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

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掲載種別：研究論文（学術雑誌）   出版者・発行元：FapUNIFESP (SciELO)  

DOI： 10.1590/1984-3143-ar2022-0127

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bbrc.2021.10.077

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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

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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.

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記述言語：日本語   出版者・発行元：(公社)日本繁殖生物学会  

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記述言語：日本語   出版者・発行元：(公社)日本繁殖生物学会  

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DOI： 10.1101/cshperspect.a035162

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2018-130

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/biolre/ioy214

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DOI： 10.1242/jcs.225441

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/asj.12044

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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

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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

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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

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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 &gt;0.81 x 10(14)/mol sec(-1) of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence.

DOI： 10.1089/cell.2009.0111

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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

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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

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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

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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

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

DOI： 10.1016/j.ceca.2009.04.004

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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

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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

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DOI： 10.1017/S0967199407004480

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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

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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&lt;0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P&lt;0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P&lt;0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P&lt;0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P&lt;0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.

DOI： 10.1262/jrd.18162

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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 &lt; 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P &lt; 0.05) higher than in those without caffeine at 3 h post-injection. in addition, the rate of development to the blastocyst stage after activation was significantly (P &lt; 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.

DOI： 10.1530/rep.1.00644

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

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

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

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

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記述言語：日本語   出版者・発行元：岡山実験動物研究会  

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語   出版者・発行元：岡山実験動物研究会  

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

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

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記述言語：日本語   出版者・発行元：(一社)日本IVF学会  

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

【目的】体細胞クローン技術は，分化した体細胞ゲノムをリプログラミングし個体発生に導くことができるが，同時に不完全なリプログラミングに起因する広範囲な遺伝子発現異常を伴う。我々は以前，マイクロアレイ解析からマウス体細胞クローン胚においてX染色体上の遺伝子が高頻度で発現異常を起こしていることを突き止めたが，これら発現異常遺伝子が発生の進行に伴いどのような挙動を示すかは未だ不明である。そこで本研究では，マウス体細胞クローン胚においてX染色体上の遺伝子発現を可視化し，その時空間的な変化を追跡することでX染色体リプログラミングの評価を試みた。【方法】X染色体上遺伝子の一つであるMoesin(Msn)のプロモーターの下流にGFP遺伝子を挿入したノックインマウス（Msn&lt;sup&gt;GFP&lt;/sup&gt;）を作製した。ドナー細胞としてMsn&lt;sup&gt;GFP&lt;/sup&gt;新生仔の雄から採取したセルトリ細胞を用いて核移植を行い，体細胞クローン胚を作出した。コントロールとしてヘテロMsn&lt;sup&gt;GFP&lt;/sup&gt;雌と野生型雄の交配により得られた受精卵を採取した。胚盤胞期までの初期発生におけるGFPの蛍光発現を共焦点レーザー顕微鏡により観察し，Msnの発現を評価した。【結果】受精卵では胚盤胞期からGFPが検出され，受精後96時間では34.5％，120時間では54.5%の胚盤胞がMsnの発現を示した。約半数の胚においてMsnの発現が確認された結果はヘテロMsn&lt;sup&gt;GFP&lt;/sup&gt;と野生型雄の交配結果を反映したものと考えられる。胚盤胞をヘキスト染色した結果，全ての細胞がGFP陽性であった。一方，体細胞クローン胚では活性化後96時間で全ての胚盤胞においてGFPが検出されが，興味深いことに，体細胞クローン胚盤胞ではGFP陽性細胞の割合が受精卵由来の胚盤胞と比較して有意に低く，Msnの発現が局所的であることがわかった。これらの結果は，体細胞クローンでは個々の細胞によって遺伝子のリプログラミングの程度が異なることを示唆しており，今後は単一細胞におけるリプログラミングの詳細な解析が必要と考えられる。

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記述言語：日本語   出版者・発行元：(一社)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。しかし，体細胞クローン個体から誕生した産子には，親世代に生じた異常は認められない。果たしてクローン個体の生殖系列で，不完全なエピゲノムエラーが修正されるのであろうか？仮に，生殖細胞にエピゲノムエラーが潜在しているとすればトランスジェネレーショナルな伝達の可能性も否定できない。そこで，本研究では体細胞クローン個体が生産する精子のDNAメチロームを明らかにしようとした。【方法】 成熟雄マウス（BDF1）の尾から調整した体細胞をドナー細胞として排卵卵子（BDF1）に核移植して体細胞クローンマウスを作出した。作出された体細胞クローンは10–12週まで飼育したのち，精巣上体尾部より精子を採取し，DNAメチローム解析用試料とした。DNAメチローム解析は，SureSelect-Methyl-seq (Agillent社)を用いてライブラリーを作製後，次世代シークエンサー（HiSeq2500, Illumina社）を用いて解析した。【結果】受精卵由来雄マウス（n=2）精子と体細胞クローンマウス（n=9）の精子において計23,116のCpG領域を比較したところ，メチル化状態が異なる領域が検出された。たとえば，コントロール個体のメチル化状態が90%を越えるCpG領域で50%以下のメチル化を示すクローン個体が存在した。一方，コントロール個体のメチル化状態がいずれも30％以下のCpG領域で70%以上のメチル化を示すクローン個体も存在した。これらのCpG領域は体細胞クローンマウス精子に特有なメチル化変異である可能性が高い。また，メチル化異常を示した領域は，必ずしも共通する領域ではないが，遺伝子発現に何らかの影響を及ぼす可能性も否定できない。

DOI： 10.14882/jrds.107.0_OR1-25

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

【目的】ATPは細胞の主要なエネルギー源であり，生体内の多くの化学反応を支える。卵母細胞は，卵成熟の進行やその後の受精や発生を支持するために多くのATPを必要とすることが考えられるが，時間空間的なATP濃度の変化について詳しくは解明されていない。最近， ATP濃度を測定する方法として，蛍光共鳴エネルギー移動（FRET）の原理に基づいたバイオセンサー，ATeamが開発され，生細胞における正確なATP濃度の測定が可能となった。そこで我々は，ATeamを用いてマウス卵母細胞の成熟過程や受精後におけるATP濃度の測定を行った。【方法】ATeamをコードするcDNAから&lt;i&gt;In vitro&lt;/i&gt;転写によりRNAを合成し未成熟卵母細胞（GV卵子）にインジェクションを行った。GV卵子はIBMX存在下で5時間培養し，ATeamの蛍光発現を確認した。ATP濃度はCFPおよびYFP蛍光画像をそれぞれ取得し，YFP/CFP蛍光強度比により評価した。ATP濃度の変化を調べるためにATP合成阻害剤oligomycinとミトコンドリアの機能阻害剤CCCPを添加した。卵成熟過程におけるATP濃度の変化を調べるためにATeamを発現したGV卵子をM2培地に移し成熟培養を行った。受精後のATP濃度の変化は，ATeamを発現させた成熟卵母細胞（MII卵子）にICSIをすることにより調べた。【結果】 ATeamの蛍光発現はインジェクション5時間後に卵細胞質内全体で確認され，その後はYFP/CFP比に大きな変化が認められなかった。OligomycinおよびCCCP添加後，YFP/CFP比は速やかに低下したことからATeamが卵子内でATPセンサーとして機能していることが確認された。卵成熟過程におけるATP濃度の測定からGV期からMII期にかけてATP濃度の緩やかな低下が観察された一方で，受精後はATP濃度の大きな増加が観察されたことから，細胞内Ca2+濃度の上昇がATP産生に寄与したことが考えられる。

DOI： 10.14882/jrds.106.0.P-76.0

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

【目的】哺乳動物の受精卵では，全能性再獲得における初期化プロセスの一環として，ゲノム広範囲な脱メチル化が生じる。精子由来ゲノムの5メチル化シトシン（5mC）は受精直後急激に減少するが，これは5mC酸化活性を持つTET3により5mCが5ヒドロキシメチル化シトシン（5hmC）に変換される現象であることが証明された。本研究では，卵子における5hmCの存在の有無および局在について検証した。【方法】新生仔，成長期，性成熟雌マウスから非成長期卵，成長期卵および成長完了卵を採取して供試した。また，DNAメチル基転移酵素補因子（&lt;i&gt;Dnmt3L&lt;/i&gt;）ヘテロあるいはホモ欠損雌マウスの成長完了卵，および野生型精子とのIVFにより得られた前核期受精卵を用いた。固定処理後，5mCおよび5hmC抗体処理・核染色し，共焦点レーザー顕微鏡により蛍光を観察した。また，卵子の5hmC修飾因子と予測される&lt;i&gt;Tet3&lt;/i&gt;の発現を定量的RT-PCRにより解析した。【結果】免疫染色の結果，5mCシグナルは15日齢の成長期卵より検出された。一方，5hmCシグナルは非成長期および成長期卵においては検出されなかったが，成長完了以降ではほぼ全ての卵子で検出された（89/91）。この5hmCシグナルはPropidium iodideによる核染色領域と一致していた。定量的RT-PCRの結果，&lt;i&gt;Tet3&lt;/i&gt;の発現は10日齢の成長期卵から上昇し始め，成長完了卵では3倍の発現を示した。5hmC修飾が&lt;i&gt;Dnmt3L&lt;/i&gt;依存的に生成されるかを検証するため，&lt;i&gt;Dnmt3L&lt;/i&gt;欠損成長完了卵および受精卵を用いた免疫染色を行った。その結果，&lt;i&gt;Dnmt3L&lt;/i&gt;欠損において卵子核および雌性前核の5mCシグナル強度は野生型と比較して70％低下したのに対し，5hmCシグナルに明確な差はなかった。これらの結果から，卵子形成過程における5hmCは5mCとは異なり，成長完了卵で修飾されることが明らかになった。卵子での5hmC獲得は受精後の初期発生過程における転写活性及びエピゲノム変化に関与することが予想される。

DOI： 10.14882/jrds.106.0.OR1-33.0

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：英語   出版者・発行元：日本哺乳動物卵子学会  

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

DOI： 10.1274/jmor.30.41

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記述言語：英語   出版者・発行元：(一社)日本卵子学会  

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

DOI： 10.1274/jmor.30.41

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：日本哺乳動物卵子学会  

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

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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

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その他リンク： http://agriknowledge.affrc.go.jp/RN/2010834004

記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

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

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記述言語：日本語   出版者・発行元：日本繁殖生物学会  

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

DOI： 10.14882/jrds.105.0_5

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記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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記述言語：日本語  

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

DOI： 10.1274/jmor.29.155

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記述言語：日本語  

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記述言語：英語   出版者・発行元：日本卵子学会  

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

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記述言語：日本語   出版者・発行元：日本繁殖生物学会  

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

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：(一社)日本生殖医学会  

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記述言語：日本語   出版者・発行元：(株)養賢堂  

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記述言語：日本語   出版者・発行元：日本胚移植研究会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

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記述言語：日本語   出版者・発行元：(公社)日本畜産学会  

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記述言語：日本語   出版者・発行元：(公社)日本獣医学会  

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DOI： 10.14882/jrds.98.0.100.0

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記述言語：日本語   出版者・発行元：(一社)日本繁殖生物学会  

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

DOI： 10.14882/jrds.98.0.163.0

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出版者・発行元：日本繁殖生物学会  

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

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記述言語：日本語   出版者・発行元：(公社)日本畜産学会  

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記述言語：日本語   出版者・発行元：(一社)日本臓器保存生物医学会  

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記述言語：日本語   出版者・発行元：(一社)日本臓器保存生物医学会  

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記述言語：日本語   出版者・発行元：(公社)日本畜産学会  

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記述言語：日本語   出版者・発行元：(一社)日本臓器保存生物医学会  

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

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記述言語：日本語   出版者・発行元：(公社)北海道獣医師会  

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記述言語：日本語   出版者・発行元：(一社)日本臓器保存生物医学会  

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記述言語：日本語   出版者・発行元：(株)日本医学館  

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記述言語：日本語   出版者・発行元：(株)日本医学館  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER SCIENCE INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

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