Updated on 2021/10/22

写真a

 
WAKAI Takuya
 
Organization
Environmental and Life Science Associate Professor
Position
Associate Professor
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Degree

  • 博士(農学) ( 東北大学 )

Research Interests

  • Mitochondria

  • Oocyte

  • Calcium signaling

  • Embryo development

  • Fertilization

  • Organelle

  • Endoplasmic Reticulum

Research Areas

  • Life Science / Animal production science

  • Life Science / Animal production science

  • Life Science / Obstetrics and gynecology

  • Life Science / Developmental biology

  • Life Science / Applied molecular and cellular biology

  • Life Science / Cell biology

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Research History

  • Okayama University   環境生命科学研究科   Associate Professor

    2014.12

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  • Tokyo University of Agriculture   Faculty of Applied Bio-Science

    2011.4 - 2014.11

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  • マサチューセッツ州立大学アムハースト校   博士研究員

    2007.7 - 2011.3

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Professional Memberships

  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

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  • JAPAN SOCIETY FOR OVA RESEARCH

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  • SOCIETY FOR REPRODUCTION AND DEVELOPMENT

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Papers

  • Ca<sup>2+</sup> Signaling and Homeostasis in Mammalian Oocytes and Eggs. Reviewed

    Wakai T, Mehregan A, Fissore RA

    Cold Spring Harbor perspectives in biology   2019.8

  • Removal of cumulus cells around 20 h after the start of in vitro maturation improves the meiotic competence of porcine oocytes via reduction in cAMP and cGMP levels. Reviewed

    Ferré-Pujol P, Nguyen XK, Nagahara T, Bui TTM, Wakai T, Funahashi H

    The Journal of reproduction and development   65 ( 2 )   177 - 182   2019.4

  • XY oocytes of sex-reversed females with a Sry mutation deviate from the normal developmental process beyond the mitotic stage†. Reviewed

    Sakashita A, Wakai T, Kawabata Y, Nishimura C, Sotomaru Y, Alavattam KG, Namekawa SH, Kono T

    Biology of reproduction   100 ( 3 )   697 - 710   2019.3

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    Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    <title>Abstract</title>
    The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry− mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry− females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry− PGCs was associated with aberrant β-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry− oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as “Swyer syndrome,” in which patients with an XY karyotype present as typical females and are infertile.

    DOI: 10.1093/biolre/ioy214

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  • Constitutive IP<sub>3</sub>R1-mediated Ca<sup>2+</sup> release reduces Ca<sup>2+</sup> store content and stimulates mitochondrial metabolism in mouse GV oocytes. Reviewed

    Wakai T, Fissore RA

    Journal of cell science   132 ( 3 )   2019.2

  • Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles Reviewed

    Tra M. T. Bui, Khank X. Nguyen, Asako Karata, Pilar Ferre, Minh T. Tran, Takuya Wakai, Hiroaki Funahashi

    REPRODUCTION FERTILITY AND DEVELOPMENT   29 ( 10 )   1902 - 1909   2017.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CSIRO PUBLISHING  

    The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) on the meiotic and developmental competence of porcine oocytes from small follicles (SF; 0.5-3mmdiameter). When cumulusoocyte complexes (COCs) from medium-sized follicles (MF; 3-6mm diameter) and SF were cultured for IVM, the maturation rates were significantly higher for oocytes from MF than SF. Concentrations of VEGF in the medium were significantly higher for COCs cultured from MF than SF. When COCs from SF were exposed to 200 ngmL(-1) VEGF during the first 20 h of IVM, the maturation rate improved significantly and was similar to that of oocytes derived from MF. The fertilisability of oocytes was also significantly higher than that of VEGF-free SF controls. Following parthenogenetic activation, the blastocyst formation rate improved significantly when SF COC culture was supplemented with 200 ngmL(-1) VEGF, with the rate similar to that of oocytes from MF. The results of the present study indicate that VEGF markedly improves the meiotic and developmental competence of oocytes derived from SF, especially at a concentration of 200 ngmL(-1) during the first 20 h of IVM.

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  • Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation Reviewed

    Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   63 ( 2 )   191 - 197   2017.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

    The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

    DOI: 10.1262/jrd.2016-156

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  • Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes Reviewed

    Pilar Ferre, Tra Mi Thi Bui, Takuya Wakai, Hiroaki Funahashi

    THERIOGENOLOGY   86 ( 7 )   1705 - 1710   2016.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes. (C) 2016 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2016.05.024

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  • DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion Reviewed

    Tasuku Koike, Takuya Wakai, Yuko Jincho, Akihiko Sakashita, Hisato Kobayashi, Eiji Mizutani, Sayaka Wakayama, Fumihito Miura, Takashi Ito, Tomohiro Kono

    BIOLOGY OF REPRODUCTION   94 ( 6 )   128   2016.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC STUDY REPRODUCTION  

    The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

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  • A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium Reviewed

    Sumire Ioki, Qing-Shan Wu, Osamu Takayama, Hideyuki H. Motohashi, Takuya Wakai, Hiroaki Funahashi

    THERIOGENOLOGY   85 ( 3 )   428 - 433   2016.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 mu M), higher sildenafil concentrations (25 and 250 mu M) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 mu M sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro. (c) 2016 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.theriogenology.2015.09.013

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  • Mitochondrial dynamics controlled by mitofusins define organelle positioning and movement during mouse oocyte maturation Reviewed

    Takuya Wakai, Yuichirou Harada, Kenji Miyado, Tomohiro Kono

    MOLECULAR HUMAN REPRODUCTION   20 ( 11 )   1090 - 1100   2014.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Mitochondria are abundant in fully grown mammalian oocytes with a unique spherical morphology, but the mechanisms controlling mitochondria behavior are not well understood. Here we describe for the first time the control of mitochondrial behavior in mouse oocytes by a fusion/fission mechanism. Mitofusins (Mfn1 and Mfn2) and OPA1 proteins are required for outer and inner mitochondrial membrane fusion, respectively, whereas Drp1 is the key regulator of mitochondrial fission. We show that mouse oocytes express the Mfn1, Mfn2, Opa1 and Drp1 proteins, both in immature and mature oocytes at similar levels. Overexpression of Mfn1 or Mfn2 causes marked mitochondrial aggregation, particularly in the perinuclear region during meiotic progression. Tracking of mitochondria with chromosomes or endoplasmic reticulum (ER) throughout oocyte maturation demonstrates that Mfn1 and Mfn2-promoted mitochondrial aggregation disturbs the spatiotemporal dynamic of the chromosomes and ER, respectively. Our findings suggest that organelle dynamics are co-ordinately controlled during meiotic division, and an imbalance of mitochondrial fusion/fission leads to disorganization of the organelle compartments.

    DOI: 10.1093/molehr/gau064

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  • Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth Reviewed

    Akihiko Sakashita, Hisato Kobayashi, Takuya Wakai, Yusuke Sotomaru, Kenichiro Hata, Tomohiro Kono

    GENES TO CELLS   19 ( 8 )   629 - 636   2014.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.

    DOI: 10.1111/gtc.12164

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  • Forced expression of DNA methyltransferases during oocyte growth accelerates the establishment of methylation imprints but not functional genomic imprinting Reviewed

    Satoshi Hara, Takashi Takano, Tsugunari Fujikawa, Munehiro Yamada, Takuya Wakai, Tomohiro Kono, Yayoi Obata

    HUMAN MOLECULAR GENETICS   23 ( 14 )   3853 - 3864   2014.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analyzed DNA methylation imprints. The results showed that 2- to 4-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing (ng) oocytes versus fully grown oocytes derived from wild-type mice, but the analyzed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in ng oocytes is insufficient for methylation imprints, and imprinted regions are resistant to DNMT3A/DNMT3L in ng oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1 and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn and Impact was observed in ng oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.

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  • Long exposure to mature ooplasm can alter DNA methylation at imprinted loci in non-growing oocytes but not in prospermatogonia Reviewed

    Yayoi Obata, Takuya Wakai, Satoshi Hara, Tomohiro Kono

    REPRODUCTION   147 ( 1 )   H1 - H6   2014.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BIOSCIENTIFICA LTD  

    DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However, the mechanisms underlying gamete-specific imprinting remain unclear. In this study, we investigated whether male and female gametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the niche surrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fused with enucleated fully grown oocytes and exposed to the ooplasm for 5-6 days, the DNA methylation status of the reconstituted oocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest that the imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. By contrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grown oocytes and the reconstituted oocytes were then cultured for 5-6 days, the Igf2r, Kcnq1ot1 and, unexpectedly, H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from 5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes the chromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs and that mechanisms for maintaining non-methylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.

    DOI: 10.1530/REP-13-0359

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  • Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs Reviewed

    Takuya Wakai, Nan Zhang, Peter Vangheluwe, Rafael A. Fissore

    JOURNAL OF CELL SCIENCE   126 ( 24 )   5714 - 5724   2013.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:COMPANY OF BIOLOGISTS LTD  

    Changes in the intracellular concentration of free calcium ([ca(2+)](i)) regulate diverse cellular processes including fertilization. In mammalian eggs, the [ca(2+)](i) changes induced by the sperm unfold in a pattern of periodical rises, also known as [ca(2+)](i) oscillations. The source of ca(2+) during oscillations is the endoplasmic reticulum ([ca(2+)](ER)), but it is presently unknown how [ca(2+)](ER) is regulated. Here, we show using mouse eggs that [ca(2+)](i) oscillations induced by a variety of agonists, including PLC zeta, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [ca(2+)](ER) and cause differential effects on the refilling and overall load of [ca(2+)](ER). We also found that ca(2+) influx is required to refill [ca(2+)](ER), because the loss of [ca(2+)](ER) was accelerated in medium devoid of ca(2+). Pharmacological inactivation of the function of the mitochondria and of the ca(2+)-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [ca(2+)](ER), especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [ca(2+)](i) rises, promotes initiation of oscillations and enhances refilling of [ca(2+)](ER). Collectively, our results provide novel insights on the regulation of [ca(2+)] ER oscillations, which underlie the unique ca(2+)-signalling system that activates the developmental program in mammalian eggs.

    DOI: 10.1242/jcs.136549

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  • Ca2+ influx and the store-operated Ca2+ entry pathway undergo regulation during mouse oocyte maturation Reviewed

    Banyoon Cheon, Hoi-Chang Lee, Takuya Wakai, Rafael A. Fissore

    MOLECULAR BIOLOGY OF THE CELL   24 ( 9 )   1396 - 1410   2013.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC CELL BIOLOGY  

    In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca2+ stores ([Ca2+](ER)), a process that requires Ca2+ influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca2+](ER) can regulate Ca2+ influx via store-operated Ca2+ entry (SOCE). We find that during maturation, as [Ca2+](ER) increases, Ca2+ influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE-stromal interaction molecule 1 (Stim1) and Orai1- and expression of human (h) Stim1 increases Ca2+ influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca2+ levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca2+ throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca2+ influx and SOCE are regulated during maturation and that alteration of Ca2+ homeostasis undermines maturation in mouse oocytes.

    DOI: 10.1091/mbc.E13-01-0065

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  • High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice Reviewed

    Hisato Kobayashi, Takayuki Sakurai, Fumihito Miura, Misaki Imai, Kentaro Mochiduki, Eikichi Yanagisawa, Akihiko Sakashita, Takuya Wakai, Yutaka Suzuki, Takashi Ito, Yasuhisa Matsui, Tomohiro Kono

    GENOME RESEARCH   23 ( 4 )   616 - 627   2013.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

    Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genome-wide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germline-specific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.

    DOI: 10.1101/gr.148023.112

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  • Ca2+ homeostasis and regulation of ER Ca2+ in mammalian oocytes/eggs Reviewed

    Takuya Wakai, Rafael A. Fissore

    CELL CALCIUM   53 ( 1 )   63 - 67   2013.1

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    Language:English   Publisher:CHURCHILL LIVINGSTONE  

    The activation of the developmental program in mammalian eggs relies on the initiation at the time of fertilization of repeated rises in the intracellular concentration of free calcium ([Ca2+](i)), also known as [Ca2+](i) oscillations. The ability to mount the full complement of oscillations is only achieved at the end of oocyte maturation, at the metaphase stage of meiosis II (MII). Over the last decades research has focused on addressing the mechanisms by which the sperm initiates the oscillations and identification of the channels that mediate intracellular Ca2+ release. This review will describe the up-to-date knowledge of other aspects of Ca2+ homeostasis in mouse oocytes, such as the mechanisms that transport Ca2+ out of the cytosol into the endoplasmic reticulum (ER), the Ca2+ store of the oocyte/egg, into other organelles and also those that extrude Ca2+. Evidence pointing to channels in the plasma membrane that mediate Ca2+ entry from the extracellular milieu, which is required for the persistence of the oscillations, is also discussed, along with the modifications that these mechanisms undergo during maturation. Lastly, we highlight areas where additional research is needed to obtain a better understating of the molecules and mechanisms that regulate Ca2+ homeostasis in this unique Ca2+ signaling system. (C) 2012 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.ceca.2012.11.010

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  • Molecular characteristics of horse phospholipase C zeta (PLC) Reviewed

    Kana Sato, Takuya Wakai, Yasunari Seita, Akiko Takizawa, Rafael A. Fissore, Junya Ito, Naomi Kashiwazaki

    ANIMAL SCIENCE JOURNAL   84 ( 4 )   359 - 368   2013

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    A sperm-specific phospholipase C (PLC), PLCzeta (PLC), is thought to underlie the initiation of calcium ([Ca2+]i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLC have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLC (ePLC) in mouse oocytes. ePLC was cloned from testis using RT-PCR. The expression of ePLC messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLC complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca2+]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLC into mouse oocytes showed that ePLC had the highest [Ca2+]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLC. The nuclear translocation ability of ePLC was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLC has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.

    DOI: 10.1111/asj.12044

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  • Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation Reviewed

    Takuya Wakai, Veerle Vanderheyden, Sook-Young Yoon, Banyoon Cheon, Nan Zhang, Jan B. Parys, Rafael A. Fissore

    JOURNAL OF CELLULAR PHYSIOLOGY   227 ( 2 )   705 - 717   2012.2

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    At the time of fertilization, an increase in the intracellular Ca2+ concentration ([Ca2+]i) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP3R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca2+ release. The sensitivity of IP3R1, that is, its Ca2+ releasing capability, is increased during oocyte maturation so that the optimum [Ca2+]i response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP3R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca2+ concentration ([Ca2+]ER). Here, we evaluated using mouse oocytes how each of these factors affected IP3R1 sensitivity. The capacity for IP3-induced Ca2+ release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP3R1 sensitivity was underpinned by an increase in [Ca2+]ER and receptor phosphorylation(s) but not by changes in IP3R1 cellular distribution, as inhibition of the former factors reduced Ca2+ release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca2+]ER and IP3R1 phosphorylation during maturation enhance IP3R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP3R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca2+ homeostasis also shape the pattern of oscillations in mammalian eggs. J. Cell. Physiol. (C) 2011 Wiley Periodicals, Inc.

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  • Caffeine Alleviates the Deterioration of Ca2+ Release Mechanisms and Fragmentation of In Vitro-Aged Mouse Eggs Reviewed

    Nan Zhang, Takuya Wakai, Rafael A. Fissore

    MOLECULAR REPRODUCTION AND DEVELOPMENT   78 ( 9 )   684 - 701   2011.9

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    The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca2+](i)) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca2+ release is not well known. Here, we investigated if the function of IP(3)R1, the major Ca2+ release channel at fertilization, was undermined in in vitro-aged mouse eggs. Wefound that in aged eggs, IP(3)R1 displayed reduced function as many of the changes acquired during maturation that enhance IP(3)R1 Ca2+ conductivity, such as phosphorylation, receptor reorganization and increased Ca2+ store content ([Ca2+] ER), were lost with increasing postovulatory time. IP(3)R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP(3)R1 function and maintained [Ca2+] ER content. Caffeine also maintained mitochondrial membrane potential, as measured by JC-1 fluorescence. We therefore conclude that [Ca2+] i responses in aged eggs are undermined by reduced IP(3)R1 sensitivity, decreased [Ca2+] ER, and compromised mitochondrial function, and that addition of caffeine ameliorates most of these agingassociated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs. Mol. Reprod. Dev. 78: 684-701, 2011. (C) 2011 Wiley-Liss, Inc.

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  • Ca2+ Signaling During Mammalian Fertilization: Requirements, Players, and Adaptations Reviewed

    Takuya Wakai, Veerle Vanderheyden, Rafael A. Fissore

    COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY   3 ( 4 )   2011.4

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    Changes in the intracellular concentration of calcium ([Ca2+](i)) represent a vital signaling mechanism enabling communication among cells and between cells and the environment. The initiation of embryo development depends on a [Ca2+](i) increase(s) in the egg, which is generally induced during fertilization. The [Ca2+](i) increase signals egg activation, which is the first stage in embryo development, and that consist of biochemical and structural changes that transform eggs into zygotes. The spatiotemporal patterns of [Ca2+](i) at fertilization show variability, most likely reflecting adaptations to fertilizing conditions and to the duration of embryonic cell cycles. In mammals, the focus of this review, the fertilization [Ca2+](i) signal displays unique properties in that it is initiated after gamete fusion by release of a sperm-derived factor and by periodic and extended [Ca2+](i) responses. Here, we will discuss the events of egg activation regulated by increases in [Ca2+](i), the possible downstream targets that effect these egg activation events, and the property and identity of molecules both in sperm and eggs that underpin the initiation and persistence of the [Ca2+](i) responses in these species.

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  • Anomalous Oxygen Consumption in Porcine Somatic Cell Nuclear Transfer Embryos Reviewed

    Satoshi Sugimura, Masaki Yokoo, Ken-ichi Yamanaka, Manabu Kawahara, Satoru Moriyasu, Takuya Wakai, Takashi Nagai, Hiroyuki Abe, Eimei Sato

    CELLULAR REPROGRAMMING   12 ( 4 )   463 - 474   2010.8

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    Oxygen consumption reflects overall metabolic activity of mammalian embryos. We measured oxygen consumption in individual porcine somatic cell nuclear transfer (SCNT) and in vitro-fertilized (IVF) embryos by modified scanning electrochemical microscopy. Oxygen consumption in IVF embryos rapidly increased at day 5 of the blastocyst stage (D5BL). IVF embryos that consumed &gt;0.81 x 10(14)/mol sec(-1) of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence.

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  • Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes Reviewed

    Junya Ito, Tomoko Yoshida, Yasushi Kasai, Takuya Wakai, Jan B. Parys, Rafael A. Fissore, Naomi Kashiwazaki

    ANIMAL SCIENCE JOURNAL   81 ( 1 )   34 - 41   2010

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    During fertilization in mammalian species, a sperm-induced intracellular Ca(2+) signal ([Ca(2+)](i)) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP(3)R1), the channel responsible for Ca(2+) release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP(3)R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP(3)R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP(3)R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34(cdc2) kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP(3)R1 phosphorylation, although inactivation of p34(cdc2) kinase by roscovitine dramatically reduced IP(3)R1 phosphorylation. Neither inhibitor affected total expression of IP(3)R1. Altogether, our results show that IP(3)R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.

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  • Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos Reviewed

    Satoshi Sugimura, Ken-ichi Yamanaka, Manabu Kawahara, Takuya Wakai, Masaki Yokoo, Eimei Sato

    ANIMAL SCIENCE JOURNAL   81 ( 1 )   48 - 57   2010

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    We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.

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  • Acetylation Level of Histone H3 in Early Embryonic Stages Affects Subsequent Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos Reviewed

    Ken-ichi Yamanaka, Satoshi Sugimura, Takuya Wakai, Manabu Kawahara, Eimei Sato

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 6 )   638 - 644   2009.12

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    Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into art embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of historic on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 +/- 2.7 and 56.6 +/- 2.7 vs. 43.5 +/- 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitro-fertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs.

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  • Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source Reviewed

    Tatsuo Kawarasaki, Kazuhiko Uchiyama, Atsushi Hirao, Sadahiro Azuma, Masayoshi Otake, Masatoshi Shibata, Seiko Tsuchiya, Shin Enosawa, Koichi Takeuchi, Kenjiro Konno, Yoji Hakamata, Hiroyuki Yoshino, Takuya Wakai, Shigeo Ookawara, Hozumi Tanaka, Eiji Kobayashi, Takashi Murakami

    JOURNAL OF BIOMEDICAL OPTICS   14 ( 5 )   054017   2009.9

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    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation. (C) 2009 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3241985]

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  • Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation Reviewed

    Veerle Vanderheyden, Takuya Wakai, Geert Bultynck, Humbert De Smedt, Jan B. Parys, Rafael A. Fissore

    CELL CALCIUM   46 ( 1 )   56 - 64   2009.7

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    Egg activation and further embryo development require a sperm-induced intracellular Ca(2+) signal at the time of fertilization. Prior to fertilization, the egg&apos;s Ca(2+) machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca(2+) releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1), which is responsible for most of this Ca(2+) release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP(3)R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP(3)R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T(2656) as a major Plk1 site on IP(3)R1. We therefore propose that the initial increase in IP(3)R1 sensitivity during oocyte maturation is underpinned by IP(3)R1 phosphorylation at an MPM-2 epitope(s). (C) 2009 Elsevier Ltd. All rights reserved.

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  • Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs Reviewed

    Ken-ichi Yamanaka, Satoshi Sugimura, Takuya Wakai, Manabu Kawahara, Eimei Sato

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 3 )   299 - 304   2009.6

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    We evaluated the developmental competence of somatic cell nuclear transfer (SCNT) embryos using in vitro embryo culture systems. Embryos were cultured in NCSU-23, NCSU-23 supplemented with essential and non-essential amino acids (NCSU-23aa) or modified PZM-5 supplemented with BSA instead of PVA (mPZM-5). The rates of blastocyst formation were significantly higher in the mPZM-5 group than in the other groups, regardless of the method of embryo production (38.0 vs. 25.3 or 29.1% for IVF, 18.2 vs. 8.7 or 9.40% for SCNT, respectively). The mean cell numbers of IVF and SCNT blastocysts were also significantly higher in mPZM-5 than in the other groups (62.0 vs. 42.3 or 43.0 for IVF, 46.5 vs. 29.4 or 31.3 for SCNT, respectively). Next, the embryos were cultured in mPZM-5 from days 0 to 4 and then in mPZM-5 (P/P), NCSU-23 (P/N) or NCSU-23aa (P/Naa) until day 6. The rates of blastocyst formation were similar among the 3 two-step culture systems in both embryo groups (36.2, 34.2, and 33.6% for IVF, 20.8, 14.1, and 17.2% for SCNT, respectively). The mean cell number in the IVF and SCNT blastocysts was significantly lower in P/N than in P/P and P/Naa (46.5 vs. 63.5 and 68.7 for IVF, 29.3 vs. 45.5 and 39.7 for SCNT, respectively). Next, we examined the effect of media on apoptosis in IVF and SCNT blastocysts. The apoptosis indices in the blastocysts derived from either NCSU-23 or mPZM-5 were analyzed by TUNEL assay. The apoptosis index of the SCNT blastocysts was significantly lower in mPZM-5 than in NCSU-23 (8.8 vs. 13.6%), whereas no such difference was observed between groups in the IVF embryos (51 vs. 4.4%). These data suggested that SCNT embryos were more easily affected by culture environment compared with IVF embryos, offering the possibility to further enhance the developmental competence of SCNT embryos by developing more appropriate culture conditions in pigs.

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  • Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries Reviewed

    Takuya Wakai, Satoshi Sugimura, Ken-Ichi Yamanaka, Manabu Kawahara, Hiroshi Sasada, Hozumi Tanaka, Asako Ando, Eiji Kobayashi, Eimei Sato

    CLONING AND STEM CELLS   10 ( 2 )   249 - 261   2008.6

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    For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.

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  • Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs. Reviewed

    Sugimura S, Kawahara M, Wakai T, Yamanaka K, Sasada H, Sato E

    Zygote (Cambridge, England)   16 ( 2 )   153 - 159   2008.5

  • Induction of estrus in pubertal miniature gilts Reviewed

    Takuya Wakai, Hozumi Tanaka, Ken-ichi Yamanaka, Satoshi Sugimura, Hiroshi Sasada, Manabu Kawahara, Eiji Kobayashi, Eimei Sato

    ANIMAL REPRODUCTION SCIENCE   103 ( 1-2 )   193 - 198   2008.1

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    Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs. (C) 2007 Elsevier B.V. All rights reserved.

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  • Effect of activation treatments on actin filament distribution and in vitro development of miniature pig somatic cell nuclear transfer embryos Reviewed

    Ken-ichi Yamanaka, Satoshi Sugimura, Takuya Wakai, Takehisa Shoji, Jin Kobayashi, Hiroshi Sasada, Eimei Sato

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   53 ( 4 )   791 - 800   2007.8

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    In the present study, we investigated the effect of activation treatments on the actin filament distribution and in vitro development of somatic cell nuclear transfer (SCNT) embryos in miniature pigs. We combined three activation methods, ionomycin (ION), electrical stimulation (ES), and cycloheximide treatment (CH), to prepare seven activation treatments (ION, ES, CH, ION + CH, ION + ES, ES + CH and ION + ES + CH). First, we investigated the activation rate of oocytes and in vitro development of parthenotes. The activation rates of the oocytes in the ION, ES, CH, ION + CH, ION + ES, ES + CH, and ION + ES + CH groups were 42.9, 51.3, 0.0, 82.1, 80.6, 78.1 and 78.6%, respectively, showing that the rates of the combined treatment groups were significantly higher (P&lt;0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P&lt;0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P&lt;0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P&lt;0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P&lt;0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.

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  • Recent patents of TGF-beta family and VEGF associated with ovarian follicular development in mammals. Reviewed

    Shimizu T, Abe Y, Wakai T, Hoshino Y, Miyamoto A, Sato E

    Recent patents on DNA & gene sequences   1 ( 3 )   195 - 199   2007

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  • Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer Reviewed

    M Kawahara, T Wakai, KI Yamanaka, J Kobayashi, S Sugimura, T Shimizu, H Matsumoto, JH Kim, H Sasada, E Sato

    REPRODUCTION   130 ( 3 )   351 - 357   2005.9

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    When the nucleus in G0/G1 phase is transferred to an enucleated oocyte by nuclear transfer (NT), its nuclear envelope is broken, followed by condensation of chromosome structure by maturation promoting factor (MPF). This morphological remodeling of the transferred interphase nucleus seems to be essential for subsequent development of NT embryos. in this study, we treated porcine NT embryos with caffeine, which has been reported to increase MPF activity, to keep their MPF level high during NIT. When 2.5 mM caffeine was added to the handling medium, the proportion of NT embryos showing condensed chromosome increased significantly (P &lt; 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P &lt; 0.05) higher than in those without caffeine at 3 h post-injection. in addition, the rate of development to the blastocyst stage after activation was significantly (P &lt; 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.

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  • Trim‐Away法を用いたマウス卵母細胞におけるDrp1の機能解析

    月向はるな, 野村瑠莉, 舟橋弘晃, 若井拓哉

    Journal of Mammalian Ova Research   36 ( 1 )   S44 - S44   2019.4

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  • 着床前胚発生におけるミトコンドリア分裂因子Drp1の機能解析

    野村瑠莉, 月向はるな, 舟橋弘晃, 若井拓哉

    Journal of Mammalian Ova Research   36 ( 1 )   S43 - S43   2019.4

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  • 体外成熟時のブタ卵母細胞のオートファジー能は由来する小中卵胞で異なる

    小野千由貴, 若井拓哉, 舟橋弘晃

    Journal of Mammalian Ova Research   36 ( 1 )   S56 - S56   2019.4

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  • Dendra2蛍光タンパク質を用いた初期胚ミトコンドリアの細胞間分配の解析

    若井拓哉, 野村瑠璃

    Journal of Reproduction and Development   64 ( Suppl Japanese Issue )   j105 - j105   2018.9

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    &lt;p&gt;【目的】細胞分裂時,ミトコンドリアは二つに娘細胞に大まかに均等分配される。最近,哺乳動物幹細胞の細胞分裂時に,新しくできたミトコンドリアが幹細胞側へ積極的に分配されることが報告され,ミトコンドリアの非対称的な分配が幹細胞性の維持や細胞分化に重要であることが示唆されている。本研究では,初期胚の細胞間でミトコンドリアの分配に非対称性が存在するか検証するために,任意の卵割段階の特定の細胞においてミトコンドリアを標識し,胚盤胞期までの発生過程でそのミトコンドリアの追跡を試みた。【方法】ミトコンドリアターゲットシグナルを付加したDendra2(Dendra2-mt)をコードするcDNAから&lt;i&gt;In vitro&lt;/i&gt;転写によりRNAを合成し,前核期のマウス受精卵の細胞質にマイクロインジェクションを行った。ミトコンドリアでの緑色蛍光を確認後,4細胞期の単一の割球に405 nmダイオードレーザーの励起光を照射し,緑色から赤色への蛍光変換を誘起した。体外胚培養を継続し,桑実胚期および胚盤胞期に赤色蛍光を示す細胞の分布を観察した。【結果および考察】Dendra2-mtを発現させた初期胚の単一割球へ励起光を照射し,特定の細胞においてミトコンドリアを標識することが可能であることが示された。照射による発生能の低下は見られなかったが,幾つかの胚では胚全体が赤色蛍光を示すものが存在したことから,励起条件は更なる検討が必要である。4細胞期で標識したミトコンドリアを追跡した結果,桑実胚期では31%,胚盤胞期では36%の細胞に寄与することが分かった。また,胚盤胞期の内部細胞塊と栄養外胚葉で明確な寄与の差は認められなかった。&lt;/p&gt;

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  • 初期胚におけるミトコンドリア分裂因子Drp1の役割

    野村瑠莉, 若井拓哉, 舟橋弘晃

    日本卵子学会誌   3 ( 1 )   S58 - S58   2018.4

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  • ブタ顕微授精卵での精子頭部膨化・雄性前核形成不全の解析

    林雄平, 若井拓哉, 舟橋弘晃

    日本卵子学会誌   3 ( 1 )   S45 - S45   2018.4

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  • 受精後のDNAメチル化リプログラミングにおける種間差異

    澤田友季乃, 舟橋弘晃, 若井拓哉

    岡山実験動物研究会報   ( 34 )   76‐77 - 77   2018.4

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  • Epidermal Growth Factorはヒト初期卵胞の体外発育を促進する

    高山修, 奥平裕一, 若井拓哉, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本卵子学会誌   2 ( 1 )   S58 - S58   2017.4

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  • ウシ精巣からの精原幹細胞採取の試み

    永原知樹, 若井拓哉, 舟橋弘晃

    岡山実験動物研究会報   ( 33 )   50 - 50   2017.4

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  • ブタ卵母細胞中のミトコンドリア細胞内分布の変化

    葛原大貴, 若井拓哉, 舟橋弘晃

    岡山実験動物研究会報   ( 33 )   50‐51 - 51   2017.4

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  • 異なる還元剤による精子前処理がICSIブタ卵での雄性前核形成に及ぼす影響

    林雄平, 若井拓哉, 舟橋弘晃

    日本畜産学会大会講演要旨   122nd   156 - 156   2017.3

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  • Mitochondrial dynamics in mammalian oocytes

    若井拓哉

    岡山大学農学部学術報告(Web)   106   39‐42 (WEB ONLY)   2017.2

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  • Mitochondrial dynamics in mammalian oocytes

    若井 拓哉

    岡山大学農学部学術報告   ( 106 )   39 - 42   2017.2

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    Subcellular distribution of mitochondria are reorganized during development of the oocyte into afertilizable egg. In growing oocytes, mitochondria are heterogeneously distributed in the cytoplasm, whereas they diffuse throughout the cytoplasm in fully-grown oocytes. GFP-labeled mitochondria demonstrate that oocyte maturation involves dynamic redistribution of the mitochondria, whose process is associated with microtubule organization. These spatio-temporal regulations of mitochondria in oocytes may be an important process in the preparation for fertilization and subsequent embryonic development.

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  • 減数分裂と卵割におけるミトコンドリアの細胞間分配

    若井拓哉

    日本卵子学会誌   1 ( 1 )   S5   2016.4

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  • マウス卵子におけるミトコンドリア

    若井 拓哉

    岡山実験動物研究会報   ( 32 )   56 - 57   2016.4

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  • 精子・卵子研究から生殖補助医療への新たな展開 減数分裂と卵割におけるミトコンドリアの細胞間分配

    若井 拓哉

    日本卵子学会誌   1 ( 1 )   S5 - S5   2016.4

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  • マウス卵子におけるミトコンドリアの特性

    若井拓哉

    岡山実験動物研究会報   ( 32 )   9‐11 - 11   2016.4

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  • IVM中のブタ小中卵胞由来COC,裸化卵母細胞,卵丘細胞内のcAMPおよびcGMP量

    奥平裕一, 若井拓哉, 舟橋弘晃

    Journal of Reproduction and Development   61 ( Suppl Japanese Issue )   J91 - j91   2015.9

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    【目的】体外成熟 (IVM)中の細胞内cAMPとcGMP量の劇的な変化は,卵母細胞の減数分裂再開に大きく関与している。本研究は,卵丘細胞-卵母細胞複合体 (COC),裸化卵母細胞 (DO)および卵丘細胞塊 (CC)内のcAMPとcGMP量のIVM中の変動を,小卵胞 (SF:直径1–3 mm)および中卵胞 (MF:3–6 mm)間で比較した。【方法】SFまたはMFからCOCsを採取し,24時間IVMを行った(eCG,hCG,dibutyryl cAMP添加mPOM中で20時間培養,その後4時間はそれらを不含のmPOM中で培養)。IVM培養開始0,10,20,24時間に,COCsを回収し,COC,DO,CCそれぞれのcAMPとcGMP量をELISA法で測定した。また,IVM培養開始0, 10, 20時間に,COCあたりの卵丘細胞数を測定し,卵丘細胞あたりのcAMPとcGMP量を推定した。【結果】IVM中のSFおよびMF由来DOのcAMPとcGMP量に差は無かった。MF由来のCOCとCCのcAMP量は,IVM培養開始後20時間にSF由来のそれらより有意に高かった (P &lt; 0.05)。また,IVM培養開始後20時間のSF由来CCのcAMP量は,IVM培養開始後10時間のそれより,有意に低下した (P &lt; 0.01)。MF由来のCOCとCCのcGMP量は,IVM培養開始後0および10時間にSF由来のそれらより有意に高かった (P &lt; 0.05)。IVM中のMF由来のCOC中の卵丘細胞数は,SF由来のそれより有意に多かった (P &lt; 0.01)。IVM培養開始後10時間のSF由来卵丘細胞の推定cAMP量は,MF由来のそれより有意に高かった (P &lt; 0.01)。IVM培養開始0時間のMF由来卵丘細胞の推定cGMP量は,SF由来のそれより有意に高かった (P &lt; 0.01)。以上の結果から,IVM時のSFおよびMF由来COC中の卵丘細胞内cAMPとcGMP量の変動パターンは大きく異なっていることが明らかになった。

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  • ヒト小卵胞および中卵胞由来卵丘細胞‐卵母細胞複合体の体外成熟能

    奥平裕一, 高山修, BUI Thi Tra Mi, FERRE Pilar, 本橋秀之, 若井拓哉, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

    日本IVF学会雑誌   18 ( 2 )   81 - 81   2015.9

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  • Moesin遺伝子発現の可視化による体細胞クローン胚におけるX染色体リプログラミングの評価

    若井拓哉, 森園倫成, 渡辺大士, 外丸祐介, 神田暁史, 塩沢誠司, 河野友宏

    Journal of Reproduction and Development   60 ( Suppl Japanese Issue )   J142 - j142   2014.8

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  • 体細胞クローンマウス精子のDNAメチレーションエラー

    河野友宏, 若井拓哉, 水谷英二, 小林久人, 若山清香, 坂下陽彦, 三浦史仁, 伊藤隆司

    Journal of Reproduction and Development   60 ( Suppl Japanese Issue )   J75 - j75   2014.8

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  • マウス初期胚における5hmCおよびH3K9me2の挙動とDNA脱メチル化制御機構

    坂下陽彦, 中島芽生, 若井拓哉, 小林久人, 井関陽介, 河野友宏

    Journal of Mammalian Ova Research   31 ( 2 )   S78 - S78   2014.4

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  • 哺乳類卵子における小胞体Ca<sup>2+</sup>オシレーションの制御

    若井拓哉, 河野友宏, FISSORE Rafael

    Journal of Mammalian Ova Research   31 ( 2 )   S32   2014.4

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  • 体細胞クローンマウス精子のDNAメチル化リプログラミング

    若井拓哉, 水谷英二, 小林久人, 若山清香, 坂下陽彦, 伊藤隆司, 三浦史仁, 河野友宏

    日本分子生物学会年会プログラム・要旨集(Web)   37th   3W8-6(3P-0172) (WEB ONLY)   2014

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  • マウス生殖細胞・初期胚のDNAメチローム解析

    小林久人, 坂下陽彦, 若井拓哉, 小池佐, 佐野賢, 河野友宏

    日本分子生物学会年会プログラム・要旨集(Web)   37th   2W6-5(2P-0005) (WEB ONLY)   2014

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  • マウス卵母細胞におけるATP濃度の可視化と測定

    若井拓哉, 今村博臣, 河野友宏

    Journal of Reproduction and Development   59 ( Suppl Japanese Issue )   J140 - j140   2013.8

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    DOI: 10.14882/jrds.106.0.P-76.0

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  • マウス卵子形成過程における5ヒドロキシメチル化シトシンのダイナミクス

    坂下陽彦, 小林久人, 井関陽介, 若井拓哉, 外丸祐介, 河野友宏

    Journal of Reproduction and Development   59 ( Suppl Japanese Issue )   J77 - j77   2013.8

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    DOI: 10.14882/jrds.106.0.OR1-33.0

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  • マウス卵母細胞の成熟過程におけるミトコンドリア動態

    若井拓哉, 河野友宏

    Journal of Mammalian Ova Research   30 ( 2 )   S87 - S87   2013.4

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  • 着床前マウスセルトリクローン胚におけるEIF1Aのタンパク発現解析(EIF1A Expression in Sertoli Cell Nuclear Transfer Embryos during Preimplantation Development)

    曹 峰, 若井 拓哉, 渡辺 大士, 河野 友宏

    Journal of Mammalian Ova Research   30 ( 1 )   41 - 45   2013.4

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    これまでの我々の網羅的なトランスクリプトーム解析は、体細胞核移植胚(SCNT胚)における細胞核の再プログラム機構を理解するために重要な情報を提供した。その一つとして、セルトリ細胞核移植胚(SeCNT胚)の着床前発生段階では、Eif1a遺伝子発現が低く抑えられていることが明らかにされた。しかしながら、EIF1Aタンパク質の発現レベルは明らかにされていない。そこで本研究では、我々は免疫染色とウェスタンブロット法によって着床前までの胚発生各段階におけるEIF1Aタンパク質の発現レベルを解析した。その結果EIF1Aタンパク質は卵核胞期(GV期)卵子、成熟卵子(MII期卵子)および着床前各時期の胚において常に一定量発現していることが明らかにした。重要なことに、EIF1Aの局在と発現レベルは、SeCNT胚と体外受精胚でほぼ同様で、母性由来のEIF1Aが着床前のSeCNT胚発生全体を通じて存在することが明らとなった。これらの結果により、Eif1aの遺伝子発現抑制はSeCNT胚の発生能に直接関与していないことが示唆された。(著者抄録)

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  • EIF1A expression in sertoli cell nuclear transfer embryos during preimplantation development

    Feng Cao, Takuya Wakai, Hiroshi Watanabe, Tomohiro Kono

    Journal of Mammalian Ova Research   30 ( 1 )   41 - 45   2013

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    Global transcriptome studies have provided important information advancing our understanding of nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos. The expression of the Eif1a gene is down-regulated in mouse Sertoli cell nuclear transfer (SeCNT) embryos during the preimplantation stages, however its protein expression level remains unclear. Here we observed the protein expression level of EIF1A by immunofluorescence and western blot analysis during the preimplantation stages in mouse SeCNT embryos. The results reveal that EIF1A protein is constantly expressed in germinal-vesicle and metaphase II oocytes and preimplantation stage embryos. Importantly, the localization and expression level of EIF1A were similar in control in vitro fertilized embryos and SeCNT embryos. Thus, since maternally derived EIF1A persisted throughout preimplantation development, repression of Eif1a is not likely to be involved in the developmental potency of mouse SeCNT embryos. © 2013 Japanese Society of Ova Research.

    DOI: 10.1274/jmor.30.41

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  • マウス卵子形成過程における5‐hydroxymethylcytosineのダイナミクス

    坂下陽彦, 小林久人, 井関陽介, 若井拓哉, 外丸祐介, 河野友宏

    日本分子生物学会年会プログラム・要旨集(Web)   36th   2P-0679 (WEB ONLY)   2013

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  • Mitochondrial Localization in Mouse Oocytes

    WAKAI Takuya

    Journal of mammalian ova research   29 ( 4 )   155 - 160   2012.10

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    卵母細胞が受精可能な卵子へと発育する過程でミトコンドリアの細胞内局在は大きく変動する。そこで,その局在の詳細を明らかにするために成長および成熟過程の卵母細胞におけるミトコンドリアの生細胞観察を行った。成長期の卵母細胞におけるミトコンドリアは細胞膜直下および核周辺に特に集中しているのに対して,成長を完了した卵母細胞では細胞質全体に拡散して分布しており,同一の組胞周期にある卵母細胞においても成長段階に応じてミトコンドリアの細胞内局在は異なることが分かった。また,成熟過程における卵母細胞のミトコンドリアをGFPでラベルし経時的に追跡したところ,局在のダイナミックな再構築が観察され,さらにそれは微小管の影響を受けていることが分かった。こうしたミトコンドリアの時間・空間的な制御は受精やその後の発生を支持するために必要と考えられる。

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  • 【卵子のエネルギー代謝-ミトコンドリア機能について-】マウス卵母細胞におけるミトコンドリアの細胞内局在

    若井 拓哉

    Journal of Mammalian Ova Research   29 ( 4 )   155 - 160   2012.10

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    卵母細胞が受精可能な卵子へと発育する過程でミトコンドリアの細胞内局在は大きく変動する。そこで、その局在の詳細を明らかにするために成長および成熟過程の卵母細胞におけるミトコンドリアの生細胞観察を行った。成長期の卵母細胞におけるミトコンドリアは細胞膜直下および核周辺に特に集中しているのに対して、成長を完了した卵母細胞では細胞質全体に拡散して分布しており、同一の細胞周期にある卵母細胞においても成長段階に応じてミトコンドリアの細胞内局在は異なることが分かった。また、成熟過程における卵母細胞のミトコンドリアをGFPでラベルし経時的に追跡したところ、局在のダイナミックな再構築が観察され、さらにそれは微小管の影響を受けていることが分かった。こうしたミトコンドリアの時間・空間的な制御は受精やその後の発生を支持するために必要と考えられる。(著者抄録)

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  • マウス始原生殖細胞におけるDNAメチローム解析

    小林久人, 櫻井隆順, 三浦史仁, 今井美咲, 望月研太郎, 柳澤永吉, 坂下陽彦, 若井拓哉, 鈴木穣, 伊藤隆司, 松居靖久, 河野友宏

    Journal of Reproduction and Development   58 ( Suppl Japanese Issue )   J102 - j102   2012.8

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    【目的】DNAメチル化は哺乳類生殖細胞分化の過程でダイナミックに変化するエピゲノム修飾の一つであり、各配偶子におけるゲノムインプリント確立・外来性リピート配列の抑制などの重要な役割を担う。また、生殖細胞の最終形態である精子・卵子ではまったく異なるメチル化パターンを示す。しかし、そのようなメチル化の性差が生じる各領域のメチル化・脱メチル化のターゲティング機構は明らかにされていない。我々は雌雄始原生殖細胞の包括的なDNAメチル化マップ(DNAメチローム)を作製し、生殖細胞形成過程におけるDNAメチロームの性差を詳細にプロファイリングした。【方法】胎齢10.5-16.5日齢Oct4-GFPマウス胚からFACS法によるソーティングによりマウス始原生殖細胞を雌雄それぞれ2000-4000細胞回収した。微量サンプルからの調整が可能となるPost-Bisulfite Adaptor Tagging(PBAT)法により、DNAメチローム解析用のDNAライブラリーを作製し、HiSeq2000(Illumina)を用いて高速シークエンスを行った。【結果】解析した胎齢を通して雌雄生殖細胞間の全体的なCpGメチル化の性差がみられた。胎齢10.5日から13.5日にかけて、X染色体上のCpGアイランドならびにインプリント制御領域(ICR)のメチル化の消去とともにゲノムワイドな脱メチル化がみられる一方、CpG-richな一部のレトロトランスポゾンにおいて高度なメチル化が残っていることが明らかとなった。また、16.5日齢において父方ICRのメチル化の上昇がみられるが、母方ICRの一部も、雌雄始原生殖細胞間のメチル化差異領域として同定することができた。本発表では生殖細胞におけるDNAメチル化の性差についてより詳細に解説し、機能的性差との関わりについて議論する。

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  • Maternal RNF12 inhibits developmental reprogramming of somatic cell in mice

    FUKUDA Atsushi, WAKAI Takuya, WATANABE Hiroshi, KONO Tomohiro

    The Journal of Reproduction and Development Supplement   105 ( 0 )   j92 - j92   2012.8

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    【目的】マウス体細胞クローン胚は胚盤胞期以降急激に発生能が低下する。これまでに,単一クローン胚盤胞からのトランスクリプトーム解析から,&lt;i&gt;Xist&lt;/i&gt;遺伝子の発現異常によるX染色体不活性化(XCI)が全能性獲得の障害となることが明らかとなった。体細胞核のリプログラミングは核内局在性の母性因子が重要な役割を果たすことが知られている。そこで,本研究では量依存的Xist活性化因子である母性RNF12に着目し,体細胞由来の&lt;i&gt;Xist&lt;/i&gt;活性化及び,全能性へのリプログラミングに及ぼす影響を検証した。【方法】核移植のレシピエント卵子にはB6D2F1系統マウス(8-12週齢),ドナー細胞にはB6CBF1系統(新生仔)のセルトリ細胞をそれぞれ用いた。母性RNF12の発現抑制にはsiRNAを用いたノックダウン(KD)法を用いた。母性RNF12抑制卵子より,セルトリ細胞クローン胚(mRnf12KD SeCNTs)を作出した。KSOM培地で胚盤胞期まで体外培養し,定量PCR法を用いた遺伝子発現解析を行った。免疫染色解析(H3K27me3)でXCIの状態を評価した。クローン胚の一部は子宮へ移植し,体内発生能を調べた。【結果】mRnf12KD SeCNTs胚盤胞期における&lt;i&gt;Xist&lt;/i&gt;発現量は,コントロール区の6倍以上発現抑制された(P&lt;0.01)。さらに,胚盤胞期におけるH3K27me3陽性細胞の割合も,mRnf12KD SeCNTsでは有意に減少した(37.2%vs7.9%)。また,母性Rnf12が60%以上存在する卵子を用いた場合,&lt;i&gt;Xist&lt;/i&gt;発現量に有意な減少はみられなかった。移植試験の結果,コントロール区では2.2%(2/93)が至ったのに対し,mRnf12KD SeCNTsでは8.7%(9/103)が産仔に至り,有意に発生能が向上した(P&lt;0.05)。以上の結果より,SeCNTsにおけるドナー細胞核の&lt;i&gt;Xist&lt;/i&gt;活性化は母性RNF12依存的であり,母性RNF12は全能性へのリプログラミングを阻害する因子であることが明らかとなった。

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  • マウス卵子の減数分裂におけるH3K27me3の伝達機構

    若井拓哉, 河野友宏

    Journal of Mammalian Ova Research   29 ( 2 )   S55 - S55   2012.4

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  • マウス卵母細胞の成熟過程におけるミトコンドリアの動態

    若井拓哉, 河野友宏

    日本分子生物学会年会プログラム・要旨集(Web)   35th   4P-0399 (WEB ONLY)   2012

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  • Mitochondrial localization in mouse oocytes

    Takuya Wakai

    Journal of Mammalian Ova Research   29 ( 4 )   155 - 160   2012

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    Intracellular distribution of mitochondria varies during development of the oocyte into a fertilizable egg. Here we demonstrate a change in the distribution of mitochondria during oocyte growth and maturation in living mouse oocytes. In growing oocytes, mitochondria concentrated into subcortical and perinuclear areas, whereas it diffusely distributed throughout the cytoplasm in fully-grown oocytes, indicating the variation of mitochondrial distribution in the same G2/M cell-cycle stages. GFP-labeled mitochondria reveal that oocyte maturation involves dynamic reorganization of the mitochondria, and whose process is associated with microtubule organization. These spatio-temporal regulations of mitochondria in oocytes may be important process in the preparation of fertilization and subsequent embryonic development.

    DOI: 10.1274/jmor.29.155

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  • マウス始原生殖細胞におけるDNAメチローム解析

    小林久人, 櫻井隆順, 三浦史仁, 今井美咲, 望月研太郎, 柳澤永吉, 坂下陽彦, 若井拓哉, 鈴木穣, 伊藤隆司, 松居靖久, 河野友宏

    日本分子生物学会年会プログラム・要旨集(Web)   35th   1W1I-5 (WEB ONLY)   2012

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  • Mitochondrial Localization in Mouse Oocytes

    Wakai Takuya

    Journal of Mammalian Ova Research   29 ( 4 )   155 - 160   2012

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    Intracellular distribution of mitochondria varies during development of the oocyte into a fertilizable egg. Here we demonstrate a change in the distribution of mitochondria during oocyte growth and maturation in living mouse oocytes. In growing oocytes, mitochondria concentrated into subcortical and perinuclear areas, whereas it diffusely distributed throughout the cytoplasm in fully-grown oocytes, indicating the variation of mitochondrial distribution in the same G2/M cell-cycle stages. GFP-labeled mitochondria reveal that oocyte maturation involves dynamic reorganization of the mitochondria, and whose process is associated with microtubule organization. These spatio-temporal regulations of mitochondria in oocytes may be important process in the preparation of fertilization and subsequent embryonic development.&lt;br&gt;

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  • University of Massachusetts Amherst

    WAKAI Takuya, Fissore Rafael

    The Journal of Reproduction and Development Supplement   104 ( 0 )   215 - 215   2011.8

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    Ca&lt;SUP&gt;2+&lt;/SUP&gt; is the universal signal for the egg activation and initiation of embryo development in all species studied to date. The sarcoendoplasmic reticulum Ca&lt;SUP&gt;2+&lt;/SUP&gt; ATPase (SERCA), which is located in the endoplasmic reticulum (ER) membrane, imports Ca&lt;SUP&gt;2+&lt;/SUP&gt; into the lumen of the ER. Despite the pivotal role of SERCA in Ca&lt;SUP&gt;2+&lt;/SUP&gt; homeostasis in the cell, its function has not been investigated in mammalian oocytes. Herein we demonstrate that SERCA2b plays crucial roles in an increase in the ER Ca&lt;SUP&gt;2+&lt;/SUP&gt; stores during oocyte maturation and subsequent Ca&lt;SUP&gt;2+&lt;/SUP&gt; oscillations after egg activation. The inhibition of SERCA by cyclopiazonic acid (CPA), a specific inhibitor of SERCA, prevented the accumulation of Ca&lt;SUP&gt;2+&lt;/SUP&gt; into ER during oocyte maturation. Expression of GFP-tagged SERCA2b isoform showed that SERCA2b is redistributed during oocyte maturation to form an organized reticular network in mature oocytes. The overexpression of SERCA2b increased the oocyte&#039;s ER Ca&lt;SUP&gt;2+&lt;/SUP&gt; sequestering ability. Furthermore, the direct ER Ca&lt;SUP&gt;2+&lt;/SUP&gt; measurement by a FRET-based Ca&lt;SUP&gt;2+&lt;/SUP&gt; sensor reveals that SERCA is required for Ca&lt;SUP&gt;2+&lt;/SUP&gt; oscillations via the rifiling of ER Ca&lt;SUP&gt;2+&lt;/SUP&gt;. Collectively, our results suggest that the increase in ER Ca&lt;SUP&gt;2+&lt;/SUP&gt; stores via SERCA2b may underpin the robust Ca&lt;SUP&gt;2+&lt;/SUP&gt; release from the ER at fertilization. In addition, the pump&#039;s activity makes possible and the refilling of the transiently depleted Ca&lt;SUP&gt;2+&lt;/SUP&gt; stores to maintain the persistent Ca&lt;SUP&gt;2+&lt;/SUP&gt; oscillations required for the initiation of embryo development.

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  • Vitrification of immature oocytes in domestic and endangered wild animals

    佐藤英明, 若井拓哉, 星野由美

    岩谷直治記念財団研究報告書   30   1 - 2   2007.8

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  • 初期胚発生におけるミトコンドリアの機能解析

    杉村智史, 横尾正樹, 山中賢一, 青野展也, 若井拓哉, 佐々田比呂志, 阿部宏之, 佐藤英明

    日本生殖医学会雑誌   52 ( 1/2 )   62 - 62   2007.4

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  • 医療目的の遺伝子改変家畜開発の到達点と今後

    若井拓哉, 佐藤英明

    畜産の研究   61 ( 4 )   437 - 441   2007.4

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  • ミニブタ体細胞クローン個体作出成功に関わる要因解析

    杉村 智史, 山中 賢一, 若井 拓哉, 佐藤 英明

    日本胚移植学雑誌   29 ( 1 )   10 - 13   2007.1

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  • 単為発生卵との共移植による体細胞クローンミニブタの作出

    山中賢一, 杉村智史, 若井拓哉, 庄司孟央, 佐々田比呂志, 田中穂積, 小林英司, 佐藤英明

    日本畜産学会大会講演要旨   106th   86 - 86   2006.3

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  • ミニブタ体細胞クローン個体作出成功に関わる要因解析

    杉村智史, 山中賢一, 若井拓哉, 佐藤英明

    日本胚移植学雑誌   29 ( 1 )   10 - 13   2006.1

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  • ミャンマー連邦の畜産行政

    田中穂積, 若井拓哉, 金川弘司

    北海道獣医師会雑誌   49 ( 9 )   366 - 370   2005.9

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  • 自治医科大学におけるブタを用いた医学教育と研究

    田中穂積, 和久井亨, 若井拓哉, 吉野浩之, 河野正樹, 岡田真樹, 筒井真理子, 鳥取潤一, 小林英司

    日本獣医学会学術集会講演要旨集   140th   224 - 224   2005.8

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  • レシピエント卵子の体外成熟培養時間がミニブタ体細胞核の再構築およびクローンはいの発生に及ぼす影響

    杉村智史, 若井拓哉, 山中賢一, 庄司孟央, 佐々田比呂志, 佐藤英明

    Journal of Reproduction and Development   51 ( Supplement )   J104   2005.8

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  • アクチンフィラメントを指標としたミニブタ再構築はい活性化方法の最適化

    山中賢一, 杉村智史, 庄司孟央, 若井拓哉, 佐々田比呂志, 佐藤英明

    日本畜産学会大会講演要旨   105th   12   2005.8

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  • 低単位の性腺刺激ホルモンによるミニブタの発情誘起

    若井拓哉, 田中穂積, 山中賢一, 杉村智志, 筒井真理子, 鳥取潤一, 佐々田比呂志, 佐藤英明, 小林英司

    Journal of Reproduction and Development   51 ( Supplement )   J135 - j135   2005.8

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    【目的】トランスジェニックブタや体細胞クローンブタを作出するためには、ドナーブタの過剰排卵処置やレシピエントブタの発情周期の同期化などの発情周期を確実に制御することが重要である。しかしながら、ミニブタでは発情誘起に関する報告は少ない。そこで本研究ではミニブタにおける発情誘起方法を確立するために、低単位のPMSGとhCGの併用投与による発情誘起を試みた。【方法】(実験1)供試ブタは未経産のクラウン系ミニブタ(8ヵ月齢)、メキシカンへアレス4頭(5~10ヵ月齢)、中国系小型ブタ2頭(9ヵ月齢)および三元交雑ミニブタ4頭(4.5~9.5ヵ月齢)を用いた。それぞれPMSG 300 IU+hCG 150 IUを筋肉内注射し発情の発現を調べた。PMSGとhCG投与後 72時間にhCG 150 IUを筋肉内注射し、その後40から48時間に卵巣の状態を観察した。(実験2)三元交雑ミニブタ8頭(6~9.5ヵ月)を用い、処理区AではPMSG 300 IU+hCG 150 IU、処理区BではPMSG 200 IU+hCG 100 IUを投与し72時間後にそれぞれhCG 150 ,750 IUを投与した。hCG投与後43~45時間に卵管を還流し排卵数を調べた。またクラウン系ミニブタ6頭(10.5~14.5ヵ月)を用い、処理区AおよびBで誘起された発情期に雄豚と交配後、発情周期の回帰と超音波診断を指標として妊娠の成立を判定した。【結果】(実験1)正常な発情周期を持った個体でホルモン投与後3~4日にクラウン系の75%、その他の系統では100%が発情を示した。一方、性成熟前の個体では発情の発現が認められなかった。また、排卵点の数は系統間で差が見られなかった。(実験2)処理区Aでは回収した卵のうち77%が成熟卵子であったが、処理区Bでは回収した卵の全てが成熟卵子であった。また、誘起された発情期に交配したところ、処理区Aでは4頭中2頭が妊娠し、処理区Bでは1頭中1頭が妊娠した。

    DOI: 10.14882/jrds.98.0.163.0

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  • Effect of duration for in vitro maturation of recipient oocytes on nuclear remodeling and embryo development after SCNT in miniature pigs.

    Sugimura Satoshi, Wakai Takuya, Yamanaka Kenichi, Syoji Takehisa, Sasada Hiroshi, Sato Eimei

    The Journal of Reproduction and Development Supplement   98 ( 0 )   100 - 100   2005.8

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    【目的】我々は,レシピエント卵子の体外成熟培養時間および顕微操作とそれに伴う時間の経過がレシピエント卵子のMPF活性レベルを低下させる一因であることを報告した(本学会第95回大会)。そこで今回は,MPF活性レベルに左右されると考えられるドナー細胞核の再構築およびクローン胚の発生に及ぼすレシピエント卵子の体外成熟培養時間の影響を調べた。【方法】食肉処理場由来のブタ卵巣から未成熟卵母細胞を採取し,体外成熟培養した。成熟培養後36,40,44および52時間に第一極体放出が確認できた成熟卵子をレシピエント卵子とした。ドナー細胞にはミニブタ胎子線維芽細胞を用いた。ドナー細胞を卵細胞質内に注入し,注入後3時間にイオノマイシン 20分間,120v/mm 60&amp;micro;secの直流パルス5秒間隔2回,およびサイトカラシンD添加シクロヘキシミド5時間の条件で活性化しクローン胚を作製した。ドナー細胞注入後3時間で早期染色体凝集(PCC)率,免疫細胞化学的手法による紡錘体と染色体の形成様式および体外発生培養後6時間で前核様核形成率を調べた。さらに,発生培養後2日で卵割率,7日で胚盤胞期胚発生率を調べた。【結果】PCC率は成熟培養時間の経過に伴い低下する傾向を示した。培養36および44時間では多くのドナー細胞核が正常な紡錘体を形成したのに対し,培養44および52時間では異常な紡錘体を形成したものが多く観察された。前核様核形成率はすべての培養時間で有意な差は認められなかったが,培養36および40時間で2前核様核を形成したクローン胚が多かった。顕微操作後もMPF活性レベルが高度に維持されていた成熟培養36および40時間では胚盤胞期胚発生率が他の培養区に比べ高い傾向であった。以上の結果,ミニブタ体細胞クローン胚ではレシピエント卵子の高いMPF活性レベルがPCC形成に必要であり,その後のドナー細胞核の正常な核の再構築を誘導することが示唆された。

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  • アクチンフィラメントを指標としたミニブタ再構築胚活性化方法の最適化

    山中 賢一, 杉村 智史, 庄司 孟央, 若井 拓哉, 佐々田 比呂志, 佐藤 英明

    日本畜産学会大会講演要旨集   105回   12 - 12   2005.8

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  • ミャンマー連邦の畜産

    田中穂積, 若井拓哉, 金川弘司

    北海道獣医師会雑誌   49 ( 7 )   13 - 17   2005.7

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  • ブタ核移植技術の現状と応用

    若井 拓哉, 佐藤 英明, 田中 穂積, 小林 英司

    Organ Biology   11 ( 2 )   169 - 169   2004.5

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  • 実験動物としての豚の現況

    田中穂積, 若井拓哉, 吉野浩之, 椎貝正太, 小林英司

    Organ Biology   11 ( 1 )   33 - 40   2004.4

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  • カフェイン添加によるMPF活性の低下抑制およびドナー細胞の条件がミニブタ再構築はいの発生に及ぼす影響

    山中賢一, 川原学, 若井拓哉, 杉村智史, 佐々田比呂志, 佐藤英明

    日本畜産学会大会講演要旨   103rd   104   2004.3

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  • カフェイン添加によるMPF活性の低下抑制およびドナー細胞の条件がミニブタ再構築胚の発生に及ぼす影響

    山中 賢一, 川原 学, 若井 拓哉, 杉村 智史, 佐々田 比呂志, 佐藤 英明

    日本畜産学会大会講演要旨集   103回   104 - 104   2004.3

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  • 声の広場 家畜豚の実験使用によるE型肝炎ウイルス対策

    田中穂積, 吉野浩之, 若井拓哉, 袴田陽二, 椎貝正太, 長尾慶和, 岡本宏明, 小林英司

    Organ Biology   10 ( 4 )   339 - 347   2003.12

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  • 譲渡犬の実験使用中止の動向とその対応に関するアンケート調査

    椎貝正太, 長尾慶和, 吉野浩之, 若井拓哉, 袴田陽二, 田中穂積, 小林英司

    Organ Biology   10 ( 4 )   321 - 329   2003.12

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    Language:Japanese   Publisher:(一社)日本臓器保存生物医学会  

    2002年度における大学医学部附属実験施設を対象として,中型動物を用いた実験の実態について,特に譲渡犬と家畜ブタの使用状況等に関する調査を行った.無効回答1件を含む94件から回答が得られ,回収率は82%であった.イヌは全実験施設のうち83%で使用され,現在も譲渡犬を使用している施設は17%であった.ブタは56%で使用され,そのうち家畜ブタを使用している施設は48%で,ブタ使用施設の約8割が家畜ブタを使用していた.平均使用頭数は,譲渡犬の平均78.8頭に対し,家畜ブタは平均31.6頭と少なかった.家畜ブタの年間使用頭数は,1〜10頭の施設が50%を占めており,利用数の少ない施設が大半であった.家畜ブタに対する検疫で,何らかの検疫を実施していると回答したのは51%であった

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  • 中国海南省の在来豚の現況と世界の実験用豚の趨勢

    田中穂積, 王希龍, 若井拓哉, 小林英司, 金川弘司

    北海道獣医師会雑誌   47 ( 12 )   440 - 443   2003.12

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  • 家畜豚の実験使用におけるE型肝炎ウイルス対策

    田中 穂積, 吉野 浩之, 若井 拓哉, 袴田 陽二, 椎貝 正太, 長尾 慶和, 岡本 宏明, 小林 英司

    Organ Biology   10 ( 4 )   339 - 347   2003.12

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  • ピギーバック方式を用いたブタ肝移植

    山内 仁, 吉野 浩之, 井上 成一朗, 清水 尚, 藤代 準, 若井 拓哉, 金子 隆志, 高橋 将文, 袴田 陽二, 田中 穂積, 小林 英司

    今日の移植   16 ( 6 )   635 - 636   2003.11

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    Language:Japanese   Publisher:(株)日本医学館  

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  • Non-penetrating vascular closure staples (VCS) clipを用いたブタ部分小腸移植法

    吉野 浩之, 山内 仁, 井上 成一朗, 清水 尚, 藤代 準, 若井 拓哉, 金子 隆志, 高橋 将文, 袴田 陽二, 田中 穂積, 小林 英司

    今日の移植   16 ( 6 )   637 - 638   2003.11

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  • Activation of porcine reconstituted embryos by complex treatment

    FUCHINOUE Kohei, KAWAHARA Manabu, WAKAI Takuya, SASADA Hiroshi, KYONO Koichi, SATO Eimei

    Journal of mammalian ova research = 日本哺乳動物卵子学会誌   19 ( 2 )   2002.4

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