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

PubMed

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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

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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

PubMed

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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

PubMed

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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

Web of Science

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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

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