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KUROSAKA Satoshi

    Advanced Technology Research Institute 　Associate Professor
Last Updated :2024/04/25

Researcher Information

Degree

  • Ph.D.（2002/09 Kyoto University）

J-Global ID

Research Areas

  • Life sciences / Applied molecular and cellular biology
  • Life sciences / Animal production science
  • Life sciences / Developmental biology
  • Life sciences / Cell biology

Association Memberships

  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   JAPAN EMBRYO TRANSFER SOCIETY   SOCIETY FOR REPRODUCTION AND DEVELOPMENT   American Society for Cell Biology   

Published Papers

  • Akihiro Yamada; Hikaru Toya; Mayuko Tanahashi; Misuzu Kurihara; Mari Mito; Shintaro Iwasaki; Satoshi Kurosaka; Toru Takumi; Archa Fox; Yoshimi Kawamura; Kyoko Miura; Shinichi Nakagawa
    RNA (New York, N.Y.) 28 (8) 1128 - 1143 2022/08 [Refereed]
     
    Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
  • Pavan Vedula; Satoshi Kurosaka; Brittany MacTaggart; Qin Ni; Garegin Papoian; Yi Jiang; Dawei W Dong; Anna Kashina
    eLife 10 2021/06 [Refereed]
     
    β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using β-actin 3'UTR; β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.
  • Tatsuya Nakano; Mizuki Kono; Kazuki Segawa; Satoshi Kurosaka; Yoshiharu Nakaoka; Yoshiharu Morimoto; Tasuku Mitani
    The Journal of reproduction and development 67 (2) 123 - 133 2021/04 [Refereed]
     
    Methylglyoxal (MG) is a precursor for the generation of endogenous advanced glycation end-products involved in various diseases, including infertility. The present study evaluated the motility and developmental competence after in vitro fertilization of mouse sperm which were exposed to MG in the capacitation medium for 1.5 h. Sperm motility was analyzed using an SQA-V automated sperm quality analyzer. Intracellular reactive oxygen species (ROS), membrane integrity, mitochondrial membrane potential, and DNA damage were assessed using flow cytometry. The matured oocytes were inseminated with MG-exposed sperm, and subsequently, the fertilization and embryonic development in vitro were evaluated in vitro. The exposure of sperm to MG did not considerably affect the swim-up of sperm but resulted in a deteriorated sperm motility in a concentration-dependent manner, which was associated with a decreased mitochondrial activity. However, these effects was not accompanied by obvious ROS accumulation or DNA damage. Furthermore, MG diminished the fertilization rate and developmental competence, even after normal fertilization. Collectively, a short-term exposure to MG during sperm capacitation had a critical impact on sperm motility and subsequent embryonic development after fertilization. Considering that sperm would remain in vivo for up to 3 days until fertilization, our findings suggest that sperm can be affected by MG in the female reproductive organs, which may be associated with infertility.
  • Kazuo Yamagata; Kouhei Nagai; Hiroshi Miyamoto; Masayuki Anzai; Hiromi Kato; Kei Miyamoto; Satoshi Kurosaka; Rika Azuma; Igor I Kolodeznikov; Albert V Protopopov; Valerii V Plotnikov; Hisato Kobayashi; Ryouka Kawahara-Miki; Tomohiro Kono; Masao Uchida; Yasuyuki Shibata; Tetsuya Handa; Hiroshi Kimura; Yoshihiko Hosoi; Tasuku Mitani; Kazuya Matsumoto; Akira Iritani
    Scientific reports 9 (1) e4050 - 4050 2019/03 [Refereed]
     
    The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.
  • Pavlyk I; Leu NA; Vedula P; Kurosaka S; Kashina A
    Traffic (Copenhagen, Denmark) 19 (4) 263 - 272 1398-9219 2018/04 [Refereed]
     
    β-actin plays key roles in cell migration. Our previous work demonstrated that β-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β-actin arginylation in cell migration. We found that arginylated β-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated β-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.
  • Vedula P; Kurosaka S; Leu NA; Wolf YI; Shabalina SA; Wang J; Sterling S; Dong DW; Kashina A
    eLife 6 2017/12 [Refereed]
     
    β- and γ-cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here, we tested the hypothesis that β- and γ-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of β-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking β-actin protein by editing β-actin gene to encode γ-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of β-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global 'silent code' mechanism that controls the functional diversity of protein isoforms.
  • Junling Wang; Xuemei Han; Nicolae Adrian Leu; Stephanie Sterling; Satoshi Kurosaka; Marie Fina; Virginia M. Lee; Dawei W. Dong; John R. Yates; Anna Kashina
    SCIENTIFIC REPORTS NATURE PUBLISHING GROUP 7 (1) 11323  2045-2322 2017/09 [Refereed]
     
    Alpha synuclein (alpha-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that alpha-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased alpha-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in alpha-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that alpha-syn arginylation can be a previously unknown factor that facilitates normal alpha-syn folding and function in vivo.
  • Takeshi Chujo; Tomohiro Yamazaki; Tetsuya Kawaguchi; Satoshi Kurosaka; Toru Takumi; Shinichi Nakagawa; Tetsuro Hirose
    EMBO JOURNAL WILEY 36 (10) 1447 - 1462 0261-4189 2017/05 [Refereed]
     
    NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in sub-nuclear granule-like structures. The semi-extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS. This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.
  • Jason A. West; Mari Mito; Satoshi Kurosaka; Toru Takumi; Chiharu Tanegashima; Takeshi Chujo; Kaori Yanaka; Robert E. Kingston; Tetsuro Hirose; Charles Bond; Archa Fox; Shinichi Nakagawa
    JOURNAL OF CELL BIOLOGY ROCKEFELLER UNIV PRESS 214 (7) 817 - 830 0021-9525 2016/09 [Refereed]
     
    Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.
  • R. Rai; F. Zhang; K. Colavita; N. A. Leu; S. Kurosaka; A. Kumar; M. D. Birnbaum; B. Gyorffy; D. W. Dong; M. Shtutman; A. Kashina
    ONCOGENE NATURE PUBLISHING GROUP 35 (31) 4058 - 4068 0950-9232 2016/08 [Refereed]
     
    Arginylation is an emerging post-translational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1-knockout (KO) embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell-cell contacts and chromosomal aberrations. Ate1-KO fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild-type fibroblasts. Furthermore, Ate1-KO cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is downregulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1-KO results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth.
  • Tomohito Sugiura; Shuji Matsuda; Satoshi Kurosaka; Nobuhiro Nakai; Keita Fukumoto; Tetsuya Takahashi; Hirofumi Maruyama; Kazunori Imaizumi; Masayasu Matsumoto; Toru Takumi
    FEBS JOURNAL WILEY 283 (8) 1475 - 1487 1742-464X 2016/04 [Refereed]
     
    Translocated in liposarcoma/fused in sarcoma (TLS/FUS) is an RNA-binding protein that regulates the splicing pattern of mRNA transcripts and is known to cause a type of familial amyotrophic lateral sclerosis (ALS). In the absence of TLS, Mammalian enabled (Mena), an actin-regulatory protein and a target of TLS, undergoes preferential alternative splicing. In the present study, we show that the ablation of TLS dysregulates the subcellular location and functions of Mena. When TLS knockout (KO) mouse embryonic fibroblasts (MEFs) were transfected with wild-type Mena, it no longer accumulated at focal adhesions and peripheral structures, whereas the localization of the alternatively spliced form was maintained. Additionally, the ability of Mena to suppress the motility of cells was lost in TLS KO MEFs. Moreover, Mena failed to promote neurite outgrowth in TLS KO primary neurons. Taken together, TLS is intimately involved in the local cytoskeletal dynamics surrounding Mena in both fibroblasts and neurons. The robust change in cytoskeletal dynamics, as indicated by the dysregulation of Mena in TLS KO cells, provides a new insight into the pathogenesis of certain types of ALS.
  • Paula A.B. Ribeiro; Jorge P. Ribeiro; Fábio C. Minozzo; Ivan Pavlov; Nicolae A. Leu; Satoshi Kurosaka; Anna Kashina; Dilson E. Rassier
    International Journal of Cardiology 168 (4) 3564 - 3571 0167-5273 2013/10 [Refereed]
     
    Background Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. Objective The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. Methods We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. Results In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89 ± 9 nN/μm2) when compared to WT (132 ± 11 nN/μm2), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (Kact), redevelopment (Ktr), and relaxation (Krel). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209 ± 31 nN/μm2) when compared to WT (123 ± 20 nN/μm 2). Diaphragm myofibrils of α-MHCAte1 and WT presented similar Kact, but α-MHCAte1 myofibrils presented a faster K rel (6.11 ± 0.41 s- 1 vs 4.63 ± 0.41 s - 1). Conclusion Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model. © 2013 Elsevier Ireland Ltd.
  • Satoshi Kurosaka; N. Adrian Leu; Ivan Pavlov; Xuemei Han; Paula Aver Bretanha Ribeiro; Tao Xu; Ralph Bunte; Sougata Saha; Junling Wang; Anabelle Cornachione; Wilfried Mai; John R. Yates; Dilson E. Rassier; Anna Kashina
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY ELSEVIER SCI LTD 53 (3) 333 - 341 0022-2828 2012/09 [Refereed]
     
    Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by alpha-myosin heavy chain promoter (alpha MHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6 months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover. Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects. (C) 2012 Elsevier Ltd. All rights reserved.
  • Satoshi Kurosaka; N. Adrian Leu; Fangliang Zhang; Ralph Bunte; Sougata Saha; Junling Wang; Caiying Guo; Wei He; Anna Kashina
    PLOS GENETICS PUBLIC LIBRARY SCIENCE 6 (3) 1553-7404 2010/03 [Refereed]
     
    Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.
  • Kurosaka S; Leu NA; Zhang F; Bunte R; Saha S; Wang J; Guo C, He; Kashina A
    PLoS genetics 6 (3) e1000878  1553-7390 2010/03 [Refereed]
     
    Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.
  • Nicolae Adrian Leu; Satoshi Kurosaka; Anna Kashina
    PLOS ONE PUBLIC LIBRARY SCIENCE 4 (11) e7734  1932-6203 2009/11 [Refereed]
     
    Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis-capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene-a problem that has not previously been reported for this commercial mouse strain-a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.
  • Y. H. Choi; H. D. Harding; D. L. Hartman; A. D. Obermiller; S. Kurosaka; K. J. McLaughlin; K. Hinrichs
    REPRODUCTION BIOSCIENTIFICA LTD 138 (3) 589 - 599 1470-1626 2009/09 [Refereed]
     
    The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast, in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2-3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as the in vivo-produced embryos. Levels of POU5F1, SOX2, and NANOG mRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired during in vitro culture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related to in vitro culture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species. Reproduction (2009) 138 589-599
  • Timo C. Dinger; Sigrid Eckardt; Soon Won Choi; Guadelupe Camarero; Satoshi Kurosaka; Vroni Hornich; K. John Mclaughlin; Albrecht M. Mueller
    STEM CELLS WILEY 26 (6) 1474 - 1483 1066-5099 2008/06 [Refereed]
     
    Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into panneural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential.
  • Kurosaka S; Kashina A
    Birth defects research. Part C, Embryo today : reviews 84 (2) 102 - 122 1542-975X 2008/06 [Refereed]
  • Eckardt S; Dinger TC; Kurosaka S; Leu NA; Müller AM; McLaughlin KJ
    Organogenesis 4 (1) 33 - 41 1547-6278 2008/01 [Refereed]
     
    The biological role of genomic imprinting in adult tissue is central to the consideration of transplanting uniparental embryonic stem (ES) cell-derived tissues. We have recently shown that both maternal (parthenogenetic/gynogenetic) and paternal (androgenetic) uniparental ES cells can differentiate, both in vivo in chimeras and in vitro, into adult-repopulating hematopoietic stem and progenitor cells. This suggests that, at least in some tissues, the presence of two maternal or two paternal genomes does not interfere with stem cell function and tissue homeostasis in the adult. Here, we consider implications of the contribution of uniparental cells to hematopoiesis and to development of other organ systems, notably neural tissue for which consequences of genomic imprinting are associated with a known bias in development and behavioral disorders. Our findings so far indicate that there is little or no limit to the differentiation potential of uniparental ES cells outside the normal developmental paradigm. As a potentially donor MHC-matching source of tissue, uniparental transplants may provide not only a clinical resource but also a unique tool to investigate aspects of genomic imprinting in adults.
  • Annegret Wuensch; Felix A. Habermann; Satoshi Kurosaka; Regina Klose; Valeri Zakhartchenko; Horst-Dieter Reichenbach; Fred Sinowatz; K. John McLaughlin; Eckhard Wolf
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 76 (6) 983 - 991 0006-3363 2007/06 [Refereed]
     
    The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.
  • Kurosaka S; Eckardt S; Ealy AD; McLaughlin KJ
    Cloning and stem cells 9 (4) 630 - 641 1536-2302 2007 [Refereed]
  • S Eckardt; NA Leu; S Kurosaka; KJ McLaughlin
    REPRODUCTION BIO SCIENTIFICA LTD 129 (5) 547 - 556 1470-1626 2005/05 [Refereed]
     
    Mammalian somatic cell cloning requires factors specific to the oocyte for reprogramming to succeed. This does not exclude that reprogramming continues during the zygote and cleavage stages. The capacity or role of zygotic and cleavage stages to reprogram somatic cell nuclei is difficult to assess due to the limited development of somatic cell nuclei transplanted into cytoplasts of these stages. Alternatively, tetraploid embryos have been used to study reprogramming and can be assessed for their contribution to extra-embryonic lineages. When mouse cumulus cell nuclei transgenic for Oct4-green fluorescent protein (GFP) were injected into intact two- and four-cell stage blastomeres, manipulated embryos developed into blastocysts with expression of Oct4-GFP as observed in embryos produced by nuclear transfer into metaphase II oocytes. However, only the latter contributed to extra-embryonic tissues in day 10.5 conceptuses, with the exclusion of the somatic genome in cells originating from transfer into blastomeres already at 5.5 days post conception. Somatic nuclei transferred into cleavage stage blastomeres reinitiated expression of an embyronic-specific transgene, but lacked the extent of reprogramming required for contribution to post-implantation development, even when complemented by an embryonic genome.
  • S Kurosaka; S Eckardt; KJ McLaughlin
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 71 (5) 1578 - 1582 0006-3363 2004/11 [Refereed]
     
    The POU-domain transcription factor Pou5f1 (Oct4) is restricted to pluripotent embryonic cells and the germ line of the mouse and is required for the maintenance of pluripotency of cells within the inner cell mass of the mouse blastocyst. Despite highly conserved genomic organization and regulatory regions between the mouse Oct4 gene and its bovine orthologue, bovine Oct4 protein is not restricted to the inner cell mass of blastocyst-stage embryos, suggesting that Oct4 may not be a key regulator of pluripotency in the bovine. We analyze the temporal and spatial distribution of Oct4 transcript in bovine oocytes and preimplantation-stage embryos, and in contrast to protein distribution, we find strong conservation between bovine and mouse. Oct4 transcript is present at low levels in the bovine oocyte. Similar to mouse, bovine Oct4 transcription begins one to two cell cycles after zygotic genome activation, followed by a sharp increase in transcription subsequent to compaction. Oct4 transcript is ubiquitously present in all cells of embryos at the morula stage; however, in Day 7 bovine blastocysts, Oct4 signal is not visible in the trophectoderm by in situ hybridization, indicating that transcriptional downregulation of Oct4 on differentiation is similar to that observed in mouse and other mammals. These results indicate that in contrast to protein distribution, regulation of Oct4 transcription is conserved between mammalian species.
  • 黒坂哲; 今井裕
    蛋白質 核酸 酵素 47 (13) 1804 - 1809 0039-9450 2002/10 [Refereed]
  • S Kurosaka; Y Nagao; N Minami; M Yamada; H Imai
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 67 (2) 643 - 647 0006-3363 2002/08 [Refereed]
     
    The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the Go phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the GO cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.
  • Cloned animal from somatic cells and nuclear reprogramming in nuclear transferred embryos
    S. Kurosaka; H. Imai
    Japanese Journal of Fertility and Sterility 45 (3) 13 - 17 2000 [Refereed]
  • Durcova-Hills G; Tokunaga T; Kurosaka S; Yamaguchi M; Takahashi S; Imai H
    Cloning 1 (4) 217 - 224 1520-4553 1999 [Refereed]

MISC

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
    Date (from‐to) : 2022/04 -2024/03 
    Author : 黒坂 哲
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 黒坂 哲; 安齋 政幸
     
    本研究の目的は、初期胚発生における母性タンパク質分解へのN末端則経路のかかわりを明らかにすることで、全能性獲得メカニズムの解明に迫ることである。 受精直後の胚は卵細胞質に蓄積された母性タンパク質を利用して発生を進めるが、それと同時に母性タンパク質を分解することも必要であり、これは全能性獲得の過程のひとつであると考えられている。N末端則とは、タンパク質の寿命がアミノ末端(N末端)残基に依存するという法則で、真核生物で広く保存されている。特にN末端残基がアルギニンであるペプチドは急速に分解されることが知られており、その反応はアルギニル化とよばれる翻訳後修飾により促進される。このメカニズムは、短時間に劇的な変化が必要な初期胚発生や全能性獲得の特性と合致すると思われ、N末端則経路と全能性獲得のかかわりを解明することは学術的に大きな意義をもつ。 本研究で実施することは、1) N末端則経路を介して分解される母性タンパク質の同定、2) アルギニル化が起こらない胚の解析、3) 加齢卵子の質の向上の試みの3点であり、2021年度はN末端則経路を介して分解されるタンパク質の候補として特にアルギニル化されるタンパク質の同定、生殖細胞特異的にアルギニル化が起こらないマウス（ATE1-CKOマウス）の生殖能力の検定を試みた。未受精卵および初期胚においてアルギニル化されることが予想されるタンパク質の検出に成功し、これらの中には未受精卵と初期胚で共通に検出された母性効果因子も含まれており、全能性獲得メカニズムへのアルギニル化の関与が示唆される。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Kato Hiromi
     
    Basic research was conducted to avoid dysfunction of the respiratory chain complex, which is thought to occur in interspecific nuclear transplanted embryos. Porcine eggs were treated with mitomycin C to inactivate mitochondria in the egg cells during in vitro maturation culture. After in vitro maturation culture, porcine eggs were activated parthenogenetically and cultured for 48 hours. ATP contents per parthenogenetic embryo was measured. Mitomycin C-treated egg-derived embryos have 2.1 pmol ATP, while untreated egg-derived embryos have 28.3 pmol ATP, indicating that mitomycin C treatment is effective not only for mitochondrial genomic replication but also for suppression of mitochondrial function.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/04 -2021/03 
    Author : Kurosaka Satoshi
     
    Arginylation is a posttranslational modification mediated by arginyltransferase (ATE1). Arginylation is essential for the development, however, its involvement in gametogenesis is still unknown. This study was conducted to elucidate the involvement of arginylation in the female gametogenesis. Arginylated beta-actin localized to the meiotic spindle in the oocytes at metaphase II stage. We identified arginylated proteins in the oocytes at germinal vesicle and metaphase II stages, and some of them are important for female fertility or preimplantation development. These novel and important results are significant steps to elucidate the involvement of arginylatiion in female fertility and development.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : Hosoi Yoshihiko
     
    Successful production on animals by interspecies nuclear transfer (iNT) is extremely rare. In this study, we attempted to improve iNT by using germinal vesicle (GV) stage oocytes. Guinea pig fibroblasts were transferred into mouse and porcine GV oocytes, and those iNT oocytes extruded the first polar body after in vitro maturation. However, those iNT oocytes didn't have the normal-shaped spindle, indicating that they were not "true" matured oocytes. The iiNT oocytes failed to develop beyond 2-cell stage after parthenogenetic activation, and the chromosomes of iNT oocytes didn't show the developmental potential beyond 2-cell stage after transfer into normal matured oocytes. In conclusion, our novel iNT method using GV oocytes didn't improve the development of iNT embryos.

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