研究者詳細 - 若井　拓哉

論文 - 若井　拓哉

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Exogenous expression of PGC-1α during in vitro maturation impairs the developmental competence of porcine oocytes

Son Quang Do, Hai Thanh Nguyen, Takuya Wakai, Hiroaki Funahashi

Theriogenology 228 30 - 36 2024年10月

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

Wanniarachchige Tharindu Lakshitha Fonseka, Son Quang Do, Phong Ngoc Van, Hai Thanh Nguyen, Takuya Wakai, Hiroaki Funahashi

Theriogenology 226 158 - 166 2024年9月

Hippo pathway inactivation through subcellular localization of NF2/merlin in outer cells of mouse embryos. 国際誌

Nanami Goda, Yui Ito, Shun Saito, Miyabi Suzuki, Hanako Bai, Masashi Takahashi, Takuya Wakai, Manabu Kawahara

Development (Cambridge, England) 151 ( 14 ) 2024年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.

DOI： 10.1242/dev.202639

Redistribution of fragmented mitochondria ensure symmetric organelle partitioning and faithful chromosome segregation in mitotic mouse zygotes

Haruna Gekko, Ruri Nomura, Daiki Kuzuhara, Masato Kaneyasu, Genpei Koseki, Deepak Adhikari, Yasuyuki Mio, John Carroll, Tomohiro Kono, Hiroaki Funahashi, Takuya Wakai

2024年6月

出版者・発行元：Cold Spring Harbor Laboratory

Abstract

In cleavage-stage embryos, preexisting organelles partition evenly into daughter blastomeres without significant cell growth after symmetric cell division. The presence of mitochondrial DNA within mitochondria and its restricted replication during preimplantation development makes their inheritance particularly important. While chromosomes are precisely segregated by the mitotic spindle, the mechanisms controlling mitochondrial partitioning remain poorly understood. In this study, we investigate the mechanism by which Dynamin-related protein 1 (Drp1) controls the mitochondrial redistribution and partitioning during embryonic cleavage. Deletion of Drp1 in mouse zygotes causes marked mitochondrial aggregation, and the majority of embryos arrest at the 2-cell stage. Clumped mitochondria are located in the center of mitotic Drp1-depleted zygotes with less uniform distribution, thereby preventing their symmetric partitioning. Asymmetric mitochondrial inheritance is accompanied by functionally inequivalent blastomeres with biased ATP and endoplasmic reticulum Ca²⁺levels. We also find that marked mitochondrial centration in Drp1-depleted zygotes prevents the assembly of parental chromosomes, resulting in chromosome segregation defects and binucleation. Thus, mitochondrial fragmentation mediated by Drp1 ensure proper organelle positioning and partitioning into functional daughters during the first embryonic cleavage.

Impact statement

Depletion of Dynamin-related protein 1, a key regulator of mitochondrial fission, in mouse zygotes impair symmetric organelle partitioning and chromosome segregation leading to early developmental arrest.

DOI： 10.1101/2024.06.13.598818

Negative correlations of mitochondrial DNA copy number in commercial frozen bull spermatozoa with the motility parameters after thawing

Hai Thanh Nguyen, Son Quang Do, Hiroshi Kobayashi, Takuya Wakai, Hiroaki Funahashi

Theriogenology 210 154 - 161 2023年10月

Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo. 国際誌

Yuka Ikawa, Takuya Wakai, Hiroaki Funahashi, Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki

Scientific reports 13 ( 1 ) 13050 - 13050 2023年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

DOI： 10.1038/s41598-023-40361-9

Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health

Hai Thanh Nguyen, Son Quang Do, Rukmali Athurupana, Takuya Wakai, Hiroaki Funahashi

Animal Reproduction 20 ( 3 ) 2023年

Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos

Masaya Komatsu, Hayato Tsukahara, Hanako Bai, Masashi Takahashi, Takuya Wakai, Manabu Kawahara

Biochemical and Biophysical Research Communications 584 1 - 6 2021年11月

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles.

Chiyuki Kohata-Ono, Takuya Wakai, Hiroaki Funahashi

The Journal of reproduction and development 65 ( 6 ) 527 - 532 2019年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

DOI： 10.1262/jrd.2019-112

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles

Chiyuki Kohata-Ono, Takuya Wakai, Hiroaki Funahashi

JOURNAL OF REPRODUCTION AND DEVELOPMENT 65 ( 6 ) 527 - 532 2019年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

マウス卵母細胞におけるTrim-Away法を用いたリン酸化タンパク質の分解

月向はるな, 野村瑠莉, 舟橋弘晃, 若井拓哉

The Journal of Reproduction and Development 65 ( Suppl. ) j119 - j119 2019年9月

ミトコンドリア分裂因子Drp1の阻害による着床前マウス胚の発生停止

野村瑠莉, 月向はるな, 舟橋弘晃, 若井拓哉

The Journal of Reproduction and Development 65 ( Suppl. ) j120 - j120 2019年9月

Ca²⁺ Signaling and Homeostasis in Mammalian Oocytes and Eggs. 査読

Wakai T, Mehregan A, Fissore RA

Cold Spring Harbor perspectives in biology 2019年8月

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. 査読

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

The Journal of reproduction and development 65 ( 2 ) 177 - 182 2019年4月

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年4月

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年4月

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年4月

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

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

Biology of reproduction 100 ( 3 ) 697 - 710 2019年3月

Constitutive IP₃R1-mediated Ca²⁺ release reduces Ca²⁺ store content and stimulates mitochondrial metabolism in mouse GV oocytes. 査読

Wakai T, Fissore RA

Journal of cell science 132 ( 3 ) 2019年2月

Fertilization and the signaling of egg activation

Takuya Wakai, Aujan Mehregan, Rafael A. Fissore

Encyclopedia of Reproduction 368 - 375 2018年1月

記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：Elsevier

After receiving the LH surge, a mature oocyte (egg) is collected in the oviduct of mammalian females. Following fusion of the gametes, the sperm elicits in the egg repeated calcium (Ca2++) transients known as oscillations, which trigger egg activation. Completion of egg activation renders the meiotic and transcriptionally silent egg into a mitotically competent, transcriptionally active zygote that eventually undergoes cell differentiation and embryo development. Here, we discuss the major events of egg activation and the molecular players that underlie them. We describe the universal role of Ca2++ on egg activation as well as the egg’s Ca2++ toolkit. We explain how failure of egg activation results in infertility, and the value of ICSI and genetic models in identifying the molecular causes of this infertility. Finally, we point to areas where additional research is needed and how this knowledge will allow better control of fertility.

DOI： 10.1016/B978-0-12-801238-3.64656-1

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

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年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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.

DOI： 10.1071/RD16321

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 査読

Yuichi Okudaira, Takuya Wakai, Hiroaki Funahashi

JOURNAL OF REPRODUCTION AND DEVELOPMENT 63 ( 2 ) 191 - 197 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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 査読

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

THERIOGENOLOGY 86 ( 7 ) 1705 - 1710 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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.

DOI： 10.1095/biolreprod.116.138677

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

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

THERIOGENOLOGY 85 ( 3 ) 428 - 433 2016年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

体細胞クローンマウス精子のDNAメチル化異常の検出

小池佐, 河野友宏, 神長祐子, 若井拓也, 水谷英二, 小林久人, 若山清香, 坂下陽彦, 三浦史仁, 伊藤隆司

日本繁殖生物学会講演要旨集 108 ( 0 ) P - 82-P-82 2015年

記述言語：日本語出版者・発行元：日本繁殖生物学会

【目的】生殖系列におけるリプログラミング機構は，受精を介して全能性を獲得する卵子と精子を生じさせる。体細胞クローン技術は，終末まで分化した細胞に全能性を再獲得させるが，産子への発生率は低く，頻繁に胎仔・胎盤の形成異常を伴うことが知られている。体細胞クローンマウスではエピジェネティックな異常が見られることが報告されている。本研究では，クローンマウス精子の包括的DNAメチローム解析を実施し，DNAメチル化の正常性について検討した。【方法】BDF1マウスの尾端線維芽細胞をドナーとして用い，同系統の除核卵に核移植し，7匹の正常な表現型を示すクローンマウスおよび5匹のBDF1野生型マウスから得られた精子をサンプルとした。DNAメチル化解析は，Post-bisulfite adaptor tagging法を用いたターゲットメチロームシークエンス法により実施し，メチル化に関係する領域についてのメチロームデータを作成した。23021個のCpGアイランドとそれらのアイランドショアについて，野生型から外れ値を除いて平均化したものとクローンの各個体との間でメチル化率が統計学的に有意な差異を示す領域を抽出した。【結果】既知のインプリント制御領域（ICR）のメチル化率は，リードがマッピングされた2個の父方メチル化ICRにおいて，クローン全個体で95%以上，19個の母方メチル化ICRにおいて6%以下であり，これらの領域は正常にリプログラムされていること確認された。全CpGアイランド中で統計学的に有意にメチル化率が異なる領域について，クローンが野生型に対して高メチル化となる領域が5個体に計11領域，低メチル化となる領域が3個体に計16領域検出された。さらにCpGアイランドショアについても同様の解析により，高メチル化領域が3個体に計6個，低メチル化領域が1個体に計3個検出された。本研究の結果から，生殖細胞系列におけるリプログラミングの影響を受けにくい領域の存在が示唆された。

DOI： 10.14882/jrds.108.0_P-82

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

Takuya Wakai, Yuichirou Harada, Kenji Miyado, Tomohiro Kono

MOLECULAR HUMAN REPRODUCTION 20 ( 11 ) 1090 - 1100 2014年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth 査読

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

GENES TO CELLS 19 ( 8 ) 629 - 636 2014年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

HUMAN MOLECULAR GENETICS 23 ( 14 ) 3853 - 3864 2014年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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.

DOI： 10.1093/hmg/ddu100

Long exposure to mature ooplasm can alter DNA methylation at imprinted loci in non-growing oocytes but not in prospermatogonia （成熟卵細胞質に非成長期卵母細胞核を暴露するとインプリント領域のメチル化に変化が生じるが精原細胞の核を暴露した場合では変化が生じない）査読

Obata Y, Wakai T, Hara S, Kono T

Reproduction 147 ( 1 ) H1 - H6 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

精子あるいは卵子特異的インプリント確立機構を解明する為に、卵母細胞のインプリントが確立する条件下に雄性生殖細胞を暴露した。十分成長した卵母細胞に雄性生殖細胞あるいは非成長期卵母細胞を核移植し、再構築卵胞を形成させ体外培養を実施した。その結果、精子型インプリントを確立した後の雄性生殖細胞には、もはや卵子型インプリントを書き込むことはできなかった。一方、卵子型インプリントが確立する前の非成長期卵母細胞を移植した場合、この卵母細胞ゲノムには精子型インプリントも卵子型インプリントも書き込まれることが示された。このことから、卵子特異的なインプリントは、精子特異的なインプリントが書き込まれないよう、ブロックする機構であることが示唆された。

DOI： 10.1530/REP-13-0359.

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

Yayoi Obata, Takuya Wakai, Satoshi Hara, Tomohiro Kono

REPRODUCTION 147 ( 1 ) H1 - H6 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs 査読

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

JOURNAL OF CELL SCIENCE 126 ( 24 ) 5714 - 5724 2013年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Artificial Activation of Mammalian Oocytes for Cloning: Present Status and Future Perspectives 査読

Takuya Wakai, Junya Ito, Rafael A. Fissore

Principles of Cloning: Second Edition 3 - 10 2013年10月

記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：Elsevier Inc.

Since the birth of the first cloned sheep, "Dolly", many studies have reported the successful use of cloning by somatic cell nuclear transfer in other mammalian species. In spite of these successes, the efficiency of this technique is low, with less than 10% of generated embryos resulting in offspring. Factors of genetic, epigenetic, and technical origin seem to affect the efficiency of cloning. Further, because these reconstructed embryos do not have the benefit of being activated by the sperm, egg activation relies on artificial procedures such as those delivered by chemical or physical stimuli. In general, these stimuli aim to replicate either the signal delivered by the sperm, namely, elevations in the intracellular concentration of calcium ion ([Ca2+]i), or the outcome of this signal, inactivation of the M-phase kinases that are responsible for the metaphase II arrest. In mammalian fertilization, the sperm initiates repetitive increases in [Ca2+]i that are also known as oscillations, and experimentally replicating these oscillations has been difficult to accomplish, except in mouse eggs where oscillations can be attained by replacing extracellular CaCl2 with SrCl2. In other mammalian species, in the absence of oscillations, most activation protocols incorporate chemical methods to subdue the activity of the M-phase kinases. Here, we will briefly describe oocyte activation protocols presently in use in different species, highlight some of their advantages and disadvantages, and discuss possible improvements. © 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/B978-0-12-386541-0.00001-1

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

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

MOLECULAR BIOLOGY OF THE CELL 24 ( 9 ) 1396 - 1410 2013年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

Takuya Wakai, Rafael A. Fissore

CELL CALCIUM 53 ( 1 ) 63 - 67 2013年1月

記述言語：英語出版者・発行元：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

Molecular characteristics of horse phospholipase C zeta (PLCζ). 査読

Sato K, Wakai T, Seita Y, Takizawa A, Fissore RA, Ito J, Kashiwazaki N

Animal science journal = Nihon chikusan Gakkaiho 84 ( 4 ) 359 - 368 2013年

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

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年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

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.

DOI： 10.1002/jcp.22778

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

Nan Zhang, Takuya Wakai, Rafael A. Fissore

MOLECULAR REPRODUCTION AND DEVELOPMENT 78 ( 9 ) 684 - 701 2011年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

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.

DOI： 10.1002/mrd.21366

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

Takuya Wakai, Veerle Vanderheyden, Rafael A. Fissore

COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY 3 ( 4 ) 2011年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT

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.

DOI： 10.1101/cshperspect.a006767

Anomalous Oxygen Consumption in Porcine Somatic Cell Nuclear Transfer Embryos 査読

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC

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 >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.

DOI： 10.1089/cell.2009.0111

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

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

ANIMAL SCIENCE JOURNAL 81 ( 1 ) 34 - 41 2010年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC

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.

DOI： 10.1111/j.1740-0929.2009.00699.x

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 査読

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

ANIMAL SCIENCE JOURNAL 81 ( 1 ) 48 - 57 2010年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC

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.

DOI： 10.1111/j.1740-0929.2009.00705.x

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

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

JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 ( 6 ) 638 - 644 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.20245

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

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年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPIE-SOC PHOTOPTICAL INSTRUMENTATION ENGINEERS

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]

DOI： 10.1117/1.3241985

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

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

CELL CALCIUM 46 ( 1 ) 56 - 64 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CHURCHILL LIVINGSTONE

Egg activation and further embryo development require a sperm-induced intracellular Ca(2+) signal at the time of fertilization. Prior to fertilization, the egg'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.

DOI： 10.1016/j.ceca.2009.04.004

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

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

JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 ( 3 ) 299 - 304 2009年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.20174

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

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC

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.

DOI： 10.1089/clo.2007.0045

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

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

Zygote (Cambridge, England) 16 ( 2 ) 153 - 159 2008年5月

Induction of estrus in pubertal miniature gilts 査読

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年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

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.

DOI： 10.1016/j.anireprosci.2007.04.004

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

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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<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<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<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<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<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.

DOI： 10.1262/jrd.18162

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

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

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

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

Shimizu Takashi, Abe Yasuyuki, Wakai Takuya, Hoshino Yumi, Miyamoto Akio, Sato Eimei

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

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 査読

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年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIO SCIENTIFICA LTD

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 < 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P < 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 < 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.

DOI： 10.1530/rep.1.00644

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