Faculty Profiles - WAKAI Takuya

MISC - WAKAI Takuya

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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

Language：Japanese Publisher：日本繁殖生物学会

【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，Xist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Xist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Xist発現量は，コントロール区の6倍以上発現抑制された（P<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，Xist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<0.05）。以上の結果より，SeCNTsにおけるドナー細胞核のXist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。

DOI： 10.14882/jrds.105.0_5

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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

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

Mitochondrial localization in mouse oocytes

Takuya Wakai

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

Language：Japanese

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

Scopus

J-GLOBAL

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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

Mitochondrial Localization in Mouse Oocytes

Wakai Takuya

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

Language：English Publisher：JAPANESE SOCIETY OF OVA RESEARCH

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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

Language：Japanese Publisher：日本繁殖生物学会

Ca2+ is the universal signal for the egg activation and initiation of embryo development in all species studied to date. The sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), which is located in the endoplasmic reticulum (ER) membrane, imports Ca2+ into the lumen of the ER. Despite the pivotal role of SERCA in Ca2+ 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 Ca2+ stores during oocyte maturation and subsequent Ca2+ oscillations after egg activation. The inhibition of SERCA by cyclopiazonic acid (CPA), a specific inhibitor of SERCA, prevented the accumulation of Ca2+ 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's ER Ca2+ sequestering ability. Furthermore, the direct ER Ca2+ measurement by a FRET-based Ca2+ sensor reveals that SERCA is required for Ca2+ oscillations via the rifiling of ER Ca2+. Collectively, our results suggest that the increase in ER Ca2+ stores via SERCA2b may underpin the robust Ca2+ release from the ER at fertilization. In addition, the pump's activity makes possible and the refilling of the transiently depleted Ca2+ stores to maintain the persistent Ca2+ oscillations required for the initiation of embryo development.

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Birth of somatic cell nuclear transfer(SCNT)-cloned miniature piglet following co-transfer parthenogenetic embryos

Sugimura,S, Yamanaka,K, Wakai,T, Kawahara,M, Tanaka,H, Kobayashi,J, Kobayashi,E, Sato,E

Tohoku J.Agr.Res. 59 ( 3,4 ) 51 - 61 2009

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

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

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

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

Recent progress and perspectives of the research in developming transgenic animals

Wakai,T, Sato,E

Sustainable Livestock Production and Human Welfare 61 ( 4 ) 437 - 441 2007

Evaluation of mitochondrial function in single cloned miniature pig embryos by measuring oxygen consumption

S. Sugimura, M. Yokoo, K. -I. Yamanaka, T. Wakai, H. Abe, T. Shoji, H. Sasada, J. Kobayashi, H. Abe, E. Sato

REPRODUCTION FERTILITY AND DEVELOPMENT 19 ( 1 ) 161 - 161 2007

Recent advances in understanding the morphological changes in the meiotic maturation in mammalian cumulus-oocyte complexe

Hoshino,Y, Yokoo,M, Wakai,T, Tomioka,I, Sato,E

Morphology 2 ( 1 ) 8 - 22 2007

Morphological aspects of porcine embryos resonstructed by somatic cell nuclear transfer

Wakai,T, Kawahara,M, Yamanaka,K, Hoshino,Y, Tomioka,I, Sugimura,S, Sugawara,A, Sato,E

Morphology 2 ( 1 ) 23 - 27 2007

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

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

Utility of parthenogenetic embryos as a co-transfer embryo for so nuclear transferred embryos in production of cloned miniature pigs

Sugimura,S, Yamanaka,K, Wakai,T, Shouji,T, Sasada,H, Tanaka,H, Kobayashi,J, Kobayashi,E, Sato,E

In Challenge of Animal Industry for the Wellbeing of Mankind, XIIth AAAP Animal Science Congress 1 - 3 2006

Effect of culture media on in vitro development of SCNT-cloned miniature pig embryos

Yamanaka,K, Sugimura,S, Wakai,T, Shoji,T, Kobayashi,J, Sasada,H, Sato,E

In Challenge of Animal Industry for the Wellbeing of Mankind, XIIth AAAP Animal Science Congress 2006 9 - 11 2006

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