGCs survived even 10 d after preservation. The efficiency of the in vitro development of FGCs to blastocysts was similar when they were fused with enucleated oocytes and 2-cells blastomeres after 2 to 4 d storage (27-33%) compared with that of control FGCs (56%).
  • TAKANO Hiroshi; KOYAMA Keisuke; KOZAI Chiaki; SHIMIZU Satoru; KATO Yoko; TSUNODA Yukio
    The Japanese journal of animal reproduction THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 42 (1) 61 - 65 0916-8818 1996/02 
    In vitro development and changes in the nuclei of reconstituted oocytes following fusion were investigated to determine the effects of cell cycle stage of donor nuclei on bovine nuclear transplantation. Enucleated oocytes, cultured for 44 to 46 h following maturation or preactivated for 9 h before fusion, were used as the source of recipient cytoplasm. Donor embryos were obtained from embryos matured, fertilized and cultured in vitro. Blastomeres of the embryos which had developed to the 4-cell stage at 39 h after insemination were separated. Each blastomere was cultured with aphidicolin for 2 to 4 h. These blastomeres, shortly after the next division (G1/S stage of the cell cycle), or those cultured for 6 h without aphidicolin after division (S stage of cell cycle), were fused with non-activated (Groups 1 and 3) or activated (Groups 2 and 4) oocytes. The proportion of embryos which developed into blastocysts were higher in Groups 2 (16%) and 4 (17%) than in Groups 1 (10%) and 3 (6%). The diameter of the nuclei after fusion with the recipient cytoplasm increased, but to a lesser extent in activated than in the non-activated cytoplasm. Some nuclei which had fused with non-activated cytoplasm showed nuclear envelop breakdown and premature chromosome condensation (PCC, 21%). No donor nuclei showed PCC when fused with activated cytoplasm.
  • 継代培養細胞の核移植,
    角田幸雄; 加藤容子
    ETニュースレター 18 1 - 3 1996 [Refereed]
  • H NAKANE; S TAKEUCHI; S YUBA; M SAIJO; Y NAKATSU; H MURAI; Y NAKATSURU; T ISHIKAWA; S HIROTA; Y KITAMURA; Y KATO; Y TSUNODA; H MIYAUCHI; T HORIO; T TOKUNAGA; T MATSUNAGA; O NIKAIDO; Y NISHIMUNE; Y OKADA; K TANAKA
    NATURE MACMILLAN MAGAZINES LTD 377 (6545) 165 - 168 0028-0836 1995/09 [Refereed]
     
    XERODERMA pigmentosum (XP) is an autosomal recessive disorder characterized by a high frequency of skin cancer on sun-exposed areas, and neurological complications, XP has a defect in the early step(s) of nucleotide-excision repair (NER) and consists of eight different genetic complementation groups (groups A-G and a variant)(1). We established XPA (group-A XP) gene-deficient mice by gene targeting of mouse embryonic stem (ES) cells. The XPA-deficient mice showed neither obvious physical abnormalities nor pathological alterations, but were defective in NER and highly susceptible to ultraviolet-B- or 9,10-dimethyl-1,2-benz[a]anthracene-induced skin carcinogenesis. These findings provide is vivo evidence that the XPA protein protects mice from carcinogenesis initiated by ultraviolet or chemical carcinogen, The XPA-deficient mice may provide a good in vivo model to study the high incidence of skin carcinogenesis in group A XP patients.
  • Y KATO; Y TSUNODA
    DEVELOPMENT COMPANY OF BIOLOGISTS LTD 121 (3) 779 - 783 0950-1991 1995/03 [Refereed]
     
    Chimeric embryos between fertilized eggs from F-1 (C57BLxCBA) and 15.5-16.5 days post coitum (dpc) male fetal germ cells (FGCs) from CD-1 strain (glucose phosphate isomerase, Gpi-1a/a) mice were produced by nuclear transfer. Briefly, a single FGC was fused with enucleated oocytes and activated, and the reconstituted oocytes were cultured to the 2-cell stage. The nucleus from the reconstituted 2-cell embryos was then transferred into an enucleated blastomere of the same stage embryos derived from Fl mice to produce chimeric embryos. The reconstituted 2-cell embryos, which synchronously divided to the 4-cell stage after treatment with nocodazole, were further cultured in vitro. Compacted morula and blastocysts were transferred to the uteri of pseudopregnant female mice. Some recipients were allowed to develop to term and the others were killed at mid gestation to analyze the contribution of donor FGC-derived cells. Survival to term was low with no chimeric animals. Glucose phosphate isomerase (GPI) analysis at midgestation revealed that some conceptuses had chimerism in the fetuses, trophoblast and yolk sac at day 10.5 of pregnancy. The contribution of donor cells was 37-47%, 19-65% and 12-63%, respectively. It was concluded that the nucleus from 15.5-16.5 dpc male fetal germ cells had the potency to develop into fetus, trophoblast and yolk sac after serial nuclear transfer with oocytes and fertilized embryos. The reason for the low viability of chimeric embryos is discussed.
  • Y KATO; Y TSUNODA
    DEVELOPMENT GROWTH & DIFFERENTIATION BLACKWELL SCIENCE PUBL AUSTR 37 (1) 79 - 84 0012-1592 1995/02 [Refereed]
     
    Albino mouse embryonic cells (Gpi-la/a) at 3.5-8.5 and 11.5 days were aggregated with zona cut 8-16 cell stage embryos from F-1 females (Gpi-1b/b), respectively. The aggregated embryos were transferred to pseudopregnant female mice. The recipients were allowed to go to term or were dissected at mid-gestation to assess the donor contribution in the conceptuses using glucose phosphate isomerase (GPI) analysis. The donor cells, which were previously labeled with fluorescent latex microparticles, were aggregated with embryos, and the allocation of the donor cells at the compacted morula and blastocyst stages were observed under a fluorescence microscope. When 3.5 and 4.5 day old inner-cell-mass (ICM) cells were used, fertile chimeric mice were obtained (50 and 19%, respectively), and when 5.5 days old primitive ectoderm cells were aggregated, they did not form chimeras but contributed to the fetuses, placenta and membrane after 13.5 days of pregnancy. However, cells from further stages never contributed to the conceptuses even though they were analyzed after 10.5 days of pregnancy. The labeled donor cells at these stages were not positively incorporated in the interior part of the compacted morula and the ICM of the blastocyst stage unlike the ICM at 3.5 days post-coitum after overnight culture.
  • Kato Yoko; Tsunoda Yukio
    Nihon Seishokumeneki Gakkai Zassi Japan Society for Immunology of Reproduction 9 29 - 30 2186-6406 1995
  • Yukio Tsunoda; Yoko Kato
    Journal of Reproduction and Development 41 (1) 71 - 75 1348-4400 1995 [Refereed]
     
    To improve the developmental ability of enucleated mouse oocytes that received male fetal germ cells at 15.5 days post coitum (G0 stage), the effects of parthenogenetic activation with electrical stimulation and pretreatment of germ cells with PDGF or FGF, competent factors for quiescent cells, were examined. Preliminary evaluations revealed that the best conditions for electrical activation of unfertilized eggs were 100 μsec direct current pulses at 150 V/mm followed by 2 pulses of 50 V/mm at 20 min intervals (100% for activation and 94% for development to blastocysts). Fusion rates were the same for PDGF, FGF and control groups (35 to 46%). The activation rate in the PDGF group was significantly higher (90%) than that of controls (78%). The proportion of activated eggs that developed to morulae and blastocysts was high in the PDGF group but was not significantly different from that in controls (82 vs 68%). One live fetus with 32 somites was obtained on day 10.5 of pregnancy after the transfer of eggs reconstituted with PDGF treated germ cells. Repeated electrical parthenogenetic stimulation and pretreatment of fetal germ cells with PDGF appeared to enhance the development of reconstituted eggs. © 1995, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
  • 牛における核移植
    角田幸雄; 加藤容子
    家畜人工授精, 170 13 - 19 1995 [Refereed]
  • Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development 41 (4) 345 - 351 1348-4400 1995 [Refereed]
     
    An attempt was made to use the zygotes arrested at the M phase of the cell cycle as the recipient cytoplasm for nuclear transfer, i) Zygotes which were collected 24, 28 and 32 h after hCG injection were incubated in M16 + nocodazole (3 μl/ml) for 10 to 14 h. After elimination of the drug, they were cultured in vitro for 4 days, ii) Blastocysts were cultured with nocodazole for 5 to 12 h and the mitotic index was examined. iii)M phase and G^S phase inner cell mass (ICM) cells synchronized with nocodazole and aphidicolin treatment, and fetal germ cells isolated from fetuses on days 11.5 (in mitosis) and 15.5 (G0 phase) of pregnancy were fused with enucleated M phase zygotes. The results were as follows, i) Zygotes obtained 24 h after hCG injection and treated with nocodazole for 10 to 14 h were at the M phase. After release from the drug, they developed to blastocysts at a rate similar to that of non-treated zygotes (83~87% vs 100%). ii) Mitotic index of blastocysts treated with nocodazole for 11 to 12 h was 94%. iii) Nuclear transfer into M phase zygotes did not improve the developmental ability of reconstituted embryos into blastocysts. In the present study, it was clear that mouse zygotes arrested at the M phase with nocodazole treatment, and after washing they developed to the blastocysts at the similar rate to non-treated zygotes. But, the developmental ability of reconstituted zygotes at the M phase did not support in vitro development. © 1995, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
  • Katsuhiro Ohkoshi; Yoko Kato; Yukio Tsunoda; Hiroshi Takano; Ryohei Nitta
    Journal of Mammalian Ova Research 12 (2) 127 - 130 1347-5878 1995 [Refereed]
     
    In vitro fertilized 8-16-cell stage embryos, cocultured with cumulus cells for 117 hours, were divided into two groups. Embryos in one group (control group) were further co-cultured with cumulus cells or cultured in TCM199 supplemented with 10. Embryos in the other group were used as donor nuclei for nuclear transfer. The nuclear transferred eggs were co-cultured with cumulus cells to the 8-16-cell stage. They were divided into two half of them were further co-cultured with cumulus cells and the others were cultured in TCM199 supplemented with to the blastocyst stage as control group. The proportions of embryos which developed to blastocysts in supplemented medium in both groups were not significantly different from those obtained in the co-culture system. However, the proportions of nuclear transferred eggs which developed to blastocysts in both groups were significantly different from those obtained in in vitro fertilized eggs. © 1995, JAPANESE SOCIETY OF OVA RESEARCH. All rights reserved.
  • Y KATO; Y TSUNODA
    THERIOGENOLOGY BUTTERWORTH-HEINEMANN 41 (6) 1315 - 1322 0093-691X 1994/05 [Refereed]
     
    Different numbers of CD-1 mouse zygotes(1, 5, 10, 20, 40 and 60) were cultured in 10 mu l M16 medium, in M16 medium+EDTA, in M16 medium +SOD+thioredoxin, and in CZB medium, respectively. When the zygotes, regardless of the number, were cultured with M16, no blastocysts could be obtained. The suitable ratio of embryos to 1 mu l of M16 medium+EDTA or M16 medium+SOD+thioredoxin was 1:1 or 2:1. Medium volume from 1 to 10 mu l did not affect blastocyst development when the embryo density was 1:1. However, blastocysts obtained from zygotes cultured singly had fewer cell numbers and showed inferior development to live fetuses after transfer to recipients. When CZB medium was used, suitable embryo density was not clear. The ratio of embryos to volume of culture medium was shown to be an important factor for in vitro culture of mouse zygotes.
  • Y KATO; T OGURO; Y TSUNODA
    THERIOGENOLOGY BUTTERWORTH-HEINEMANN 41 (7) 1483 - 1488 0093-691X 1994/05 [Refereed]
     
    Experiments were conducted to test the hypothesis that the nucleo/cytoplasmic ratio of mouse embryos determines the time of blastocele formation. Half the volume of 2-cell stage embryos was removed from each blastomere by micropipette to alter the nucleo/cytoplasmic ratio. Reduced embryos chose nucleo/cytoplasmic ratio increased and non-treated control embryos were cultured in vitro to compare the timing of division to the 4-cell stage and blastocele formation. Reduced 2-cell embryos formed blastoceles significantly earlier than the controls(49.0+/-2.9 vs 52.2+/-6 h) and with fewer cells, although division into the 4-cell stage was significantly delayed(11.4+/-4.4 vs 9.0+/-2.4 h). The cell number of blastocysts 70 h after treatment and developmental ability of blastocysts after transfer to pseudopregnant recipients were the same for the reduced and control groups. The present study indicates that the nucleo/cytoplasmic ratio of embryos may possibly be an important factor that determines the time of blastocele formation.
  • Kato Y.; Tsunoda Y.
    Nihon Seishokumeneki Gakkai Zassi Japan Society for Immunology of Reproduction 8 14 - 16 2186-6406 1994
  • Hiroshi Takano; Satoru Shimizu; Keisuke Koyama; Chiaki Kozai; Yoko Kato; Yukio Tsunoda
    Journal of Reproduction and Development 40 (2) 167 - 170 1348-4400 1994 [Refereed]
     
    The present study was undertaken to examine whether the bovine nuclear transferred embryos produced by in vitro culture system could develop to young after transfer to recipients. Donor 8- to 16-cell stage embryos were obtained by in vitro maturation, in vitro fertilization and in vitro culture. Follicular oocytes maturated in vitro were used as recipient oocytes for nuclear transfer. Fusion of donor blastomere with recipient oocyte was induced by electric stimulation at 44 to 46 h of maturation. Of the 398 manipulated oocytes, 261 (66%) were fused with donor blastomere. Twenty-five (10%) embryos which developed into morula to blastocyst stage after coculturing with cumulus cells in vitro. Fifteen embryos were transferred nonsurgically, 1 to 3 embryos per recipient, into the uterine horn of 9 heifers or cows on Day 7 to 9. The pregnancy rate was 44% (4/9) on Day 35 to 40. One recipient aborted on Day 60 to 70 and another on Day 50 to 60, respectively. Two normal offsprings were produced from the remaining 2 recipients. The production of calves for embryos transferred was thus low (13%). Nuclear transfer bovine embryos produced in vitro system developed to full-term. © 1994, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
  • 核移植を用いた胚操作
    角田幸雄; 加藤容子
    実験医学 12 10 - 16 1994 [Refereed]
  • Y KATO; Y TSUNODA
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 36 (2) 276 - 278 1040-452X 1993/10 [Refereed]
  • Y TSUNODA; Y KATO
    JOURNAL OF REPRODUCTION AND FERTILITY J REPROD FERTIL INC 98 (2) 537 - 540 0022-4251 1993/07 [Refereed]
     
    The developmental ability of enucleated mouse eggs that had received embryonic stem cells was examined. In a preliminary study, none of the reconstituted eggs formed a nucleus using inactivated Sendai virus (HVJ) after activation with ethanol. DC pulses were applied at 100 or 140 V mm-1 in addition to this treatment. After electrofusion, 29-40% of reconstituted eggs formed nuclei and 41-60%, 25-44%, 12-24% and 12-18% developed to two-cell and four-cell and morulae and blastocysts, respectively. The ability of reconstituted eggs to form blastocysts did not depend on the age of recipient eggs, except in cases of very young and very old eggs. Although implantation sites were observed, no live fetuses were obtained after the transfer of reconstituted eggs to the recipients.
  • H TAKANO; K KOYAMA; C KOZAI; Y KATO; Y TSUNODA
    THERIOGENOLOGY BUTTERWORTH-HEINEMANN 39 (4) 909 - 917 0093-691X 1993/04 [Refereed]
     
    The Present study was undertaken to examine the effect of the length of in vitro maturation of oocytes on the efficiency of enucleation, parthenogenetic activation and blastomere fusion by electric stimulus. In vitro development of reconstituted oocytes receiving a blastomere from 8 to 16 cell bovine embryos fertilized in vitro was investigated to assess the effect of aging of the oocytes. The Proportion of oocytes with a first polar body at 22 to 24 hours after maturation was high (80%) compared with those obtained at 16 to 18. 28 to 30 or 42 to 44 hours (50 to 75%). The success rate of enucleation significantly decreased with aging (88,85,74 and 55%). The activation rate significantly increased with the length of maturation in vitro (P<0.01) (1 to 4, 24 to 41, 57 to 70 and 80 to 87%). The proportion of oocytes fused with a blastomere from 8 to 16 cell embryos was not dependent on the age of the oocytes (54-59%). The ability of the reconstituted oocytes to develop to the 2 cell and the 8 to 16 cell stage increased with the length of maturation of recipient oocytes. When oocytes enucleated and a blastomere at 22 to 24 hours were incubated further for 22 to 23 hours until electrofusion. The proportions of oocytes which developed to the 2 cell and the 8 to 16 cell stages (74 and 17%) were similar to those obtained at 42 to 44 hours after maturation. However, only 1 to 6% of reconstituted eggs receiving a blastomere from 8 to 16 cell embryos fertilized in vitro developed into a blastocyst in vitro.
  • 角田幸雄; 加藤容子
    遺伝 裳華房 47 (2) 29 - 33 0387-0022 1993 [Refereed]
  • 生殖系列上細胞の実験的操作
    角田幸雄; 加藤容子
    アニテックス 6 269 - 273 1993 [Refereed]
  • KATO Yoko; ODAGAKI Satoshi; TSUNODA Yukio
    Journal of Mammalian Ova Research Japanese Society of Mammalian Ova Research 10 (2) 194 - 199 0916-7625 1993 [Refereed]
     
    Effects of leukemia inhibitory factor (LIF) on the developmental ability of mouse 8-cell embryos in vitro and in vivo were examined.(1) Eight-cell embryos were cultured with M16+LIF medium in vitro to examine the development to blastocysts and the attachment to culture dish.(2) Blastocysts were transferred to recipient mice following the injection of LIF into the uterine lumen to examine the number of live youngs on Day 17. The results obtained were as follows.(1) When embryos were cultured in a medium supplemented with LIF, the proportion of embryos developed to hatched blastocysts was increased. Blastocysts cultured in the presence of LIF and 10% FCS attached to the surface of the culture dish at a significantly higher frequency than control blastocysts (42vs71-90%).(2) The proportion of embryos that developed into live young was significantly increased, when the LIF was directly injected into the uterine lumen, compared with the control group (15 vs 35%).
  • KATO Yoko; YUBA Syunsuke; TANAKA Kiyoji; TOKUNAGA Tomoyuki; TSUNODA Yukio
    Nihon Chikusan Gakkaiho Japanese Society of Animal Science 64 (11) 1115 - 1120 1346-907X 1993 [Refereed]
     
    In this study, transgenic mice for xeroderma pigmentosum (XP) disease were produced with gene transferred ES cells. The ES cell line used in the present study was established from a blastocyst with C57BL/6×CBA genotype and had euploid XY chromosomes. The methods for disruption of a single allele of XPAC gene in ES cells by gene targeting will be reported separately by YUBA et al. Four lines of gene transferred ES cells were aggregated with 4-16-cell embryos from albino CD-1 strain mice. Male chimaeras obtained following transfer of aggregated embryos to recipients were mated with CD-1 females. Offspring with pigmented color were analysed by Southern blotting to confirm the presence of the mutated allele and heterozygotes were mated each other to obtain homozygotes at the mutant locus. After aggregation of 736 embryos with gene transferred ES-cells, 706 (96%) embryos developed to morulae-blastocysts and they were transferred to 51 recipient female mice. Thirty-three females produced 117 pups (14%) including 14 chimaeras (12%). The proportions of young and chimaeras obtained were significantly decreased compared with the case of control ES-cells (24 and 47%, respectively). One germ line chimaeric male was obtained. When he was mated with CD-1 females four times, 44 young was obtained including 32 (73%) heterozygous transgenic mice. Eleven couples of heterozygotes were mated 2 to 6 times and 427 young were born. After Southern blotting analysis, 111 homozygous transgenic mice (52 males and 59 females) were confirmed. In this study, it took 16 months to establish the homozygous transgenic mice since gene transferred ES-cells were aggregated with embryos. Considering the application of this method to farm animals, it was suggested that new methods to produce young originating from ES cells directly not through chimaera should be developed.
  • 加藤容子; 角田幸雄
    日本不妊学会誌 日本不妊学会 38 (4) 600 - 604 0029-0629 1993 [Refereed]
  • NITTA Ryohei; KATO Yoko; TSUNODA Yukio
    Nihon Chikusan Gakkaiho Japanese Society of Animal Science 64 (9) 904 - 908 1346-907X 1993 [Refereed]
     
    The present study was undertaken to examine the effect of medium volume and the egg density on the development of bovine embryos fertilized in vitro. The results obtained were as follows. The proportions (13.6 to 20.0%) of eggs developed to blastocysts when 2, 5, 10 or 20 eggs were cultured in 10μl medium were significantly high compared with 10tμl medium were significantly higher than those (54.0 to 85.1) obtaind in other groups due to the faster growth rate. However, the volume of medium (5 to 100μl) did not affect the developmental ability of eggs when number of eggs per medium was the same.
  • KATO Yoko; NITTA Ryohei; TAKANO Hiroshi; TSUNODA Yukio
    Nihon Chikusan Gakkaiho Japanese Society of Animal Science 64 (5) 484 - 490 1346-907X 1993 [Refereed]
     
    Effects of timing of fusion and culture medium on the development of bovine eggs receiving blastomeres from 8-to 32-cell embryos were examined in vitro. Donor embryos were produced from immature oocytes obtained from ovaries following in-vitro maturation, fertilization and culture for 3 to 4 days. Recipient oocytes were also used after in-vitro maturation for 22 hours in media supplemented with various agents. Electrofusion (750V/cm for 50μsec, DC pulses 2 times) was performed at 30 or 40 hours from maturational culture of recipient oocytes. Reconstituted eggs were co-cultured with follicular cells in TCM-199 medium supplemented with hormones (LH, FSH, estradiol) and/or SOD + thioredoxin, or in M16 medium replaced glucose with gultamine. The results obtained were as follows. Overall fusion rate was 31 to 57%. Reconstituted eggs fused at 40 hours developed to 8-cell stage onwards at the higher rate than those of fused at 30 hours (7 to 23% VS 7 to 32%). However, supplements of hormones, SOD + thioredoxin or gultamine did not improve the developmental ability of nuclear transfer eggs.
  • Y TSUNODA; Y KATO; GT ONEILL
    JOURNAL OF REPRODUCTION AND FERTILITY J REPROD FERTIL INC 96 (1) 275 - 281 0022-4251 1992/09 [Refereed]
     
    Micromanipulation techniques were used to produce reconstituted one-cell mouse embryos after the fusion of fetal male germ cells 15.5 day post coitum with enucleated secondary oocytes. At this stage of development, male fetal germ cells are arrested at G1 of mitotic interphase. Two distinct populations of germ cells, differing i n size and ploidy, were isolated from the genital ridge of a mid-term fetus. Oocytes that had received male germ cells from the population of smaller (mononuclear) germ cells developed as diploid one-cell reconstituted embryos. When the same procedures were used to produce reconstituted one-cell embryos using male fetal germ cells from a population of larger (multinucleate) cells, they exhibited ploidy of either 4x, 6x or 8x at metaphase of the first cell division. Although most reconstituted embryos (90 and 96%) developed to the two-cell stage, the proportion of embryos receiving small germ cells developed to blastocysts was much higher (62%) than that receiving large germ cells (4%). These studies indicate that not all fetal germ cells are diploid before the onset of meiosis and have identified procedures to produce reconstituted embryos from fetal germ cells that do not carry genome or chromosome anomalies.
  • Y KATO; Y TSUNODA
    JOURNAL OF REPRODUCTION AND FERTILITY J REPROD FERTIL INC 95 (1) 39 - 43 0022-4251 1992/05 [Refereed]
     
    Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12.5-14.5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.
  • Y KATO; Y TSUNODA
    THERIOGENOLOGY BUTTERWORTH-HEINEMANN 37 (4) 769 - 778 0093-691X 1992/04 [Refereed]
     
    Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F1 (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.
  • 哺乳動物胚細胞核の全能性
    角田幸雄; 加藤容子
    実験医学 10 49 - 53 1992 [Refereed]
  • ES細胞を用いたキメラマウスの新しい作製法
    徳永智之; 加藤容子; 角田幸雄
    実験医学 10 33 - 37 1992 [Refereed]
  • KATO Yoko; OHGURO Toshi; TSUNODA Yukio
    Nihon Chikusan Gakkaiho Japanese Society of Animal Science 63 (2) 157 - 161 1346-907X 1992 [Refereed]
     
    The developmental ability of tetraploid mouse embryos (4 N embryos) produced by electrofusion at the 2-cell stage was examined in vitro and in vivo. The effect of aggregating two 4 N embryos at the 2-cell stage was also examined. The results obtained were as follows.
    Tetraploid embryos developed to blastocysts at approximately the same rate as in control embryos (76 vs 65%), but they had reduced cell numbers (30±5 vs 64±11). On 11.5 days post coitum after transfer to recipients, 79% of 4 N embryos implanted but none of them had developed to fetuses. Although aggregated 4 N embryos formed blastocysts at a high rate (85%), fetuses were not observed even on 9.5 days post coitum.
    These results suggest that the poor developmental ability of 4 N embryos was not caused by reduced cell numbers.
  • Several factors affecting the nuclear formation after nuclear transfer of male fetal germ cells into enucleated eggs in the mouse
    Kato,Y; Tsunoda,Y
    Jpn.J.Fertil.Steril., 37 375 - 379 1992 [Refereed]
  • Kato Y.; Tsunoda Y.
    Nihon Seishokumeneki Gakkai Zassi Japan Society for Immunology of Reproduction 5 114 - 117 2186-6406 1991
  • 哺乳動物の核移植
    加藤容子; 角田幸雄
    実験医学 9 222 - 226 1991 [Refereed]
  • 核移植と初期胚の全能性
    角田幸雄; 加藤容子
    臨床科学, 27 754 - 760 1991 [Refereed]
  • KATO Yoko; KAMEKI Junichi; TSUNODA Yukio
    Journal of Mammalian Ova Research Japanese Society of Mammalian Ova Research 8 (1) 1 - 8 0916-7625 1991 [Refereed]
     
    The present study was undertaken to examine the effects of culture media (M16) stored by freezing (-20°C) or freeze-drying on the development of mouse zygotes or 2-cell embryos in vitro and in vivo.
    Zygotes and 2-cell embryos were obtained from the superovulated CD-1 female mice mated with CD-1 males on 19-23 or 43-46 hrs after hCG injection, respectively. Zygotes with two pronuclei were cultured for 4 days with fresh and frozen media supplemented with 100 μM EDTA. Two-cell embryos were cultured for 3 days with fresh, frozen and freeze-drying media. Some blastocysts developed from zygotes or 2-cell embryos cultured with each media were transferred into the oviducts on day 0.5 or into the uteri on day 2.5 or 3.5 of pseudopregnant mice. They were killed on day 16.5 or 17.5 to examine the number of live fetuses. Cell numbers in both trophectoderm and inner cell mass of blastocysts on freeze-drying media were also examined.
    High proportions of zygotes and 2-cell embryos cultured with frozen and freeze-drying media developed to blastocysts in vitro (59% and 86% for frozen medium, 89% for freeze-drying medium), and were not significantly differentcompared with those obtained in fresh medium (59% and 86%).
    The proportion of live fetuses after transfer of blastocysts developed from zygotes or 2-cell embryos with frozen medium (34% and 31%) were not significantly different from those obtained with fresh medium (35% and 39%, respectively). However, only 8% of embryos cultured with freeze-drying medium developed to fetuses. But, the cell numbers in trophectoderm and inner cell mass of blastocysts were not significantly different from those obtained with fresh medium.
    The present study demonstrated that M16 medium could be preserved at least for 10 months at -20°C without the decrease of its potency for the development of mouse embryos in vitro and in vivo. However, although the reason was not clear, freeze-drying of medium resulted in a drastical decrease in the percentage of embryos developing to live fetuses after transfer to recipients.
  • 高野博; 新田良平; 加藤容子; 角田幸雄
    繁殖技術会誌 13 15 - 19 1991 [Refereed]
  • Yoko Kato; Yukio Tsunoda
    Jpn.J.Anim.Reprod THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 37 (3) 225 - 230 0385-9932 1991 [Refereed]
     
    The present study was undertaken to examine the developmental potential of mouse fetal germ cells aggregated with 816-cell embryos. Germ cells were isolated from fetal gonads of albino mice on 12.5-16.5 Day post coitum. Eight-16 cell embryos were obtained from pigmented F1 (C57BL×CBA) mice. The germ cells were aggregated with the zone free embryos or injection into perivitelline space of ones. The aggregated embryos were cultured overnight in vitro and those developed to morula to blastocyst stage were transferred to the oviducts on Day 1 or the uteri on Day 3 of pseudopregnancy. The surrogate females were allowed to go to term. The proportions of aggregated embryos developed to morulae to blastocysts were high (86100%) but the proportions of those developed to term after transfer were low in both groups (21% and 28%, respectively). None of the animals born showed contribution of the phenotypes derived from donor cells, judging from the eye and coat colors. Some of the animals obtained were mated with albino partners, but did no evidence in support of the formation of germ line chimaerism has so far been obtained.
  • Yoko Kato; Kyoko Sugiyama; Yukio Tsunoda
    Jpn.J.Anim.Reprod. THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 36 (4) 235 - 239 0385-9932 1990 [Refereed]
     
    Primordial germ cells (PGCs) are the only cells which can differentiate into spermatozoa or oocytes after gametogenesis. They aquire totipotency when they are fertilized, but it is not clear that whether isolated PGCs from somatic cells have totipotency or pluripotency in mammals. The present study was undertaken to obtain the fundamental data on some characters of mouse PGCs for following studies of examination of their developmental potency using aggregation methods and nuclear transplantation. The results obtained are as follows.
    1) The morphology of genital ridges changed from an oblong to round form according to the days of pregnancy since the rate of major axis against minor one was gradually closer to 1. 2) The fact that the PGCs isolated from 12.5 days male (before meiosis) and 15.5 female (during meiosis) gonads showed the strong activity for alkaline phosphatase indicated that this marker did not decline in activity after the onset of meiosis. 3) The viability of PGCs preserved for 1 to 5 hrs at the room temperature in the drop of PBI supplemented with 10% FCS did not different from that of PGCs freshly collected.
  • Y TSUNODA; Y KATO; Y SHIODA
    GAMETE RESEARCH WILEY-LISS 17 (1) 15 - 20 0148-7280 1987/05 [Refereed]
  • クローン個体の作出からみた「核移植と核のリプログラミング
    加藤容子
    MedicalBio (9月) [Refereed]

Conference Activities & Talks

  • p53 遺伝子がもたらすエピブラスト様細胞への誘導機構  [Not invited]
    青木雪来; 加藤容子; 岡村大治
    第３９回日本受精着床学会  2021/07
  • 家畜繁殖学研究の最先端 ～基礎から臨床へ～  [Invited]
    加藤容子
    第39回日本受精着床学会  2021/07
  • GV期核置換卵の生存性向上に関わる要因  [Not invited]
    真柄怜央; 加藤容子
    第６２回日本卵子学会  2021/05
  • マウスMII期卵の凍結融解後の体外発生能に及ぼす添加物の影響  [Not invited]
    山本魁音; 加藤容子
    第６２回日本卵子学会  2021/05
  • Cloning as a Tool in the Production and Propagation of Genetically Superior Livestock  [Invited]
    Yoko Kato
    4th International Livestock Biotechnology Symposium  2019/07
  • 死亡マウス個体から回収した卵子のクオリティ調査  [Not invited]
    藤井颯; 加藤容子
    第６０回日本卵子学会  2019/05
  • Production of reconstructed oocytes and its application on genetic conservation  [Invited]
    Yoko Kato
    JAAP joint continuing education series and JSPS bridge fellowship symposium  2018/12
  • 卵子が誘導する体細胞核の全能性  [Invited]
    Yoko Kato
    第３３回日本生殖免疫学会  2018/11
  • ＧＶ置換法を用いた希少動物再生に関する基礎的研究  [Not invited]
    藤井颯; 中田雄大; 加藤容子
    第５９回日本卵子学会  2018/05
  • KSRの添加がブタ体細胞核移植卵の体外発生能に及ぼす影響  [Not invited]
    矢野未来; 加藤容子
    第５９回日本卵子学会  2018/05
  • 過剰排卵誘起方法の違いがマウス体細胞核移植卵の発生能に及ぼす影響  [Not invited]
    山下輝; 加藤容子
    第５９回日本卵子学会  2018/05
  • 体細胞クローンマウス卵子のDNAメチレーションエラー  [Not invited]
    吉岡 匠; 神長 祐子; 大畠 一輝; 加藤 容子; 小池 佐; 小林 久人; 河野 友宏
    第109回日本繁殖生物学会  2016/09
  • Evaluation of the impact of pre-exposure to PHA on developmental efficiency of porcine PA and SCNT embryos  [Not invited]
    第121回日本畜産学会  2016/03
  • マウス個体の老化が生殖能力に及ぼす影響ならびに個体老化の緩和に関する予備的検討  [Not invited]
    山口 正義; 谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  2015/09
  • ブタ精子の前処理がICSI卵の体外発生能に及ぼす影響  [Not invited]
    秦 仁樹; 谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  2015/09
  • Oct3/4，Nanog遺伝子の発現を指標としたマウス体細胞核移植胚の移植前選別の試み  [Not invited]
    大畠一輝; 谷哲弥; 加藤容子
    第108回日本繁殖生物学会  2015  宮崎
  • マウス体細胞核移植胚の発生能と胚盤胞期におけるNanogの発現様式との関連性  [Not invited]
    大畠一輝; 谷哲弥; 加藤容子
    第５６回日本卵子学会  2015  宇都宮
  • マウス体細胞核移植卵の第一卵割における不等分裂改善の試み  [Not invited]
    大畠 一輝; 谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  2014/08
  • 体外加齢ブタ未受精卵の生物学的特性と体外発生能の検討  [Not invited]
    谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  2014/08
  • 核移植研究の動向と課題  [Invited]
    加藤容子
    第５５回日本卵子学会  2014/05
  • マウス体細胞核移植胚の2細胞期における割球サイズの差異が発生能に及ぼす影響  [Not invited]
    大畠 一輝; 谷 哲弥; 加藤 容子
    Journal of Mammalian Ova Research  2014/04
  • 融解後のMG132処理がウシ凍結-融解未受精卵の体外発生能に及ぼす影響  [Not invited]
    清水 聡一郎; 谷 哲弥; 加藤 容子
    Journal of Mammalian Ova Research  2014/04
  • 核移植研究の動向と課題  [Not invited]
    加藤 容子
    Journal of Mammalian Ova Research  2014/04
  • 過剰排卵処置の有無がマウス体細胞核移植卵の発生能に及ぼす影響  [Not invited]
    松下 淳; 谷 哲弥; 加藤 容子
    日本畜産学会大会講演要旨集  2014/03
  • マウス体細胞核移植卵の第一卵割における不等分裂改善の試み  [Not invited]
    大畠一輝; 谷哲弥; 加藤容子
    第107回日本繁殖生物学会  2014  帯広
  • 体外加齢ブタ未受精卵の生物学的特性と体外発生能の検討  [Not invited]
    谷哲弥; 加藤容子
    第107回日本繁殖生物学会  2014  帯広
  • マウス体細胞核移植胚の2細胞期における割球サイズの差異が発生能に及ぼす影響  [Not invited]
    大畠一輝; 谷哲弥; 加藤容子
    第５５回日本卵子学会  2014  神戸
  • 融解後のMG132処理がウシ凍結-融解未受精卵の体外発生能に及ぼす影響  [Not invited]
    清水聡一郎; 谷 哲弥; 加藤容子
    第５５回日本卵子学会  2014  神戸
  • 受胚雌へのPlacental Lactogen(PL)投与がマウスクローン胚の体内発生能に及ぼす影響  [Not invited]
    松下 淳; 加藤 容子
    The Journal of Reproduction and Development  2013/08
  • ブタ未受精卵の体外加齢抑制法の最適化  [Not invited]
    谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  2013/08
  • マウス体細胞核移植胚の移植前選別の試み  [Not invited]
    大畠 一輝; 加藤 容子
    The Journal of Reproduction and Development  2013/08
  • マウスにおける体内受精卵および体細胞核移植胚由来一卵性双子胚の遺伝子発現  [Not invited]
    大畠 一輝; 加藤 容子
    Journal of Mammalian Ova Research  2013/04
  • 受胚雌へのプロラクチンの投与がマウス体細胞核移植卵の体内発生能に及ぼす影響  [Not invited]
    松下 淳; 加藤 容子
    Journal of Mammalian Ova Research  2013/04
  • マウス体細胞核移植由来2細胞期分離胚の培養条件の検討  [Not invited]
    大畠 一輝; 加藤 容子
    日本畜産学会大会講演要旨集  2013/03
  • 受胚雌へのPlacental Lactogen(PL)投与がマウスクローン胚の体内発生能に及ぼす影響  [Not invited]
    松下 淳; 加藤容子
    第106回日本繁殖生物学会  2013  府中
  • ブタ未受精卵の体外加齢抑制法の最適化  [Not invited]
    谷哲弥; 加藤容子
    第106回日本繁殖生物学会  2013  府中
  • マウス体細胞核移植胚の移植前選別の試み、第106回日本繁殖生物学会  [Not invited]
    大畠一輝; 加藤容子
    第106回日本繁殖生物学会  2013  府中
  • マウスにおける体内受精卵および体細胞核移植胚由来一卵性双子胚の遺伝子発現  [Not invited]
    大畠一輝; 加藤容子
    第５４回日本卵子学会  2013  東京
  • 受胚雌へのプロラクチンの投与がマウス体細胞核移植卵の体内発生能に及ぼす影響  [Not invited]
    松下 淳; 加藤容子
    第５４回日本卵子学会  2013  東京
  • マウス体細胞核移植由来2細胞期分離胚の透明帯への再挿入が分離胚の発生能に及ぼす影響  [Not invited]
    大畠 一輝; 角田 幸雄; 加藤 容子
    The Journal of Reproduction and Development  2012/08
  • ブタ卵子におけるMII期保存の試み  [Not invited]
    辻 暖永; 角田 幸雄; 加藤 容子
    The Journal of Reproduction and Development  2012/08
  • 核リプログラミング因子強発現卵による核リプログラミング能増強の試み  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2012/08
  • マニピュレーションと発生研究 マイクロマニピュレーション技術とクローン研究  [Not invited]
    加藤 容子; 角田 幸雄
    Journal of Mammalian Ova Research  2012/04
  • KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  2012/04
  • OHATA Kazuki; TSUNODA Yukio; KATO Yoko
    Journal of Mammalian Ova Research  2012/04
  • TSUJI Haruhisa; TSUNODA Yukio; KATO Yoko
    Journal of Mammalian Ova Research  2012/04
  • ラット体細胞核移植卵における第一卵割のタイミングとその後の体外発生能との関係性  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2011/08
  • SUGIMOTO Takaaki; TSUJI Yuta; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2011/04
  • MIZUMOTO Shigetoshi; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2011/04
  • メラトニンがブタ体細胞核移植卵の体外発生能に及ぼす影響  [Not invited]
    中野 真夕; 谷 哲弥; 加藤 容子; 福田 愛作; 森本 義晴; 角田 幸雄
    日本IVF学会誌  2010/09
  • ドナーとして用いる卵丘細胞の培養がラット体細胞核移植卵の体外発生能に及ぼす影響  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2010/08
  • 単為発生卵との共移植がマウス体細胞核移植(SCNT)胚の発生能に及ぼす影響  [Not invited]
    辻 優大; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2010/08
  • MIZUMOTO Shigetoshi; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2010/04
  • TSUJI Yuta; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2010/04
  • 受胚雌へのProgesterone投与がマウス体細胞核移植卵の発生能に及ぼす影響  [Not invited]
    辻 優大; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2009/08
  • 培養細胞を用いたラット体細胞核移植  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2009/08
  • NAKANO Mayu; TANI Tetsuya; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  2009/04
  • TSUJI Yuta; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  2009/04
  • ブタ体細胞核移植卵の体外発生能に及ぼすバブプロ酸の影響  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2009/03
  • 活性化付与のタイミングがラット体細胞核移植卵の前核形成および体外発生能に及ぼす影響  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2009/03
  • Cyclosporin Aの投与が体細胞核移植卵の体内発生能に及ぼす影響  [Not invited]
    辻 優大; 加藤 容子; 角田 幸雄
    Reproductive Immunology and Biology  2008/11
  • Cyclosporin Aの投与が受精卵移植後の胎子形成に及ぼす影響  [Not invited]
    辻 優大; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2008/08
  • ラット体細胞核移植卵における紡錘体の継時的観察と体外発生能の検討  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2008/08
  • MIZUMOTO Shigetoshi; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2008/04
  • TSUJI Yuta; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2008/04
  • ウシ未受精卵からの初期化因子の同定と体細胞核移植への利用  [Not invited]
    谷 哲弥; 島田 浩明; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2007/09
  • ラット単為発生卵におけるMG132とdemecolcineの影響  [Not invited]
    水本 茂利; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2007/09
  • 加藤 容子
    The Journal of Reproduction and Development  2007/09
  • KAWAMATA Miyako; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2007/04
  • LIU Guohui; KATO Yoko; TSUNODA Yukio
    Journal of Mammalian Ova Research  2007/04
  • ウシ体細胞核移植卵の初期化におけるMAPKの必要性  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2006/08
  • ウシ核移植卵母細胞の最初の卵割の時期(The timing of the first cleavage of bovine nuclear-transferred oocytes)  [Not invited]
    Amarnath Dasari; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2006/03
  • クローン胚盤胞中の形態学及びmRNA発現パターンに関する比較研究(Comparative studies on the morphology and mRNA expression pattern in cloned blastocysts)  [Not invited]
    Li Xiangping; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2006/03
  • ウシ体細胞核移植卵のスピンドルチェックポイント  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2005/08
  • クローン化ウシ胚盤胞における発生関連遺伝子発現の比較(Comparison of development-related gene expression in cloned bovine blastocysts)  [Not invited]
    Li Xiangping; Amarnath Dasari; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2005/03
  • 体細胞クローン個体作出研究の現状と問題点  [Not invited]
    角田 幸雄; 加藤 容子
    日本畜産学会大会講演要旨集  2005/03
  • YATSUKAWA Syogo; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  2004/04
  • KAWAKAMI Masahiro; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  2004/04
  • 核移植方法の違いがブタ体細胞核移植卵の体外発生能に及ぼす影響  [Not invited]
    河野 康二郎; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2004/03
  • Nuclear transfer in farm animals  [Not invited]
    加藤 容子; 角田 幸雄
    第18 回日本生殖免疫学会学術集会シンポジウム（山口）  2003/11  第18 回日本生殖免疫学会学術集会シンポジウム（山口）
     
    家畜の核移植に関する情報を提供した。
  • Nuclear transfer of embryonic and somatic cells  [Not invited]
    加藤 容子; 角田 幸雄
    XIX International Congress of Genetics, Symposium on Stem Cells and Development（オーストラリア）  2003/07  XIX International Congress of Genetics, Symposium on Stem Cells and Development（オーストラリア）
     
    胚細胞、体細胞の核移植について情報を提供した。
  • 再生医学のがん治療への応用 ES細胞からin vitroで蠕動運動する腸管をつくる  [Not invited]
    山田 高嗣; 久永 倫聖; 金広 裕道; 高木 都; 鳥橋 茂子; 加藤 容子; 角田 幸雄; 中島 祥介
    日本癌治療学会誌  2002/09
  • マウス ES 細胞の核移植；核移植方法、 細胞の遺伝的背景並びに細胞周期の影響  [Not invited]
    角田 幸雄; 薮内晶子; 加藤 容子
    第 100 回日本畜産学会 （東京）  2002/03  第 100 回日本畜産学会 （東京）
     
    核移植方法、 ES 細胞を樹立したマウスの遺伝的背景並びに細胞周期の違いがマウス ES 細胞由来核移植卵の体外並びに産子の発生能に及ぼす影響について検討した。
  • ブタ核移植卵の体外発生能に及ぼすドナー細胞及び核移植卵の培養気相条件の影響  [Not invited]
    角田 幸雄; 川上雅弘; 谷 哲弥; Yin Xi Jun; 加藤 容子
    第 100 回日本畜産学会 （東京）  2002/03  第 100 回日本畜産学会 （東京）
     
    培養中の酸素濃度の影響について検討した。
  • マウスES細胞の核移植;核移植方法,細胞の遺伝的背景ならびに細胞周期の影響  [Not invited]
    薮内 晶子; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2002/03
  • Effects of activation procedures and origin of somatic cells on the in vitro developmental ability of nuclear transferred porcine oocytes  [Not invited]
    角田 幸雄; Yin Xi Jun; 谷 哲弥; 川上雅弘; 加藤 容子; 米村功
    第 99 回日本畜産学会 （長野）  2001/09  第 99 回日本畜産学会 （長野）
     
    ブタ体細胞核移植卵の 10％が胎盤胞へ発生することを明らかにした。
  • Nuclear transfer of mouse ES cells and cumulus cells  [Not invited]
    角田 幸雄; 薮内晶子; 加藤 容子
    第 99 回日本畜産学会 （長野）  2001/09  第 99 回日本畜産学会 （長野）
     
    核移植卵の発生能に及ぼす再核置換ならびに集合の影響について
  • マウスES細胞ならびに排卵卵丘細胞の核移植;再核置換及び集合の影響  [Not invited]
    薮内 晶子; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2001/08
  • マウスES細胞の核移植:産子生産率が低い原因に関する一考案  [Not invited]
    天野 朋和; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2001/03
  • マウスES細胞の核移植によるクローンマウスの作出  [Not invited]
    天野 朋和; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2000/12
  • ドナー細胞及びレシピエント卵細胞質の細胞周期の組み合わせがウシ卵丘細胞由来核移植卵の体外発生能に及ぼす影響  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2000/12
  • 体外成熟ウサギ卵子をレシピエントにした体細胞の核移植  [Not invited]
    Yin Xi Jun; 谷 哲弥; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  2000/12
  • 高温処理胚盤胞への注入によるES細胞由来マウスの作出 特に4倍体胚盤胞との比較に関する検討  [Not invited]
    天野 朋和; 谷 哲弥; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2000/03
  • マウス排卵卵丘細胞由来核移植卵の体外発生能の検討  [Not invited]
    薮内 晶子; 谷 哲弥; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  2000/03
  • 体細胞クローン動物  [Not invited]
    角田 幸雄; 谷 哲弥; 加藤 容子
    The Journal of Reproduction and Development  1999/12
  • 輸送した卵子或いは卵巣由来のレシピエント卵子がウシ卵丘細胞核移植卵の体外発生能に及ぼす影響  [Not invited]
    谷 哲弥; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  1999/12
  • 高温処理胚盤胞への注入によるES細胞由来マウスの作出  [Not invited]
    天野 朋和; 谷 哲弥; 加藤 容子; 角田 幸雄
    日本畜産学会大会講演要旨集  1999/09
  • 体細胞クローン動物  [Not invited]
    角田 幸雄; 谷 哲弥; 加藤 容子
    日本内分泌学会雑誌  1999/09
  • YABUUCHI Akiko; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1999/04
  • MOTOSUGI Nami; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1999/04
  • AMANO Tomokazu; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1999/04
  • TANI Tetsuya; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1999/04
  • TSUNODA Yukio; KATO Yoko
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1999/04
  • 牛及びマウスの体細胞を用いた核移植の現状と問題点  [Not invited]
    角田 幸雄; 加藤 容子
    日本獣医学会学術集会講演要旨集  1999/03
  • 継代培養ウシ卵丘細胞の核移植,特にドナー細胞の細胞周期同調法の違いが核移植卵の発生能に及ぼす影響  [Not invited]
    谷 哲弥; 外丸 祐介; 加藤 順也; 加藤 容子; 角田 幸雄
    The Journal of Reproduction and Development  1998/12
  • SOTOMARU Yusuke; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1998/04
  • TANI Tetsuya; SOTOMARU Yusuke; KATO Jun-ya; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1998/04
  • OHKOSHI Katsuhiro; KATO Yoko; TSUNODA Yukio
    Journal of mammalian ova research = 日本哺乳動物卵子学会誌  1997/04
  • KATO Yoko; TSUNODA Yukio
    哺乳動物卵子学会誌 = Journal of Mammalian Ova Research  1995/04

MISC

Industrial Property Rights

Research Grants & Projects

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2022/04 -2026/03 
    Author : 加藤 容子
  • ブタ卵子(胚)を用いた新しいポータブル培 養システムの開発
    JSPS外国人招聘研究者：（長期）
    Date (from‐to) : 2016/04 -2017/01 
    Author : 加藤容子
  • JSPS外国人特別研究員事業：科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 2014/04 -2016/03 
    Author : 加藤容子
     
    ブタではヒトの臓器移植を目指した研究が多く、現在では畜産分野への貢献を目指した体細胞核移植研究は少ない。そのため、ウシやマウスとは異なり、核移植のドナーとして用いる細胞の種類と核移植卵の発生能とを結びつける系統立てた情報は少ない。そこで、本研究では、ブタで様々な組織から細胞を採取し適切な培養法を検討し、それらを用いて核移植を行い、発生能を検討する。これらの知見を得て、最終目的としてブタ体細胞核移植実験系の改善を目指す。H27年度に、海外特別研究員は、本人にとって全く新しい技術である、哺乳動物卵子・初期胚の取り扱い、体外成熟培養、単為発生、核移植、リアルタイムPCRによる遺伝子発現の検討などを順調に習得したので、H2７年度はブタ体細胞核移植の体外発生能向上を目指した研究を実施した。すなわち、体細胞核移植卵の活性化方法の検討、ドナー細胞核の除核レシピエント卵への導入方法の検討、ドナー細胞種の影響；特に幹細胞の有用性の検討、培養方法を検討した。その結果、活性化方法、ドナー核の導入方法では、検討したいずれの方法でも、胚盤胞への発生率、胚盤胞の細胞数に大差はみられなかった。また、ドナー細胞としてブタiPS細胞を用いても、体外発生能や細胞数は、体細胞を用いた場合と比較して大差が見られなかったことから、幹細胞であっても卵子細胞質内で生じる初期化が強く促進されるわけではないと示唆された。培養培地にPHAを添加したところ、体外発生率が増加する傾向がみられた。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2014/04 -2016/03 
    Author : KATO Yoko; TANI Tetsuya; TAGUCHI Misato
     
    In this study, we examined whether pre-implantation stage embryos in mammals could be freeze-dried like sperm and somatic cells. When mouse blastocysts were freeze-dried, trehalose supplemented medium for freeze-drying and isotonic solution for rehydration were effective for their blastocoele recovery. For porcine blastocysts, no blastocysts recovered their blastocoele after rehydration in any approach.
  • 日本学術振興会：科学研究費助成事業 特定領域研究
    Date (from‐to) : 2011 -2012 
    Author : 加藤 容子
     
    本研究では,平成23年度に引き続き,体細胞核の初期化を促進する天然物を探索し,各天然物同士の種々の組み合わせを含めながら,初期化を促進すると考えられるものを選び出すことを最終目的として実施した。すなわち,滋養強壮,不妊治療,傷の治療などに効果があるとされている生薬を中心とした約100種類の天然物の中から,平成16年度～19年度の基盤A研究(研究分担者)「初期化誘導活性を持つ天然物の探索;クローン個体の作出,未分化対細胞株樹立への応用」ならびに平成21～22年度の当該領域研究「天然物を利用した体細胞核の初期化促進に関する研究」の中で得られた知見に基づき,初期化の促進に有効と考えられるものがないかどうかを,マウス胚を用いて検討した。まず,前核期から胚盤胞までの体外培養時に,培地に添加して胚盤胞への発生能を向上させないかを検討した。また,胚盤胞期へ発生したものについては,細胞数が増加していないかを検討した。さらに,体細胞の培養時に培地に添加して,細胞増職能に効果がないかを検討した。その結果,これまでのところ,百数十種類の生薬を用いて調べたところ,7種類の生薬において,胚盤胞への発生能の向上,あるいは,胚盤胞期における細胞数の増加,また,体細胞の細胞増殖能の促進に効果のある可能性が示唆された。これらの生薬が,体細胞核移植卵を用いた場合に同様に発生能を向上させる効果があるのかどうか検討した結果,顕著な際は認められなかった。さらに,他の動物種の胚を用いた場合に,顕著な効果はみられず,現在検討中である。
  • 文科省：特定領域研究「生殖系列」
    Date (from‐to) : 2009 -2012 
    Author : 加藤容子
     
    体細胞核移植卵の発生能は,核移植時にヒストン蛋白質のアセチル化を修整する処理(TSA処理)や初期化に関与するタンパク質(TCTP)の導入等を行うことで,多少改善されるが,劇的な向上には結びつかず,正常産子生産率は依然として数パーセント程度である。そのため,体細胞核を正しく初期化させて正常に発生させるには,ピンポイントの救済術では不十分である可能性があると考えられる。 本研究では,体細胞全体,あるいは卵全体の潜在的な生命力を高めることを期待して,天然物を用いた初期化促進法の開発,ならびに,天然物による胚の質の改善と向上を試みた。本年度は下記を中心に実施した。 前年度に引き続き,受精卵を用いて初期化を促進する天然物ならびに胚の質を改善する天然物の探索を行った。また,前年度までの結果から効果が見られた天然物数種類を用いて,受精卵の体内発生能の検討ならびに,核移植卵の発生能に及ぼす影響を検討した。核移植法は,当研究室の常法(Cloning and stem Cells., 10,133,2008.など)に従い,それぞれの天然物添加培地を用いて培養した。そして,受精卵,核移植卵のいずれも,胚盤胞への発生能とそれらを受胚雌へ移植後の妊娠中期の胎子,あるいは,正常産子への発生能を検討した。核移植卵においては,胚盤胞への発生能に対照区と大差がみられなかった。受胚雌へ移植後の体内発生能も,受精卵,核移植卵いずれにおいても,劇的な向上はみられなかった。
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    Date (from‐to) : 2008 -2008 
    Author : 角田 幸雄; 加藤 容子
     
    犬や猫等の愛玩動物では外科的去勢手術が行われているが、簡便で確実な動物の受胎調節法が存在しないため、保護の進んだ野生動物、動物園動物、外来動物の一部では、個体数の増加の制御が困難な場合が有り、人間の生活権との間で葛藤が見られている。研究代表者らは、別の目的で実施してきた研究の中で、生薬であるオウレンがマウス初期胚の発生能を阻害する可能性があることを見いだした。そこで本研究では、オウレン抽出物が動物の避妊薬として使用できるか否か検討する目的で実施した。 1 オウレン抽出物が前核期受精卵の発生能に及ぼす影響;水で煮沸後、上清を凍結乾燥した後用いた。その結果、1mg/ml以上の濃度でオウレン抽出物を添加した培地でマウス前核期受精卵を培養すると、胚盤胞への発生が完全に阻害された。 2 卵子の受精能に及ぼす影響;種々の濃度で添加した培地でマウス未受精卵を1時間前培養し、受精能を獲得させた精巣上体精子液に移して体外受精を行った。その結果、受精と胚発生は正常におこり、卵子の受精能は阻害されないことが明らかとなった。 3 精子の受精能力に及ぼす影響;種々の濃度で添加した培地にマウス精巣上体精子を入れて受精能を獲得させ、ついで未受精卵を移して体外受精を行った。その結果、受精は正常におこり、オウレン抽出物は精子の受精能力を阻害しないことが判明した。 4 雌への投与の影響;過剰排卵雌に1mgの抽出物を7日間筋肉内投与後交配し、採卵して、胚盤胞への発生状況を調べた。その結果、PBS投与区と比べて胚盤胞への発生率に大差は見られなかった。しかしながら、オウレン投与雌では、回収卵数が有意に少なく、排卵抑制効果あるいは生体内における受精卵発生能阻害または流出効果がある可能性が示唆された。 オウレン抽出物の主要成分は、塩化ベルベリンとされている。そこで今後、市販の塩化ベルベリンを用いて、受精卵の発生能阻害作用と避妊効果が有るか否か調べる予定である。
  • 日本学術振興会：科学研究費助成事業 萌芽研究
    Date (from‐to) : 2006 -2007 
    Author : 角田 幸雄; 加藤 容子
     
    研究代表者らは、未受精卵細胞質中に、核の初期化誘導に関わる蛋白質(リン酸化TCTP)を同定し、リン酸化TCTPペプチドを導入した体細胞を核移植に用いると、クローンウシが高率に得られる事を明らかにした。そこで、マウス核移植実験系を用いてリン酸化TCTPの持つ初期化誘導能を確認する事を目的に実施した。これまでは、接着細胞であるウシ培養細胞へ不活化センダイウイルスを用いる方法(GenomeOne)でペプチドを導入し、核移植に用いてきた。しかしながら、マウス体細胞の核移植で確実に産子の得られる細胞は、浮遊細胞である卵丘細胞や短期間培養する卵胞上皮細胞に限られている。これらの細胞へGenomeOneを用いてペプチド導入を行うと、細胞融合が高率に生じた事から、前年度はペプチドの効率的導入法を検討してきた。本年度は、これらの手法を用いて、リン酸化TCTPペプチドの導入がマウス体細胞核移植卵の発生能に及ぼす影響を検討した。卵胞上皮細胞ヘリン酸化ペプチドを導入し、除核未受精卵細胞質に直接注入法を用いて核移植した。ついで、トリコスタチンA (TSA)添加培地で前培養し、TSA添加活性化培地で6時間培養して活性化後、体外で4日間培養して胚盤胞へ発生させた。つぎに、胚盤胞を受胚雌に移植して胎子への発生率を調べた。なお、対照区として、非リン酸化ペプチド導入体細胞を用いた。その結果、胚盤胞への発生率は、実験区49% (176/358)、対照区57% (104/183)と有意差が見られなかった。また、妊娠10.5日目における生存胎子の割合も両区で大差は見られなかった。リン酸化ペプチド導入を導入した体細胞におけるOct4蛋白質の発現の有無を調べたが、検出する事は出来なかった。本実験いた事による可能性が考えられた。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2004 -2007 
    Author : TSUNODA Yukio; KATO Yoko
     
    The present study was undertaken to examine natural products which have potential to reprogram somatic cells by using two procedures. 1. Screening of natural products by TG somatic cell lines; By using cell lines established from Oct4-GFP transgenic mice fetuses, natural products whose extracts have potential to induce Oct4 expression were examined. The water soluble extracts from natural products were freeze-dried, and after addition of each product at different concentrations to TG somatic cell lines, Oct4-GFP reaction was examined under fluorescence microscope. Of 506 products, 4 crud drugs showed positive reaction. Since the reaction was too weak, these crud drugs were extracted by methanol and the effects on the expression of Oct4-GFP in TG mouse somatic cells were examined. The positive reaction was observed in crud drugs Nos.212 and 368, but the reaction was weak and the expression of Oct4 was unclear at mRNA level. 2. Screening by preimplantation embryos; The natural products which have influences on the developmental potential or the timing of differentiation were examined. Although there were no products to enhance the developmental potential of zygotes to blastocysts, 5 crude drugs increased cell numbers of blastocysts more than 20%. Unexpectedly, 3 crude drugs had inhibitory effects on the developmental potential of zygotes even at 0.1 ug/ml. We are now examining whether such crude drugs have contraceptive reaction in animals.
  • 文科省：特定領域研究「幹細胞」
    Date (from‐to) : 2003 -2004 
    Author : 加藤容子
     
    前年度は,体細胞あるいはES細胞を除核未受精卵へ核移植後,胚盤胞への発生能を支持しなくなる卵細胞質を作り出す条件を検討した。今年度はその結果を参考に,発生を支持する能力を持つ卵細胞質と支持しない卵細胞質との間で2次元電気泳動等を行ない,両者間にタンパクレベルで差異があるかどうかを調べた。 方法:MII期未受精卵に電気刺激等による活性化刺激を付与した卵子を集め,活性化刺激を付与していない卵子との間で2次元電気泳動による比較をおこなった。 結果:両者の2次元電気泳動像を比較検討したところ,1カ所で著しい差異のあるスポットが見つかったので,スポットの差異に注目して詳細を検討した。その結果,それはヒトやマウス等で既知のタンパクであることが分かった。今後,そのタンパクの性質やそれが初期化に直接関与しているかをさらに詳細に検討する必要があると考えている。
  • 文科省：特定領域研究「発生システム」
    Date (from‐to) : 2002 -2003 
    Author : 加藤容子
     
    本研究では,体細胞核移植をより確実な技術にすること,また,核移植を介さない細胞の初期化法を確立することを最終目的として実施した。すなわち,卵子に含まれる初期化因子の実体を探るとともに,初期割球や培養細胞を様々な角度から受精卵に近付ける工夫を行なった。また,現在の体細胞核移植法では,クローン胚の発生率が低く,高頻度で死亡することから,正常に発生するための工夫を行なった。 本年度は以下の実験を行なった。ウシMII期末受精卵に電気刺激による活性化刺激を付与し,電気刺激を与えていない卵子と与えた卵子との間で,予備的に2次元電気泳動をおこない,タンパクレベルでの差異がみられるかどうかを条件設定した。その結果,20個の卵子を用いて,差異のあるスポットを検出することができた。 マウスMII期の卵子の回収率の高いタンパク採取法について検討した100個のMII期卵子の透明帯を除去し,その後,(1)界面活性剤や還元剤を含まないバッファ,(2)界面活性剤や還元剤を含むバッファ,(3)より強力な界面活性剤や還元剤を含むバッファで可溶化した上清を2次元電気泳動に供した。その結果,(1)界面活性剤や還元剤を含まないバッファで処理した場合に解析可能なスポットを複数見つけることができた。 現在は,ウシとマウスの卵子を用い,活性化を付与したものと付与しないものとの間におけるタンパクレベルの差異をより詳細に検討中である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2003 
    Author : TSUNODA Yukio; TANI Tetsuya; KATO Yoko
     
    Some of nuclear-transferred oocytes receiving mouse embryonic stem(ES) cells develop into fertile young. Although the developmental potential of nuclear-transferred oocytes that receive ES cells is high compared with those receive somatic cells, a large proportion die at various developmental stages and the rate of neonatal death is high. The present study examined the effect of 1)aggregation of nuclear-transferred oocytes to increase cell numbers, 2)genetic background of ES cells, 3)different nuclear transfer procedures, 4)cell cycle of ES cells, and 5)activation methods on cloning. The following results were obtained. 1)The low potential of nuclear-transferred oocytes with ES cells to develop into young was not due to the lower cell number of nuclear transferred-embryos. 2)The cloning efficiency with hybrid ES cell lines was not superior to that of an inbred ES cell line. 3)The potential of nuclear-transferred oocytes to develop into blastocysts after fusion by Sendai virus was high compared with that after direct injection. 4)The developmental potential of oocytes receiving ES cells at the M phase was higher than that of oocytes receiving ES cells at the G1 phase. Different activation protocols did not affect the potential to develop into blastocysts. Since the developmental potential of nuclear-transferred oocytes to live pups was still low in all group(1 to 4%), further methodological studies to increase the viability of nuclear-transferred oocytes are required.
  • 核移植を介さない新しい個体作出法の開発
    JSPS科学研究費助成事業：萌芽的研究
    Date (from‐to) : 2000 -2001 
    Author : 加藤容子
  • 体細胞を用いた新しい個体作出法に関する基礎的研究
    JSPS科学研究費助成事業：基盤研究B
    Date (from‐to) : 1999 -2001 
    Author : 加藤容子
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1997 -1998 
    Author : 加藤 容子
     
    本研究では,まず,未受精卵あるいは受精卵をレシピエント卵細胞質とした核移植を行なうことにより,「初期化因子」が存在すると考えられる細胞質のステージを特定し,ついで「初期化因子」の実体を明らかにすることを目的として予備的実験を行なった。本年度では,これまでにマウス卵子で観察された上記の現象が他の動物種においても共通であるかどうかを検討したのち,卵細胞質に存在する「初期化」因子を含む分画を単離する目的として予備的実験を試みた。 前年度までに,(1)マウス未受精卵細胞質には,ドナー細胞核を初期化して受精卵と同じような初期発生を誘起する因子が存在すること,(2)一旦受精した卵細胞質にはそのような因子が存在しないことを明らかにした。今年度は,他の動物種(ウシ)のサンプルを用いた核移植においても,マウスと同様の結果がみられるかどうかを検討した。その結果,マウスの核移植で見られた現象と同様の傾向が観察できた。すなわち,ウシ除核未受精卵をレシピエント卵細胞質として核移植すると,体細胞核を高率に初期化するが,一旦活性化刺激を付与された未受精卵では,初期化できないということが観察された。これらのことから,受精あるいは受精と同等の活性化刺激を付与することにより,卵細胞質中の「初期化」に関わる因子が消失することが推察された。また,卵細胞質中に存在すると考えられる初期化因子を単離するための予備実験として,細胞質の因子を効率的に回収する方法について,体細胞をモデルとして検討した。その結果,細胞を少量の2次蒸留水に浮遊し,極細注射針でホモゲナイズした後,低速遠心する方法により,核のみが沈殿した細胞質溶液を作出できることが明らかとなった。今後は,このような方法によって,卵細胞質溶液を採取し,初期化因子を失った卵細胞質に注入することによって,初期化作用が回復するか否かを検討する予定である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 加藤 容子
     
    胎子から取り出した生殖細胞をドナー細胞として核移植を行ったところ、120個の再再構築2細胞期胚が作出できた。それらの胚をさらに1晩培養後に分裂した胚105個を受精卵由来4〜8細胞期胚と集合したところ、90%が胚盤胞へ発育し、18匹の偽妊娠受胚雌に移植した。ついで、10.5〜14.5日目に開腹したところ、それぞれ、54%〜100%の胚が回収された。そのうち、正常に発育していると判定されたものは、56〜72%であった。GPI分析の結果、10.5日目の分析では、正常胚のうち1例の胎盤と胎膜にそれぞれドナー細胞の寄与がみとめられ、既に死滅した胚においても1例で寄与が見られた。12.5日目の分析では、2例の胎盤に寄与がみられ、死滅胚のうち3例にドナー細胞の寄与が見られた。14.5日目の検査でも、胎盤、胎膜にそれぞれ1例ずつ、ドナーの寄与がみられた。しかしながら、移植後分娩させたマウスにキメラは含まれていなかった。11.5日齢の生殖細胞を用いた場合も、14.5〜16.5日齢の細胞を用いた場合と同様に、受胎産物にキメリズムをみることができた。しかしながら、胎子への寄与はみられず、胚体外組織にのみその寄与がみられた。また、死滅した受胎産物で高率に寄与していたことから、14.5〜16.5日齢の細胞の場合と同様に、ドナー由来細胞が発育の過程で何らかの理由により脱落していったかあるいは、発育を阻害していることが示唆された。また、本実験では用いた日齢のドナー細胞の遺伝情報を完全に「初期化」することができなかったことが明らかであり、今後、ドナー細胞を「初期化」する方法の改善や、この「初期化」現象の機構について解明する必要があると考えられる。

Teaching Experience

  • Focus on microinjection and advanced imaging (summer school 2014, June9-14)Focus on microinjection and advanced imaging (summer school 2014, June9-14) CEITEC(Czech Republic)
  • Basic Life ScienceBasic Life Science KINDAI Univ.
  • Biotechnology in mammalsBiotechnology in mammals KINDAI Univ.
  • Experimental animalsExperimental animals KINDAI Univ.
  • EmbryologyEmbryology KINDAI Univ.

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