研究者詳細 - 若井　拓哉

論文 - 若井　拓哉

分割表示 54 件中 21 - 40 件目／全件表示 >>

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

　前のページ - 次のページ　

1
2
3