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Last Updated :2024/08/25

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Master

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

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Profile and Settings

Profile and Settings

  • Name (Japanese)

    Takahashi

  • Name (Kana)

    Masashi

  • Name

    201301045898361360

Alternate Names

Achievement

Research Interests

  • 発生   RNA干渉   初期胚   遺伝子発現   牛初期胚   牛   

Research Areas

  • Life sciences / Veterinary medicine

Research Experience

  • 2012/07 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
  • 2006/04 - 2012/06 National Agriculture and Food Research Organization
  • 2001/04 - 2006/03 独立行政法人 農業技術研究機構 九州沖縄農業研究センター 繁殖技術研究室長
  • 1998/04 - 2001/03 農水省畜産試験場繁殖部 主任研究官
  • 1998/09 - 1999/09 米国 ペンシルバニア大学 科学技術庁 在外研究員
  • 1988/04 - 1997/04 National Institute of Animal Industry research official

Education

  • 1986/04 - 1988/03  Hokkaido University

Awards

  • 2011/09 日本繁殖生物学会 技術賞
     酸化還元調節によるウシ胚の体外生産効率向上に関する研究 
    受賞者: 高橋昌志

Published Papers

  • Shinjiro Kagawa, Yoshihiro Hayashi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Molecular reproduction and development 91 (8) e23767  2024/08 
    In many mammals, including ruminants, pregnancy requires pregnancy recognition signaling molecules secreted by the conceptus; however, the mechanism underlying pregnancy establishment in cattle remains unknown. Trophoblastic vesicles (TVs) are artificially produced from the extraembryonic tissues of the elongating conceptus and may be useful tools for understanding conception. This study investigated the morphological and functional properties of TVs in comparison to those of intact conceptuses. TVs were prepared from the extraembryonic tissues of conceptuses collected 14 days after artificial insemination (AI), cryopreserved immediately after dissection, and cultured after thawing for subsequent transplantation into the uterus. The transferred TVs were collected 7 days after transplantation and compared with extraembryonic tissue samples collected from conceptuses at 21 days post-AI. The recovered TVs were 40 times longer than those of their pre-transplant counterparts. Microscopic evaluation revealed that their membrane structures consisted of trophoblast and hypoblast layers. The expression patterns of the cell differentiation markers, CDX2, SOX2, and GATA6, and interferon tau (IFNT) protein expression levels in the TVs were similar to those in control extraembryonic tissue samples. These findings suggest that TVs are capable of morphological elongation and maintain IFNT production in a similar way as original trophoblasts.
  • Nanami Goda, Yui Ito, Shun Saito, Miyabi Suzuki, Hanako Bai, Masashi Takahashi, Takuya Wakai, Manabu Kawahara
    Development (Cambridge, England) 151 (14) 2024/07/15 
    The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.
  • Shuhei Fukaya, Takeshi Yamazaki, Hayato Abe, Satoshi Nakagawa, Toshimi Baba, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Journal of Dairy Science 0022-0302 2024/07
  • 着床期子宮内インターフェロンと誘導性遺伝子
    唄 花子, 川原 学, 高橋 昌志
    細胞 (株)ニュー・サイエンス社 56 (3) 196 - 198 1346-7557 2024/03 
    妊娠率の向上は,哺乳動物に共通の課題である。反芻動物では,黄体の維持すなわち妊娠の成立にI型インターフェロン(IFN)の一種であるインターフェロン・タウ(IFNT)が必須であるため,以前よりIFNTの研究が盛んに行われてきた。最近では,ヒトを含む多くの哺乳動物でも着床過程においてI型IFNおよびその誘導性遺伝子群ISGsの発現がみられることが明らかになってきた。本稿では,哺乳動物の着床過程におけるI型IFNおよびISGsが役割を担う可能性について紹介したい。(著者抄録)
  • Masaya Komatsu, Hikaru Takuma, Shun Imai, Maiko Yamane, Masashi Takahashi, Takuto Ikegawa, Hanako Bai, Hidehiko Ogawa, Manabu Kawahara
    Scientific Reports 13 (1) 2023/12/27 
    Abstract Heteroplasmic mammalian embryos between genetically distant species fail to develop to term, preventing transmission of xenomitochondrial DNA to progeny. However, there is no direct evidence indicating the mechanisms by which species specificity of the mitochondrial genome is ensured during mammalian development. Here, we have uncovered a two-step strategy underlying the prevention of xenomitochondrial DNA transmission in mouse embryos harboring bovine mitochondria (mtB-M embryos). First, mtB-M embryos showed metabolic disorder by transient increase of reactive oxygen species at the 4-cell stage, resulting in repressed development. Second, trophoblasts of mtB-M embryos led to implantation failure. Therefore, we tested cell aggregation with tetraploid embryos to compensate for the placentation of mtB-M embryos. The 14 mtB-M embryos harboring bovine mtDNAs developed to term at embryonic day 19.5. Taken together, our results show that contamination of bovine mtDNA is prohibited by embryonic lethality due to metabolic disruption and failure of placentation, suggesting these represent xenomitochondrial elimination mechanisms in mammalian embryos.
  • Shabur Abdus Talukder, Ahmed Z Balboula, Hanako Bai, Manabu Kawahara, Koji Kimura, Masashi Takahashi
    Reproduction (Cambridge, England) 2023/10/01 
    Interferon tau (IFNT) is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post-artificial insemination (AI), after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in non-pregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.
  • Mohamed Aboul Ezz, Masashi Takahashi, Rocío Melissa Rivera, Ahmed Zaky Balboula
    Cell proliferation e13526  2023/07/07 
    Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.
  • Hanako Bai, Manabu Kawahara, Masashi Takahashi
    Theriogenology 198 183 - 193 0093-691X 2023/03
  • Eri Furukawa, Yojiro Yanagawa, Akira Matsuzaki, Heejin Kim, Hanako Bai, Masashi Takahashi, Seiji Katagiri, Shogo Higaki
    The Journal of reproduction and development 69 (2) 103 - 108 2023/02/17 
    The present study investigated the applicability of a calving prediction model based on supervised machine learning of ruminal temperature (RT) data in dairy cows. The existence of cow subgroups for prepartum RT changes was also examined, and the predictive performance of the model was compared among these subgroups. RT data were collected from 24 Holstein cows at 10 min intervals using an RT sensor system. The average hourly RT was calculated and data were expressed as residual RTs (rRT = actual RT - mean RT for the same time on the previous three days). The mean rRT decreased beginning at approximately 48 h before calving to a low of -0.5°C at 5 h before calving. However, two cow subgroups were identified: cows with a late and small rRT decrease (Cluster 1, n = 9) and those with an early and large rRT decrease (Cluster 2, n = 15). A calving prediction model was developed using five features extracted from the sensor data (indicative of prepartum rRT changes) through a support vector machine. Cross-validation showed that calving within 24 h was predicted with a sensitivity of 87.5% (21/24) and precision of 77.8% (21/27). A significant difference in sensitivity was observed between Clusters 1 and 2 (66.7 vs. 100%, respectively), while none was observed for precision. Therefore, the model based on RT data with supervised machine learning has the potential to efficiently predict calving, although improvements for specific cow subgroups are required.
  • 最先端医療の今 子宮内インターフェロンと誘導性遺伝子
    唄 花子, 川原 学, 高橋 昌志
    Medical Science Digest (株)ニュー・サイエンス社 49 (2) 88 - 90 1347-4340 2023/02 
    妊娠率の向上は,多くの哺乳動物において共通の課題である。ウシを含む反芻動物では,黄体の維持すなわち妊娠の成立にI型インターフェロン(IFN)の一種であるインターフェロン・タウ(IFNT)が必須であるため,以前よりIFNTの研究が盛んに行われてきた。最近では,ヒトを含む多くの哺乳動物でも着床過程においてI型IFNおよびその誘導性遺伝子群ISGsの発現がみられることが明らかになってきた。本稿では,哺乳動物の着床過程におけるI型IFNおよびISGsが役割を担う可能性について紹介したい。(著者抄録)
  • Khoi Thieu Ho, Ahmed Zaky Balboula, Kohei Homma, Jun Takanari, Hanako Bai, Manabu Kawahara, Khang Thi Kim Nguyen, Masashi Takahashi
    The Journal of Steroid Biochemistry and Molecular Biology 106181 - 106181 0960-0760 2022/09
  • Weihong Fan, Tengda Huang, Tian Wu, Hanako Bai, Manabu Kawahara, Masashi Takahashi
    Biology of Reproduction 0006-3363 2022/08/10 
    Abstract The zona pellucida (ZP) plays a crucial role in the process of fertilization to early embryonic development, including cellular arrangement and communication between blastomeres. However, little is known regarding the role of the ZP in pre- and post-implantation embryonic development associated with gene expression. We investigated the effect of zona pellucida removal (ZPR) on pre- and post-implantation development of mouse embryos. After ZPR of 2-cell stage embryos was performed by acid Tyrode’s solution, which is commonly used for ZP treatment, compaction occurred earlier in ZP-free (ZF) than ZP-intact (ZI) embryos. In addition, the expression of differentiation-related genes in the inner cell mass (ICM) and trophectoderm (TE) was significantly altered in ZF blastocyst compared with ZI embryos. After embryo transfer, the rate of implantation and live fetuses was lower in ZF embryos than in control embryos, whereas the fetal weight at E17.5 was not different. However, placental weight significantly increased in ZF embryos. RNA-seq analysis of the placenta showed that a total of 473 differentially expressed genes (DEGs) significantly influenced the biological process. The present study suggests that ZPR by acid Tyrode’s solution at the 2-cell stage not only disturbs the expression pattern of ICM/TE-related genes but affects the post-implantation development of mouse embryos. Overall, this study provides deeper insight into the role of the ZP during early embryonic development and the viability of post-implantation development.
  • Haruka Ukita, Takeshi Yamazaki, Satoshi Yamaguchi, Hayato Abe, Toshimi Baba, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Journal of Dairy Science 105 (8) 6947 - 6955 0022-0302 2022/06
  • Shinjiro Kagawa, Shingo Hiraizumi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Theriogenology 185 121 - 126 2022/06 
    Intracytoplasmic sperm injection (ICSI), oocyte vitrification after ovum pick-up (OPU), and in vitro maturation are reproductive technologies with incredible potential for efficient cattle production. However, the developmental competence of embryos produced by ICSI using vitrified OPU oocytes remains unknown. Here, we aimed to evaluate the developmental competence of these embryos from the early embryo period to full term. The cleavage rate in the ICSI embryos using vitrified OPU oocytes during in vitro culture was significantly lower than those in control in vitro fertilized (IVF) embryos using fresh OPU oocytes (30.9 ± 4.5% v.s. 65.9 ± 7.0%) (P < 0.05), but the proportion of blastocysts to cleaved embryos was significantly higher than those of IVF embryos using vitrified OPU oocytes (55.9 ± 10.8% v.s. 23.2 ± 9.3%) (P < 0.05). To further investigate the transcription levels of genes related to cell differentiation in ICSI embryos using vitrified OPU oocytes, the relative abundance of mRNAs (OCT4, NANOG, SOX2, CDX2, GATA3, and IFNT) was analyzed by quantitative reverse-transcription PCR. There were no significant differences in the expression levels between ICSI embryos using vitrified OPU oocytes and control IVF embryos. Finally, developmental competence to term in ICSI embryos using vitrified OPU oocytes was examined by embryo transfer, and two healthy calves were born. These findings confirmed that ICSI and vitrification decrease developmental rates in vitro, but both procedures can lead to full-term development of bovine embryos. These results demonstrate that ICSI embryos using vitrification OPU oocytes are viable for cattle production.
  • Ahmed Z Balboula, Mansour Aboelenain, Miki Sakatani, Ken-Ichi Yamanaka, Hanako Bai, Takahiro Shirozu, Manabu Kawahara, Abd Elraouf O Hegab, Samy M Zaabel, Masashi Takahashi
    Genes 13 (2) 2022/02/10 
    Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.
  • Shun SAITO, Hiroki AKIZAWA, Eri FURUKAWA, Yojiro YANAGAWA, Hanako BAI, Masashi TAKAHASHI, Manabu KAWAHARA
    Journal of Reproduction and Development 0916-8818 2022
  • Hanako BAI, Manabu KAWAHARA, Masashi TAKAHASHI, Kazuhiko IMAKAWA
    Journal of Reproduction and Development 0916-8818 2022
  • Masaya Komatsu, Hayato Tsukahara, Hanako Bai, Masashi Takahashi, Takuya Wakai, Manabu Kawahara
    Biochemical and biophysical research communications 584 1 - 6 2021/12/20 
    GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression.
  • Hiroki Akizawa, Shun Saito, Nanami Kohri, Eri Furukawa, Yoshihiro Hayashi, Hanako Bai, Masashi Nagano, Yojiro Yanagawa, Hayato Tsukahara, Masashi Takahashi, Shinjiro Kagawa, Ryouka Kawahara-Miki, Hisato Kobayashi, Tomohiro Kono, Manabu Kawahara
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 35 (10) e21904  2021/10 
    Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.
  • Khoi Thieu Ho, Kohei Homma, Jun Takanari, Hanako Bai, Manabu Kawahara, Khang Thi Kim Nguyen, Masashi Takahashi
    Scientific reports 11 (1) 18175 - 18175 2021/09/13 
    Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.
  • Weihong Fan, Misato Homma, Renliang Xu, Hiroki Kunii, Hanako Bai, Manabu Kawahara, Toshikazu Kawaguchi, Masashi Takahashi
    Biochemical and biophysical research communications 577 116 - 123 2021/09/07 
    The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.
  • Yoshihiro Hayashi, Shun Saito, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Theriogenology 175 69 - 76 2021/09/02 
    Cellular differentiation induces various morphological changes, including elongation, in mitochondria. Preimplantation embryos have round-shaped mitochondria, characteristic of undifferentiated cells. However, there is controversy regarding the precise mitochondrial morphology in blastocyst embryos, which are generated from two cell lineages: undifferentiated inner cell mass (ICM) and differentiated trophectoderm (TE). This study attempted to precisely determine mitochondrial morphology in these two blastocyst regions. Transmission electron microscopy analyses were conducted using more than 1000 mitochondria from blastocyst embryos. No significant differences were observed in the configuration of mitochondrial cristae and frequencies of hooded mitochondria, which are specific to embryos of livestock animals, between the ICM and TE. To accurately compare mitochondrial roundness between the ICM and TE, oblateness was calculated based on both the major and minor axes. Average oblateness was significantly greater in the TE than in the ICM (P < 0.01). These results indicate tissue-specific mitochondrial maturation with complete elongation in the TE at the blastocyst stage. Since mitochondrial elongation is closely associated with cellular metabolism and differentiation, the present study provides new insights for better understanding of early embryonic development in cattle.
  • 第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係
    古川 瑛理, 高橋 昌志, 松崎 明, 唄 花子, 永野 昌志, 片桐 成二, 柳川 洋二郎
    日本畜産学会大会講演要旨集 (公社)日本畜産学会 129回 117 - 117 1342-4688 2021/09
  • 第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係
    古川 瑛理, 高橋 昌志, 松崎 明, 唄 花子, 永野 昌志, 片桐 成二, 柳川 洋二郎
    日本畜産学会大会講演要旨集 (公社)日本畜産学会 129回 117 - 117 1342-4688 2021/09
  • Hirona Murata, Hiroki Kunii, Kazuya Kusama, Toshihiro Sakurai, Hanako Bai, Manabu Kawahara, Masashi Takahashi
    Biology of reproduction 105 (5) 1114 - 1125 2021/07/22 
    Heat stress adversely affects the reproductive function in cows. Although a relationship between heat stress and oxidative stress has been suggested, it has not been sufficiently verified in bovine endometrial epithelial cells. Here, we investigated whether oxidative stress is induced by heat stress in bovine endometrial epithelial cells under high temperature. Luciferase reporter assays showed that the reporter activity of heat shock element (HSE) and antioxidant responsive element (ARE) was increased in endometrial epithelial cells cultured under high temperature compared to that in cells cultured under basal (thermoneutral) temperature. Also, nuclear factor, erythroid 2 like 2 (NFE2L2), a master regulator of cellular environmental stress response, stabilized and the expression levels of antioxidant enzyme genes increased under high temperature. Immunostaining confirmed the nuclear localization of NFE2L2 in endometrial epithelial cells cultured under high temperature. Quantitative polymerase chain reaction analysis showed that the expression levels of representative inflammatory cytokine genes, such as prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin 8, were significantly decreased in endometrial epithelial cells cultured under high temperature compared to those in cells cultured under basal temperature. Thus, our results suggest that heat stress induces oxidative stress, whereas NFE2L2 plays a protective role in bovine endometrial epithelial cells cultured under heat stress conditions.
  • Hiroki Kunii, Tomoaki Kubo, Natsuki Asaoka, Ahmed Z Balboula, Yu Hamaguchi, Tomoya Shimasaki, Hanako Bai, Manabu Kawahara, Hisato Kobayashi, Hidehiko Ogawa, Masashi Takahashi
    Biochemical and biophysical research communications 569 179 - 186 2021/07/09 
    An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.
  • Shun Saito, Shota Yamamura, Nanami Kohri, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Biochemical and biophysical research communications 555 140 - 146 2021/05/28 
    WW domain-containing transcription regulator 1 (WWTR1) is one of the primary effectors in the Hippo pathway, which plays essential roles in cell differentiation into trophectoderm (TE) and inner cell mass cell lineages at the blastocyst stage. However, little is known about the roles of WWTR1 in preimplantation development. The present study aimed to explore the significance of WWTR1 expression in preimplantation development using an mRNA knockdown (KD) system in bovine embryos. We first quantitated WWTR1 expression at protein and mRNA levels from fertilization to blastocyst stage. WWTR1 proteins gradually shifted from extranuclear localization during the 16-cell stage to nuclear localization by morula stage. WWTR1 mRNA expression was also transiently upregulated at the 16-cell stage. WWTR1 KD efficiently repressed WWTR1 expression at protein and mRNA levels. The WWTR1 KD embryos developed to the blastocyst stage at rates equivalent to those of controls, but TE cell numbers were significantly decreased. Representative TE-expressed genes, including CDX2 and IFNT were also significantly decreased in WWTR1 KD blastocysts. These results provide the first demonstration that WWTR1 expression is responsible for normal TE cell development in preimplantation embryos.
  • Shota Yamamura, Nanami Goda, Hiroki Akizawa, Nanami Kohri, Ahmed Z Balboula, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Developmental biology 468 (1-2) 14 - 25 2020/09/15 [Refereed][Not invited]
     
    A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.
  • Ahmed Z Balboula, Karen Schindler, Tomoya Kotani, Manabu Kawahara, Masashi Takahashi
    Molecular human reproduction 26 (9) 689 - 701 2020/09/01 [Refereed][Not invited]
     
    As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.
  • Nanami Kohri, Hiroki Akizawa, Sakie Iisaka, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Biochemical and biophysical research communications 528 (4) 713 - 718 2020/08/06 [Refereed][Not invited]
     
    Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle.
  • Ahmed Z Balboula, Mansour Aboelenain, Jianye Li, Hanako Bai, Manabu Kawahara, Mohammed A Abdel-Ghani, Masashi Takahashi
    Reproduction (Cambridge, England) 159 (6) 757 - 766 2020/06 [Refereed][Not invited]
     
    Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.
  • H. Bai, H. Hiura, Y. Obara, M. Kawahara, M. Takahashi
    Journal of Dairy Science 103 (8) 7531 - 7534 0022-0302 2020/05 [Refereed][Not invited]
  • Jianye Li, Ahmed Zaky Balboula, Mansour Aboelenain, Takashi Fujii, Satoru Moriyasu, Hanako Bai, Manabu Kawahara, Masashi Takahashi
    The Journal of reproduction and development 66 (1) 83 - 91 2020/02/14 [Refereed][Not invited]
     
    The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.
  • Jianye Li, Mana Maeji, Ahmed Zaky Balboula, Mansour Aboelenain, Takashi Fujii, Satoru Moriyasu, Hanako Bai, Manabu Kawahara, Masashi Takahashi
    The Journal of reproduction and development 66 (1) 9 - 17 0916-8818 2020/02/14 [Refereed][Not invited]
     
    Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.
  • Guanglei Li, Jianye Li, Yu Otsuka, Shuai Zhang, Masashi Takahashi, Koji Yamada
    Materials (Basel, Switzerland) 13 (3) 2020/02/03 [Refereed][Not invited]
     
    We developed an easily accessible boron-dipyrromethene (BODIPY)-based fluorogenic probe, which we named LD-TB. This probe emits bright fluorescence in oil; when compared with aqueous solution, a significant enhancement of fluorescence brightness is observed. Cellular experiments confirmed that the probe stains the lipid droplets (LDs) specifically in both live and fixed cells, providing background-free images. Compared with Nile Red dye, a commonly used LD marker, LD-TB showed superior photostability. The sharp absorption and emission bands enable its multicolor imaging with blue and green probes. Importantly, the probe has proved to have low toxicity and is compatible with cell fixation. Our research provides a promising new fluorogenic probe for specific imaging of LDs.
  • Hanako Bai, Haruka Ukita, Manabu Kawahara, Tomohiro Mitani, Eri Furukawa, Yojiro Yanagawa, Naoto Yabuuchi, Heejin Kim, Masashi Takahashi
    Animal science journal = Nihon chikusan Gakkaiho 91 (1) e13474  2020/01 
    Heat stress negatively affects reproductive functions in cows. Increased temperature disturbs fetal development in utero. However, the effect of heat stress on uterine endometrial tissues has not been fully examined. Using qPCR analysis, we measured the mRNA expression of various molecular markers in uterine endometrial tissue of dairy cows from Hokkaido, Japan, in winter and summer. Markers examined were heat shock proteins (HSPs), antioxidant enzymes (catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase 4), inflammatory cytokines, and interferon stimulated genes. Our results showed heat stress, body and milk temperatures were higher during summer than during winter. Expression levels of HSP27, HSP60, and HSP90 mRNA, and of catalase and copper/zinc superoxide dismutase mRNA were lower in summer than in winter. Tumor necrosis factor alpha expression was higher in summer than in winter. In conclusion, summer heat stress may reduce the expression of HSPs, affecting the levels of inflammatory cytokines in bovine uterine endometrial tissue.
  • Al-Nur Md Iftekhar Rahman, Seiya Yamashita, Md Rashedul Islam, Taisuke Fujihara, Hayato Yamaguchi, Manabu Kawahara, Masashi Takahashi, Hideyuki Takahashi, Takafumi Gotoh, Nobuhiko Yamauchi
    Animal science journal = Nihon chikusan Gakkaiho 91 (1) e13350  2020/01 [Refereed][Not invited]
     
    This study investigated the effect of type-I interferon (IFN) on the expression of matrix metalloproteinases (MMPs) of the bovine endometrial stromal cells (BES) and epithelial cells (BEE). The cells were separated and purified from the caruncles and cultured in DMEM/F-12 containing 10% fetal bovine serum. Spheroids were generated by using ascorbate. Zymograms of the supernatant showed that BEE predominantly expressed MMP-9, whereas MMP-2 was expressed in BES and homo-spheroids. While MMPs expression was not detected in hetero-spheroids. Real-time quantitative PCR revealed that type-I IFN and P4 suppressed the gene expression of MMP-2 and MMP-9 in hetero-spheroids, respectively. On the other hand, gelatin zymography analysis of the supernatant showed that type-I IFN strongly promote the clearance of MMPs. While zymograms of the MMPs stocked in the hetero-spheroids were significantly reduced by type-I IFN. Phenylmethanesulfonyl fluoride and leupeptin (both are serine proteinase inhibitors) significantly repressed the clearance of MMP-2 and MMP-9 induced by type-I IFN. Moreover, collagen fibers in hetero-spheroids significantly decreased after the treatment with type-I IFN. In conclusion, it was suggested that type-I IFN participate in the tissue remodeling by regulation the clearance of MMPs.
  • Nanami Kohri, Hiroki Akizawa, Sakie Iisaka, Hanako Bai, Yojiro Yanagawa, Masashi Takahashi, Masaya Komatsu, Masahito Kawai, Masashi Nagano, Manabu Kawahara
    The Journal of biological chemistry 294 (50) 19209 - 19223 0021-9258 2019/12/13 [Refereed][Not invited]
     
    Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to a posteriori externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.
  • Kohei Oikawa, Takeshi Yamazaki, Satoshi Yamaguchi, Hayato Abe, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Theriogenology 135 33 - 37 0093-691X 2019/09/01 [Refereed][Not invited]
     
    Conception rate with the use of sexed semen is lower than that with the use of conventional semen, posing a major problem in the dairy industry. The aim of this study was to understand the risk factors that affect the conception rate after artificial insemination (AI) with conventional and sexed semen using field data. The records of the first insemination in Holstein heifers with conventional (n = 41,857) and sexed semen (n = 45,465) in Hokkaido, Japan were analyzed. The mean conception rate after AI from 2012 to 2016 was 56.9% with conventional semen and 47.3% with sexed semen. A multivariable logistic regression model including the effects of year, heifer age, time of the year, semen type, service sire, and their interactions was used to evaluate the interaction effect of heifer age and time of the year by semen type on the conception rate. In the analysis using heifer age, we found that heifers inseminated with sexed semen were approximately 21 days younger than those inseminated with conventional semen. Interestingly, in early, warmer months (Jun, Jul, and Aug), the conception rate after AI with sexed semen significantly decreased compared with that after AI with conventional semen (P < 0.01). Our results showed that more careful implementation of AI is required for a stable conception using sexed semen, particularly during warmer months.
  • 性選別精液による乳牛人工授精の受胎率に関するロジスティック解析
    及川 康平, 山崎 武志, 山口 諭, 阿部 隼人, 唄 花子, 高橋 昌志, 川原 学
    北海道畜産草地学会報 北海道畜産草地学会 7 (2) 21 - 21 2187-5391 2019/08
  • Kouki Shiina, Masaya Komatsu, Fumi Yokoi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    FASEB bioAdvances 1 (7) 393 - 403 2019/07 [Refereed][Not invited]
     
    Oocyte spindle transfer (OST) is a potent reproductive technology used for mammals that enables the spindle in a deteriorated oocyte at the metaphase of the second meiotic division (MII) to serve as the genetic material for producing descendants. However, whether postnatal growth is achieved via OST using developmentally deteriorated MII oocytes remains unclear. At 16 h after human chorionic gonadotropin administration, denuded MII oocytes immediately after retrieval from oviducts (0 h-oocytes) were used for in vitro fertilization (IVF) as controls. For IVF using postovulatory-aged oocytes, the 0 h-oocytes were further incubated for 12 h and 24 h (12 h- and 24 h-oocytes). These mouse oocytes served as a model for assessing the postnatal growth of individuals produced via OST from developmentally deteriorated oocytes. The embryos from 12 h- and 24 h-oocyte spindles exhibited high rates of development up to the neonatal stage as good as the non-manipulated controls. However, the mice derived from the 24 h-oocyte spindles displayed heavier body weights and greater feed consumption than both controls and mice derived from 12 h-oocyte spindles. Our results demonstrate the feasibility of OST as a potent reproductive technology and its limitation in the use of excessively aged postovulatory oocytes in mammalian reproduction.
  • Bai H, Shabur TMA, Kunii H, Itoh T, Kawahara M, Takahashi M
    The Journal of reproduction and development 65 (4) 313 - 318 0916-8818 2019/05 [Refereed][Not invited]
  • Akizawa H, Yanagawa Y, Nagano M, Bai H, Takahashi M, Kawahara M
    Animal science journal = Nihon chikusan Gakkaiho 90 (1) 49 - 54 1344-3941 2019/01 [Refereed][Not invited]
     
    In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.
  • Toshiyuki Suzuki, Ryosuke Sakumoto, Ken-Go Hayashi, Takatoshi Ogiso, Hiroki Kunii, Takahiro Shirozu, Sung-Woo Kim, Hanako Bai, Manabu Kawahara, Koji Kimura, Masashi Takahashi
    The Journal of reproduction and development 64 (6) 495 - 502 0916-8818 2018/12/14 [Refereed][Not invited]
     
    Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.
  • Iwasaki W, Yamanaka K, Sugiyama D, Teshima Y, Briones-Nagata MP, Maeki M, Yamashita K, Takahashi M, Miyazaki M
    Scientific reports 8 (1) 14273  2045-2322 2018/09 [Refereed][Not invited]
     
    We fabricated a simple microfluidic device for separation of bovine oocytes based on the oocyte quality to improve the conception rate of in vitro fertilization (IVF) by using good quality oocytes. The microfluidic device separates oocytes based on sedimentation rate differences in a sucrose buffer, which is dependent on oocyte quality. The microfluidic device has a 700 mu m width, 1 mm height, and 10 mm long separation channel. Oocytes were injected from the upper half of the separation channel, and they flowed while sinking. The outlets of the separation channel were divided into upper and lower chambers. Good quality oocytes settled faster than poor quality oocytes in sucrose buffer; therefore, good quality oocytes were collected from the lower outlet. We performed IVF after the microfluidic separation of oocytes. The developmental rate to blastocysts of oocytes collected from the lower outlet was significantly higher than those collected from the upper outlet (36.0% vs. 14.1%). This result was comparable to that in the BCB staining method performed as a comparison method (BCB+ : 35.7%, BCB-: 15.4%). These findings indicate that our microfluidic device could be applied to oocyte separation and contribute to improvement of in vitro embryo production system.
  • Hiroki Kunii, Keisuke Koyama, Tsukino Ito, Toshiyuki Suzuki, Ahmed Z Balboula, Takahiro Shirozu, Hanako Bai, Masashi Nagano, Manabu Kawahara, Masashi Takahashi
    Journal of dairy science 101 (9) 8396 - 8400 0022-0302 2018/09 [Refereed][Not invited]
     
    In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.
  • Dulal C. Kabiraz, Kinichi Morita, Kazuhira Sakamoto, Masashi Takahashi, Toshikazu Kawaguchi
    Talanta 186 521 - 526 0039-9140 2018/08/15 [Refereed][Not invited]
     
    This study proposed the filtration method for removal of inhibitors from real urine samples for immunoassay without centrifuge. Although the inhibitors could not be removed by the physical filtration, the carboxyl group terminated silica effectively removed the inhibitors. In a low pH, antibody formed aggregation due to the protonation. We propose to adjust pH of the sample solution by adding a phosphate buffer solution (pH 7.5). As a result of pretreatment, the SPR immunosensing achieved the SPR signal of 45 mdeg and a low limit of detection with 100 ppq (100 fg mL−1).
  • Abdel-Ghani MA, Yanagawa Y, Balboula AZ, Sakaguchi K, Kanno C, Katagiri M, Takahashi M, Nagano M
    Reproduction, Fertility and Development 31 (2) 272 - 281 2018/08 [Refereed][Not invited]
     
    In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17β-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.
  • Ken-ichi Yamanaka, Hafiza Khatun, Junki Egashira, Ahmed Zaky Balboula, Hideki Tatemoto, Miki Sakatani, Naoki Takenouchi, Yasuhiko Wada, Masashi Takahashi
    Theriogenology 114 293 - 300 0093-691X 2018/07/01 [Refereed][Not invited]
     
    Heat stress can cause significant reproductive dysfunction in mammals and previous studies report that expression and activity of cathepsin B (CTSB), a lysosomal cysteine protease, is negatively correlated with the developmental competence of bovine oocytes and embryos. However, the relationship between heat shock (HS) and CTSB remains largely unknown. Here, we investigated the effects of HS during IVF and early embryonic stages of IVC on CTSB activity and developmental competence in bovine embryos. HS (40 °C for 6 h during IVF and 20 h during IVC) caused a significant increase in CTSB activity irrespective of the developmental stage or duration of HS. The developmental rate to the blastocyst stage was also significantly decreased by HS. Additionally, HS during IVC significantly increased the number of apoptotic cells in blastocysts. Notably, these HS-induced changes in blastocyst development and quality were significantly improved by inhibition of CTSB activity, indicating a key role for CTSB. These results showed that CTSB activity plays an essential role in HS-induced dysfunction in bovine embryo development, and that inhibition of this activity could enhance the developmental competence of heat-shocked embryos.
  • Li G, Otsuka Y, Matsumiya T, Suzuki T, Li J, Takahashi M, Yamada K
    Materials (Basel, Switzerland) 11 (8) 2018/07 [Refereed][Not invited]
  • Yamanaka KI, Yamashita K, Khatun H, Wada Y, Tatemoto H, Sakatani M, Takenouchi N, Takahashi M, Watanabe S
    Animal science journal = Nihon chikusan Gakkaiho 89 (10) 1406 - 1414 1344-3941 2018/07 [Refereed][Not invited]
  • Md Abdus Shabur Talukder, Ahmed Zaky Balboula, Takahiro Shirozu, Sung Woo Kim, Hiroki Kunii, Toshiyuki Suzuki, Tsukino Ito, Koji Kimura, Masashi Takahashi
    Reproduction (Cambridge, England) 155 (6) 515 - 528 1470-1626 2018/06 [Refereed][Not invited]
     
    In ruminants, interferon-tau (IFNT)-mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy.
  • Hiroki Akizawa, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Shinjiro Kagawa, Hiroaki Nagatomo, Manabu Kawahara
    Reproduction 155 (6) 563 - 571 1470-1626 2018/06 [Refereed][Not invited]
     
    The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that theTEAD4mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;CDX2,GATA2andCCN2, in theTEAD4-knockdown (KD) blastocysts. These expression levels significantly decreased in theTEAD4KD blastocysts compared with controls. Of these downregulated genes, theCCN2expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;CDX2,GATA2andTEAD4in theCCN2KD blastocysts. Strikingly, theCCN2KD blastocysts showed the downregulation ofCDX2,GATA2andTEAD4. Furthermore, the ratio of TE-to-ICM cell numbers in theCCN2KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation ofCCN2expression thoroughTEAD4in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation ofTEAD4andCCN2is required for TE development with appropriate gene expression in bovine blastocysts.
  • Kohta Kikuchi, Keisuke Sasaki, Hiroki Akizawa, Hayato Tsukahara, Hanako Bai, Masashi Takahashi, Yasuo Nambo, Hiroshi Hata, Manabu Kawahara
    The Journal of reproduction and development 64 (1) 57 - 64 0916-8818 2018/02/27 [Refereed][Not invited]
     
    Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5'- and 3'-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses.
  • Pregnancy-induced expression of interferon-stimulated genes in cervical and vaginal mucosal membrane.
    Kunii H, Koyama K, Ito T, Suzuki T, Balboula A.Z, Shirozu T, Bai H, Nagano M, Kawahara M, Takahashi M
    Journal of Dairy Science 101 1 - 5 2018 [Refereed][Not invited]
  • Kohta Kikuchi, Keisuke Kozai, Takuo Hojo, Miki Sakatani, Kiyoshi Okuda, Hanako Bai, Manabu Kawahara, Masashi Takahashi
    Journal of Reproduction and Development 64 (2) 193 - 197 1348-4400 2018 [Refereed][Not invited]
     
    We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.
  • Miki Sakatani, Kenichi Yamanaka, Ahmed Zaky Balboula, Masashi Takahashi
    ANIMAL SCIENCE JOURNAL 88 (12) 1934 - 1942 1344-3941 2017/12 [Refereed][Not invited]
     
    The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine invitro-produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and invitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0=day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P<0.05). Second, the effect of sperm pre-incubation for 4h on subsequent invitro fertilization was assessed using different sperm genetic backgrounds. The pre-incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0 degrees C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.
  • Wenjing Yan, Chihiro Kanno, Eiki Oshima, Yukiko Kuzuma, Sung Woo Kim, Hanako Bai, Masashi Takahashi, Yojiro Yanagawa, Masashi Nagano, Jun-ichi Wakamatsu, Manabu Kawahara
    ANIMAL REPRODUCTION SCIENCE 185 195 - 204 0378-4320 2017/10 [Refereed][Not invited]
     
    Improving sperm motility and viability are major goals to improve efficiency in the poultry industry. In this study, the effects of supplemental dietary turmeric by-product (TBP) from commercial turmeric production on sperm motility, viability, and antioxidative status were examined in domestic fowl. Mature Rhode Island Red roosters were divided into two groups controls (group C) without TBP administration and test subjects (group T) fed a basal diet supplemented with 0.8 g of TBP/day in a temperature-controlled rearing facility (Experiment 1) and 1.6 g/day under heat stress (Experiment 2) for 4 weeks. In Experiment 1, TBP dietary supplementation increased the sperm motility variables straight-line velocity, curvilinear velocity, and linearity based on a computer-assisted semen analysis, 2 weeks following TBP supplementation. In Experiment 2, using flow cytometry, sperm viability at 3 and 4 weeks following TBP supplementation was greater in Group T than C, and this increase was consistent with a reduction in reactive oxygen species (ROS) production at 2 and 4 weeks. The results of both experiments clearly demonstrate that dietary supplementation with TBP enhanced sperm motility in the controlled-temperature conditions as well as sperm viability, and reduced ROS generation when heat stress prevailed. Considering its potential application in a range of environments, TBP may serve as an economical and potent antioxidant to improve rooster fertility.
  • 妊娠初期の牛におけるリソソームカテプシンと白血球中リソソームの評価(Evaluation of lysosomal cathepsins and lysosomes in bovine blood leukocytes during early pregnancy)
    Talukder Md Abdus Shabur, Balboula Ahmed Z, 岩野 弘暉, 鈴木 惇文, 伊藤 月乃, 唄 花子, 川原 学, 高橋 昌志
    日本畜産学会大会講演要旨集 123回 84 - 84 1342-4688 2017/09
  • Masahiro Kaneda, Masashi Takahashi, Ken-ichi Yamanaka, Koji Sait, Masanori Taniguchi, Satoshi Akagi, Shinya Watanabe, Takashi Nagai
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 63 (4) 365 - 375 0916-8818 2017/08 [Refereed][Not invited]
     
    Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG]/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKNIIC was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.
  • Takahiro Shirozu, Hiroki Iwano, Takatoshi Ogiso, Toshiyuki Suzuki, Ahmed Z Balboula, Hanako Bai, Manabu Kawahara, Koji Kimura, Hitomi Takahashi, Bai Rulan, Sung-Woo Kim, Yojiro Yanagawa, Masashi Nagano, Kazuhiko Imakawa, Masashi Takahashi
    The Journal of reproduction and development 63 (3) 211 - 220 0916-8818 2017/06/21 [Refereed][Not invited]
     
    Interferon tau (IFN-τ) is a ruminant-specific type I IFN secreted by a conceptus before its attachment to the uterus. IFN-τ induces the expression of IFN-stimulated genes (ISGs) via the type I IFN receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits in the endometrium. However, expression patterns of IFNARs during the estrous cycle have not been reported. We hypothesized that the response to a type I IFN changes along with IFNARs and the IFN-regulatory factors (IRFs) driving transcription of IFN signal-related genes and modulating a type I IFN signal during the estrous cycle. We investigated the estrous cycle stage-dependent type I IFN induction of ISGs and expression patterns of IFN signal-related genes in bovine endometrial tissues. Endometrial tissue pieces collected from bovine uteri at each estrous stage (early, mid, and late) were cultured with or without recombinant bovine IFN-α or concentrated pregnant uterine flushing (PUF) on day 18 after confirming the presence of a conceptus. IFN-α and PUF each significantly increased the expression of ISGs in endometrial tissues. The induction levels of the typical ISGs (MX1-a and ISG15) were significantly higher at the mid stage and correlated with high expression of IRFs at the mid stage. The immunostaining of IFNARs showed strong fluorescence intensities in luminal and glandular epithelia at the early and mid stages. Collectively, these results suggest that the endometrium exhibits estrous cycle stage-dependent responsiveness to type I IFN that may be associated with the expression of IFNARs and IRFs for pregnancy recognition.
  • Ahmed Z. Balboula, Cecilia S. Blengini, Amanda S. Gentilello, Masashi Takahashi, Karen Schindler
    BIOLOGY OF REPRODUCTION 96 (6) 1197 - 1209 0006-3363 2017/06 [Refereed][Not invited]
     
    During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT) attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes. Summary Sentence Maternal RNA contained in mouse oocytes regulates localized AURKC-CPC activity independent of its role in translation to support meiotic maturation.
  • Mansour Aboelenain, Ahmed Zaky Balboula, Manabu Kawahara, Abd El-Monem Montaser, Samy Moawad Zaabel, Sung-Woo Kim, Masashi Nagano, Masashi Takahashi
    THERIOGENOLOGY 91 127 - 133 0093-691X 2017/03 [Refereed][Not invited]
     
    Recently, inhibition of cathepsin B (CTSB) activity during in vitro maturation (IVM) and culture (NC) improved the developmental competence and quality of bovine oocytes and embryos. E-64 is a widely used inhibitor to inhibit CTSB activity, however, E-64 inhibits not only CTSB activity but also the activities of other proteases including cathepsin L (CTSL), papain, calpain, and trypsin. Pyridoxine, the catalytically active form of vitamin B6, plays a crucial role in several cellular processes and has the ability to inhibit CTSB activity. However, whether pyridoxine has an improving effect during IVM of bovine oocytes is still unknown. In this study, we investigated the effect of pyridoxine supplementation during IVM on the developmental competence of bovine oocytes and the quality of the produced blastocysts. Supplementation of pyridoxine to the maturation medium significantly decreased the activity of CTSB in both bovine cumulus cells and oocytes. Moreover, pyridoxine improved both the blastocyst and hatched blastocyst rates. In addition, the presence of pyridoxine during IVM also significantly improved the quality of the produced embryos by increasing the total cell number as well as decreasing the CTSB mRNA expression and apoptotic rate. These results indicate that pyridoxine is a promising tool to improve the developmental competence of bovine oocytes and subsequent embryo quality. (C) 2017 Elsevier Inc. All rights reserved.
  • Wataru Yamazaki, Tomoko Amano, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (40) 20924 - 20931 0021-9258 2016/09 [Refereed][Not invited]
     
    Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes inDNAmethylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals.
  • Hiroki Akizawa, Hiroaki Nagatomo, Haruka Odagiri, Nanami Kohri, Nobuhiko Yamauchi, Yojiro Yanagawa, Masashi Nagano, Masashi Takahashi, Manabu Kawahara
    MOLECULAR REPRODUCTION AND DEVELOPMENT 83 (6) 516 - 525 1040-452X 2016/06 [Refereed][Not invited]
     
    A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P< 0.05) -which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle.
  • Keisuke Kozai, Shota Tokuyama, Anna Z. Szostek, Yuko Toishi, Nobuo Tsunoda, Kazuyoshi Taya, Miki Sakatani, Masashi Takahashi, Yasuo Nambo, Dariusz J. Skarzynski, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda
    REPRODUCTION 151 (5) 517 - 526 1470-1626 2016/05 [Refereed][Not invited]
     
    In mares, prostaglandin F-2 alpha (PGF(2 alpha)) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF(2 alpha) can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 mu g) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P-4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF(2 alpha) (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF(2 alpha) significantly stimulated (P < 0.05) PGF(2 alpha) production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF(2 alpha) synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF(2 alpha) auto-amplification system in mares.
  • Miki Sakatani, Masashi Takahashi, Naoki Takenouchi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 62 (2) 201 - 207 0916-8818 2016/04 [Refereed][Not invited]
     
    Recently, weak estrous behavior was assumed to be the cause of a decline in breeding efficiency in cattle. The present study investigated the effect of measuring the vaginal temperature on the detection of estrus in Japanese Black cows. First, the effect of hormone administration to cows with a functional corpus luteum on the vaginal temperature was evaluated by continuous measurement using a temperature data logger. After 24 h of cloprostenol (PG) treatment, the vaginal temperature was significantly lower than on day 7 after estrus, and the low values were maintained until the beginning of estrus (P < 0.05). The cows that received PG and exogenous progesterone (CIDR) did not show a temperature decrease until the CIDR was removed. This finding suggested that the vaginal temperature change reflected the progesterone concentration. The rate of detection of natural estrus was lower for a pedometer than for the vaginal temperature (P < 0.05); synchronization of estrus resulted in a high estrus detection rate regardless of the detection method. In a subsequent experiment, the effect of vaginal temperature measurement and the use of a pedometer on estrus detection was evaluated in the cool and hot seasons. The average activities during non-estrus and the activity increase ratio (estrus/non-estrus) changed according to season (P < 0.01, P < 0.05). However, the average vaginal temperatures during estrus and non-estrus were not affected by season. The estrus detection rate of the pedometer was lower in summer and lower than that obtained using the vaginal temperature. These results indicated that vaginal temperature measurement might be effective for detecting estrus regardless of estrous behavior.
  • 牛卵細胞と着床前胚におけるリソソームカテプシンの動態(Dynamics of lysosomal cathepsin in bovine oocyte and preimplantation embryos)
    Li Jianye, 前地 真奈, 郡 七海, Aboelenain Mansour, Balboula Ahmed-Zaky, 金 星佑, 成 煥厚, 唄 花子, 川原 学, 高橋 昌志
    日本畜産学会大会講演要旨集 121回 190 - 190 1342-4688 2016/03
  • Hiroaki Nagatomo, Nanami Kohri, Hiroki Akizawa, Yumi Hoshino, Nobuhiko Yamauchi, Tomohiro Kono, Masashi Takahashi, Manabu Kawahara
    ANIMAL SCIENCE JOURNAL 87 (3) 457 - 461 1344-3941 2016/03 [Refereed][Not invited]
     
    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle.
  • Keisuke Sasaki, Aiko Tanaka, Hiroaki Nagatomo, Hidehiko Ogawa, Ken Kobayashi, Masashi Takahashi, Manabu Kawahara
    Journal of Genital System & Disorders 05 (01) 2325-9728 2016 [Refereed][Not invited]
  • Takahiro Shirozu, Keisuke Sasaki, Manabu Kawahara, Yojiro Yanagawa, Masashi Nagano, Nobuhiko Yamauchi, Masashi Takahashi
    The Journal of reproduction and development 62 (1) 29 - 35 0916-8818 2016 [Refereed][Not invited]
     
    MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants-namely, MX1-a and MX1B. In ruminants, IFN-τ-a type I IFN-is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14-18, 25-40, 50-70, 80-100, and 130-150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14-18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.
  • Wataru Yamazaki, Masashi Takahashi, Manabu Kawahara
    ZYGOTE 23 (6) 874 - 884 0967-1994 2015/12 [Refereed][Not invited]
     
    Eukaryotic species commonly contain a diploid complement of chromosomes. The diploid state appears to be advantageous for mammals because it enables sexual reproduction and facilitates genetic recombination. Nonetheless, the effects of DNA ploidy on mammalian ontogeny have yet to be understood. The present study shows phenotypic features and expression patterns of imprinted genes in tripronucleate diandric and digynic triploid (DAT and DGT) mouse fetuses on embryonic day 10.5 (E10.5). Measurement of crown-rump length revealed that the length of DGT fetuses (1.87 +/- 0.13 mm; mean +/- standard error of the mean) was much smaller than that of diploid fetuses (4.81 +/- 0.05 mm). However, no significant difference was observed in the crown-rump length between diploid and DAT fetuses (3.86 +/- 0.43 mm). In DGT fetuses, the expression level of paternally expressed genes, Igf2, Dlk1, Ndn, and Peg3, remained significantly reduced and that of maternally expressed genes, Igf2r and Grb10, increased. Additionally, in DAT fetuses, the Igf2 mRNA expression level was approximately twice that in diploid fetuses, as expected. These results provide the first demonstration that imprinted genes in mouse triploid fetuses show distinctive expression patterns independent of the number of parental-origin haploid sets. These data suggest that both DNA ploidy and asymmetrical functions of parental genomes separately influence mammalian ontogeny.
  • Hiroaki Nagatomo, Hiroki Akizawa, Ayari Sada, Yasunori Kishi, Ken-ichi Yamanaka, Tetsuya Takuma, Keisuke Sasaki, Nobuhiko Yamauchi, Yojiro Yanagawa, Masashi Nagano, Tomohiro Kono, Masashi Takahashi, Manabu Kawahara
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (4) 159 - 171 0047-1917 2015/11 [Refereed][Not invited]
     
    There has been no work on spatiotemporal transcriptomic differences of blastocysts using in vivo- and in vitro-derived, and somatic cell nuclear transfer (SCNT) embryos. Here, we first compared the lineage-differentially transcriptomic profiles of in vivo- and in vitro-derived embryos by microarray analysis using divided into inner cell mass (ICM)-and trophectoderm (TE)-side samples, as well as those derived from SCNT in order to explore lineage-differentially expressed genes that are associated with preimplantation development in cattle. The transcriptomic profiles of the ICM-specific and TE-specific genes were similar between in vitro-derived embryos and in vivo-derived embryos, whereas SCNT embryos exhibited unusual lineage-differentially gene expression regulation at the blastocyst stage. The genes expressed in a spatiotemporal manner between developmentally normal in-vivo derived blastocysts and developmentally abnormal SCNT blastocysts might play critical roles for preimplantation development. Comparing spatial expression dynamics of bovine blastocyst under three different procedures revealed that CHTA was expressed in ICM-side samples of all the embryo types. CIITA is known as the master regulator of major histocompatibility complexes (MHC) class II genes that express in antigen-presenting cells but its biological function in preimplantation embryo is still unknown in mammals. Knockdown of CIITA expression in in vitro-derived embryos did not affect cleavage, but disrupted development of embryos into the blastocyst stage. These findings provide the novel transcriptomic information on blastocyst formation, raising the possibility that immune function-related gene directly plays important roles in bovine preimplantation development.
  • Manabu Kawahara, Shiori Koyama, Satomi Iimura, Wataru Yamazaki, Aiko Tanaka, Nanami Kohri, Keisuke Sasaki, Masashi Takahashi
    SCIENTIFIC REPORTS 5 14512  2045-2322 2015/09 [Refereed][Not invited]
     
    Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals.
  • Saiko Sugawara, Toshihiko Ito, Shiori Sato, Yuki Sato, Akira Sasaki, Tomokazu Fukuda, Ken-ichi Yamanaka, Miki Sakatani, Masashi Takahashi, Masayuki Kobayashi
    BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY 62 (2) 164 - 172 0885-4513 2015/03 [Refereed][Not invited]
     
    Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro(32) to Leu(206)) proteins without a secretory signal peptide at the aminoterminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser(54) and Leu(55) in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu(55) to Leu(206)) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs. (C) 2014 International Union of Biochemistry and Molecular Biology, Inc.
  • Miki Sakatani, Kenichi Yamanaka, Ahmed Z. Balboula, Naoki Takenouchi, Masashi Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 82 (1) 36 - 47 1040-452X 2015/01 [Refereed][Not invited]
     
    Low pregnancy rates during the summer are due, in part, to reduced fertilization. Given that elevated temperature is associated with this season, we investigated the effect of heat stress during fertilization using an in vitro model. Three experiments were performed to determine the mechanism by which exposure to elevated temperature disrupts fertilization. Oocytes were fertilized for 6hr at 38.5 degrees C or 41.0 degrees C or 40.0 degrees C with non-pre-incubated sperm, or for 6hr at 38.5 degrees C with sperm that had been pre-incubated at 38.5 degrees C or 41.0 degrees C for 4hr. In each experiment, zygotes were cultured at 38.5 degrees C in 5% CO2 and 5% O-2. Rates of cleavage and blasocyst formation were reduced when fertilization occurs at elevated temperatures. The percent of sperm classified as alive, using fluorescein diacetate labeling, was decreased by pre-incubation and fertilization at 40.0 degrees C. Although no difference was observed in sperm penetration rate, polyspermy tended to be increased by heat stress during fertilization. The zona pellucidae of zygotes formed following fertilization at 40.0 degrees C for 6hr were more sensitive to digestion with pronase. Furthermore, these zygotes exhibited higher hydrogen peroxide levels, measured by 2,7-dihydrodichlorofluorescein diacetate staining, and showed increased transcript abundance for HSPA1A, a gene involved in the heat-shock response, but decreased transcript abundance for UCHL1, a gene involved in preventing polyspermy. Results indicate that heat stress during fertilization is lethal to sperm, and causes oxidative stress, altered transcript abundance, and a defective block to polyspermy in the zygote. Thus, an increase in polyspermy is likely one cause of the reduced competency of zygotes fertilized under elevated temperatures to develop to the blastocyst stage. Mol. Reprod. Dev. 82: 36-47, 2015. (c) 2014 Wiley Periodicals, Inc.
  • Mansour Aboelenain, Manabu Kawahara, Ahmed Zaky Balboula, Abd El-Monem Montasser, Samy Mowaed Zaabel, Kiyoshi Okuda, Masashi Takahashi
    The Journal of reproduction and development 61 (3) 229 - 36 0916-8818 2015 [Refereed][Not invited]
     
    Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.
  • Kazuki Yamagami, Nobuhiko Yamauchi, Kaiyu Kubota, Sho Nishimura, Vishwajit Sur Chowdhury, Kenichi Yamanaka, Masashi Takahashi, Shoji Tabata, Masa-aki Hattori
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 (6) 468 - 475 0916-8818 2014/12 [Refereed][Not invited]
     
    The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles ofFoxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hoimones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant HedgehOg protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.
  • Keisuke Kozai, Takuo Hojo, Shota Tokuyama, Anna Z Szóstek, Masashi Takahashi, Miki Sakatani, Yasuo Nambo, Dariusz J Skarzynski, Kiyoshi Okuda
    The Journal of reproduction and development 60 (2) 150 - 4 0916-8818 2014/04/24 [Refereed][Not invited]
     
    Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.
  • Hitomi Takahashi, Makoto Tsunazaki, Takashi Hamano, Masashi Takahashi, Kiyoshi Okuda, Shigeki Inumaru, Akira Okano, Masaya Geshi, Makoto Hirako
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (3) 447 - 451 0916-7250 2014/03 [Refereed][Not invited]
     
    Bovine interferon (bIFN) tau plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mari (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFN tau (rbIFN tau) on prostaglandin (PG) F-2 alpha synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFN tau and OT were shown to suppress PGF(2 alpha) production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFN tau, Bm-rbIFN tau (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFN tau was suggested to have the same bioactivity as native IFN tau.
  • Daisuke Sugiyama, Yuki Teshima, Kenichi Yamanaka, Maria Portia Briones-Nagata, Masatoshi Maeki, Kenichi Yamashita, Masashi Takahashi, Masaya Miyazaki
    ANALYTICAL METHODS 6 (1) 308 - 311 1759-9660 2014 [Refereed][Not invited]
     
    We investigate the behaviour of a simple microfluidic device designed to separate particles based on density. The device consists of a separation-channel with three inlet and two outlet channels. The particle samples were loaded in the middle of the main channel. The results of separation experiments using model particles provide clear evidence that this approach can be used to achieve good separation of particles of close density populations. Particle separation was found more favourable with low flow rates. Forces exerted on particles were modelled by Stokes' law and found to be consistent with experimental results. One advantage of this microfluidic system is low exposure of the sample to hydrodynamic shear stresses compared with conventional methods. This device may be adopted to precisely handle single cells and easily interface with other tools and separation techniques.
  • A. Z. Balboula, K. Yamanaka, M. Sakatani, M. Kawahara, A. O. Hegab, S. M. Zaabel, M. Takahashi
    REPRODUCTION 146 (4) 407 - 417 1470-1626 2013/10 [Refereed][Not invited]
     
    Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 degrees C for 22 h (control group) or at 38.5 degrees C for 5 h followed by 41 degrees C for 17 h (heat shock group) either with or without 1 mu M E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 mu M E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.
  • Hiroaki Nagatomo, Shinjiro Kagawa, Yasunori Kishi, Tetsuya Takuma, Ayari Sada, Ken-ichi Yamanaka, Yasuyuki Abe, Yasuhiko Wada, Masashi Takahashi, Tomohiro Kono, Manabu Kawahara
    BIOLOGY OF REPRODUCTION 88 (6) doi: 10.1095/biolreprod.113.10  0006-3363 2013/06 [Refereed][Not invited]
     
    Mice and cattle use distinct pathways for the first cell segregation into inner cell mass (ICM) and trophectoderm (TE) lineages at the blastocyst stage. However, limited knowledge is available regarding the reliable transcriptional networks that orchestrate the complex developmental processes at this stage in nonrodent species. In order to elucidate the site-dominant transcriptomic properties of bovine blastocysts, we separated cell samples into the ICM and TE using both mechanical and chemical methods and performed in silico prescreening for candidate genes that were site-dominantly expressed in bovine blastocysts. We further performed quantitative real-time PCR and in situ hybridization using the site-specific cell samples. As a result, we identified seven ICM-dominant genes and five TE-dominant genes not found in earlier studies. Our findings provide novel insights into the mechanism of cell-fate specification in the pre-implantation bovine embryo.
  • Akira Aono, Hiroaki Nagatomo, Tetsuya Takuma, Rika Nonaka, Yoshitaka Ono, Yasuhiko Wada, Yasuyuki Abe, Masashi Takahashi, Tomomasa Watanabe, Manabu Kawahara
    THERIOGENOLOGY 79 (8) 1146 - 1152 0093-691X 2013/05 [Refereed][Not invited]
     
    The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 +/- 2.1% vs. 65.0 +/- 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method. (C) 2013 Elsevier Inc. All rights reserved.
  • Saiko Sugawara, Toshihiko Ito, Sho Sato, Mari Yokoo, Yuki Mori, Kano Kasuga, Ikuo Kojima, Tomokazu Fukuda, Ken-ichi Yamanaka, Miki Sakatani, Masashi Takahashi, Masayuki Kobayashi
    ANIMAL SCIENCE JOURNAL 84 (3) 275 - 280 1344-3941 2013 [Refereed][Not invited]
     
    Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro32-Leu206) compared with the sequence previously reported. In the present study, HisbFGF4, a 6x histidine-tagged bovine FGF4 (Pro32-Leu206), was produced in Escherichiacoli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly25-Leu206) produced in E.coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E.coli utilizing its structural gene elucidated by us.
  • Miki Maehara, Hitomi Matsunari, Kasumi Honda, Kazuaki Nakano, Yasuhiro Takeuchi, Takahiro Kanai, Taisuke Matsuda, Yukina Matsumura, Yui Hagiwara, Norihisa Sasayama, Akio Shirasu, Masashi Takahashi, Masahito Watanabe, Kazuhiro Umeyama, Yutaka Hanazono, Hiroshi Nagashima
    BIOLOGY OF REPRODUCTION 87 (6) 133  0006-3363 2012/12 [Refereed][Not invited]
     
    In vitro matured (IVM) oocytes have been used to create genetically modified pigs for various biomedical purposes. However, porcine embryos derived from IVM oocytes are very cryosensitive. Developing improved cryopreservation methods would facilitate the production of genetically modified pigs and also accelerate the conservation of genetic resources. We recently developed a novel hollow fiber vitrification (HFV) method; the present study was initiated to determine whether this new method permits the cryopreservation of IVM oocyte-derived porcine embryos. Embryos were created from the in vitro fertilization of IVM oocytes with frozen-thawed sperm derived from a transgenic pig carrying a humanized Kusabira-Orange (huKO) gene. Morula-stage embryos were assigned to vitrification and nonvitrification groups to compare their in vitro and in vivo developmental abilities. Vitrified morulae developed to the blastocyst stage at a rate similar to that of nonvitrified embryos (66/85, 77.6% vs. 67/84, 79.8%). Eighty-eight blastocysts that developed from vitrified morulae were transferred into the uteri of three recipient gilts. All three became pregnant and produced a total of 17 piglets (19.3%). This piglet production was slightly lower, albeit not significantly, than that of the nonvitrification group (27/88, 30.7%). Approximately half of the piglets in the vitrification (10/17, 58.8%) and nonvitrification (15/27, 55.6%) groups were transgenic. There was no significant difference in the growth rates among the piglets in the two groups. These results indicate that the HFV method is an extremely effective method for preserving cryosensitive embryos such as porcine in vitro maturation/fertilization-derived morulae.
  • Hitomi Matsunari, Miki Maehara, Kazuaki Nakano, Yuka Ikezawa, Yui Hagiwara, Norihisa Sasayama, Akio Shirasu, Hisayoshi Ohta, Masashi Takahashi, Hiroshi Nagashima
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 (5) 599 - 608 0916-8818 2012/10 [Refereed][Not invited]
     
    Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 mu l of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/group). The HFV method was also proven to be effective for vitrifying porcine in vitro- and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs.
  • Keisuke Kozai, Takuo Hojo, Masashi Takahashi, Tomas J. Acosta, Yasuo Nambo, Kiyoshi Okuda
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 (4) 393 - 397 0916-8818 2012/08 [Refereed][Not invited]
     
    Although circulating progesterone (P-4) levels tend to change with the season, little is known about the seasonal changes of P-4 synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (similar to N32 degrees), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P-4 concentration, was used to evaluate the seasonal changes in P-4 synthesis. The luteal P-4 concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase (3 beta-HSD) were lowest during early winter and increased during late winter. These results suggest that P-4 synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P-4 synthesis-related proteins in mares.
  • M. Sakatani, N. V. Alvarez, M. Takahashi, P. J. Hansen
    JOURNAL OF DAIRY SCIENCE 95 (6) 3080 - 3091 0022-0302 2012/06 [Refereed][Not invited]
     
    The goal was to understand the role of heat shock at the zygote stage in causing infertility. Culture at 40 degrees C reduced the percentage of inseminated oocytes that became a morula or blastocyst by d 6 or that were a blastocyst at d 8. An additional experiment was done to test whether effects of heat shock occur early in development or at the time of morula formation. Exposure to 40 degrees C for 24 h decreased development to the blastocyst stage if exposure was at the zygote stage [8 to 32 h postinsemination (hpi)] but not if exposure occurred at the morula stage (116 to 140 hpi). To test effect of oxygen concentration; inseminated oocytes were cultured at 40 degrees C for 12 or 24 h in either air (20.95% O-2; high oxygen) or a 5% (vol/vol) O-2 environment (low oxygen) that approximates the partial oxygen pressure of the reproductive tract. Blastocyst development was reduced by 40 degrees C for 12 or 24 h under both atmospheres and was higher for embryos cultured in low oxygen than for embryos cultured in high oxygen. Examination of cell numbers at 72 hpi indicated that heat shock reduced developmental potential of embryos by reducing competence to complete cleavage divisions after first cleavage. Changes in expression of genes involved in heat shock and oxidative stress were measured to determine whether zygotes are more susceptible to heat shock because of reduced capacity for transcription. Heat shock was performed for 24 h at the 1-cell stage (expression examined in 2-cell embryos) or at d 5 (examined in morulae). Heat shock increased amounts of steady-state mRNA for HSPA1A but not for HSP90AA, SOD1, or CAT. We observed a tendency for a stage x temperature interaction for HSPA1A because the difference in expression between 38.5 and 40 degrees C was greater for morulae than for 2-cell embryos. The amount of HSPA1A mRNA was less for morulae that were heat shocked than for 2-cell embryos cultured at 38.5 degrees C. Heat shock at a temperature and oxygen tension similar to those seen in vivo can disrupt developmental competence of bovine zygotes. Increased susceptibility of the early embryo compared with the morula to heat shock was not due to reduced HSPA1A mRNA because amounts were higher for 2-cell embryos than for morulae.
  • Masashi Takahashi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 (1) 1 - 9 0916-8818 2012/02 [Refereed][Not invited]
     
    Many factors affect development of mammalian preimplantation embryos in vitro. It is well known that in vitro development of bovine embryos is highly affected by culture condition including energy source, growth factors, or gas environment. Many efforts have been made towards the suitable environments which can successfully support embryo development in vitro. For a rapid growth and differentiation, embryo requires energy by utilizing ATP, NADPH with oxygen molecules. These energy substrates are produced from the electron transport chain ill the mitochondria. In addition to energy production, reactive oxygen species (ROS) are also generated as by-product of such energy production system. ROS production is sensitively controlled by the balance of oxidizing and reducing status and affected by several antioxidant enzymes such as superoxide dismutase (SOD), Catalase, glutathione peroxidase (GPx) or low molecular weight thiols such as glutathione (GSH). Imbalance of oxidation and reduction causes production of excess ROS, which causes the developmental arrest, physical DNA damage, apoptosis induction or lipid peroxidation. Environmental oxygen condition during embryo culture also highly affects embryo development as well as intracellular redox balance. Several studies have revealed that regulation of intra- and extra- cellular reducing environment by reducing excess ROS by using antioxidants, reducing oxygen concentration are effective for improving embryo development. Also, recent studies have demonstrated the difference in gene expression affected by oxidative stress. This review briefly summarizes the effects of ROS and the role of redox balance on preimplantation embryos for improving the efficiency of in vitro production of mammalian embryos.
  • Miki Sakatani, Ahmed Z. Balboula, Kenichi Yamanaka, Masashi Takahashi
    ANIMAL SCIENCE JOURNAL 83 (5) 394 - 402 1344-3941 2012 [Refereed][Not invited]
     
    This study investigated the effect of summer heat environment on estrous cycles and blood antioxidant levels in Japanese Black cows. A total of 13 non-lactating Japanese Black cows (summer: 9, winter: 4) were examined. Body temperature was measured rectally and intravaginally using a thermometer and data logger, respectively. Estrous behavior was monitored using a radiotelemetric pedometer that recorded walking activity. Rectal temperatures were higher during summer than winter (P < 0.001). There was an acute increase in vaginal temperature at the onset of estrus during winter but such an increase was not observed during summer. Walking activity during estrus decreased dramatically in the summer compared to the winter. Duration of estrous cycle was longer in summer (23.4 days, P < 0.05) than winter (21.5 days), and the subsequent rise in progesterone concentrations following estrus tended to be delayed in summer. The level of thiobarbituric acid reactive substances (TBARS) in peripheral blood cells was higher during summer (P < 0.05), while the levels of superoixde dismutase (SOD), glutathione peroxidase (GPx) and glutathione were lower (P < 0.05). These results indicate that high ambient temperature during summer increases both body temperature and oxidative stress, and also reduces signs of estrus in Japanese Black cows.
  • YAMANAKA Ken-ichi, KANEDA Masahiro, INABA Yasushi, SAITO Koji, KUBOTA Kaiyu, SAKATANI Miki, SUGIMURA Satoshi, IMAI Kei, WATANABE Shinya, TAKAHASHI Masashi
    Animal science journal 82 (4) 523 - 530 1344-3941 2011/08/01 [Not refereed][Not invited]
     
    Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.
  • YAMANAKA Ken-ichi, SAKATANI Miki, KUBOTA Kaiyu, BALBOULA Ahmed Zaky, SAWAI Ken, TAKAHASHI Masashi
    Journal of Reproduction and Development 日本繁殖生物学会 57 (3) 393 - 402 0916-8818 2011/06/01 [Not refereed][Not invited]
     
    For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.
  • Ken Sawai, Masashi Takahashi, Takashi Fujii, Satoru Moriyasu, Hiroki Hirayama, Akira Minamihashi, Tsutomu Hashizume, Sadao Onoe
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 57 (2) 236 - 241 0916-8818 2011/04 [Refereed][Not invited]
     
    DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.
  • Kazuaki Nakano, Hitomi Matsunari, Naoki Nakayama, Buko Ogawa, Mayuko Kurome, Masashi Takahashi, Mitsuhito Matsumoto, Hitoshi Murakami, Yuji Kaji, Hiroshi Nagashima
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 57 (2) 312 - 316 0916-8818 2011/04 [Refereed][Not invited]
     
    The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.
  • A. Z. Balboula, K. Yamanaka, M. Sakatani, A. O. Hegab, S. M. Zaabel, M. Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 77 (12) 1031 - 1039 1040-452X 2010/12 [Refereed][Not invited]
     
    Recently, the activity of cathepsins B was found to be correlated inversely with the developmental competence of bovine oocytes. In this study, we investigated (1) the role of intracellular cathepsin B expression and developmental competence as well as the quality of bovine preimplantation embryos, and (2) the effect of cathepsin B inhibitor (E-64) during in vitro culture (IVC) on the development and quality of bovine embryos. After in vitro fertilization (IVF) followed by IVC for 7 days, good and poor quality embryos classified by morphology and developmental rate on days 2, 4, and 7 were assessed for cathepsin B expression and activity. To investigate the effect of cathepsin B inhibition on embryonic development, putative zygotes were cultured with or without E-64, followed by evaluation of cleavage and blastocyst rates on days 2 and 7, respectively. Embryonic quality was evaluated by both TUNEL staining and total cell number in day-7 blastocysts. In each developmental stage, cathepsin B expression and activity were significantly higher in poor quality embryos than good quality ones. Moreover, addition of E-64 during IVC significantly increased both the blastocyst rate and the total cell number. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in day-7 blastocysts. These results indicate that cathepsin B activity can be useful as a marker for inferior quality embryos. Moreover, inhibition of cathepsin B greatly improves the developmental competence of preimplantation embryos and increases the number of good quality embryos.
  • Marta I. Siemieniuch, Magdalena Majewska, Masashi Takahashi, Miki Sakatani, Karolina Lukasik, Kiyoshi Okuda, Dariusz J. Skarzynski
    REPRODUCTIVE BIOLOGY 10 (3) 249 - 256 1642-431X 2010/11 [Refereed][Not invited]
     
    We determined the transcript content of three genes involved in the metabolism of glucocorticoids (GC) in bovine in vitro fertilized embryos (2-blastomere stage until hatched blastocyst), trophoblast as well as the oviduct (Day 2-4 of the estrous cycle) and endometrium (Day 16 of the cycle and pregnancy). Since mRNA expression of the glucocorticoid receptor and two enzymes responsible for GC production (11 beta-HSD1 and 2) was demonstrated in the embryos in all pre-implantation stages as well as in the endometrium and oviduct, it is suggested that GC may serve as auto-/paracrine factors in the development of bovine pre-implantation embryos. Reproductive Biology 2010 10 3: 249-256.
  • A. Z. Balboula, K. Yamanaka, M. Sakatani, A. O. Hegab, S. M. Zaabel, M. Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 77 (5) 439 - 448 1040-452X 2010/05 [Refereed][Not invited]
     
    Recently, the quantity of cathepsin transcripts in cumulus cells was found to be associated with low-developmental competence of bovine oocytes In the present study, we investigated (1) the relation between cathepsin B activity and the quality of in vitro-matured cumulus oocyte complexes (IVM COCs) and denuded oocytes and (2) the effect of a cathepsin B inhibitor (E-64) on embryo development and quality The activity of cathepsin B was evaluated in IVM COCs and denuded oocytes After maturation of COCs with or without E-64, followed by in vitro fertilization, zygotes were cultured for 8 days Cleavage and blastocyst rates were evaluated on days 2 and 8, respectively. Quality of embryos was evaluated by differential staining of day 8 blastocysts TUNEL staining was conducted on IVM COCs and blastocysts Cathepsin B activity was clearly detected in the low-quality oocytes, and in the cumulus cells of both high- and low-quality oocytes This latter activity was diminished by addition of E-64. The presence of E-64 during IVM also significantly increased both the blastocyst rate and the total cell number, and improved blastocyst quality associated with a significant increase of trophoectoderm cells TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in both the cumulus cell layer of matured oocytes and blastocysts These results indicate that cathepsin B activity can be a useful marker of oocyte quality Furthermore, inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high-quality embryos.
  • Mineto Tani, Takuya Hayashida, Kouichiro Tomokawa, Yasuaki Mito, Daisuke Funakoshi, Chikako Tani, Miki Sakatani, Masashi Takahashi, Go Kitahara, Shunichi Kamimura
    JOURNAL OF VETERINARY MEDICAL SCIENCE 72 (5) 627 - 629 0916-7250 2010/05 [Refereed][Not invited]
     
    In order to improve the reproductive performance during the summer period, embryo transfer (ET; Japanese black embryo) following artificial insemination (AI; Holstein semen: ETFAI) was conducted in dairy cows in south-western Japan (n=56). The conception rate was improved in cows with ETFAI compared with conventional AI, which served as the control (n=195; 30.4% vs. 13.8%, P < 0.01). However, higher fetal loss was observed in ETFAI compared with the controls (38.1% vs. 7.4%, P < 0.05). Four cases of twin pregnancy resulted in 2 singletons and a set of twins. There was no difference in the plasma progesterone level on d0 or d7 (d0=AI), but rather lower rectal temperature was observed on d7 or d8 (38.7 degrees C vs. 39.4 degrees C and 38.8 degrees C vs. 39.1 degrees C, P < 0.05) in pregnant cows compared with those that were open. ETFAI could improve reproductive performance in dairy cows during the summer period in southwestern Japan.
  • Kaiyu Kubota, Nobuhiko Yamauchi, Kazuki Yamagami, Sho Nishimura, Takafumi Gobaru, Ken-Ichi Yamanaka, Chris Wood, Tomoki Soh, Masashi Takahashi, Masa-Aki Hattori
    Cell and Tissue Research 340 (2) 389 - 395 0302-766X 2010/05 [Refereed][Not invited]
     
    Ovarian steroid hormones, progesterone (P4), and estradiol (E2) strictly regulate the endometrial tissue remodeling required for successful embryo implantation. Indian hedgehog (Ihh) is up-regulated by P4 and critically mediates uterine receptivity in the mouse. However, the regulation of Ihh expression during the implantation period still remains unclear. The present study was conducted to elucidate the mechanism of the steroidal regulation in the expression of Ihh and Gli1, the mediator of the Ihh pathway. Ihh mRNA was expressed in the rat uterus on 3.5-5.5 days post-coitus (dpc), while Gli1 expression transiently increased at 3.5 dpc but decreased significantly on 5.5 dpc (P< 0.001). In delayed implantation, the expression of Ihh was induced by the implantation-induced E2 treatment in the primed rat uterus. In contrast, expression of Gli1 was significantly decreased by E2 treatment (P=0.016). In the case of ICI182.780 (ICI) treatment, Ihh expression was eliminated by ICI, whilst Gli1 expression increased. These results suggest that Ihh expression is maintained at a high level until the initiation of implantation, while the expression of Gli1 is decreased just prior to the initiation of implantation depending on the E2 action. This observation aids in the understanding of the Ihh signaling pathway mediating uterine remodeling for implantation. © 2010 Springer-Verlag.
  • Ken Sawai, Masashi Takahashi, Satoru Moriyasu, Hiroki Hirayama, Akira Minamihashi, Tsutomu Hashizume, Sadao Onoe
    CELLULAR REPROGRAMMING 12 (1) 15 - 22 2152-4971 2010/02 [Refereed][Not invited]
     
    The epigenetic reprogramming of the donor cell nucleus is an important factor in the development of embryos and production of normal offspring derived by somatic cell nuclear transfer (NT-SC). During early development, a dramatic reduction in methylation levels occurs in mouse. In early embryos, this process makes it possible to erase gamete-specific methylation patterns and induce de novo methylation at defined developmental time-points. To clarify changes in DNA methylation in bovine NT-SC embryos, we examined satellite I sequences in bovine embryos derived in vivo (Vivo) and by NT-SC at the blastocyst (BC) and elongated (EL) stages. Because the EL stage embryo consists of the embryo disc (ED) and trophectoderm (TE), the methylation status of each part was analyzed with respect to the progress of differentiation. DNA methylation levels in Vivo embryos were increased during the elongation stage. In contrast, DNA methylation levels in NT-SC embryos remained unchanged in the ED and significantly decreased in the TE. Real-time PCR analysis showed that Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower than that in Vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. Our results suggest that the aberrant methylation level of bovine NT-SC embryos in the satellite I region is corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates.
  • YAMANAKA Ken-ichi, BALBOULA Ahmed Zaky, SAKATANI Miki, TAKAHASHI Masashi
    The journal of reproduction and development Society for Reproduction and Development 56 (1) 60 - 67 0916-8818 2010/02/01 [Not refereed][Not invited]
     
    A highly methylated genome, like a somatic donor cell, is observed in somatic cell nuclear transfer (SCNT) embryos. The aberrant DNA methylation status causes global gene expression failure, resulting in low developmental competence of SCNT embryos. In addition, recent studies have uncovered the relationship between DNA methylation status and reprogramming efficiency. Because DNA methylation is performed by DNA methyltransferases (DNMTs), developing a technique which specifically inhibits DNMTs is necessary for further SCNT studies. In the present study, we examined the potential use of RNA interference for knockdown of DNMT mRNA in bovine fibroblast cells that were commonly used as karyoplast donors in SCNT studies. We designed three siRNAs corresponding to DNMT1, DNMT2 and DNMT3a mRNA. In Experiment 1, to optimize transfection conditions, fluorescence and cell viability after transfection were evaluated at different concentrations of transfection reagent using a FITC-labeled nonsilencing control siRNA. Although fluorescence was observed in all groups transfected except for the negative control group, transfection with a higher concentration of transfection reagent significantly decreased in cell viability (P<0.05). In Experiment 2, the amount of DNMT mRNA was measured by real-time PCR at 0, 48 and 96 h after siRNA transfection into the cells. The levels of each DNMT mRNA were significantly decreased at 48 and 96 h after transfection (P<0.01). Furthermore, decreased expression of DNMT1 protein was confirmed by western blotting. In Experiment 3, the DNA methylation statuses were analyzed in each of the siRNA-transfected groups. The DNMT1 siRNA-transfected group had a significantly decreased level of DNA methylation (P<0.05), but the other groups did not. Our data demonstrate that RNA interference with siRNA can be analyzed the function and expression of DNMT genes in bovine fibroblast cells. The present study provides useful information for further SCNT studies.
  • Hanako Bai, Toshihiro Sakurai, Min-Su Kim, Yoshikage Muroi, Atsushi Ideta, Yoshito Aoyagi, Hiromi Nakajima, Masashi Takahashi, Kentaro Nagaoka, Kazuhiko Imakawa
    MOLECULAR REPRODUCTION AND DEVELOPMENT 76 (12) 1143 - 1152 1040-452X 2009/12 [Refereed][Not invited]
     
    Expression of interferon-tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT-PCR analysis between bovine trophoblast CT-1 and Mardin-Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT-1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (-631 to +59 bp) of bovine IFNT gene (bIFNT, IFN-tau-c1), over-expression of GATA2/GATA3 did not affect the transcription of bIFNT-reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up-regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT-1 cells, endogenous bIFNT gene transcription was up-regulated by over-expression of GATA2 or GATA3, but down-regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast-specific regulation of bIFNT gene transcription.
  • Toshihiro Sakurai, Atsushi Sakamoto, Yoshikage Muroi, Hanako Bai, Kentaro Nagaoka, Kazuhiro Tamura, Toru Takahashi, Kazuyoshi Hashizume, Miki Sakatani, Masashi Takahashi, James D. Godkin, Kazuhiko Imakawa
    BIOLOGY OF REPRODUCTION 80 (6) 1223 - 1231 0006-3363 2009/06 [Refereed][Not invited]
     
    Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.
  • 吉ざわ 努, 平子 誠, 下司 雅也, 高橋 昌志, 永井 卓
    日本胚移植学雑誌 = Japanese journal of embryo transfer 31 (2) 105 - 118 1341-2965 2009/05/19 [Not refereed][Not invited]
  • 高橋 昌志, 阪谷 美樹
    畜産技術 畜産技術協会 0 (645) 2 - 9 0389-1348 2009/02 [Not refereed][Not invited]
  • 高橋 昌志, 山中 賢一, 阪谷 美樹
    日本胚移植学雑誌 = Japanese journal of embryo transfer 31 (1) 9 - 17 1341-2965 2009/01/28 [Not refereed][Not invited]
  • 松本 光史, 阪谷 美樹, 井上 寛暁, 村上 斉, 高橋 昌志, 梶 雄次
    日本養豚学会誌 = The Japanese journal of swine science 45 (4) 271 - 271 0913-882X 2008/12/25 [Not refereed][Not invited]
  • SAKATANI Miki, YAMANAKA Kenichi, KOBAYASHI Shuji, TAKAHASHI Masashi
    Journal of Reproduction and Development 日本繁殖生物学会 54 (6) 496 - 501 0916-8818 2008/12/01 [Not refereed][Not invited]
     
    Heat shock is known to increase the mortality of early stage embryos, but the exact mechanism is unclear. In the present study, we investigated the possibility that the increased mortality is caused by heat shock-generated reactive oxygen species (ROS). The level of ROS was controlled by using β-mercaptoethanol (β-ME), a scavenger of ROS. In vitro-produced 8-cell stage embryos were cultured at 38.5 C or heat-shocked by exposure to 41 C for 6 h with 0, 10 and 50 μM β-ME. Intracellular ROS levels were measured by a fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA), and intracellular reduced form of glutathione (GSH) contents were estimated by another fluorescent dye, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin. Total glutathione content was estimated by the glutathione recycling assay. On day 8 after insemination, heat shock decreased the percentage of embryos that developed to the blastocyst stage and increased intracellular ROS levels, but there was no significant effect on the GSH and total glutathione contents. In contrast, β-ME significantly decreased ROS levels in heat-shocked embryos and increased the GSH and total glutathione concentrations. Ten μM β-ME significantly improved the viability of heat-shocked embryos. β-ME caused no detrimental effects when it was added at normal culture temperature (38.5 C). These results indicate that ROS is the primary cause of increased embryonic mortality in heat-shocked early stage embryos.
  • Toshihiro Sakurai, Yoshikage Muroi, Masashi Takahashi, Toru Takahashi, Kazuyoshi Hashizume, Alan Ealy, James Godkin, Kentaro Nagaoka, Kazuhiko Imakawa
    BIOLOGY OF REPRODUCTION 141 - 141 0006-3363 2008 [Refereed][Not invited]
  • 松本 光史, 阪谷 美樹, 井上 寛暁, 村上 斉, 高橋 昌志, 梶 雄次
    日本養豚学会誌 = The Japanese journal of swine science 44 (4) 224 - 224 0913-882X 2007/12/25 [Not refereed][Not invited]
  • KOBAYASHI Shu-ichi, SAKATANI Miki, KOBAYASHI Shuji, OKUDA Kiyoshi, TAKAHASHI Masashi
    Journal of Reproduction and Development 日本繁殖生物学会 53 (6) 1305 - 1311 0916-8818 2007/12/01 [Not refereed][Not invited]
     
    Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F2α (PGF 2α) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF2α concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF2α concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF2α concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.
  • C. Suzuki, K. Yoshioka, M. Sakatani, M. Takahashi
    Zygote 15 (4) 317 - 324 0967-1994 2007/11 [Refereed][Not invited]
     
    We previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and hypotaurine (HT) during in vitro culture improved embryo development. This study examined the effects of Gln and HT on in vitro development, intracellular oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilization (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2mM Gln and 5mMHT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control) (2) Gln (3) HT or (4) Gln+ HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the intracellular GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the intracellular H 2O 2content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the development of porcine embryos by improvement of intracellular oxidative status. © 2007 Cambridge University Press.
  • HASHIYADA Yutaka, TANIGUCHI Masanori, FUJII Yoichi, MIYACHI Rie, WATANABE Akiyuki, KOZAI Chiaki, TAKAHASHI Hitomi, OKADA Masato, SUGAWARA Toru, FUJII Mitutaka, YOKOTA Masami, URATA Hirofumi, TAKAHASHI Masashi, IMAI Kei
    日本胚移植学雑誌 = Japanese journal of embryo transfer 29 (3) 114 - 124 1341-2965 2007/09/21 [Not refereed][Not invited]
  • HASHIYADA Yutaka, FUJII Yoichi, TANIGUCHI Masanori, MIYACHI Rie, WATANABE Akiyuki, URATA Hirofumi, SUGAWARA Toru, YOKOTA Masami, KOZAI Chiaki, FUJII Mitutaka, TAKAHASHI Masashi, TAKAHASHI Hitomi
    日本胚移植学雑誌 = Japanese journal of embryo transfer 29 (3) 106 - 113 1341-2965 2007/09/21 [Not refereed][Not invited]
  • Miki Sakatani, Ikuo Suda, Tomoyuki Oki, Shu-ichi Kobayashi, Shuji Kobayashi, Masashi Takahashi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 53 (3) 605 - 614 0916-8818 2007/06 [Refereed][Not invited]
     
    The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0 = day of IVF) with 0, 0.1, 1 and 10 mu g/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 mu g/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P < 0.05) and an increase in intracellular ROS (P < 0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 mu g/ml anthocyanins significantly (P < 0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P < 0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 mu g/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.
  • 岡野 彰, 高橋 ひとみ, 高橋 昌志, 下司 雅也
    Bulletin of National Institute of Livestock and Grassland Science 農業技術研究機構畜産草地研究所 0 (7) 1 - 7 1347-0825 2007/03 [Not refereed][Not invited]
     
    野生動物やヒトにおいて，エストロジェン様化学物質がエストロジェンのアゴニストあるいはアンタゴニストとしてエストロジェンレセプターと結合競合するために，内分泌かく乱物質として生殖障害を引き起こすことが報告されている。一方，ブタ子宮内膜におけるエストロジェンとエストロジェン様化学物質がエストロジェンレセプターへの結合競合を示すか否かについての報告はこれまで無い。そのため，我々はブタ子宮内膜におけるエストロジェンレセプターへのエストロジェンと5つのエストロジェン様化学物質の結合競合を明らかにしようとした。我々は子宮内膜におけるエストロジェンレセプターの性状を明らかにするため，放射レセプター測定法を実施した。子宮内膜の細胞質および核分画の調整は，1.5mM EDTA，1mM dithiothreitolを含む低張の1.5mMトリス-HCl緩衝液を用いて行った。放射レセプター測定法の最適条件を決定するため，両分画と放射性リガンドである3H-estradiol-17β(E2)を4，20および37℃で0.5，2，4，8および24時間反応させた。その結果，エストロジェンレセプターが子宮内膜の両分画に存在することが確認され，3H-E2の結合量は，分画のタンパク質量に依存し，4℃で24時間あるいは37℃で2時間の反応で最大に達した。エストロジェンレセプターの結合定数は，放射レセプター測定法の結果をもとにSctachard分析で解析した。結合定数は，発情周期の時期によって変動し，KdおよびBmaxは，それぞれ0.12～0.49nMと0.12～0.015nmol/mg proteinの範囲で推移した。3H-E2とE2および各種エストロジェン様化学物質(diethylstilbestrol: DES，p,p-isopropylidenediphenol: Bis， p-n-nonylphenol: Non，daidzein: Da，coumestrol: Coum，bis(2-ethylhexyl)phthalate: Ph)との間での結合競合を検討した結果，エストロジェンレセプターはE2およびエストロジェン様化学物質と種々の程度での親和性を示した。高い結合親和性は，Des，E2，Nonの順であった。Coum，Bis，DaそしてPhの結合親和性は，E2に比べて1,000から10,000分の1程度と低かった。
  • Shirou Matsumoto, Kenji Okumura, Akira Ogata, Yuichiro Hisatomi, Ayumi Sato, Kiyoko Hattori, Mitsuhito Matsumoto, Yuji Kaji, Masashi Takahashi, Tetsuro Yamamoto, Kimitoshi Nakamura, Fumio Endo
    Cloning and Stem Cells 9 (2) 176 - 190 1536-2302 2007 [Refereed][Not invited]
     
    Tissue stem cells participate in the repopulation of tissue after injury. Tissue injury stimulates the normally quiescent tissue stem cells to differentiate and proliferate, in the process of replacing and/or repairing the damaged cells, and hence effecting tissue regeneration. The salivary glands retain the ability for frequent regeneration. Previously, we isolated progenitor cells from the injured salivary glands of mice and rats that differentiated into hepatic and pancreatic lineages. The isolated progenitors were CD49f-positive and intracellular laminin-positive, and proliferated on type I collagen while maintaining their multipotency. In this study, we analyzed the tissue stem cells induced by ligating the main excretory duct of the salivary gland in swine. After duct ligation of the gland, acinar cells receded due to apoptosis, and epithelial cells subsequently proliferated. We cultured cells obtained from the duct-ligated salivary gland and purified the cells by limited dilution. The isolated cells were positive for CD29, CD49f, intracellular laminin, AFP, CK19, CK18, and Thy-1(CD90), and weakly positive for c-Kit (CD117). After three-dimensional formation, the cells expressed insulin and albumin. We designated the cells as swine salivary gland-derived progenitor cells. Gene expression of insulin and albumin was significantly increased (five-fold) and that of insulin was also increased (3.8-fold) with differentiation medium with nicotinamide and/or GLP-1 treatment in spherical culture. The expressions of albumin and insulin were 1/10-fold and 1/4-fold compared to porcine hepatocytes and pancreatic endocrine cells. The differentiated SGP cells could release insulin, which were stimulated by glucose and potassium. These results indicate that swine SGP cells could differentiate into hepatocytes and β-cells, functionally. Swine SGP cells were useful tools for therapy and analyzing endodermal regenerative models in large animals. © Mary Ann Liebert, Inc.
  • 高橋 昌志, 阪谷 美樹, 小林 修司, 小林 修一, 澤井 健, 志賀 一穂
    日本胚移植学雑誌 = Japanese journal of embryo transfer 28 (3) 112 - 117 1341-2965 2006/09/22 [Not refereed][Not invited]
  • A Okano, H Kishi, H Takahashi, M Takahashi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 52 (2) 301 - 306 0916-8818 2006/04 [Refereed][Not invited]
     
    Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.
  • OKANO Akira, KISHI Hisashi, TAKAHASHI Hitomi, TAKAHASHI Masashi
    Journal of Reproduction and Development Japanese Society of Animal Reproduction 52 (2) 301 - 306 0916-8818 2006/04/01 [Not refereed][Not invited]
     
    Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.
  • KOBAYASHI Shuji, KANEYAMA Kanako, SAKATANI Miki, TAKAHASHI Masashi, SAITO Norio, YONAI Miharu
    Jpn. J. Embryo Transfer 27 (2) 52 - 58 1341-2965 2005/05/16 [Not refereed][Not invited]
  • H Takahashi, M Takahashi, H Nagaya, M Hirako, K Sawai, A Minamihashi, S Inumaru, Y Yokomizo, M Geshi, A Okano, K Okuda
    THERIOGENOLOGY 63 (4) 1050 - 1060 0093-691X 2005/03 [Refereed][Not invited]
     
    A radioimmunoassay (RIA) was developed for quantification of bovine interferon (blFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of blFN tau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau. (c) 2004 Elsevier Inc. All rights reserved.
  • TAKAHASHI Masashi, TAKAHASHI Misa, HAMANO Seizo, TAKAHASHI Hitomi, OKANO Akira
    Journal of Reproduction and Development Japanese Society of Animal Reproduction 51 (1) 47 - 57 0916-8818 2005/02/01 [Not refereed][Not invited]
     
    We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin alpha5beta1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin alpha5beta1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of alpha5 and beta1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin-mediated manner.
  • H Nagaya, T Kanaya, H Kaki, Y Tobita, M Takahashi, H Takahashi, Y Yokomizo, S Inumaru
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1395 - 1401 0916-7250 2004/11 [Refereed][Not invited]
     
    We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.
  • M Sakatani, SI Kobayashi, M Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 67 (1) 77 - 82 1040-452X 2004/01 [Refereed][Not invited]
     
    We investigated the effects of heat shock on developmental competence of bovine embryos and intracellular oxidative state. After in vitro fertilization, embryos were exposed to heat shock at 41degreesC for 6 hr on days 0, 2, 4, and 6, respectively. On day 2, cleavage rate was not significantly different in all groups. However, the percentage of embryos developing to blastocyst stage after exposure to heat shock on day 0 (18.8+/-4.3%) and day 2 (23.6+/-3.7%) were significantly decreased compared with control (37.5+/-4.0%), day 4 (40.0+/-7.4%), and day 6 (38.1+/-2.0%). In addition, the total cell number of blastocysts was significantly decreased by heat shock on day 0 (107.5+/-6.6) and day 2 (112.8+/-5.7) compared with the control (143.2+/-9.4). To evaluate intracellular oxidative state by heat shock, embryos exposed to heat shock on days 0, 2, 4, and 6 were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescence of oxidized DCHFDA by reactive oxygen species (ROS) was detected under fluorescent microscope. The intensity of fluorescence was significantly increased when embryos were exposed to heat shock on days 0 and 2. However, heat shock on day 4 and day 6 did not increase the fluorescence intensity. These results indicate that (1) heat shock to earlier stage embryos causes a decrease in development to blastocysts and cell proliferation and (2) the decrease in development by heat shock could be involved in an increase of intracellular oxidative stress.
  • TAKAHASHI Masashi, TAKAHASHI Hitomi, HAMANO Seizo, WATANABE Satoko, INUMARU Shigeki, GESHI Masaya, OKUDA Kiyoshi, YOKOMIZO Yuichi, OKANO Akira
    Journal of Reproduction and Development 日本繁殖生物学会 49 (4) 297 - 305 0916-8818 2003/08/01 [Not refereed][Not invited]
     
    The effect of interferon-τ on in vitro development of bovine embryos was investigated. After in vitro fertilization, embryos developed to the morula stage were cultured for 3 days in TCM-199 or CR1 medium containing BSA or FCS supplemented with or without recombinant IFN-τ produced by a baculovirus expression system. Addition of baculovirus-expressed IFN-τ (100 ng/ml) significantly promoted development to the blastocyst stage in both culture media. Addition of E. coli expressed IFN-τ (2 μg/ml) also significantly promoted the embryonic development. Supplementation of BSA or FCS did not affect the growth-promoting effect of IFN-τ. To determine whether the growth-promoting effect of IFN-τ is related to the interferon type I receptors that bind to type I interferon such as IFN-α, embryos were cultured with IFN-α. Although IFN-α significantly promoted the development, a much higher concentration (25 μg/ml) was required than IFN-τ. A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA encoded type-I IFN receptor subunit from morula to blastocyst stage embryos. The overall results suggest a novel function for IFNs in promoting embryonic development and the effect may be related to type-I IFN receptor expressed in the early stages of preimplantation embryos.
  • M Takahashi, T Nagai, N Okamura, H Takahashi, A Okano
    BIOLOGY OF REPRODUCTION 66 (3) 562 - 567 0006-3363 2002/03 [Refereed][Not invited]
     
    The effects of beta-mercaptoethanol (beta-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O-2 supplemented with beta-ME. Addition of beta-ME significantly (P<0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O-2, to almost the same rates when they were cultured in 5% O-2. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P<0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P<0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.
  • S Watanabe, T Kokuho, H Takahashi, M Takahashi, T Kubota, S Inumaru
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 (7) 5090 - 5093 0021-9258 2002/02 [Refereed][Not invited]
     
    We investigated the ability of a baculo-virus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type N-glycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man(3)GlcNAc(2)(+/-Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated beta-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.
  • OGAWA Hidehiko, TAKAHASHI Masashi, TAKAHASHI Hitomi, OKANO Akira
    Journal of Reproduction and Development 47 (2) 83 - 89 0916-8818 2001/04/01 [Not refereed][Not invited]
     
    分娩後子宮修復過程におけるブタ子宮における組織学的変化を，子宮内膜上皮細胞の形態と膜型細胞外マトリックスの局在とから解明した。分娩後1、13、20および31日の泌乳中の雌ブタと分娩後28日に離乳させた3日後の雌ブタから子宮を摘出し、凍結切片を作製した。子宮組織における細胞外マトリックスの局在を明らかにする為に、抗ラミニン抗体および抗IV型コラーゲン抗体を用いて免疫染色を行った。その結果、泌乳中の子宮では細胞外マトリックスの局在は子宮内膜上皮細胞下に迂曲していたのに対し、離乳後の子宮では未経産の子宮と同様、基底膜上に一層に局在していた。以上の結果から、分娩後子宮修復は、離乳後速やかに起ると考えられた。
  • M Takahashi, K Keicho, H Takahashi, H Ogawa, RM Schultz, A Okano
    THERIOGENOLOGY 54 (1) 137 - 145 0093-691X 2000/07 [Refereed][Not invited]
     
    The correlation of oxidative stress on development and DNA damage in bovine embryos was investigated by the comet assay (single-cell microgel electrophoresis), an effective technique for detecting single-strand DNA breakage. After in vitro maturation and fertilization, one-cell stage embryos without cumulus cells were cultured for 8 days in SOF medium containing amino acids plus 5% FCS under low (5%) and atmospheric (20%) oxygen concentration. After 8 days of culture, the extent of blastocyst formation was significantly decreased (P<0.001) when embryos were cultured under 20% oxygen concentration (5.8 +/- 2.4%) when compared to embryos cultured under 5% oxygen concentration (35.1 +/- 6.7%). At the day 3 of development, DNA damage of individual embryos cultured under 5% or 20% oxygen concentration was measured by the comet assay, which entails microgel electrophoresis that can readily detect damaged DNA. After measuring the DNA damage in individual embryos by the comet assay, the length (149.9 +/- 15.3 mu m) of the migrating DNA fragment that is indicative of damaged DNA was significantly increased (P<0.001) in the embryos cultured under 20% oxygen concentration when compared to embryos cultured in 5% oxygen concentration (42.3 +/- 7 mu m). The length of damaged DNA in more than 50% of embryos was less than 50 mu m. when embryos were cultured under 5% oxygen concentration. In contrast, the distribution of damaged DNA shifted to the more damaged extent when embryos were cultured under 20% oxygen concentration. These results demonstrate that the retardation in bovine embryo development than in likely due oxidative stress as a consequence of the higher atmospheric oxygen concentration is positively correlated with an increase in the extent of DNA damage. Moreover, these results demonstrate that the comet assay is a useful method to evaluate embryo culture conditions. (C) 2000 by Elsevier Science Inc.
  • M Takahashi, N Saka, H Takahashi, Y Kanai, RM Schultz, A Okano
    MOLECULAR REPRODUCTION AND DEVELOPMENT 54 (1) 1 - 7 1040-452X 1999/09 [Refereed][Not invited]
     
    DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single-strand breaks in DNA, a significant increase in DNA damage was detected in individual one-cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one-cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO2 in air at 37 degrees C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one- and two-cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro, (C) 1999 Wiley-Liss, Inc.
  • H Takahashi, M Kuwayama, S Hamano, M Takahashi, A Okano, H Kadokawa, T Kariya, T Nagai
    THERIOGENOLOGY 46 (6) 1009 - 1015 0093-691X 1996/10 [Refereed][Not invited]
     
    Experiments were conducted to assess the effect of B-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (IVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 mu M beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fail (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P<0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54%). Blastocysts ranked as A or B after transportation in medium with or without 150 mu M beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.
  • A Okano, K Okuda, M Takahashi, D Schams
    Animal Reproduction Science 41 (1) 61 - 70 0378-4320 1996/01 [Refereed][Not invited]
     
    Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using I-125-labeled OT antagonist [d(CH2)(5),Tyr(Me)(2),Thr(4),Tyr-NH29]-vasopressin, [Thr(4),Gly(7)]-OT, OVT, OT) and with four peptides related to oxytocin ([Arg(7)]-vasopressin, [Thr(4),Gly(7)]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile(3)]-pressinoic acid (tocinoic acid)). The dissociation constant (K-d) of endometrial OT receptors on D0 (0.59 +/- 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 +/- 0.21; D15, 0.60 +/- 0.14 nM; mean +/- SEM). In the early luteal stage (D5), K-d (2.41 +/- 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K-d values were 3.25 +/- 0.29 nM on D5 and 2.44 +/- 0.44 nM on D15. Binding site concentration (B-max) on D0 (910.0 +/- 25.1 fmol mg(-1) protein) was significantly higher than on D5 and D10 (D5, 322.5 +/- 71.7; D10, 147.5 +/- 25.8 fmol mg(-1) protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 +/- 82.6; D15, 315.0 +/- 20.1 fmol mg(-1) protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B-max values were constant and similar to that on D5 of the early luteal stage. Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.
  • H. Kadokawa, M. Minezawa, Y. Yamamoto, M. Takahashi, K. Shimada, H. Takahashi, T. Kariya
    Theriogenology 44 (2) 295 - 306 0093-691X 1995/07/15 [Refereed][Not invited]
     
    Heterosexual chimerism among singleton females produced by multiple nonsexed embryo transfer (MNET singleton females) was investigated using chromosome typing and PCR (polymerase chain reaction)-amplification of male-specific DNA (msDNA). Of the 22 animals tested, 21 were classified as normal by both methods (i.e., showing no male cells among 100 metaphase spreads in chromosome typing and being msDNA negative in PCR). No morphological abnormalities of the genital organs were observed among 19 MNET single females. One MNET singleton female was, however, classified as a freemartin by PCR (male-specific DNA positive), but it was classified as normal cytogenetically. This individual probably had a low degree of heterosexual chimerism, and it seems that the chimerism derived from MNET was difficult to diagnose by chromosome typing, although it was detectable by PCR. The genital organs of this individual (15-mo-old Aberdeen Angus) were normal in form (both external and internal) and size. However, a very small structure, resembling seminiferous tubule, was found in the left ovary. It may be concluded that most MNET singleton females are expected to have normal reproductive function. © 1995.
  • Seizo Hamano, Masashige Kuwayama, Masashi Takahashi, Akira Okano, Naomichi Okamura
    Journal of Reproduction and Development 40 (4) 355 - 359 1348-4400 1994 [Refereed][Not invited]
     
    The present study investigated effects of β-merchaptoethanol (β-ME) on the development of bovine embryos derived from in-vitro matured and fertilized (IVM-IVF) oocytes. IVM oocytes were inseminated with frozen-thawed bovine sperm, and at 24, 48 and 72 h after insemination, 2-, 8- and 16-cell embryos, respectively, were transferred to TCM-199 medium containing 5% fetal bovine serum and 0, 5, 10, 20, 50 or 100 μm β-ME without a cumulus cell monolayer or with a cumulus cell monolayer but without addition of β-ME as a control. All embryos were subsequently incubated such that the total duration of culture was 8 days. The addition of low concentrations (5 or 10 μM) of β-ME significantly enhanced the development of all the embryos to the blastocyst stage. High concentrations (20, 50 or 100 μM) of β-ME were effective only for the development of 8-and 16-cell embryos, but not of 2-cell embryos, to the blastocyst stage. Compared with embryos in the co-culture system (control), 2-cell embryos that were cultured with β-ME but without a cumulus cell monolayer showed significantly lower rates of development to the blastocyst stage. However, even though without a cumulus cell monolayer, 8-cell embryos showed the same rates of development to the blastocyst stage as controls when low concentrations of β-ME (5 or 10 μM) were added to the medium. Furthermore, significantly higher rates of development of embryos to the blastocyst stage were obtained when 16-cell embryos were cultured in medium supplemented with 20 μM β-ME than that of controls. Twenty blastocysts developed from 16 cell IVM-IVF embryos in the medium containing 20 μM β-ME were transferred to 20 recipients, and 11 of them became pregnant, resulting in 10 calves. These results indicate that β-ME is effective for blastocyst development of embryos that are cultured without a cumulus cell monolayer, and the effect is remarkable for embryos of advanced stages. © 1994, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.
  • M TAKAHASHI, T NAGAI, S HAMANO, M KUWAYAMA, N OKAMURA, A OKANO
    BIOLOGY OF REPRODUCTION 49 (2) 228 - 232 0006-3363 1993/08 [Refereed][Not invited]
     
    The purpose of this investigation was to determine the effect of beta-mercaptoethanol (beta-ME) and cysteamine, low-molecular-weight thiol compounds, on the development and intracellular glutathione content of bovine embryos obtained by in vitro fertilization of in vitro-matured oocytes. Embryos developed to the 6-8-cell stage after in vitro fertilization were cultured without feeder cells in TCM-199 containing 10% fetal calf serum with or without beta-ME or cysteamine. The percentages of embryos that developed to the blastocyst and hatched blastocyst stages were significantly higher in medium containing beta-ME or cysteamine. Also, total intracellular glutathione levels were higher for embryos cultured in the medium with beta-ME or cysteamine than for those cultured in medium without thiol compounds. Moreover, when buthionine sulfoximine, a specific inhibitor of glutathione synthesis, was added to medium containing thiol compounds, there was a reduction both in the development of embryos to the blastocyst stage and in intracellular glutathione content. These results indicate that the inclusion of low-molecular-weight thiol compounds aids the in vitro development of bovine embryos without feeder cells and that the effect of thiol compounds is mediated through the increase of intracellular glutathione levels.
  • Akira Okano, Masashi Takahashi
    Journal of Reproduction and Development 38 (3) 203 - 209 1348-4400 1992 [Refereed][Not invited]
     
    The elongated conceptuses were collected from pregnant sows on Day 14 by flushing uteri with PBS. Conceptuses cultured in vitro in M-199 at 37C, under an atmosphere of 5% CO2 in air, formed trophoblastic vesicles by the following day. The trophoblastic vesicles were cultured continuously in vitro and subjected to successive subculture, where the free-floating trophoblastic cells again formed small trophoblastic vesicles. The trophoblastic vesicles of all stages of culture showed similar patterns of the protein synthesis. The elongated conceptus and trophoblastic vesicle synthesized β-fetoprotein and retinol-binding protein. While culturing trophoblastic vesicles in vitro, fairly large amounts of estradiol-17β (E2) and progesterone were synthesized. Possibilities that E2 acts as a signal for the recoginition of pregnancy were discussed. © 1992, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.

MISC

  • Hayashi, Yoshihiro, Edure, Taichi, Bai, Hanako, Takahashi, Masashi, Kawahara, Manabu  Memories of the Research Faculty of Agriculture, Hokkaido University  39-  1  -6  2023/02/13  
    Embryo transfer is a widespread assisted reproductive technique for the production of livestock. In general, the blastocyst-stage embryos used for transfer are produced in vivo or in vitro. However, little is known about differences in mitochondrial ultrastructure between in vivo and in vitro blastocysts in mammals. This study aimed to compare the mitochondrial ultrastructure between in vivo and in vitro mouse blastocysts using transmission electron microscopy (TEM). TEM analyses were performed using more than 1,000 mitochondria from blastocyst embryos. No significant difference was observed in mitochondrial roundness. However, the average major axes (nm) and areas (nm2) were significantly greater in the in vitro embryos than in the in vivo embryos (p＜0.01). Furthermore, the average electron density was significantly decreased in the in vitro embryos compared to the in vivo embryos (p＜0.01). Taken together, we conclude that mitochondria in in vitro blastocysts show an increase in size and a decrease in electron density compared with those in in vivo blastocysts. This implies that mitochondrial functions are deteriorated in in vitro blastocysts compared to in vivo blastocysts.
  • 唄 花子, 川原 学, 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer / 日本胚移植技術研究会 編  43-  (2)  85  -94  2022/02
  • 楠野莉奈子, HO Khoi, HO Khoi, 本間康平, 高成準, 唄花子, 川原学, 高橋昌志  日本畜産学会大会講演要旨  130th-  2022
  • 古川瑛理, 檜垣彰吾, 松崎明, 唄花子, 高橋昌志, 片桐成二, 柳川洋二郎  日本畜産学会大会講演要旨  130th-  2022
  • 高橋昌志, 高橋昌志  臨床獣医  40-  (13)  2022
  • 高橋昌志  Dairyman  72-  (5)  2022
  • 唄花子, 川原学, 高橋昌志  化学と生物  60-  (6)  2022
  • 唄 花子, 川原 学, 高橋 昌志  68-  (12)  695  -702  2021/12
  • 宇喜多遥, 山崎武志, 山口諭, 阿部隼人, 馬場俊見, 唄花子, 高橋昌志, 川原学  日本畜産学会大会講演要旨  129th-  2021
  • BAI Hanako, KUNII Hiroki, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  114-  P-56  -P-56  2021  
    【目的】暑熱ストレスは，ウシの卵巣機能や胚発生などに影響を与え，繁殖性を低下させることが知られるが，母体子宮に対する影響については知見が少ない。我々は以前，ウシ子宮内膜上皮細胞において，暑熱負荷培養により酸化ストレスが誘導されることを報告している（第112回日本繁殖生物学会大会）。一方，誘導される酸化ストレスへの応答について詳細は不明である。本研究では，暑熱負荷培養時に誘導される酸化ストレス応答経路を検証した。【方法】と場由来のウシ子宮組織から子宮内膜上皮細胞を単離，培養した。細胞は牛の平常時の体温である38.5℃または暑熱時の体温である40.5℃の暑熱条件下で培養した。ルシフェラーゼレポーターアッセイ法により，熱ショック応答経配列（HSE）の活性，抗酸化剤応答配列（ARE）の活性および細胞内ストレス応答に主要な役割を担う転写制御因子であるNFE2L2の安定性を評価した。リアルタイムPCRおよび免疫染色法により，NFE2L2，その上流因子であるKEAP1および標的因子であるサイトカインの発現解析を行った。【結果と考察】暑熱負荷培養により，ウシ子宮内膜上皮細胞においてHSEおよびAREの活性， NFE2L2の安定性が有意に増加した。KEAP1，NFE2L2の遺伝子発現量に有意な変化はみとめられなかった。暑熱負荷によりKEAP1の細胞内局在は変化がみられなかった一方で，NFE2L2の局在は細胞質から核へと変化していた。このとき一部のサイトカイン遺伝子の発現量には低下がみとめられた。以上より，暑熱時のストレス応答として，KEAP1-NFE2L2-ARE経路が働くことにより，抗酸化作用および抗炎症作用を示す可能性が示唆された。暑熱負荷による悪影響を明らかにするためには，より長期，あるいは強い暑熱負荷により応答性にどのような変化があるか今後検証する必要がある。
  • KUNII Hiroki, KUBO Tomoaki, ASAOKA Natsuki, SHIMASAKI Tomoya, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  114-  P-98  -P-98  2021  
    【目的】我々は，簡便かつ非侵襲的に採取可能な腟底部粘膜組織（Vaginal mucus; VM）において，授精後17日（D17)-D18で妊娠特異的にISG15，IFIT1発現が増加し，MUC16発現が低下することを見出した。そこで，これらの遺伝子発現レベルを指標として，RT-LAMP法と機械学習の応用により簡易迅速な早期妊娠判定モデルの作成を検討した。【方法】AI実施ホルスタイン種搾乳牛からD17-D18に綿棒を用いて簡易迅速にVMサンプルを採取した（非妊娠確定：n=40，妊娠確定：n=40）。採取サンプルから簡易的にRNAを抽出し，ISG15，IFIT1およびMUC16を判定指標としてRT-LAMP法を実施した。吸光度が設定閾値を最初に超えた時間（Threshold time; TT）をサンプルごとに算出し，各サンプルのISG15，IFIT1およびMUC16のそれぞれにおけるTTデータセットを作成した。最初に，遺伝子ごとにROC曲線を描画し曲線下面積（AUC）を求めた後，最適なカットオフ値を算出し，単一指標での判定精度を評価した。次に，TTデータセットのうち50頭を訓練データ，30頭を評価データとして分割し，訓練データを用いて機械学習法（LightGBM）による妊娠予測モデルを作成した。最後に，作成したモデルを評価データに適用し，モデル性能を評価した。【結果】D17-D18における妊娠判定性能は，単一指標による判定では，IFIT1が最も高く（AUC=0.88），感度92.5%，特異度75.0%であった。一方で，ISG15およびMUC16の判定性能はIFIT1と比較して低く，AUCはそれぞれ0.69および0.58であった。複合指標で作成したモデルの場合，IFIT1とMUC16の組み合わせが最も高く，感度93.3%，特異度86.7%であった。以上より，本研究で作成した遺伝子組合わせモデルにより，D17-D18のVMサンプルを用いた簡便な早期妊娠判定が可能であることが示唆された。
  • 高橋昌志, 高橋昌志  繁殖技術  41-  (1)  3  -8  2021
  • カワグチ トシカズ, サトウ ヨウスケ, タカハシ マサシ  67-  64  -66  2020/03
  • HO Thieu Khoi, HOMMA Kohei, TAKANARI Jun, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  113-  (0)  P  -123-P-123  2020  

    [Introduction] Heat shock protein 70 (HSP70) is well known as a heat shock (HS) induced protein that has function as intracellular chaperones for other proteins to help the cells against the stress condition. Although HS is common to induce HSP70 expression to add stress-resistant ability to the cells, HS causes the toxicity to cells and tissues such as increasing reactive oxygen species (ROS). Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from by-product of asparagus, was found to induce HSP70 expression without HS and regulating cellular redox balance in human cells. However, effect of EAS on reproductive cells is unknown. In the present study, we investigated the effect of EAS on HSP70 induction and antioxidant defense system in bovine cumulus cells. [Materials and Methods] Bovine cumulus cells were treated with various concentration of EAS (0.5, 1 and 5 mg/ml) for 6 h at 38.5°C followed by sampling to analyze gene and protein expression of HSP70 as well as gene expressions related to antioxidant system. Besides, intracellular ROS and reduced form of glutathione (GSH) were detected and quantified by using fluorescent dyes. [Results] EAS significantly increased gene expression of HSP70 whereas no effect to HSP27 and 90. Moreover, protein expression of HSP70 was also increased by EAS. Besides, EAS decreased intracellular ROS generation and increased GSH synthesis significantly with enhancement in the gene expressions of antioxidant enzymes such as The Cu,Zn superoxide dismutase, Peroxiredoxin, Glutamate Cysteine Ligase as well as Nuclear factor erythroid 2-related factor 2 transcription factor that contribute to keep intracellular antioxidant status with GSH synthesis and scavieging ROS. These results suggest that EAS has beneficial effect to bovine cumulus cells by improving HSP70 expression and antioxidant defense system under non-heat shock condition.

  • KUNII Hiroki, KUBO Tomoaki, ASAOKA Natsuki, SHIMASAKI Tomoya, KOYAMA Keisuke, KIMURA Koji, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  113-  (0)  P  -65-P-65  2020  

    【目的】ウシの着床過程では，授精後18日前後をピークとして，胚の栄養外胚葉からインターフェロン・タウ（IFNT）が分泌される。IFNTは母体の子宮内膜に作用し，JAK-STAT経路を介してIFN誘導性遺伝子（ISGs）の発現を誘導する。我々はこれまでに，子宮外組織である子宮頸部粘膜組織（CMM）においても妊娠特異的なISGsの高発現を見出した。しかし，IFNTの存在が直接的に示されているのは子宮内のみであり，IFNTがCMMにおけるISGs発現にどのように関与するのかは不明である。そこで本研究では，CMMへのIFNT移行の有無，ならびにCMMにおけるISGsの発現機序を明らかにすることを目的とした。【方法】非妊娠（np）および妊娠（p）ホルスタイン種搾乳ウシ由来CMMを，AI実施後14日目（d14），18日目（d18）および25日目（d25）において低侵襲的に採取後，RNAおよびタンパク質を抽出した。IFNTの検出は，抗IFNT抗体を用いウェスタンブロッティング（WB）にて行った。加えて，np-, p-CMMと共培養したnp-ウシ末梢血白血球（PBL）におけるISG15発現を解析し，p-CMMのI型IFN活性を評価した。次に，定量PCRによりJAK-STAT経路関連因子の継時的な遺伝子発現動態を，またWBにより着床前におけるSTAT1活性化状態を評価した。【結果】p-d18のCMM由来タンパク質成分より，IFNTのバンドが検出された。加えて，CMM-PBL共培養実験では，np-d18のCMMと比較して，p-d18のCMMと共培養したPBLにおけるISG15発現が顕著に増加した。さらに，p-d18のCMMでは，STAT1, STAT2, IRF9発現および，JAK-STAT経路の活性化を示すリン酸化STAT1の発現が顕著に増加した。以上の結果から，子宮頸部における着床前特異的ISGs発現は，胚由来因子IFNTが子宮外へ移行し，JAK-STAT経路を活性化することにより誘導されることが明らかとなった。

  • SHIMASAKI Tomoya, TAKAHASHI Masashi, KUBO Tomohide, KUNII Hiroki, HURUYAMA Keisuke, HAMAGUCHI Yu, ASAOKA Natsuki, BAI Hanako, KAWAHARA Manabu, OGAWA Hidehiko  The Journal of Reproduction and Development Supplement  113-  (0)  P  -66-P-66  2020  

    【目的】反芻動物では着床前の受胎産物がインターフェロン・タウ（IFNT）を分泌し，子宮内膜でIFN誘導性遺伝子群（ISGs）を誘導する。我々は，これまでにISG15などの代表的ISGsが子宮頸管粘膜組織においても妊娠18日に子宮内膜での発現に匹敵する発現増加を示すことを明らかにした。子宮頸管粘膜（CMF）のサンプリングは簡便かつ生体に対して低侵襲的であるため，この知見を基に人工授精後最初に発情が回帰する21日までの早期妊娠判定への応用が進められている。一方，ISGsを指標とした場合の判定精度を上げる際には，ウイルス感染によるIFN経路の活性化による非妊娠ISGsの増加可能性の考慮も必要であり，そのため，IFN経路に依存しない妊娠応答遺伝子の探索が必要である。我々がこれまで実施したCMFのRNAシーケンス解析では，妊娠時18日の発現上昇遺伝子の多くがISGsであった一方，妊娠時の発現低下遺伝子群にはIFNTのような共通の制御因子を持つと考えられるものが存在しなかった。そのため，本研究では子宮頸管粘膜組織における妊娠時発現低下遺伝子の検出を目的とした。【方法】人工授精（AI）実施から14，18，24日が経過したホルスタイン種乳用牛の生体からCMFを綿棒で軽くこすり取ることで採取し，AI後30日及び40日の超音波診断による妊娠診断結果より各サンプルを「非妊娠区」「妊娠区」に分別した。採取したCMFからRNA抽出およびcDNA合成を行った。これまでのRNAシーケンス解析により，妊娠18日における発現低下を示した遺伝子を複数選択し，それらについて作成したプライマーを用いてAI後14，18，24日それぞれのサンプルでリアルタイムPCRを行い各遺伝子の発現量を測定した。【結果と考察】リアルタイムPCRの結果，AI後18日の妊娠時発現低下遺伝子としてIGFBP3，FOS，EGR1を検出し，これらの遺伝子が妊娠判定の指標遺伝子として有用である可能性を示した。

  • BAI Hanako, MITANI Tomohiro, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  113-  (0)  P  -70-P-70  2020  

    【目的】夏季の暑熱ストレスは，家畜の繁殖性に悪影響を及ぼす。暑熱ストレスにより，ウシの卵巣機能，卵子や胚発生などに異常をきたし，人工授精後の受胎率は低下することが報告されている。また近年，北海道という比較的冷涼な地域においても夏季の受胎率低下が報告されており，温暖化の影響は深刻化していることが懸念される。一方，母体子宮組織は胚受容に必須であるが，暑熱ストレスが母体子宮組織に及ぼす影響は知見が少ない。本研究では，ウシ子宮内膜組織を夏季および冬季にわけて遺伝子発現の差異を検証した。【方法】と場由来のウシ子宮から子宮内膜組織を採取した。採取した組織からRNA抽出，cDNA合成を行い，リアルタイムPCRにより，熱ショックタンパク質，抗酸化酵素，インターフェロン誘導性遺伝子および炎症性サイトカインの発現量解析を行った。【結果】熱ショックタンパク質HSP27, 60, 90および抗酸化酵素であるカタラーゼおよびCuZnSODは，夏季に採取した子宮内膜組織において，冬季に採取したものと比べ発現が有意に低かった。炎症性サイトカインであるIL1Bの発現量は夏季に採取した子宮内膜組織において，冬季に採取したものと比べ有意に高く，TNFA発現は増加傾向であった。インターフェロン誘導性遺伝子の発現量に変化はみとめられなかった。これらの結果から，長時間の暑熱負荷により，ウシ子宮内膜上皮細胞における熱ショックタンパク質および抗酸化酵素の発現は低下し，炎症性サイトカインの発現は増加することが示唆された。

  • 及川康平, 山崎武志, 山口諭, 阿部隼人, 唄花子, 高橋昌志, 川原学  北海道畜産草地学会報  7-  (2)  2019
  • 山崎武志, 及川康平, 山口諭, 阿部隼人, 唄花子, 高橋昌志, 川原学  農研機構北海道農業研究センター成果情報(Web)  2019-  2019
  • LI Jianye, AHMED Balboula Zaky, FUJII Takashi, MORIYASU Satoru, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  112-  (0)  P  -56-P-56  2019  

    We have demonstrated that the levels of lysosomal Cathepsin B (CTSB) transcript was significantly higher in trophectoderm (TE) than inner cell mass (ICM) of bovine blastocyst, whereas CTSB activity was significantly higher in ICM than TE. In the present study, we investigated the opposite expression pattern of CTSB gene and protein in blastocyst. CTSB protein distribution in IVF derived blastocyst was achieved by immunostaining. Supported by activity, CTSB protein was clearly localized in the cytoplasm and significantly higher in ICM than TE. Therefore, we hypothesized the opposite expression pattern was caused by the different turnover of CTSB by consuming of mRNA in ICM and TE. On day7, the blastocysts were treated by cycloheximide (CHX) for 2 h to block protein synthesis followed by re-cultured in KSOM. After that, total RNA amount was detected by Acridine Orange (AO) staining in whole blastocyst. CHX treatment increased the total RNA amount, especially in ICM. In the next experiment, CTSB was quantified in separated ICM and TE by micro blade dissection after CHX treatment. CTSB abundance was significantly increased in CHX-treated ICM compared to control ICM, whereas no difference in CHX-treated TE than control TE. Importantly, the increasing ratio of CTSB transcript was significantly increased in ICM than TE between CHX-treated and control group. Taken together, these results suggest that low CTSB level in ICM than TE is not caused the down regulation of CTSB gene, but the high turnover of CTSB used for protein synthesis and enzymatic activity for potential role of differentiation.

  • 高橋昌志, 高橋昌志  家畜人工授精  (304)  3  -7  2019  [Not refereed][Not invited]
  • 香西 圭輔, 徳山 翔太, Szostek Anna Z, 登石 裕子, 角田 修男, 田谷 一善, 阪谷 美樹, 高橋 昌志, 南保 泰雄, Skarzynski Dariusz J, 山本 ゆき, 木村 康二, 奥田 潔  馬の科学 = Equine science  56-  (4)  282  -293  2019  [Not refereed][Not invited]
  • MURATA Hirona, KOMATSU Seimei, BAI Hanako, KAWAHARA Manabu, MASASHI Takahashi  The Journal of Reproduction and Development Supplement  112-  (0)  P  -75-P-75  2019  [Not refereed][Not invited]
     
    <p>【目的】夏季の暑熱ストレスに伴い引き起こされる酸化ストレスは，人工授精受胎率低下など，家畜の繁殖性に悪影響を及ぼすことが知られている。これまでに，ウシの卵子や卵巣の機能，胚発生などに異常をきたすことが報告されているが，母体子宮に関しては暑熱ストレスにより酸化ストレスが引き起こされるかどうかは不明である。一方子宮は，妊娠成立過程において着床の場となる重要な組織であり，正常な受胎の前提条件として，子宮が健全であることが必須である。本研究では，子宮においても暑熱ストレスに伴い酸化ストレスが引き起こされるかを検証した。【方法】と場由来のウシ子宮組織から子宮内膜上皮細胞を単離，培養した。単離した上皮細胞を牛の平常時の体温である38.5℃および暑熱時の体温である40.5℃の暑熱条件下で12時間培養した後，CellROX® Green Reagentを用いて，マイクロプレートリーダーでの蛍光強度測定および蛍光顕微鏡により活性酸素種（ROS）を検出した。また，同様に培養した細胞からRNA抽出，cDNA合成を行い，リアルタイムPCRにより，酸化ストレスの指標である抗酸化酵素の遺伝子; <i>SOD</i>（<i>スーパーオキシドジスムターゼ</i>），<i>GPX</i>（<i>グルタチオンペルオキシダーゼ</i>），<i>CAT</i>（<i>カタラーゼ</i>）の発現量解析を行った。<i>SOD</i>については，<i>CuZnSOD</i>および<i>MnSOD</i>の2種類の遺伝子を解析した。【結果と考察】蛍光強度には暑熱負荷による影響はなかった。蛍光シグナルの観察では細胞の核と細胞質の両方において強い蛍光シグナルがみとめられた。抗酸化酵素の遺伝子の発現量は，<i>MnSOD</i>，<i>GPX</i>，<i>CAT</i>遺伝子の発現量に有意な影響はみられなかったが，<i>CuZnSOD</i>遺伝子の発現量は暑熱負荷により有意に増加した。これらの結果から，12時間の暑熱負荷培養により，ウシ子宮内膜上皮細胞で酸化ストレスが引き起こされたことが示唆された。</p>
  • ASAOKA Natsuki, KIMURA Koji, TAKAHASHI Masashi, KUNII Hiroki, KOYAMA Keisuke, KUBO Tomoaki, HAMAGUCHI Yu, OGAWA Hidehiko, KOBAYASHI Hisato, BAI Hanako, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  112-  (0)  P  -71-P-71  2019  [Not refereed][Not invited]
     
    <p>【目的】反芻動物特異的な妊娠認識物質であるインターフェロン・タウ（IFNT）は着床前後期には母体子宮内膜においてIFN誘導遺伝子群（ISGs）の発現を誘導する。ISGsは妊娠認識や着床に関与するとともに，早期受胎判定としての利活用も期待されている。我々はこれまでに，ISGsの一種である<i>ISG15, MX1, MX2</i>が外子宮口内腔および腟底部壁の粘膜組織においても妊娠特異的な高発現誘導を示すことを明らかにしたが，子宮外組織における他の妊娠応答遺伝子に関する知見はない。そこで子宮外組織における網羅的な遺伝子発現解析をもとにウシ子宮外組織における妊娠応答遺伝子の探索と妊娠検出を目的とした。【方法】人工授精（AI）実施後，18日後のホルスタイン種乳用牛から採取した外子宮口粘膜（CM）における発現解析をRNA-seqにより行い，候補遺伝子としてinterferon-induced protein with tetratricopeptide repeats 1（<i>IFIT1</i>）を選出した。AI後14，18，24日目に同組織の採取を行い，妊娠の成否はAI後30日目と45日目の妊娠診断で判断した。またIFNT誘導性を検証するために，食肉公社から採取した非妊娠ウシCM組織を採取・細切後，組み換えウシIFNTを添加して24時間培養し<i>IFIT1発現を解析し</i>た。【結果】AI後18日目に採取したCMにおいては，<i>IFIT1</i>が非妊娠サンプルと比較して妊娠サンプルで発現量の増加傾向がみられた。さらに14，18，24日目における<i>IFIT1</i>の遺伝子発現の経時的変化を調べたところ，IFNT産生ピークである18日目を頂点とした挙動を示した。また，<i>IFIT1</i>のIFNT誘導性については，IFNT添加CMで発現量が増加する傾向があった。これらの結果から妊娠初期のCMにおける<i>IFIT1発現には</i>IFNTの関与ならびに早期妊娠応答の新たな指標の可能性が示唆された。</p>
  • NIWA Yuto, BALBOULA Ahmed Zaky, FUJII Takashi, LI Jianye, MORIYASU Satoru, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  112-  (0)  P  -122-P-122  2019  [Not refereed][Not invited]
     
    <p>【目的】カテプシンB（CTSB）はリソソームプロテアーゼであり，細胞内消化や細胞死に関与する。我々は，これまでにウシ卵子・胚の品質の程度や暑熱曝露の有無によるCTSBの動態変化を明らかにし，卵子・胚の品質や障害指標としての利用可能性を提示した。CTSBを指標としたガラス化保存による障害評価はウシ卵子やヒツジ卵子で報告されているが，ウシ胚盤胞では未報告である。ウシ胚盤胞では，ガラス化保存を含む凍結保存により，胚内側の内部細胞塊（ICM）と比べて，胚外側の栄養外胚葉（TE）における高頻度のDNA損傷が報告されていることから，ガラス化保存ウシ胚盤胞のTEにおける障害評価が重要であると考えられる。本研究ではウシ胚盤胞のTEにおいて，ガラス化保存による障害とCTSBの関連性解明を目的とした。【方法】体外受精・発生させたウシ胚盤胞をガラス化保存し，加温・回復培養に供した後の生存胚を実験に用いた。まず，Magic Red®によるCTSB活性検出とTUNEL染色をガラス化保存胚盤胞全体で行った。その後，定量的な解析を行うため，ブレードを用いた顕微操作により胚盤胞をICMとTEに切断分離し，単離したTEにおけるCTSB活性の測定およびqPCRによる<i>CTSB</i>遺伝子の発現解析を行った。また，CTSBとアポトーシスとの関連が報告されていることから，アポトーシス関連遺伝子も同時に発現解析を行った。【結果】ガラス化によってウシ胚盤胞全体のDNA損傷レベルが上昇した。また，無処理対照胚と比較して，ガラス化保存胚盤胞のTEにおけるCTSB活性の上昇が観察され，この結果は単離したTE単独においても同様であった。さらに，ガラス化保存後単離TEにおいて，<i>CTSB</i>遺伝子とアポトーシス関連遺伝子（<i>BAX</i>, <i>CASPASE-9</i>,<i> CASPASE-3</i>）の発現上昇が確認された。これらの結果から，ガラス化保存によりウシ胚盤胞のTEにおける<i>CTSB</i>の遺伝子発現および細胞内活性が上昇し，ミトコンドリアを介したアポトーシスが亢進されたことが示唆された。</p>
  • SUZUKI Toshiyuki, KIMURA Koji, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi  Hokkaido Journal of Livestock and Grassland Science  7-  (1)  7  -15  2019  [Not refereed][Not invited]
     

    Interferon-tau (IFN-τ:195 aa) is a unique protein secreted only from ruminant and plays a very important role of pregnancy recognition. However, the source of IFN-τ is recombinant IFN-τ using E.coli expression system and it is very difficult to obtain. Therefore basic research and application research for improving pregnancy is highly limited. Besides, use of recombinant IFN-τ for field application is strictly regulated by biosafety and public consensus. Therefore, the purpose of this research is to discover the active site of IFN-τ by using of synthetic peptides as a substitute for recombinant IFN-τ. Eleven peptides (long chain: 27-28 aa, short chain: 7-17 aa) were chemically synthesized from amino acid sequence of IFN-τ. To confirm the agonistic activity of the synthetic peptides, single or mixed peptides were added to cultured bovine endometrium stromal cells to detect gene expression of interferonstimulated genes (ISGs). Recombinant IFN-τ was also added as a positive control. Next experiment was performed to confirm the antagonistic activity of the synthetic peptides. Single or mixed peptides was added to stromal cells with recombinant IFN-τ followed by detecting of ISGs expression. IFN-τ significantly stimulated ISGs expression, whereas all peptides did not stimulate. Besides, addition of single or mixed peptides with IFN-τ showed no inhibitory effect. Overall results suggest that synthetic peptides would not have sufficient agonistic and antagonistic ability or lost the binding ability to IFN receptor by the possible structural change or no activity site having.

  • Wataru Iwasaki, Kenichi Yananaka, Daisuke Sugiyama, Yuki Teshima, Masatoshi Maeki, Maria Portia, P. Briones-Nagata, Kenichi Yamashita, Masashi Takahashi, Masaya Miyazaki  Proc. Micro TAS 2018  3-  1727  -1728  2018/11  [Refereed][Not invited]
     
    We fabricated a microfluidic device for separation of bovine oocytes based on its quality to improve the conception rate of in vitro fertilization by using good quality oocytes. The microfluidic device separates oocytes based on sedimentation rate differences in a sucrose buffer, depending on the oocyte quality. We performed IVF after the microfluidic separation of oocytes. The developmental rate to blastocyst of oocytes collected from the lower outlet was significantly higher than those collected from the upper outlet. These findings indicate that our microfluidic device could be applied to contribute to the improvement of the in vitro embryo production system.
  • 岩崎渉, 山中賢一, 永田マリアポーシャ, 真栄城正寿, 山下健一, 高橋昌志, 宮崎真佐也, 宮崎真佐也  化学とマイクロ・ナノシステム学会研究会講演要旨集  38th-  83  2018/10/30  [Not refereed][Not invited]
  • 着床前ウシ頸管および外子宮口周辺粘膜におけるインターフェロン誘導遺伝子の発現
    国井宏樹, 伊藤月乃, 小木曽貴季, 鈴木惇文, 古山敬祐, Md. Abdus, Shabur Talkder, Balboula Ahmed Zaky, 唄 花子, 川原 学, 永野昌志, 木村康二, 高橋昌志  北海道牛受精卵移植研究会会報  37-  17  -17  2018/08  [Not refereed][Not invited]
  • Kou Nakahara, Shinya Sakuma, Manabu Kawahara, Masashi Takahashi, Fumihito Arai  Journal of Microelectromechanical Systems  27-  (3)  464  -471  2018/06/01  [Not refereed][Not invited]
     
    The aim of this paper is to measure the changes in the mechanical characteristics of the zona pellucida (ZP) of an oocyte over time. To measure the mechanical characteristics such as Young's modulus during cultivation, it is necessary to achieve the cultivation and the measurement of the mechanical characteristics of target oocytes. Generally, measurement systems are constructed on a microscope for observing the deformation of the oocytes, but it is difficult to integrate such a system with a conventional incubator. Therefore, we propose a cell carrier chip for the time-lapse mechanical characterization. The cell carrier chip has a probe as well as a force sensor to measure the mechanical characteristics of the ZP. In addition, the chip enables us to carry the target oocyte from the measurement system to the incubator. To calculate the Young's modulus of the ZP, we used an elastic spherical shell model and measured the Young's modulus of the ZP of cryopreserved oocytes at 0.5, 1, 2, 3, 4, 5, and 6 h after being thawed. The result indicates that the Young's modulus of the ZP tends to increase with the cultivation time. [2017-0133]
  • 秋沢宏紀, 唄花子, 高橋昌志, 川原学  日本畜産学会大会講演要旨  124th-  213  -213  2018/03/28  [Not refereed][Not invited]
  • 川口 俊一, カビラズ デュラル, 森田 金市, 高橋 昌志  Proceedings of the Chemical Sensor Symposium  63-  34  -36  2018/03  [Not refereed][Not invited]
  • 川口俊一, KABIRAZ Dulal, 森田金市, 高橋昌志  電気化学会大会講演要旨集(CD-ROM)  85th-  ROMBUNNO.1B12  2018/02/23  [Not refereed][Not invited]
  • 唄花子, 川原学, 高橋昌志  栄養生理研究会報  62-  (1)  19  -25  2018  [Not refereed][Not invited]
     
    一般的なイメージとして、精子と卵子が受精することにより生命が始まると考えられている。しかし、受精卵(胚)の半数近くは、母体子宮に着床する前に死滅してしまい、妊娠成立に至らない。ウシの人工授精受胎率についても低下を続けており、現在では50%を下回っている。これは畜産農家にとって大きな経済的損失である。この着床前の胚死滅の原因の一つとして、母体の「妊娠認識不足」が考えられている。妊娠認識とは、胚から分泌されるシグナルにより、妊娠に必要な黄体機能が維持されるように働く過程である。反芻動物では、着床前の胚が分泌するインターフェロン・タウ(IFNT)がその役割を担う。ここでは、反芻動物における妊娠成立機構、特にIFNTについての知見を紹介したい。
  • OIKAWA Kohei, YAMAZAKI Takeshi, YAMAGUCHI Satoshi, ABE Hayato, BAI Hanako, TAKAHASHI Masashi, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  111-  (0)  P  -93-P-93  2018  [Not refereed][Not invited]
     

    【目的】性選別精液とは，X染色体を持つ精子（雌）とY染色体を持つ精子（雄）のDNA含量の違いから，特定染色体を持つ精子を高率に選別したものである。酪農において，X染色体性選別精液の利用による計画的雌畜生産は，経営面および育種改良面においてメリットが大きい。しかし，一般に，性選別精液の受胎率は低く，通常精液の75から80%といわれている。性選別精液の受胎率向上のためには，通常精液と性選別精液の性質の違いを把握した適切な使用が求められる。受胎率を含む繁殖形質は遺伝率が低く，環境要因による影響が大きいことから，環境要因から受ける影響の理解が極めて重要になる。よって本研究では，性選別精液の環境要因側の特性を理解すべく，道内の酪農家で実施された過去4年間の人工授精（AI）成績を含むフィールドデータを分析した。【方法】北海道酪農検定検査協会で集積された道内ホルスタイン種雌牛の個体繁殖成績を使用した。分析対象は，2012から2015年までの国産乳牛精液による未経産牛69,857頭分の初回授精記録とした。分析環境要因は，授精年，授精月，および授精月齢とした。【結果と考察】通常精液，性選別精液ともに6から9月にかけて受胎率が低下していた。各精液種の受胎率低下をより詳細に分析するため，各月の受胎率と全月平均受胎率の差を分析すると，7および8月では，性選別精液のみで有意な低下がみられた。一般に，受胎の成否には，精子と卵母細胞の受精可能時間が重複するタイミングでのAIが重要となる。しかし，母体は暑熱ストレスを受けると発情の微弱化や乱れが生じ，AI適期の把握が困難となる。夏期におけるAI適期の齟齬は，受胎率の低下を引き起こすことが知られているが，性選別精液は通常精液に比べて精子生存性が低く，受胎不成立の頻度が高まったと推測された。以上より，性選別精液を用いた7および8月のAI受胎率は，通常精液に比べ，より顕著に低下することが判明した。

  • KOMATSU Seimei, SUZUKI Toshiyuki, KUNII Hiroki, KAWAHARA Manabu, KIMURA Koji, TAKAHASHI Masashi, BAI Hanako  The Journal of Reproduction and Development Supplement  111-  (0)  P  -38-P-38  2018  [Not refereed][Not invited]
     

    【目的】夏期の暑熱ストレスは，人工授精受胎率低下を引き起こすなど，家畜繁殖性に悪影響を与えることが知られている。これまでに，ウシの卵子や卵巣，胚に対する暑熱ストレスの影響は報告されているが，母体子宮への影響は不明である。ウシを含む反芻動物の妊娠認識および成立はインターフェロン・タウ（IFNT）が役割を担う。IFNTは孵化後の胚から産生され，子宮内膜上の I 型IFN受容体（IFNAR）を介して働き，黄体退行を抑制する。また，インターフェロン誘導性因子（ISGs）の発現を誘導する。本研究ではこの妊娠認識に着目し，子宮内膜上皮細胞におけるIFNT応答性を暑熱負荷培養条件下で評価し，子宮への暑熱ストレスの影響を検証した。【方法】食肉検査場由来ウシ子宮から子宮内膜組織を採取し，上皮細胞を単離，培養した。単離した上皮細胞をウシの平常時の体温である38.5 ℃および暑熱時の体温である40.5 ℃の暑熱条件下で3，6，12時間培養後，RNA抽出，cDNA合成を行い，定量PCRにより暑熱ストレス指標である熱ショックタンパク質（HSP）群の発現を解析した。また，上皮細胞を同様の暑熱負荷条件下にて組換えウシIFNT（500 IU）を添加し12時間培養した。各処理区において定量PCRによるISGs（ISG15, MX1a）およびIFNAR（IFNAR1, IFNAR2）の発現解析を行った。【結果と考察】HSPのmRNA発現量は，12時間の暑熱負荷によりHSP27, 60, 90の全てが対照区と比べ有意に上昇した。この結果から，12時間の暑熱処理により，細胞への十分なストレス負荷が確認された。ISGs の発現はIFNT添加により有意に増加したが，暑熱負荷による影響はみられなかった。また，IFNAR発現へのIFNT添加および暑熱の影響もみとめられなかった。以上より，培養子宮上皮細胞でのIFNAR発現およびIFNTによるISGs（ISG15, MX1a）発現への暑熱ストレスの影響は少ないことが示唆された。

  • YAMAMURA Shota, KOHRI Nanami, AKIZAWA Hiroki, BAI Hanako, TAKAHASHI Masashi, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  111-  (0)  OR1  -28-OR1-28  2018  [Not refereed][Not invited]
     

    【目的】哺乳動物胚は桑実期から胚盤胞期にかけて内部細胞塊（ICM）と栄養外胚葉（TE）へ分化する。この過程において接触阻害を担うシグナル伝達系Hippo経路が重要な役割を果たすことがマウス胚で知られている。Hippo経路のエフェクターである転写コアクチベーターYAPとTAZが転写因子TEAD4と協調することにより，TEマーカーであるCdx2の転写を制御する。ウシ胚でもHippo経路がICM/TE分化に関与するといわれているが，詳細はわかっていない。さらに，哺乳類初期胚におけるYAPの解析は多いものの，YAPのパラログであるTAZについての知見は乏しい。そこで本研究では，ウシ初期胚におけるTAZ発現の役割を調べることを目的とした。【方法】食肉検査場由来のウシ卵巣から卵丘細胞−卵母細胞複合体を吸引採取し，定法に従い体外成熟，体外受精および体外培養に供しウシ胚を作出した。定量PCR，免疫染色により，卵母細胞および初期胚におけるTAZ遺伝子およびタンパク質の発現動態を調べた。続いて，TAZを標的としたshRNA発現ベクターを1細胞期胚に顕微注入しTAZ発現抑制（KD）胚を作出した。また，定量PCRおよび免疫染色により胚盤胞期におけるTEAD4やCDX2などのICM/TE分化関連遺伝子のmRNA発現，およびCDX2タンパク質のシグナル強度の変化を調べた。【結果】ウシ初期胚発生過程においてTAZ mRNA発現レベルは16細胞期で最も高く，TAZタンパク質発現は，16細胞期以降で核内での強いシグナルが観察された。TAZ KD胚の胚盤胞期までの発生率は，コントロールと比較し有意差は認められなかった。TEAD4およびCDX2発現レベルはコントロールと比較しTAZ KD胚で有意に低下し，CDX2タンパク質もシグナルの減衰が認められた。以上の結果から，ウシ初期胚発生におけるTAZ遺伝子およびタンパク質発現動態および，TAZ遺伝子発現抑制が分化関連遺伝子の発現に影響を及ぼすことが明らかとなった。

  • BAI Hanako, TALUKDER Shabur Md Abdus, KUNII Hiroki, ITO Tsukino, KAWAHARA Manabu, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  111-  (0)  P  -86-P-86  2018  [Not refereed][Not invited]
     

    【目的】乳牛における周産期疾病の予防は重要な課題である。特に分娩前後3週間の移行期において，分娩に伴う代謝変動や免疫状態の変化が研究，報告されている。本研究では，分娩がウシ母体に及ぼす影響を評価するため，分娩前後の末梢血単核球を用いてストレスおよび免疫関状態を評価することを目的とした。【材料と方法】北海道大学農場で飼養しているホルスタイン雌牛を用いた。分娩前後24時間および分娩後一週間の三時点で頚静脈から血液を採取し，密度勾配遠心法により末梢血単核球（PBMCs）を分離した。PBMCsからRNA抽出，cDNA合成を行い，定量PCRによりストレス関連遺伝子および炎症性サイトカイン遺伝子の発現解析を行った。また，同様に得られたPBMCsをマイトジェン（ConcanavalinA, ConA）添加および無添加条件で培養し，72時間後に幼若化反応をMTTアッセイ法により検証した。【結果と考察】抗酸化酵素GPX4, CuZnSOD, および炎症性サイトカインIL-2, IL-6, TNFAの発現が，分娩後に採取したPBMCsにおいて分娩前に採取したものと比較して増加していた。PBMCsの増殖性自体は試験期間を通して変化はみとめられなかった。一方，リンパ球の幼若化反応は，分娩直後のウシから採取したPBMCsでは，ConAの刺激下においても変化はみとめられなかったが，分娩直後および一週間後に有意に上昇していた。本研究より，分娩後24時間以内においてストレスおよび免疫関連遺伝子の発現が変化すること，および分娩前においては免疫応答能が低下しており，分娩後の回復を示唆する結果を得た。

  • 国井宏樹, 伊藤月乃, 鈴木惇文, 小木曽貴季, BALBOULA Ahmed Z, 唄花子, 永野昌志, 川原学, 高橋昌志  日本畜産学会大会講演要旨  123rd-  61  -61  2017/09/06  [Not refereed][Not invited]
  • KABIRAZ Dulal, 森田 金市, 川口 俊一, 高橋 昌志  Proceedings of the Chemical Sensor Symposium  62-  67  -69  2017/09  [Not refereed][Not invited]
  • 秋沢宏紀, 唄花子, 高橋昌志, 川原学  北海道畜産草地学会報  5-  (2)  21  -21  2017/08/24  [Not refereed][Not invited]
  • 塚原隼人, 菊池康太, 秋沢宏紀, 唄花子, 高橋昌志, 南保泰雄, 秦寛, 川原学  北海道畜産草地学会報  5-  (2)  21  -21  2017/08/24  [Not refereed][Not invited]
  • Natsumi Takei, Ahmed Balboula, Masashi Takahashi, Tomoya Kotani  MECHANISMS OF DEVELOPMENT  145-  S92  -S93  2017/07  [Not refereed][Not invited]
  • 椎名浩己, 唄花子, 高橋昌志, 川原学  日本卵子学会誌  1-  (1)  S48  -S48  2016/04/01  [Not refereed][Not invited]
  • SUZUKI Toshiyuki, TAKAHASHI Masashi, SHIROZU Takahiro, IWANO Hiroki, OGISO Takatoshi, YAMAUCHI Nobuhiko, YANAGAWA Yojiro, NAGANO Masashi, BAI Hanako, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  109-  (0)  OR1  -1-OR1-1  2016  [Not refereed][Not invited]
     

    【目的】ウシの子宮組織機能は発情周期及び着床期において絶えず変化している。特に，受精後14–18日では胚からIFN-τが分泌され，黄体退行が阻止されることで妊娠が維持される。そのため，この期間は妊娠認識とともに着床・妊娠の準備のための期間でもある。細胞内のタンパク質分解・再利用に関わるオートファジー機構は異化，細胞増殖，分化など，細胞内の代謝・環境変化に関わることが知られている。生殖組織においても，ステロイドホルモン依存的なオートファジー動態の変化がマウス子宮組織で見られる。しかし，ウシにおいては，オートファジーと子宮組織との関わりは未解明である。そこで，本研究では発情周期及び着床前期のウシ子宮組織におけるオートファジー及び，関連の深いリソソームカテプシンの動態を明らかにすることを目的とした。【方法】食肉検査場由来のウシ子宮を卵巣所見及び頸管粘液の状態・インピーダンス値を指標として，発情期，黄体前期，黄体中期，黄体後期のステージに分け，子宮内膜小丘間組織を採取した。また，AI後，14日及び18日で子宮灌流により胚を確認できたウシの子宮内膜組織を採取した。採取した子宮内膜組織からmRNAを抽出し，オートファジー関連因子（ATG3，ATG5，ATG7，LC3α，LC3β，mTOR，Beclin1）及びリソソームカテプシン群（CTSB，CTSD，CTSL，CTSZ）の遺伝子発現定量解析を行った。【結果】発情周期のウシ子宮内膜小丘間組織におけるオートファジー関連因子の遺伝子発現に変動は見られなかったが，妊娠14日及び18日ではLC3αの有意な上昇が見られた。また，リソソームカテプシン群については，発情周期間での発現動態に差は見られなかったものの，妊娠14日及び18日でCTSZの有意な上昇が見られた。以上の結果よりウシ子宮におけるオートファジー-リソソームカテプシンは，妊娠特異的に変動し，着床に向けた子宮改編への関与が示唆された。

  • SHIROZU Takahiro, SUZUKI Toshiyuki, IWANO Hiroki, OGISO Takatoshi, KIM Sung Woo, BAI Hanako, KAWAHARA Manabu, KIMURA Koji, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  109-  (0)  P  -14-P-14  2016  [Not refereed][Not invited]
     

    【目的】着床前のウシ子宮では，胚より分泌されるインターフェロンタウ（IFN-τ）が，IFNAR1（R1）とIFNAR2（R2）の二量体で構成されるI型IFN受容体を介したJAK/STAT経路によってIFN誘導性遺伝子（ISGs）の発現を誘導する。しかし，ウシ子宮におけるIFN-τシグナルの受容体サブユニット依存的なISGs発現調節機構は不明である。本研究では，ウシ子宮内膜上皮細胞におけるI型IFN受容体サブユニットの発現抑制がIFN-τ刺激による遺伝子発現へ及ぼす影響を検証した。【材料と方法】食肉処理場より採材したウシ子宮内膜組織由来の初代培養細胞を実験に供試した。細胞にR1またはR2のsiRNAを導入しRNA干渉した。導入24時間後のR1，R2の発現抑制効果を定量PCRで検証した。さらに，siRNA導入24時間後に組換えウシIFN-τを添加し，12および24時間後の1）抗ウイルス能（MX1，2，ISG15），2）IFNシグナル伝達制御（STAT1，2，IRF1，2，3，9），3）I型IFN受容体発現の維持（COPS5）および4）着床時の細胞増殖（TGF-β1，2）に関連する遺伝子群の発現量を定量PCRで解析した。【結果と考察】R1またはR2のsiRNA導入によるRNA干渉はR1，R2の発現をそれぞれ特異的に抑制した。R1またはR2の発現抑制はIFN-τによるMX1，2，ISG15，STAT1，2，IRF1，2，3，9，COPS5発現の誘導を有意に抑制した。特に，R2と比べてR1抑制時に高い発現抑制効果を示した。TGF-β1，2の発現については，R1，R2の発現抑制による影響は見られず，JAK/STATシグナルから直接制御されない可能性が示唆された。【結論】IFN-τのIFNARを介したJAK/STAT経路にはR1依存的なシグナル伝達機構が存在することが明らかとなった。

  • YAMAZAKI Wataru, AMANO Tomoko, BAI Hanako, TAKAHASHI Masashi, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  109-  (0)  OR1  -27-OR1-27  2016  [Not refereed][Not invited]
     

    【目的】哺乳類において三倍体や四倍体の多倍体胚は胎生致死となる。また，片親性発現を示すインプリント遺伝子の正常な発現は哺乳類の個体発生に必須であり，過去にマウス三倍体胎子におけるインプリント遺伝子発現異常を確認している（Yamazaki et al., 2015）。インプリント遺伝子発現の主要な制御機構として，父母ゲノムの性特異的なDNAメチル化修飾が知られ，メチル化修飾を受ける領域はメチル化可変領域（DMR）と呼ばれる。植物ではゲノム多倍体化によりDNAメチル化レベルが変化するが，哺乳類ゲノムでは倍数性とDNAメチル化レベルの関係は不明である。そこで，本研究では，マウス四倍体および三倍体胎子について，インプリント遺伝子のDMRメチル化レベルと発現レベルを解析した。【方法】マウス四倍体胚は二細胞期胚における電気融合法により作出した。三倍体胚は前核期卵の核移植により作出した。胎齢10.5日で回収した胎子からRNA，DNAを抽出し，H19，Gtl2，Igf2r，Grb10，Zim1，Igf2，Dlk1，Peg3，Snrpn，Ndn，Ipwの11インプリント遺伝子の定量PCRを行った。さらに，バイサルファイトシークエンスにより4か所のDMR（H19-，IG-，Igf2r-，Snrpn-）のメチル化解析を実施し，アリル発現解析（Igf2，Gtl2，Dlk1）も行った。【結果および考察】マウス四倍体胎子のインプリント遺伝子発現レベルを二倍体胎子と比較すると，9遺伝子で発現レベルが有意に変化していた。しかしながら，解析した4か所のDMRメチル化レベルは，四倍体，三倍体胎子共に性特異的なメチル化様式を維持していた。また，四倍体胎子におけるアリル発現解析においても，解析した3遺伝子については片親性発現を維持していた。これらの結果から，マウス胚の多倍体化はインプリント遺伝子発現には影響を及ぼすものの，DMRメチル化状態，および，片親性発現の維持には影響を及ぼさないことが明らかとなった。

  • 高橋昌志  繁殖技術  35-  (3)  43  -48  2015/12  [Not refereed][Not invited]
  • 高橋昌志  食肉に関する助成研究調査成果報告書  33-  204  -209  2015/11  [Not refereed][Not invited]
  • 田中愛子, 飯村さとみ, 小山詩織, 山崎渉, 佐々木恵亮, 高橋昌志, 川原学  日本畜産学会大会講演要旨  120th-  71  2015/09/11  [Not refereed][Not invited]
  • 秋沢宏紀, 長友啓明, 定郁里, 岸靖典, 山中賢一, 詫摩哲也, 佐々木恵亮, 山内伸彦, 柳川洋二郎, 永野昌志, 河野友宏, 高橋昌志, 川原学  日本畜産学会大会講演要旨  120th-  70  2015/09/11  [Not refereed][Not invited]
  • 小木曽貴季, 岩野弘暉, 白水貴大, 川原学, 高橋昌志  日本畜産学会大会講演要旨  120回-  71  -71  2015/09  [Not refereed][Not invited]
  • 岩野弘暉, 小木曽貴季, 白水貴大, 川原学, 枝重圭祐, 高橋昌志  日本畜産学会大会講演要旨  120回-  71  -71  2015/09  [Not refereed][Not invited]
  • 高橋昌志  LIAJ News (Web)  (153)  6‐8 (WEB ONLY)  2015/07/25  [Not refereed][Not invited]
  • 高橋昌志  臨床獣医  33-  (5)  12-17,9  2015/05/01  [Not refereed][Not invited]
  • 阪谷美樹, 竹之内直樹, 高橋昌志  九州沖縄農業試験研究の成果情報(Web)  2015-  WEB ONLY  2015  [Not refereed][Not invited]
  • 手島裕貴, 杉山大輔, 真栄城正寿, 岩崎渉, 山下健一, 山中賢一, 高橋昌志, 宮崎真佐也  化学とマイクロ・ナノシステム学会研究会講演要旨集  30th-  41  2014/10/02  [Not refereed][Not invited]
  • 小山詩織, 飯村さとみ, 高橋昌志, 川原学  北海道畜産草地学会報  2-  138  2014/03/31  [Not refereed][Not invited]
  • 菊地康太, 香西圭輔, 法上拓生, 徳山翔太, 阪谷美樹, 川原学, 南保泰雄, 奥田潔, 高橋昌志  日本畜産学会大会講演要旨  118th-  226  2014/03/27  [Not refereed][Not invited]
  • 白水貴大, 川原学, 高橋昌志  北海道畜産草地学会報  2-  137  -137  2014/03  [Not refereed][Not invited]
  • 阪谷美樹, 竹之内直樹, 山中賢一, 高橋昌志  九州沖縄農業試験研究の成果情報(Web)  2014-  WEB ONLY  2014  [Not refereed][Not invited]
  • 山中賢一, 水落絢子, 阪谷美樹, 竹之内直樹, 高橋昌志, 和田康彦  Journal of Reproduction and Development  107-  (0)  P  -78-P-78  2014  [Not refereed][Not invited]
     
    【目的】近年，夏季の高温環境が家畜の繁殖性を大きく低下させることが生産現場で深刻な問題となっている。その1つとして，暑熱ストレスによるウシ胚の損耗が挙げられる。我々は，これまで体外成熟および体外受精時の暑熱ストレスがリソソーム内に存在するシステインプロテアーゼであるカテプシンB活性を上昇させること，さらにそれらの活性を阻害することによって発生能が改善されることを報告してきた（第106回日本繁殖生物学会大会）。そこで，暑熱ストレス下で起こる胚の発生停止機構の一端を明らかにすることを目的とし，今回は体外発生時の暑熱ストレスによっても同様の現象が起こりうるのかについて検討を行った。【方法】実験1では，体外発生時の暑熱ストレスに対する耐性の変化を調べるために通常の体外培養温度(38.5℃)で培養した対照区，暑熱ストレス(40.0℃)を培養後1，2，4および6日目に24時間付加した暑熱ストレス（HS）区をそれぞれ設け，胚の体外発生率を比較した。実験2では，暑熱ストレスとカテプシンB活性との関係を明らかにするため，実験1で体外発生率が低下した暑熱区の胚におけるカテプシンB活性の測定を蛍光基質を用いて行った。実験3では，カテプシンB活性の制御が暑熱ストレスを受けた胚の発生能に及ぼす影響を検討するために，HS区の胚をカテプシンB阻害剤であるE-64を添加した培地で培養し，それらのカテプシンB活性および体外発生率を調べた。【結果】実験1の結果，発生培養後1および2日目の胚では他の発生ステージの胚と比較して暑熱ストレスに対する耐性が低かった。また実験2の結果から，暑熱ストレスを受けた胚は対照区と比較してカテプシンB活性が高いことが明らかとなった。そこで実験3では，カテプシンB活性阻害剤E-64の効果を検証したところ，E-64添加区の胚ではカテプシンB活性がHS区の胚と比較して抑制され，さらに，胚盤胞形成率の改善がみられた。以上の結果から，暑熱ストレスによる胚発生能低下にカテプシンBの活性が関与する可能性が示された。
  • 田中愛子, 佐々木恵亮, 高橋昌志, 川原学  Journal of Reproduction and Development  107-  (0)  P  -62-P-62  2014  [Not refereed][Not invited]
     
    【目的】Toll-like receptor(TLR)ファミリーはウイルスの構成成分を認識する自然免疫応答必須な病原体レセプターであり，その一員であるTLR9は細菌やウイルスの非メチル化CpG DNAを認識することで炎症反応を誘導する。しかし，哺乳類卵母細胞および初期胚における発現動態はほとんど調べられていない。そこで本研究では，マウス初期胚発生におけるTLR9の発現動態を明らかにすることを目的とし，卵母細胞および初期胚において定量PCRと免疫染色による発現解析を行った。【方法】過剰排卵を誘起したC57BL/6N系統の雌マウスの卵管膨大部より卵丘細胞卵子複合体を回収し，体外受精および体外培養に供した。採取したMII期卵母細胞，1細胞期胚，2細胞期胚，4細胞期胚，8細胞期胚，桑実期胚および胚盤胞期胚について，定量PCRおよび免疫蛍光染色によりTLR9の発現動態を調べた。【結果】定量PCRの結果，8細胞期胚を除く全てのステージにおいてTlr9のmRNAが検出され，各発生ステージの発現レベルをMII期卵母細胞と比較したところ，1細胞期胚で有意に高く，2細胞期以降の全てのステージでは有意に低くなっていた(P < 0.05)。免疫染色の結果，TLR9タンパク質は，mRNAと異なり全てのステージにおいて主に細胞質中で蛍光シグナルが観察された。さらに，前核期1細胞期胚での雄性および雌性前核において細胞質中よりも弱い蛍光シグナルが観察された。また，胚盤胞期胚では壁栄養膜細胞において顆粒状のパターンを示して局在する傾向が見られた。以上の結果から，マウス卵母細胞および初期胚においてTLR9のmRNA発現が確認され，タンパク質の局在パターンとして体細胞の知見と同様に主に細胞質中に存在することが示された。
  • 白水貴大, 川原学, 木村康二, 高橋ひとみ, 柳川洋二郎, 永野昌志, 白汝嵐, 今川和彦, 高橋昌志  Journal of Reproduction and Development  107-  (0)  OR2  -24-OR2-24  2014  [Not refereed][Not invited]
     
    【目的】ウシMX1はI型インターフェロン(IFN)-α/β誘導性遺伝子であり，MX1Bというスプライシング変異体が存在する。我々は第2回北海道畜産草地学会において，子宮組織におけるMX1とMX1B mRNAが妊娠中期と比べて妊娠初期で有意に高い発現を示すことを報告した。このことから，MX1とMX1Bの発現には，妊娠認識時にのみ胚より産生されるIFN-τが深く関わることが示唆された。IFN-τはI型IFNに属するが，IFN-α/βとは異なる一過性の産生パターンを示す。本研究では，MX1とMX1BのI型IFN応答性差異の有無を明らかにする目的で，ウシ子宮組織培養系を用いてIFN-τまたはIFN-αによるMX1とMX1B mRNAの発現応答を比較し検証した。さらに，発情周期の違いによる両遺伝子のIFN応答性の差異について，I型IFN受容体発現と併せて検証した。【材料と方法】黄体所見をもとに前期，中期，後期に分けた食肉処理場由来のウシ非妊娠子宮から内膜組織を採取した。これらの組織小片を1時間前培養後，抗ウイルス活性を合わせたIFN-τを含む濃縮妊娠子宮灌流液(D18)またはウシ組換えIFN-α添加で培養し，12および24時間後のMX1とMX1B mRNAの発現量を定量PCRで調べた。また，採取直後の子宮組織におけるI型IFN受容体IFNAR1とIFNAR2の発現量も定量PCRで調べた。【結果と考察】子宮灌流液(D18)またはIFN-α添加によってMX1とMX1Bともに発現が誘導され，MX1Bに対しMX1が有意に高い発現量を示したが，両添加区間で応答性に差はみられなかった。発情周期別で比較した結果，12時間培養後のMX1とMX1Bの発現量ならびに採取直後の子宮組織でのIFNAR1とIFNAR2の発現量は後期で低い傾向がみられた。以上より，発情周期の違いで子宮内膜におけるI型IFN受容体の発現量が変化し，それに伴いI型IFNによるMX1とMX1Bの発現応答性に差異が生じる可能性が示唆された。
  • 山崎渉, 河野友宏, 高橋昌志, 川原学  Journal of Reproduction and Development  107-  (0)  OR1  -15-OR1-15  2014  [Not refereed][Not invited]
     
    【目的】哺乳類の正常な個体発生は雌雄からなる二倍体(Diploid)であることが必須である。一方，三倍体の個体発生については，哺乳類の場合，正常な個体発生は行われず，マウスでは胎齢11.5日で胎生致死となる。哺乳類の正常な個体発生には父母由来ゲノムから性特異的に発現するインプリント遺伝子の正常な発現が必要である。本研究ではマウス三倍体胚であるDiandric triploid: DATとDigynic triploid: DGTのインプリント遺伝子発現解析を行った。また，DGT胚の一セットの雌ゲノムに非成長期卵母細胞(ng卵子)を用いた三倍体胚(ng-DGT)を作製し，哺乳類個体発生におけるインプリント遺伝子と核ゲノム倍数性の関係について検証した。【方法】BDF1マウスを用い，体外受精もしくは活性化処理により作出した前核期胚を用意し，核移植によりDAT胚，DGT胚を作出した。作出した胚は体外培養-胚移植に供し，胎齢10.5日で胎子を回収し，母性発現: H19，Gtl2，Igf2r，Grb10，並びに，父性発現: Igf2，Dlk1，Ndn，Peg3のインプリント遺伝子発現を定量PCRによって解析した。また，ng-DGT胚は，定法の二母性胚作出過程で体外受精を行い作出し，通常の三倍体胚と同様に発生能を検証した。【結果および考察】マウス三倍体胎子におけるインプリント遺伝子発現についてDiploidと比較すると，DAT胎子ではIgf2の発現レベルが有意に増加していた。また，DGT胎子は，全ての父性発現インプリント遺伝子とH19において有意な発現レベルの低下がみられ，Igf2rとGrb10では発現レベルが有意に増加していた。一方，ng-DGT胎子の頭尾長を調べた結果，DAT胎子とDGT胎子の中間のサイズであった。これらのことから，マウス三倍体胎子の発生はインプリント遺伝子と核ゲノム倍数性の両面から特徴づけられていることが示唆された。
  • 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer  35-  (2)  45  -53  2013/05/28  [Not refereed][Not invited]
  • 高橋昌志  日本胚移植学雑誌  35-  (2)  45  -53  2013/05/28  [Not refereed][Not invited]
  • NAGATOMO Hiroaki, KAGAWA Shinjiro, SADA Ayari, KISHI Yasunori, TAKAHASHI Masashi, KONO Tomohiro, KAWAHARA Manabu  Journal of mammalian ova research = 日本哺乳動物卵子学会誌  30-  (2)  S35  2013/04/01  [Not refereed][Not invited]
  • SANO Nagisa, SASAYAMA Norihisa, SIRASU Kazuo, OHTA Hisayoshi, SAKATANI Miki, NAGASHIMA Hiroshi, KAWAHARA Manabu, TAKAHASHI Masashi  Journal of mammalian ova research = 日本哺乳動物卵子学会誌  30-  (2)  S73  2013/04/01  [Not refereed][Not invited]
  • 長友啓明, 加川真二郎, 定郁里, 岸靖典, 高橋昌志, 河野友宏, 川原学  Journal of Mammalian Ova Research  30-  (2)  S35  2013/04/01  [Not refereed][Not invited]
  • 佐野渚, 笹山典久, 白数昭雄, 太田久由, 阪谷美樹, 長嶋比呂志, 川原学, 高橋昌志  Journal of Mammalian Ova Research  30-  (2)  S73  2013/04/01  [Not refereed][Not invited]
  • 定郁里, 長友啓明, 加川真二郎, 高橋昌志, 川原学  北海道畜産草地学会報  1-  89  2013/03/29  [Not refereed][Not invited]
  • 佐野渚, 笹山典久, 白数昭雄, 太田久由, 長嶋比呂志, 川原学, 高橋昌志  北海道畜産草地学会報  1-  90  2013/03/29  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志, 竹之内直樹  日本畜産学会大会講演要旨  116th-  172  2013/03/28  [Not refereed][Not invited]
  • 菅原彩子, 佐藤匠, 伊藤俊彦, 高橋利清, 西宮弘, 加藤真姫子, 伊藤隆, 横尾正樹, 横尾万里, 森祐貴, 春日和, 小嶋郁夫, 山中賢一, 阪谷美樹, 高橋昌志, 小林正之  日本畜産学会大会講演要旨  116th-  175  2013/03/28  [Not refereed][Not invited]
  • 櫻井伸行, 藤井貴志, 平山博樹, 陰山聡一, 山中賢一, 高橋昌志, 橋爪力, 澤井健  日本畜産学会大会講演要旨  116th-  161  2013/03/28  [Not refereed][Not invited]
  • 高橋昌志  麻布大学雑誌  24-  (24)  140  -140  2013/01/31  [Not refereed][Not invited]
  • 高橋ひとみ, 綱崎誠, 濱野貴史, 高橋昌志, 奥田潔, 犬丸茂樹, 岡野彰, 下司雅也, 平子誠  農研機構畜産草地研究所成果情報(Web)  2013-  2013
  • 高橋昌志  北海道畜産草地学会報  1-  47  -54  2013  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志, 竹之内直樹  Journal of Reproduction and Development  106-  (0)  P  -81-P-81  2013  [Not refereed][Not invited]
     
    【背景】初期胚発生にエピジェネティックな遺伝子発現制御が密接に関わっており，DNAメチル化パターンやTen eleven translocation genes (TETs)による5メチルシトシンの水酸化が受精の成立や正常胚発生に重要なことが指摘されている。ウシでは高温成熟条件が卵子成熟や初期胚発生を阻害するが，これらの発生阻害とエピゲノム状態に関する報告はほとんどない。そこで高温成熟条件がウシ卵子および体外受精後の発生胚におけるTETsとDNMTsの発現パターンを解析した。 【方法】屠場由来卵巣より採取した卵子を体外成熟(38.5℃)，受精させて得られた成熟卵子，zygote，2，8細胞期胚，桑実胚，胚盤胞各15個からmRNAを抽出し，定量PCRにてDNAメチル基修飾に関連する遺伝子（TET2, TET3, DNMT1, DNMT3A, DNMT3B）のステージ別発現量をΔCT法（成熟卵子=1）で解析した。また，通常区（38.5℃），高温区（40℃）で成熟培養したウシ受精卵について同様の遺伝子発現と体外受精による発生能に関して検討を行った。 【結果・考察】胚の発生ステージ依存的に遺伝子発現に差が認められた。すなわち，TET2,3並びにDNMT1は成熟卵子期～8細胞期まで高発現であり，桑実胚，胚盤胞での発現は有意に低かった。一方DNMT3Aは8細胞期，胚盤胞期で高く，DNMT3Bは成熟卵子，zygote，胚盤胞で高い発現を示した。高温成熟培養により卵子成熟率，発生培養2日目の8細胞期以上発生率および7日目の胚盤胞発生率は有意に低下した。また，TET2は処理区間の差が認められなかったが，zygoteで高温区のTET3, DNMTsの発現量が有意に増加し，反対に8細胞期胚ではTET3の発現量が有意に低下した。以上の結果から高温成熟培養によりウシ初期受精卵のエピゲノム状態も影響を受ける可能性が示唆された。現在も研究を継続中である。本研究は平成24年度旗影会研究助成金で実施された。
  • 高橋昌志, 阪谷美樹, 川原学, 竹之内直樹  Journal of Reproduction and Development  106-  (0)  OR2  -33-OR2-33  2013  [Not refereed][Not invited]
     
    【目的】ウシを含む雌個体の発情発来時には，一過性に体温が上昇することが知られており，LHサージによる排卵との関連が調べられている。演者らは腟内に留置したデータロガーによる連続体温計測並びに直腸温度の定期計測によって深部体温と腟内温度の両方で発情時の体温上昇パターンを明らかにしている。しかし，深部体温や腟内温度の長期連続計測には労力や衛生的な問題があり，非侵襲的な体温情報取得の必要がある。発情時や疾病時の体温情報を非侵襲的に得るための手法として赤外線サーモグラフィー(Infrared Thermography:IRT)が注目されている。そこで本研究では，発情に伴う体表面温度変化を計測し，計測部位による温度データ取得の可否および，非発情，発情時の温度変化を捉えることを目的とした。【方法】九農研内で飼養されている黒毛和牛繁殖雌牛群について，1～2周期にかけての発情を乗駕行動，外陰部充血腫脹ならびに頸管粘液の電気インピーダンス (EI) 値を元に確認した。体表面温度の計測にはIRTを用い，発情期および非発情期の牛について外陰部および頭部側面の二か所で距離と角度を同一にして撮影し，赤外線画像を得た。画像データは計測ソフトウエアを用いて温度表示範囲を統一表示後，計測領域内の最高温度値を体温値とした。【結果】乗駕行動，発情粘液の漏出とEI値の低下によって発情を確認した個体では，外陰部周辺の表面温度最高値は非発情期の個体と比べて有意(P<0.01)に高かった。また，頭部についても温度最高値が得られた眼球白目部分の温度平均は発情期の個体で有意（P<0.05）に高かった。撮影対象部位の体毛に覆われた部分では発情，非発情の違いは検出できなかった。加えて，日中，太陽光に照らされた個体では，太陽光の輻射熱が優位になることで正確な体温情報を得ることは困難であった。本結果から，適切な撮影部位の選択および輻射熱の影響を考慮することで，IRTによる非侵襲的な体温情報を得ることが可能であることが示唆された。
  • 徳山翔太, 香西圭輔, 登石裕子, 角田修男, 田谷一善, 阪谷美樹, 高橋昌志, 南保泰雄, 奥田潔  Journal of Reproduction and Development  106-  (0)  P  -86-P-86  2013  [Not refereed][Not invited]
     
    【目的】哺乳類において子宮で産生されるPGFは主要な黄体退行因子である。最近，私達はウシ子宮内膜においてPGFがPGF産生を自己増幅的に促進することを報告した。ウマでは，ウシと比較し少量のPGF投与でも黄体が退行することから，我々はウマ子宮にPGF自己増幅機構が存在しているという仮説を立て，この仮説を証明するため以下の実験を行った。【方法】1)中期黄体を有するウマにPGF類縁体のEstrumate (d,l-cloprostenol: 250 mcg/ml)を1 ml投与した。投与から3日目まで定期的に採血し，血液中progesterone (P4)濃度および血清中PGF metabolite (PGFM)濃度を測定した。2)発情周期を通じた子宮内膜組織におけるPGF receptor (FPr) mRNA発現量を調べた。3)子宮内膜組織および上皮細胞，間質細胞にPGF (0.1 μM)およびPGF合成酵素阻害剤のindomethacin (Indo: 10 μM)を単独または組み合わせて添加し2時間培養した。組織片および細胞を洗浄後，培養液を交換し，2時間培養後の培養上清中PGF濃度を測定した。4)子宮内膜上皮細胞および間質細胞にPGF (0.1 μM)を添加し4時間培養後，上皮細胞および間質細胞におけるPGF関連遺伝子発現量を調べた。【結果】1)血液中P4濃度はEstrumate投与2時間後に半減し，投与24時間後で約1/10量まで減少した。血清中PGFM濃度はEstrumate投与45分後に一過性の上昇を示し，投与16時間以降はピークよりも高い値を示し続けた。2)黄体後期の子宮内膜組織のFPr mRNA発現量は黄体初期および退行期と比較し高かった。3)子宮内膜組織および上皮細胞，間質細胞においてPGF添加によりPGF産生が刺激された。また，IndoはPGF添加によるPGF産生刺激作用を抑制した。4)子宮内膜上皮細胞および間質細胞においてPGFはcyclooxygenase (COX)-2 mRNA発現を刺激した。COX-2はPGF合成酵素の一つのため，ウマ子宮においてPGFはCOX-2発現を増加させることでPGF産生を刺激していると考えられる。本研究の結果より，ウマにおいて少量のPGF投与でも黄体が退行する理由は，子宮にPGF自己増幅機構が存在するためと推察される。
  • 佐々木恵亮, 高橋昌志, 川原学  Journal of Reproduction and Development  106-  (0)  OR1  -13-OR1-13  2013  [Not refereed][Not invited]
     
    【目的】脊椎動物はウイルスに対して複雑な自然免疫機構を保持する。免疫細胞においてはウイルスレセプターが種々の病原体構成分子を認識し，インターフェロンや炎症性サイトカインによる炎症反応を引き起こす。しかしながら，哺乳動物の初期胚における抗ウイルス応答機構は不明である。本研究では，マウス初期胚における抗ウイルス応答の有無を検証することを目的とし，RNAウイルス感染を認識するレセプターRetinoic acid-inducible gene-I（RIG-I）を介する抗ウイルス応答を解析した。【方法】過剰排卵を誘起したC57BL/6N系統の雌マウスの卵管膨大部より卵丘細胞卵子複合体を回収し，体外受精および体外培養に供した。得られたMII期卵母細胞，1細胞期胚，2細胞期胚，4細胞期胚，8細胞期胚，桑実胚および胚盤胞期胚について，qRT-PCRおよび免疫染色法によりRIG-Iの発現を検出した。採取したMII期卵母細胞にGFP発現組換え水疱性口内炎ウイルス（VSV）を感染させ，体外受精および体外培養に供した。VSV感染胚は1細胞期および2細胞期の時点で回収し，qRT-PCRによりRIG-Iおよび炎症性サイトカインであるInterleukin 1β（IL1B）の発現レベルを調べた。【結果】qRT-PCRおよび免疫染色法の結果，MII期卵母細胞から胚盤胞期胚においてRIG-Iの発現が確認され，とくに2細胞期胚までに強い発現が認められた。MII期卵母細胞へのVSV感染の結果，感染胚の胚盤胞期胚までの発生率は有意に低下した（P<0.05）。VSV感染によりRIG-Iの発現レベルは一過性に増加したが，IL1Bは感染の有無に関わらず検出限界以下であった。以上のことから，VSV感染胚においてRIG-I発現は上昇するものの，炎症反応による抗ウイルス応答は示さないと考えられた。現在，感染による他のサイトカイン類への影響，さらにRIG-IのリガンドであるPoly(I:C)のマイクロインジェクションによる解析を進めている。
  • 山中賢一, 樫澤彩, 和田康彦, 阪谷美樹, 竹之内直樹, 高橋昌志  Journal of Reproduction and Development  106-  (0)  P  -113-P-113  2013  [Not refereed][Not invited]
     
    【目的】我々はこれまで，リソソーム内でタンパク質分解に関わる酵素であるカテプシンBの活性とウシ体外受精胚の発生能との間に負の相関があることを明らかにしてきた。一方，暑熱ストレスにより受精胚の発生能が低下することがこれまでに報告されているが，カテプシンB活性の関与について調べた報告はない。したがって，今回は体外受精時の暑熱ストレスがウシ体外受精胚の発生能およびカテプシンB活性に及ぼす影響について検討を行った。さらに，カテプシンB活性の抑制により暑熱ストレスを受けた胚の発生阻害回避の可能性についても検討を行った。【方法】対照区，暑熱ストレス(HS)区，暑熱ストレス＋カテプシンB阻害剤E-64添加(E-64;濃度:1, 5, 10 μM)区を設定し，対照区は38.5℃，HSおよびE-64区は40.0℃で体外受精を行った。媒精完了後，卵丘細胞を剥離し，38.5℃，5％CO2の条件下で発生培養を行った。培養後2日目にMagic Red Cathepsin B Detection Kitを用いてカテプシンＢ活性の検出を行った。また，発生率は分割率と胚盤胞形成率をそれぞれ培養後2日目，8日目に観察することで算出した。さらに，胚盤胞期胚は二重染色およびTUNEL染色により細胞構成数およびアポトーシス陽性細胞の検出により品質評価を行った。【結果】発生率はHS区において対照区と比較して分割率，胚盤胞形成率ともに有意に低下した。一方，5 μM添加E-64区で分割率，胚盤胞形成率および総細胞数がHS区と比較して有意に増加し，対照区と同等の成績であった。さらに，各実験区のカテプシンB活性を調べたところ，HS区では対照区と比較して有意に高く，5 μM添加 E-64添加区では，HS区と比較して有意に低かった。以上の結果から，暑熱ストレスによるカテプシンB活性の増加が胚発生阻害を引き起こす原因の一つであるという可能性が示された。
  • 定郁里, 長友啓明, 高橋昌志, 川原学  Journal of Reproduction and Development  106-  (0)  P  -82-P-82  2013  [Not refereed][Not invited]
     
    【目的】哺乳類の初期胚は胚盤胞期において細胞分化が明確化する。すなわち，将来胎子組織となる内部細胞塊(ICM)と，胚体外組織を形成する栄養外胚葉(TE)の2種の細胞群に分化し，それぞれの細胞に特有の遺伝子発現を示すようになる。これまでに我々は，ウシ胚盤胞期胚においてICMあるいはTE特異的な発現遺伝子パターンを明らかにするために部位特異的発現を示す遺伝子群をマイクロアレイにより決定した。本研究では，これらの遺伝子群のうち，定量PCRおよびIn situ hybridization法によってTE特異的な発現が新たに確認されたConnective tissue growth factor (CTGF )について着目し，初期胚における発現動態を解析した。体細胞の研究において，CTGFはCDX2の上流で機能するTEAD4の標的遺伝子であると考えられているが，ウシ胚での発現の挙動は明らかになっていない。そこで，TEAD4に関しても発生過程における発現動態を解析した。【方法】屠場由来の卵巣より採取した卵子を体外成熟，受精，培養に供した。体外受精後の発生胚を8–16細胞期，桑実期，胚盤胞期の3ステージに分け，1ユニット10個として各時期で3ユニットずつサンプリングした。各サンプル胚からRNAを抽出後，cDNAを合成し，定量PCRに供してmRNA発現レベルの比較を行った。【結果および考察】CTGFは8–16細胞期から拡張胚盤胞期にかけて約10倍に発現レベルが有意に上昇した(P<0.01)。一方，TEAD4についても同様に8–16細胞期から胚盤胞期にかけて約20倍に発現レベルが上昇し，有意差が認められた(P<0.01)。CTGFおよびTEAD4遺伝子ともに，8–16細胞期から胚盤胞期にかけて発現レベルが上昇したことから，胚盤胞期胚でのCTGFのTE細胞特異的発現にTEAD4が関連していることが示唆された。
  • 山崎渉, 馬狩柚子, 河野友宏, 高橋昌志, 川原学  Journal of Reproduction and Development  106-  (0)  P  -36-P-36  2013  [Not refereed][Not invited]
     
    【目的】現在マウスゲノム上で同定されている父性メチル化インプリント領域は1番，7番，9番，そして12番染色体の4箇所のみである。本研究では，父性ゲノムを用いない二母性マウス胚と野生型(WT)胚を用いて新規父性メチル化インプリント遺伝子の有無について検証した。二母性胚では7番および12番染色体上の2領域以外の父性メチル化領域はデフォルトの状態にあるため，それ以外の父性メチル化インプリント遺伝子については母性ゲノム様の発現パターンを示すと考えられる。このことから，既知4領域以外に父性メチル化領域が存在していれば，WT胚と比較して二母性胚では発現差がみられると考えられる。そこで，二母性胚とWT胚でマイクロアレイ解析を行い，発現差がみられた遺伝子について多型解析を実施し，発現アリルを決定した。【方法】初めに，胎齢12.5日における，7番染色体のみ遺伝子発現を補正したng⊿7ch/fg，12番染色体のみ発現を補正したng⊿12ch/fg，そして双方において遺伝子発現を補正したng⊿Double/fgの各二母性胚とWT胚の遺伝子発現をマイクロアレイによって解析し，2倍以上の発現差がある遺伝子をピックアップした。次に，それらについて，JF/Ms(J)およびC57BL/6N(B)系統間で多型を調べ，J×BおよびB×J間のF1胎齢12.5日胚の発現遺伝子について多型解析を実施し，発現アリルを決定した。【結果および考察】全タイプの二母性胚とWT胚において遺伝子発現差を比較し，発現差があり，かつ，発現の増加および低下の傾向が同じ遺伝子を調べた結果，35遺伝子が確認された。これらの遺伝子のうち多型が検出された33遺伝子について発現アリル調べた結果，全て両アリル発現を示すことが明らかとなった。そのため，全身性発現を示す父性メチル化インプリント遺伝子は既知4領域のみであることが示唆された。今後，標的組織を絞って組織特異的な発現を比較していく必要があると考えられる。
  • 徳山翔太, 香西圭輔, 法上拓生, 阪谷美樹, 高橋昌志, 奥田潔  日本内分泌学会雑誌  88-  (2)  743  2012/09/20  [Not refereed][Not invited]
  • 手島裕貴, 杉山大輔, BRIONES‐NAGATA Maria, Portia P, 山下健一, 山中賢一, 高橋昌志, 宮崎真佐也  化学工学会秋季大会研究発表講演要旨集(CD-ROM)  44th-  ROMBUNNO.XA1P43  2012/08/19  [Not refereed][Not invited]
  • 吉ざわ努, 平子誠, 下司雅也, 高橋昌志, 永井卓  日本胚移植学雑誌  34-  (2)  63  -68  2012/05/31  [Not refereed][Not invited]
  • 吉ざわ 努, 平子 誠, 下司 雅也, 高橋 昌志, 永井 卓  日本胚移植学雑誌 = Japanese journal of embryo transfer  34-  (2)  63  -68  2012/05/31  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹  化学とマイクロ・ナノシステム研究会講演要旨集  25th-  1  2012/05/17  [Not refereed][Not invited]
  • 手島裕貴, 杉山大輔, PORTIA Briones, Patena Maria, 山下健一, 山中賢一, 高橋昌志, 前田英明, 宮崎真佐也  化学とマイクロ・ナノシステム研究会講演要旨集  25th-  30  2012/05/17  [Not refereed][Not invited]
  • 香西 圭輔, 法上 拓生, 高橋 昌志  馬の科学 = Equine science  49-  (4)  268  -273  2012  [Not refereed][Not invited]
  • SAKATANI Miki, TAKAHASHI Hitomi, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  105-  (0)  1104  -1104  2012  [Not refereed][Not invited]
     
    【目的】 高泌乳牛では泌乳と採食量の負のエネルギーバランスにより血中の遊離脂肪酸（NEFA）が高濃度となることが知られている。同様に卵胞液中のNEFAも高濃度となり卵子の品質を低下させることが示唆されている。そこで本研究では，体外培養系を用いた高NEFA濃度の卵子成熟環境下が卵子成熟および胚発生に及ぼす影響について，卵丘細胞のプロジェステロン（P4）分泌に注目して検討した。  【方法】 屠場由来牛卵巣より採取した卵丘細胞卵子複合体を0.75％BSA加M199を基礎培地とし，脂肪酸(ステアリン酸，パルミチン酸，オレイン酸)を計0 (FA0), 150 (FA1x), 300 (FA2x), 450 μM (FA3x)添加した成熟培養液にて38.5℃ 22h成熟培養後，成熟率を検討した。また体外受精後SOF-BE1にて発生培養を行い，分割率，胚盤胞発生率を検討した。さらに成熟開始0，5，22 h後に卵丘細胞からｍRNAを抽出し，P4並びにステロイド分泌に関連する遺伝子(STAR，P450，PGR，PGRMC1，HSPB1)の発現をリアルタイムqPCRにて検討した。【結果・考察】成熟終了後の成熟率はFA1xで高く（78.9%）FA0x，FA3xで低かったが（71.1%，71.4%）有意な差は認められなかった。また胚盤胞発生率も同様にFA1xが高く，NEFA濃度が高くなると低くなる傾向が認められた。一方、卵子の成熟に密接に関わるステロイドホルモン産生に重要な遺伝子については，STARの発現が0hに対し5h，22hで低下したが，他のP450，PGRMC1，HSPB1は成熟時間が進むほどに発現量が増え，PGRは成熟開始5hで発現量が最大であった。しかしながら，脂肪酸濃度による遺伝子発現量の差は認められなかった。以上より成熟培養中の高NEFA濃度は卵子の品質を低下させ，胚発生を阻害する可能性が示唆されたものの，卵子品質への卵丘細胞P4産生能の関与は少ないものと考えられた。
  • KOZAI Keisuke, HOJO Takuo, TAKAHASHI Masashi, SAKATANI Miki, ACOSTA Tomas, NAMBO Yasuo, OKUDA Kiyoshi  The Journal of Reproduction and Development Supplement  105-  (0)  1096  -1096  2012  [Not refereed][Not invited]
     
    【目的】多くの哺乳動物において黄体は妊娠の成立・維持に必須の progesterone (P4) を分泌する。妊娠が不成立の場合，次の発情を回帰するため黄体は退行する。黄体の退行は機能的および構造的退行からなる。構造的退行は黄体細胞のアポトーシスによるものと知られているが，機能的退行機構には不明な点が多く残されている。げっ歯類の不完全性周期において，黄体細胞に P4 を生物活性のない 20α-dihydroprogesterone (20α-OHP) に代謝する酵素である 20α-hydroxysteroid dehydrogenase (20α-HSD) が発現するため，黄体は P4 分泌能を持たないとされている。ウマにおいては，aldo-keto reductase (AKR) superfamily の一つである AKR1C23 が 20α-HSD 活性を有すること，また黄体組織での 20α-OHP の存在が示されている。本研究はウマ黄体の機能的退行機構研究の一環として，黄体組織における AKR1C23 発現を検討した。【材料および方法】ウマの繁殖期である 4-7 月に熊本県の食肉センターより黄体を採取し，肉眼的所見により黄体初期，黄体中期，黄体後期および黄体退行期の 4 ステージに分類し、組織を解析に用いた。実験 1: 黄体周期を通じた組織中 P4 および 20α-OHP 濃度を EIA により測定した。実験 2: 黄体周期を通じた AKR1C23 mRNA 発現を定量的 RT-PCR 法により調べた。【結果】実験 1: 組織中 P4 濃度は黄体中期において最も高く，黄体後期および黄体退行期にかけて有意に減少した。一方，組織中 20α-OHP 濃度は黄体後期において他の周期に比べ有意に高かった。実験 2: AKR1C23 mRNA 発現量は黄体後期において黄体初期に比べ有意に高く，また黄体退行期において黄体初期および黄体中期に比べ有意に高かった。【考察】本研究において，黄体後期に組織中 P4 濃度の減少および 20α-OHP 濃度の増加，ならびに AKR1C23 mRNA 発現の増加が見られたことから，ウマ黄体の機能的退行には AKR1C23 による P4 の 20α-OHP への代謝が関与していると示唆された。
  • SUGAWARA Saiko, KASUGA Kano, KOJIMA Ikuo, YAMANAKA Kenichi, SAKATANI Miki, TAKAHASHI Masashi, KOBAYASHI Masayuki, SATO Sho, TAKAHASHI Toshikiyo, NISHINOMIYA Hiroshi, KATOH Makiko, ITOH Ryu, YOKOO Masaki, YOKOO Mari, MORI Yuki  The Journal of Reproduction and Development Supplement  105-  (0)  1004  -1004  2012  [Not refereed][Not invited]
     
    【目的】マウスの胚発生において，線維芽細胞増殖因子4 (FGF4) は極栄養外胚葉および胚体外外胚葉の増殖を促進することにより胎盤形成に関与する，重要な細胞増殖因子であることが示されている。また，体細胞クローンウシ胚においてFGF4遺伝子の発現に異常が認められることが報告された。すなわち，ウシの正常な胚発生とFGF4遺伝子の発現には関連性が指摘されている。本研究では，黒毛和種牛，日本短角種牛，ホルスタイン種牛に由来するFGF4構造遺伝子の全塩基配列を決定し，既に報告されているウシFGF4構造遺伝子 (GenBank accession no. U15969，品種不明) と比較した。【材料および方法】少なくとも2世代前までに血縁関係が認められない黒毛和種雌牛（2頭），日本短角種雌牛（2頭），ホルスタイン種雌牛（3頭）から採血し，遠心分離によって得た白血球層，および黒毛和種雄牛（1頭）の凍結融解精子よりゲノムDNAを調製した。調製したゲノムDNA（8頭）を鋳型として，PCRによりFGF4構造遺伝子を含むDNA断片（約2.2 kb）を増幅した。その後，PCR産物の全塩基配列を決定した。【結果および考察】FGF4構造遺伝子領域の塩基配列を比較したところ，黒毛和種牛（3頭），日本短角種牛（2頭），ホルスタイン種牛（3頭）の塩基配列およびFGF4タンパク質一次構造（推定）は完全に一致した。GenBank登録配列（U15969）と比較した場合，U15969にはアミノ酸の置換（1カ所），挿入（1カ所），欠失（1カ所）を導く塩基配列の変異を見出した。以上の結果より，黒毛和種牛，日本短角種牛，ホルスタイン種牛において共通し，しかも新規な塩基配列を示すウシFGF4構造遺伝子の存在が示された。なお，品種が明らかであるウシFGF4構造遺伝子の全塩基配列の決定は本報告が初めてである (Sato et al., Anim. Sci. J., 2012) 。現在，ウシFGF4遺伝子の応用について検討をすすめている。
  • KAGAWA Shinjiro, NAGATOMO Hiroaki, TAKAHASHI Masashi, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  105-  (0)  106  -106  2012  [Not refereed][Not invited]
     
    ウシ胚盤胞期胚におけるTEAD4およびYAPの発現動態に関する研究

    ○加川真二朗，長友啓明，高橋昌志，川原学 (北大院農)

    【目的】哺乳類の個体発生における最初の分化は胚盤胞期胚に起こり，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。この分化調節機構についてマウス胚では解析が進んでおり，Hippo pathway下流の転写コアクチベーターであるYes-associated protein(Yap)が重要な役割を果たすことがわかっている。すなわち，胚のそれぞれの割球における転写因子Tead4の細胞内局在を，Yapが直接変化させることにより分化を制御している。しかし，ウシ胚でも同一の仕組みで分化が制御されているかは不明である。そこで，ウシ胚におけるICM/TE分化制御機構を探るため，ウシTEAD4(bTEAD4)遺伝子の胚盤胞期胚における発現解析を行い，さらにウシYAP(bYAP)遺伝子の塩基配列を解析した。【方法】体外受精により作出したウシ胚盤胞期胚からICMおよびTEの部分胚を採取した。これらのサンプルに通常の全体胚(Whole)を加え，3種のサンプルより抽出したtotal RNAを用いて，bTEAD4の定量PCRを行った。また，RT-PCRでbYAP遺伝子cDNAをクローニングし塩基配列を決定したのち，マウスYap(mYap)のデータベースと比較した。【結果および考察】ICM，TE，Whole胚でのbTEAD4の発現を定量PCRで比較した結果，TEでの発現レベルがICMおよびWholeサンプルの発現レベルよりも高い値を示した。また，決定したbYAP塩基配列からタンパク質一次構造を予測しマウスと比較した結果，ウシではN末端領域が欠損していることが判明した。この欠損領域はTeadファミリー転写因子との結合領域であり，mYapタンパク質局在に重要な役割を果たすリン酸化部位も含まれている。以上の結果から，ウシ胚では，マウス胚のようなTead-Yapの直接的な相互作用を介した機構とは異なる様式でICM/TEの細胞分化が制御されている可能性が示された。
  • TAKAHASHI Masashi  Reprod. Med. Biol.  11-  (1)  37  -47  2012/01/01  [Not refereed][Not invited]
  • 高橋昌志  農林水産技術研究ジャーナル  34-  (9)  28  -33  2011/09/01  [Not refereed][Not invited]
  • TAKAHASHI Masashi  農林水産技術研究ジャーナル  34-  (9)  28  -33  2011/09/01  [Not refereed][Not invited]
  • 高橋昌志  Journal of Reproduction and Development  57-  (Suppl Japanese Issue)  J101  2011/08/20  [Not refereed][Not invited]
  • YAMANAKA Ken-ichi, KANEDA Masahiro, INABA Yasushi, SAITO Koji, KUBOTA Kaiyu, SAKATANI Miki, SUGIMURA Satoshi, IMAI Kei, WATANABE Shinya, TAKAHASHI Masashi  Animal science journal  82-  (4)  523  -530  2011/08/01  [Not refereed][Not invited]
     
    Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.
  • 高橋昌志, 阪谷美樹  畜産技術  (674)  7-13,1(1)  2011/07/01  [Not refereed][Not invited]
  • 高橋 昌志, 阪谷 美樹  畜産技術  0-  (674)  7  -13,図巻頭1p  2011/07  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志  養牛の友  (423)  34  -37  2011/06/01  [Not refereed][Not invited]
  • 阪谷 美樹, 高橋 昌志  養牛の友  (423)  34  -37  2011/06  [Not refereed][Not invited]
  • 牛における核移植胚作出と胚の品質評価のためのマニュアル：胚の品質評価法
    高橋昌志, 山中賢一  畜産草地研究所技術レポート  (9)  28  -48  2011/03  [Not refereed][Not invited]
  • 高橋昌志, BALBOULA Ahmed Z, 阪谷美樹, 山中賢一  日本胚移植学雑誌  33-  (1)  46  2011/01/19  [Not refereed][Not invited]
  • M. Tani, C. Tani, K. Tomokawa, D. Funakoshi, M. Sakatani, M. Takahashi, G. Kitahara, S. Kamimura  REPRODUCTION FERTILITY AND DEVELOPMENT  23-  (1)  179  -179  2011  [Not refereed][Not invited]
  • IWAZAWA Makoto, ACOSTA Tomas J, SAKUMOTO Ryosuke, TAKAHASHI Masashi, OKUDA Kiyoshi  The Journal of Reproduction and Development Supplement  104-  (0)  312  -312  2011  [Not refereed][Not invited]
     
    【目的】ウシにおいて夏場の気温の上昇は暑熱ストレス (HS) として卵子の質，卵胞の活性および血中のプロジェステロン (P4) に影響を与えることで夏季不妊の一因となることが報告されている。しかし，妊娠の成立維持に必須の内分泌器官である黄体の機能に及ぼす HS の影響について明らかではない。多くの細胞は高温において，ヒートショックプロテイン (HSP) 70 および HSP90 のような HSP ファミリーの遺伝子発現を増加すると報告されている。本研究は，ウシの夏季不妊の原因と黄体機能に及ぼす HS との関係を明らかにする目的で培養系における黄体細胞の機能発現に及ぼす高温負荷の影響ついて検討した。【方法】中期黄体を有する卵巣から単離した黄体細胞を 37.5℃ (通常の温度)，39.0℃ (穏やかな高温) および 41.0℃ (過酷な高温) で 12 または 24 時間培養後，以下の実験に供した。1) HSP70 および HSP90 mRNA 発現量を半定量的 RT-PCR 法で測定し，高温条件が黄体細胞に HS を与えているかどうかを確認した。2) 24 時間培養後の黄体細胞を 0.1%トリプシンを用いて再懸濁後，トリパンブルー染色により細胞生存率を通常の条件と比較した。3) 培養上清中の P4，プロスタグランジン (PG) F2α および PGE2 濃度を EIA により測定し，通常の条件と比較した。【結果】1) 39.0℃，12 時間培養は HSP70 mRNA 発現を有意に刺激したが HSP90 mRNA 発現には影響しなかった。また，41.0℃，24 時間培養は HSP70 および HSP90 mRNA 発現を有意に刺激した。これらの結果から，本実験系における高温条件は培養黄体細胞にとって HS 条件であることを確認した 。2) 各温度区の黄体細胞生存率に有意な差はなかった。3) 39.0℃および 41.0℃における 12 ならびに 24 時間培養は P4 および PGF2α 産生を有意に刺激した。一方，41.0℃，24 時間培養は PGE2 産生を有意に刺激した。本培養系において，黄体細胞の機能発現に及ぼす高温負荷の影響は低いことならびに夏季不妊との関係も低いことが示唆された。
  • KOZAI Keisuke, HOJO Takuo, NAMBO Yasuo, TAKAHASHI Masashi, ACOSTA Tomas J, OKUDA Kiyoshi  The Journal of Reproduction and Development Supplement  104-  (0)  311  -311  2011  [Not refereed][Not invited]
     
    【目的】ウマは長日性季節繁殖動物である。これまでに血中プロジェステロン (P4) 濃度を指標とした黄体機能に及ぼす季節の影響に関して多くの報告がなされてきた。しかし、一貫した結果は得られておらず、黄体機能の季節変化は未だ明らかとされていない。本研究では、黄体組織中 P4 濃度ならびに steroidogenic acute regulatory protein (StAR)、P450 cholesterol side-chain cleavage enzyme (P450scc)、3β-hydroxysteroid dehydrogenase (3β-HSD) および luteinizing hormone receptor (LHCGR) 遺伝子発現を指標として季節間の黄体機能の違いを検討した。【方法】4 月、7 月、9-10 月、12 月および 1-2 月に熊本県の食肉検査所 (北緯 37°) よりウマ黄体を採取し、肉眼的所見により黄体初期、黄体中期および黄体退行期の 3 ステージに分類した。実験 1: 各季節について発情周期を通じた組織中 P4 濃度を EIA により測定した。また、黄体中期における組織中 P4 濃度を季節間で比較した。実験 2: 黄体中期における StAR、P450scc および 3β-HSD mRNA 発現を定量的 RT-PCR 法により解析し季節間で比較した。実験 3: 黄体中期における LHCGR mRNA 発現を定量的 RT-PCR 法により解析し季節間で比較した。【結果】実験1: いずれの季節においても組織中 P4 濃度は黄体中期において最も高かった。また 1-2 月において、4 月、7 月および 12 月と比べ高かった。実験 2: StAR mRNA 発現は 4 月において 12 月と比べ、7 月において 9-10 月および 12 月と比べ高かった。P450scc mRNA 発現は 4 月において 9-10 月および 12 月と比べ、また7 月および 1-2 月において 12 月と比べ高かった。3β-HSD mRNA 発現は 4 月において 9-10 月および 12 月と比べ高かった。実験 3: LHCGR mRNA 発現は他の季節に比べ 1-2 月において高かった。【考察】ウマ黄体機能は季節変化を示すことが示唆された。1-2 月における組織中 P4 濃度の上昇は LHCGR 発現の上昇と関係があると考えられる。
  • NAGATOMO Hiroaki, KAWAHARA Manabu, TAKUMA Tetsuya, KAGAWA Shinjirou, KISHI Yasunori, YAMANAKA Kenichi, SOU Hou, TAKAHASHI Masashi, KONO TOMOHIRO, WATANABE Tomomasa  The Journal of Reproduction and Development Supplement  104-  (0)  1062  -1062  2011  [Not refereed][Not invited]
     
    【目的】胚盤胞期胚は，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。さらに，TEはICMへの隣接の有無から極栄養外胚葉(pTE)と壁栄養外胚葉(mTE)に分類される。ウシ胚では，これらの細胞での遺伝子発現プロファイルは十分に理解されていない。第103会大会では全体胚(whole)，および，顕微操作により機械的に分断した部分胚(ICM側: ICM + pTE; TE側: mTE)の解析結果を報告した。しかし、ICM単体のサンプルでの解析が欠けていたため，今回はTEを完全に除去したICMのサンプルを採取して遺伝子発現解析を追加した。さらに，ICM，TEサンプル間で発現差のあった遺伝子についてリアルタイムPCRを行った。【方法】ウシ胚盤胞期胚TEを界面活性剤で除去したICMサンプルを使用し、定法により調整してマイクロアレイに供した。解析結果を，主成分分析やオントロジー分析を行い、各サンプル間での遺伝子発現パターンの相違を調べ，発現差のみられた肝細胞核因子4A(HNF4A)などの遺伝子についてリアルタイムPCRを行った。【結果】マイクロアレイの結果，主成分分析により遺伝子発現パターンを比較したところ，4種類の部分胚，すなわちmTE，whole，ICM+pTE，ICMにおいてそれぞれ特異的な遺伝子発現パターンを示した。とくに，mTEとICMの発現パターンが大きく異なっていた。また、pTEとmTEを比較すると、pTEで発現が顕著に上昇している遺伝子は3遺伝子のみであった。ICMとmTEにおいて，それぞれ特異的に発現レベルが有意に上昇していた遺伝子について成長因子や転写因子の機能的な側面から15個ずつ選出した。ICMで発現が上昇していた遺伝子中には，HNF4Aのような転写因子が含まれており，mTEと比較して17倍以上の差がみられた。同様の動向を示す遺伝子も含めて，リアルタイムPCRによる発現解析も行った。今回、ウシ胚盤胞期胚で部位毎に分別したサンプルを用い、ICMとTEで発現差のみられた遺伝子群から、機能的側面も考慮し、候補遺伝子を絞り込んだ。今後更なる解析によりウシ胚盤胞期胚における分化関連遺伝子探索に役立てたい。
  • NAKANO Kazuaki, SASAYAMA Norihisa, SHIRASU Akio, OHTA H, TAKAHASHI Masashi, NAGASHIMA Hiroshi, MASTUNARI Hitomi, MAEHAEA M, TAKEUCHI Y, OGAWA Buko, MATSUDA Taisuke, KANAI Takahiro, HONDA Kasumi, HAGIWARA Yui  The Journal of Reproduction and Development Supplement  104-  (0)  1115  -1115  2011  [Not refereed][Not invited]
     
    【目的】ブタの品種・系統の多様性の維持に，胚の凍結保存が有効である。本研究は，我が国固有の登録種であるトウキョウXを対象として，ブタ初期胚凍結保存法としての中空糸法の有効性を検証することを目的とした。【方法】1000-1500iu eCG/750-1500iu hCGによる過排卵処置を施した未経産トウキョウX雌(約11ケ月齢)から，hCG投与後約125時間に4-8細胞期-桑実期胚を採取した。桑実期胚は回収直後に，4-8細胞期胚は17-30時間培養後に，桑実期-初期胚盤胞期に発達したものをガラス化に供した。胚のガラス化保存には修正した中空糸法(松成ら，2010)を用いた。まず，胚を7.5% ethylen glycol(EG)及び7.5% DMSOを含む液中で5分間平衡し，その間に胚を中空糸デバイスに収容した。次に胚を保持する中空糸膜を15% EG，15% DMSO，0.5M sucroseを含むガラス化液に1分間浸漬した後，液体窒素に直接投入した。胚の融解は38.5°Cに温めた融解液(1M sucroseを含む)に中空糸膜を浸漬することで行い，その後，希釈液(0.5M sucroseを含む)，洗浄液に移し，凍害保護剤を除去した。融解後，20-34時間培養して得られた後期桑実期-胚盤胞期胚をレシピエントブタに外科的に移植した(21-26個/頭)。【結果】4頭のドナーブタから得られた73個の胚をガラス化保存した結果，72個(98.6%)が生存し，融解後，後期桑実期-胚盤胞期胚に発達した。それらを3頭のレシピエントブタに移植したところ，全頭が妊娠し，合計29頭の産仔(29/72, 40.3%)が得られた(内死産1)。各レシピエントブタの産仔数は9-10頭の範囲であった。生存産仔の離乳時までの体重増加はトウキョウXの自然交配産仔と同等であった。【考察】中空糸法はブタ生体由来胚のガラス化保存に有効であり，高い再現性を持って高効率な産仔作出が可能であることが示された。以上のことから中空糸法はブタの品種・系統の保存の実用的方法となり得ると考えられる。
  • MAEHARA Miki, SHIRASU Akio, TAKAHASHI Masashi, WATANABE Masahito, UMEYAMA Kazuhiro, HANAZONO Yuraka, NAGASHIMA Hiroshi, HONDA Kasumi, NAKANO Kazuaki, MATSUNARI Hitomi, TAKEUCHI Yasuhiro, KANAI Takahiro, MATSUDA Taisuke, HAGIWARA Yui, SASAYAMA Norihisa  The Journal of Reproduction and Development Supplement  104-  (0)  1037  -1037  2011  [Not refereed][Not invited]
     
    【目的】中空糸法が、ブタ体外成熟・受精胚の凍結保存に有効であることを、培養および移植試験に基づいて実証することを目的とした。【方法】ブタ卵丘卵子複合体をHP-POM液で培養し、体外成熟卵を得た。それらをYoshiokaら(2008)の方法に準じて体外受精(IVF)に供し、一部は電気的活性化により単為発生を誘導した。IVFにはKusabira-Orange遺伝子導入ブタの凍結精巣上体精子を用いた。IVF胚及び単為発生胚をPZM-5液で96時間培養し、形態的に正常な桑実期胚を選別して、ガラス化区と対照の非ガラス化区に二分した。胚のガラス化は修正した中空糸法(松成ら,2010)を用いた。操作の基本液には20%仔牛血清含20mM Hepes緩衝TCM199液を用いた。5-22個の胚を収容した中空糸膜を7.5%ethylene glycol(EG)、7.5%DMSOを含む平衡液中に5分間、15%EG、15%DMSOを含むガラス化液中に1分間保持した後、液体窒素に投入しガラス化した。融解には38.5°Cの1M sucrose溶液を用い、段階的に希釈・洗浄した。融解胚を約40時間培養し、胚盤胞への発達を評価した。さらにガラス化区、非ガラス化区各々から得られた胚盤胞を同数ずつ3頭のレシピエント雌(25-32個/頭)に移植し、産仔への発達を比較した。【結果】6回の反復実験において、IVF胚の桑実胚への発達率は平均46.4%(559/1206)であった。IVF桑実胚のガラス化後の生存率は高く、77.2%(88/114)が胚盤胞 (d-6)へ発達したが、非ガラス化区に比べるとやや低い成績であった(92.0%,127/138;P<0.05)。単為発生桑実胚を用いた比較でも同様の傾向が見られ、ガラス化胚の胚盤胞(d-7)への発達率(79.0%,49/62)は非ガラス化胚よりわずかに低かった(90.3%,56/62;有意差なし)。両区のIVF胚盤胞(d-5,6)を各々3頭に移植した結果、全頭が妊娠し、これまでに得られた産仔作出成績はガラス化区で4/32(12.5%)及び8/31(25.8%)、非ガラス化区で9/32(28.1%)及び9/31(29.0%)であった(１頭は妊娠継続中)。【結論】中空糸法の使用によって、体外成熟・受精ブタ桑実胚からの高効率な産仔作出が可能となった。本研究はJST CRESTの助成を受けた。
  • KOBAYASHI Yoshihiko, WAKAMIYA Kaori, SAKUMOTO Ryosuke, ACOSTA Tomas J, TAKAHASHI Masashi, OKUDA Kiyoshi  The Journal of Reproduction and Development Supplement  104-  (0)  224  -224  2011  [Not refereed][Not invited]
     
    【目的】卵管において，排卵前後にピークを迎える弛緩および収縮運動は，精子，未受精卵および受精卵の移送に必須である。この弛緩および収縮運動には，卵管上皮細胞が産生する prostaglandin (PG) E2 および PGF2α が関与している。ウシにおいて，夏の暑熱ストレス(HS) は受胎率を低下させる一因であることが知られている。Heat shock protein (HSP) は熱ストレスにより一時的に発現が増強される。HSP90 は PGE2 合成酵素のひとつである cytosolic PGES (cPGES) と結合し，cPGES の酵素活性を高めることが報告されており，HS が HSP90 発現の増加を介して PGE2 産生を増加させ，卵管の弛緩に影響を及ぼすことも考えられるが，詳細は明らかにされていない。本研究では，卵管の弛緩および収縮運動に及ぼす HS の影響の一端を明らかにする目的で，ウシ卵管上皮細胞培養系を用いて PGs 合成に及ぼす培養温度の影響について検討した。【方法】排卵後 0-3 日目のウシ卵管膨大部および峡部から上皮細胞を単離，播種し，コンフルエントに達した後，通常の培養温度 (37.5℃) を対照区，39.0℃ および 41.0℃ を HS 処理区として 24 時間培養後，培養上清中 PGs 濃度を EIA により検討した。また，PGs の合成酵素である PGESs，PGFSs および PGE2 を PGF2α に転換する酵素である carbonyl reductase 1 ならびに HSP90 mRNA 発現量を定量的 RT-PCR 法により検討した。【結果】HS 処理区において，膨大部および峡部における PGE2 濃度が増加するとともに cPGES および HSP90 mRNA 発現量が増加した。PGF2&alpha 濃度およびその他の酵素の mRNA 発現量は変化しなかった。以上の結果より，夏の HS が卵管膨大部および峡部上皮細胞の cPGES 発現を刺激し，PGE2 産生が増加することが示唆された。また，この PGE2 産生の増加は，HSP90 発現が刺激されることによる cPGES 活性の増強に起因する可能性が示された。この HS による PGE2 の増加は，胚移送の際の卵管運動に影響を及ぼすのかもしれない。
  • 高橋 昌志  Bio九州  (196)  15  -18  2010/10  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志  九州沖縄農業研究成果情報  (25)  161  -162  2010/09/03  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志  九州沖縄農業研究成果情報  (25)  163  -164  2010/09/03  [Not refereed][Not invited]
  • 谷千賀子, 友川浩一郎, 舩越大資, 阪谷美樹, 高橋昌志, 北原豪, 谷峰人, 上村俊一  日本獣医学会学術集会講演要旨集  150th-  268  2010/09/01  [Not refereed][Not invited]
  • 高橋 昌志, 阪谷 美樹, 山中 賢一  畜産草地研究所研究資料  (10)  33  -38  2010/07  [Not refereed][Not invited]
     
    体細胞クローン牛の報告が成されて以降、10年を超え、その作成手法や胚発生効率向上の研究が全世界で実施されてきている。我が国においても国公立研究機関、民間や大学で精力的に遂行されている。特に、我が国のクローン研究は他の国とは異なり、乳肉生産を最終目的として、その実施が成されており、これまでに600頭近い産子個体の作出数が報告されている。しかしながら、その作出効率は依然として低く、「安心安全」を求める消費者への理解は未だ遠いという状況にある。体細胞クローン個体作出効率向上は、実験動物も含めてその基礎的研究が進んでおり、新たな局面からのアプローチが待たれるところである。近年、体細胞クローン研究において、分化した体細胞をドナーとすることで、通常受精を経た初期胚に見られるようなゲノムの初期化が十分に起こらないために、正常な遺伝子調節が成されていないことがその原因の一つとしてとらえられている。この不全現象を｢後性的遺伝子修飾機構：エピジェネティクス」の観点からDNAの修飾・発現機構不全を解明、評価、および制御することで、より受精胚に近い発生・分化条件を整える研究が期待される。本稿では、クローン胚のエピジェネティクス機構をDNAのメチル化の観点から検出し、その状況を評価することと共に、メチル化制御に関与する因子を制御する手法としてのRNA干渉を用いた新たな調節技術研究の知見についての紹介を行う。
  • 高橋昌志, 阪谷美樹, 山中賢一  畜産草地研究所研究資料  (10)  33  -38  2010/07  [Not refereed][Not invited]
  • 高橋 昌志, 阪谷 美樹, 山中 賢一  畜産草地研究所研究資料  0-  (10)  33  -38  2010/07  [Not refereed][Not invited]
     
    体細胞クローン牛の報告が成されて以降、10年を超え、その作成手法や胚発生効率向上の研究が全世界で実施されてきている。我が国においても国公立研究機関、民間や大学で精力的に遂行されている。特に、我が国のクローン研究は他の国とは異なり、乳肉生産を最終目的として、その実施が成されており、これまでに600頭近い産子個体の作出数が報告されている。しかしながら、その作出効率は依然として低く、「安心安全」を求める消費者への理解は未だ遠いという状況にある。体細胞クローン個体作出効率向上は、実験動物も含めてその基礎的研究が進んでおり、新たな局面からのアプローチが待たれるところである。近年、体細胞クローン研究において、分化した体細胞をドナーとすることで、通常受精を経た初期胚に見られるようなゲノムの初期化が十分に起こらないために、正常な遺伝子調節が成されていないことがその原因の一つとしてとらえられている。この不全現象を「後性的遺伝子修飾機構: エピジェネティクス」の観点からDNAの修飾・発現機構不全を解明、評価、および制御することで、より受精胚に近い発生・分化条件を整える研究が期待される。本稿では、クローン胚のエピジェネティクス機構をDNAのメチル化の観点から検出し、その状況を評価することと共に、メチル化制御に関与する因子を制御する手法としてのRNA干渉を用いた新たな調節技術研究の知見についての紹介を行う。
  • 吉ざわ努, 平子誠, 下司雅也, 高橋昌志, 永井卓  畜産技術  (659)  2  -6  2010/04/01  [Not refereed][Not invited]
  • 阪谷美樹, BALBOULA Ahmed, 菅原晃美, 山中賢一, 高橋昌志  日本畜産学会大会講演要旨  112th-  75  2010/03/28  [Not refereed][Not invited]
  • 西村翔, 諌山慧士郎, 久保田海雄, 山中賢一, 高橋ひとみ, 宗知紀, 木崎景一郎, 山内伸彦, 高橋昌志, 橋爪一善, 服部眞彰  日本畜産学会大会講演要旨  112th-  76  2010/03/28  [Not refereed][Not invited]
  • 久保田海雄, 合原崇文, 西村翔, 宗知紀, 山中賢一, 木崎景一郎, 山内伸彦, 高橋昌志, 橋爪一善, 服部眞彰  日本畜産学会大会講演要旨  112th-  76  2010/03/28  [Not refereed][Not invited]
  • YAMANAKA Ken-ichi, BALBOULA Ahmed Zaky, SAKATANI Miki, TAKAHASHI Masashi  The journal of reproduction and development  56-  (1)  60  -67  2010/02/01  [Not refereed][Not invited]
     
    A highly methylated genome, like a somatic donor cell, is observed in somatic cell nuclear transfer (SCNT) embryos. The aberrant DNA methylation status causes global gene expression failure, resulting in low developmental competence of SCNT embryos. In addition, recent studies have uncovered the relationship between DNA methylation status and reprogramming efficiency. Because DNA methylation is performed by DNA methyltransferases (DNMTs), developing a technique which specifically inhibits DNMTs is necessary for further SCNT studies. In the present study, we examined the potential use of RNA interference for knockdown of DNMT mRNA in bovine fibroblast cells that were commonly used as karyoplast donors in SCNT studies. We designed three siRNAs corresponding to DNMT1, DNMT2 and DNMT3a mRNA. In Experiment 1, to optimize transfection conditions, fluorescence and cell viability after transfection were evaluated at different concentrations of transfection reagent using a FITC-labeled nonsilencing control siRNA. Although fluorescence was observed in all groups transfected except for the negative control group, transfection with a higher concentration of transfection reagent significantly decreased in cell viability (P<0.05). In Experiment 2, the amount of DNMT mRNA was measured by real-time PCR at 0, 48 and 96 h after siRNA transfection into the cells. The levels of each DNMT mRNA were significantly decreased at 48 and 96 h after transfection (P<0.01). Furthermore, decreased expression of DNMT1 protein was confirmed by western blotting. In Experiment 3, the DNA methylation statuses were analyzed in each of the siRNA-transfected groups. The DNMT1 siRNA-transfected group had a significantly decreased level of DNA methylation (P<0.05), but the other groups did not. Our data demonstrate that RNA interference with siRNA can be analyzed the function and expression of DNMT genes in bovine fibroblast cells. The present study provides useful information for further SCNT studies.
  • 吉ざわ努, 平子誠, 下司雅也, 高橋昌志, 永井卓  肉用牛研究会報  (88)  46  -48  2010/01/22  [Not refereed][Not invited]
  • 吉〓 努, 平子 誠, 下司 雅也, 高橋 昌志, 永井 卓  肉用牛研究会報  88-  (0)  46  -48  2010/01/22  [Not refereed][Not invited]
  • 阪谷美樹, 山中賢一, 高橋昌志  日本胚移植学雑誌  32-  (1)  13  -17  2010/01/19  [Not refereed][Not invited]
  • 阪谷 美樹, 山中 賢一, 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer  32-  (1)  13  -17  2010/01/19  [Not refereed][Not invited]
  • 鍋西久, 西元俊文, 高橋昌志, 森田哲夫, 續木靖浩, 芦澤幸二  日本獣医師会三学会年次大会講演要旨集  2009-  68  2010/01/07  [Not refereed][Not invited]
  • 高橋昌志, 山中賢一  農研機構畜産草地研究所成果情報(Web)  2010-  2010
  • Miki Sakatani, Ahmed Zaky Balboula, Ken-ichi Yamanaka, Masashi Takahashi  BIOLOGY OF REPRODUCTION  107  -108  2010  [Not refereed][Not invited]
  • AKAGI Satoshi, MATSUKAWA Kazutsugu, YAMANAKA Kenichi, TAKAHASHI Masashi, KANEDA Masahiro, MIZUTANI Eiji, SOMFAI Tamas, WATANABE Shinya, KUBO Masanori, HASHIYADA Yutaka  The Journal of Reproduction and Development Supplement  103-  (0)  149  -149  2010  [Not refereed][Not invited]
     
    【目的】我々は日本畜産学会第111回大会においてウシ体細胞核移植胚へのスクリプタイド（SCR）処理により胚盤胞形成率が改善されることを報告した。本研究では、ウシ核移植においてドナー細胞と胚へのSCR処理が体外発生能に及ぼす影響、胚へのSCR処理がDNAのメチル化に及ぼす影響およびSCR処理核移植胚の移植後の受胎能について調べた。【方法】ドナー細胞として成牛雌線維芽細胞を用いた。ドナー細胞あるいは活性化後の発生培地へ5 nM のSCR を20時間添加し、核移植胚の体外発生能を調べた。また、発生した７日目（核移植日を0日）の胚盤胞期胚（胚へのSCR処理と無処理のみ）をサテライトI 領域のメチル化解析または胚移植による受胎能調査に用いた。【結果】核移植胚へのSCR処理により胚盤胞期への発生率が有意に高くなったが、ドナー細胞のみの処理やドナーと胚への両方の処理では有意な増加は認められなかった。また、胚盤胞期の細胞数に及ぼすSCR処理による効果は認められなかった。胚盤胞期胚のサテライトI領域のメチル化についてはSCR処理胚と無処理胚との間で相違は認められなかった。SCR処理胚と無処理胚を各６頭の受胚牛に移植を行った結果、35日目の超音波妊娠診断によりSCR処理胚では４頭、無処理胚では１頭の受胎が認められた。しかしながら、90日目までにSCR処理胚受胎牛の３頭、無処理胚の１頭について流産が発生した。SCR処理胚受胎牛1頭については帝王切開により40.７kgのクローン産子を分娩したが、クローンは出生６時間後に死亡した。以上の結果から核移植胚への5 nMのSCR処理は、胚盤胞期への発生率を改善し、産子の生産は可能であったが、体細胞核移植胚で頻発する移植後の流産や生後直死がSCR処理胚においても高頻度で発生し、体細胞クローン牛生産率を改善することはできなかった。
  • NAKANO Kazuaki, SASAYAMA Norihisa, SHIRASU Akio, OHTA Hisayoshi, TAKAHASHI Masashi, NAGASHIMA Hiroshi, MATSUNARI Hitomi, MAEHAEA Miki, TAKEUCHI Yasuhiro, OGAWA Buko, FUJIWARA Tsukasa, IKEZAWA Yuka, HONDA Kasumi, HAGIWARA Yui  The Journal of Reproduction and Development Supplement  103-  (0)  161  -161  2010  [Not refereed][Not invited]
     
    【目的】ブタMII期卵及び初期胚のガラス化保存に対する中空糸法の有効性を検証することを目的とした。【方法】ブタ体外成熟(IVM)由来のMII期卵, 単為発生桑実期胚, 体外受精(IVF)桑実期胚, 及び桑実期-胚盤胞期の生体回収胚をガラス化の対象とした。MII期卵には細胞質内脂肪顆粒除去(delipation)及び, 紡錘体安定化剤(paclitaxel)処理を施した。MII期卵及び単為発生桑実期胚のガラス化には, 中空糸法とCryotop法を用いた。平衡, ガラス化, 融解, 希釈, 洗浄液の基本培地には20%の仔ウシ血清を添加した20mM Hepes緩衝TCM199液を用いた。MII期卵は7.5%及び15% ethylene glycol(EG)存在下で段階的(各30秒)に平衡し, 30% EGと0.5M sucroseを含む液に1分間浸漬した後, 液体窒素に投入してガラス化した。胚は7.5% EG及び7.5% DMSOを含む液中で5分間平衡し, 15% EG, 15% DMSO, 0.5M sucrose存在下に1分置いた後にガラス化した。融解には38.5°Cの1M sucrose液を用い, 卵, 胚ともに段階的に希釈・洗浄した。融解後のMII期卵の生存性は, 電気刺激付与後の単為発生能によって, また単為発生及びIVF桑実期胚の生存性は胚盤胞への発生能によって判定した。生体由来胚は融解後20時間培養後に, 発情同期化したレシピエントブタに移植し, 産仔への発生能を検証した。【結果】中空糸法でガラス化されたMII期卵の胚盤胞形成率(13/38, 34.2%)は, Cryotop法を用いた場合(10/40, 25.0%)より向上する傾向があったが, 両者間に有意な差は見られなかった。単為発生桑実期胚の融解後の胚盤胞形成率は中空糸法(48/65, 73.8%)の使用によってCryotop法(25/65, 38.5%)に比して有意に向上した(P<5)。中空糸法でガラス化されたIVF桑実期胚の胚盤胞形成率(30/40, 75.0%)は, 非ガラス化胚の成績(25/30, 83.3%)と同等であった。また, 25個の生体由来ガラス化胚をレシピエントブタに移植したところ, 9頭の産仔(36.0%, 死産1頭含む)が得られた。以上のことから, 中空糸法はブタMII期卵及び初期胚のガラス化保存に有効であることが明らかとなった。
  • NAGATOMO Hiroaki, KISHI Yasunori, TAKUMA Tetsuya, YAMANAKA Kenichi, SO Ho, WADA Yasuhiko, TAKAHASHI Masashi, KONO Tomohiro, KAWAHARA Manabu  The Journal of Reproduction and Development Supplement  103-  (0)  29  -29  2010  [Not refereed][Not invited]
     
    【目的】胚盤胞期胚は，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。さらに，TEはICMへの隣接の有無から極栄養外胚葉(pTE)と壁栄養外胚葉(mTE)に分類される。ウシ胚では，これらの細胞での遺伝子発現プロファイルは十分に理解されていない。そこで本研究では，ウシ胚盤胞期胚の部位ごとに細胞を分別してマイクロアレイ法による網羅的遺伝子発現解析を行った。【方法】マイクロアレイ解析には灌流採取した体内由来胚(vivo胚)，体外受精由来胚(vitro胚)，体細胞クローン胚(SCNT胚)の3種類について，透明帯を除去した全体胚，および，ICM側(ICM + pTE)とTE側(mTE)を顕微操作により機械的に分断した部分胚を使用した。定法により調整したサンプルをマイクロアレイにかけて，主成分分析(PCA)や解析サイトFatiGOでのオントロジー分析を行い各サンプル間での遺伝子発現パターンの相違を調べた。【結果】マイクロアレイの結果，PCAにより遺伝子発現パターンを俯瞰したところ，それぞれの部分胚においてvivoおよびvitro胚由来では近似した発現パターンを示した。しかし，SCNT胚由来ではそれらとは異なる発現パターンを示し，この傾向はオントロジー分析の結果でも同様であった。ICM側およびTE側部分胚で有意に発現レベルが異なっていた遺伝子を調べたところ，vivo胚で984個，vitro胚で2279個，SCNT胚では2599個であった。これらの結果から，胚盤胞期胚においてICM側とTE側の部分胚を使ったマイクロアレイによる網羅的遺伝子発現解析により部位特異的に発現する遺伝子群を探索できる可能性が示された。
  • WAKAMIYA Kaori, KOBAYASHI Yoshihiko, Acosta Tomas J., TAKAHASHI Masashi, OKUDA Kiyoshi  The Journal of Reproduction and Development Supplement  103-  (0)  69  -69  2010  [Not refereed][Not invited]
     
    【目的】Prostaglandins(PGs) は，生殖機能の調節に重要な役割を果たしている生理活性物質であり，多岐の作用が知られている。なかでも，PGE2は初期胚の発育環境を最適に保つ因子であることが報告されている一方で，子宮内膜から分泌される PGF2α(PGF) は胚の生存および発育を抑制し，妊娠率を低下させることが知られている。また，PGE2およびPGFはともに，受精卵の移送に重要な卵管の収縮運動に関係していると考えられている。ウシにおいて，夏期の気温上昇による暑熱ストレス (heat stress; HS) は，卵胞の発育異常による排卵障害，初期胚の死滅などを引き起こし，妊娠率を低下させることが報告されている。さらに，HSは初期胚の発育の場である卵管に影響を与え，初期胚の生存性に影響を与える可能性も考えられる。本研究では，卵管の機能に及ぼすHSの影響を明らかにするために，培養ウシ卵管上皮細胞を用いて以下の検討を行った。
    【方法】排卵後 0-5 日の卵管から単離した卵管上皮細胞を播種し，1)細胞接着後，培養液を交換した時間を 0 日とし，通常の培養温度(37.5℃)を control 区，39℃ (HS39) および 41℃ (HS41) をHS 処理区とした。HS 処理 1-4 日後に細胞を採取し，DNA assay により卵管上皮細胞の増殖を検討した。2) コンフルエントに達した卵管上皮細胞を control 区，HS39およびHS41で培養した。HS 処理 4，24，48 時間後の培養上清中PGE2およびPGF濃度をEIAにより測定した。なお，PGs 濃度は DNA (µg) あたりに換算した。
    【結果】1) Control 区と比較して HS41 で細胞増殖率が有意に低下した (P<0.05)。2) PGE2濃度は HS 処理 4 時間後に control 区と比較してHS41 で有意に減少し，PGF濃度は HS 処理 24 時間後に control 区と比較して HS39 で増加した(P<0.05)。本研究において，HS は卵管上皮細胞の増殖率を低下させ，卵管内における受精卵移送を困難にするとともに，PGE2 濃度の減少および PGF 濃度を増加することにより，初期胚の発達に悪影響を与え，繁殖率を低下させている可能性が示された。
  • MATSUNARI Hitomi, HAGIWARA Yui, TAKAHASHI Masashi, NAGASHIMA Hiroshi, MAEHARA Miki, IKEZAWA Yuka, NAKANO Kazuaki, OCHIAI KEIKO, TAKEUCHI Yasuhiro, HONDA Kasumi, SASAYAMA Norihisa, SHIRASU Akio  The Journal of Reproduction and Development Supplement  103-  (0)  160  -160  2010  [Not refereed][Not invited]
     
    【目的】我々が開発した中空糸ガラス化法(高橋, 2008)は、微量のガラス化液中に多数の胚を収容し得る特徴を持つ。本研究では、中空糸ガラス化法を用いて、多数のマウス胚を同時にガラス化保存した際の生存性を検証することを目的とした。【方法】BDF1 系マウスの1細胞期、2細胞期、桑実期、胚盤胞期胚、および老化促進モデルマウス(SAMP1)の2細胞期胚を用いた。10–29個の胚を、内径185μm、膜厚15μm、長さ30–40 mm、ポアサイズ約15nmのセルロースアセテート製中空糸(HFV–device)に吸引した。胚を保持するHFV–deviceを、平衡液(7.5% ethylene glycol: EG, 7.5% DMSOを含む)中に5–15分、さらにガラス化液(15% EG, 15% DMSOを含む)中に1–2分保持した後、液体窒素に投入した。HFV–deviceを37.5°Cの融解液(1M sucrose含)に投入して胚を融解後、順次希釈液(0.5M sucrose)および洗浄液に移し換えた。HFV–deviceより回収した胚はCZB液にて培養し、体外発生能を検証した。1細胞期で凍結した胚の一部は、偽妊娠マウス(ICR strain)の卵管内に移植し、胎仔への発生能を検証した。また、製品化用試作品を用いて、BDF1マウスおよびSAMP1マウスの2細胞期胚を、1–5カ月間ガラス化保存し、融解後の体外発生能を非ガラス化区と比較した。【結果】BDF1胚では、胚の全ステージにおいて、融解後の胚盤胞形成率は92.0(46⁄50)–100%(50⁄50)であった。1細胞期にガラス化された胚を卵管移植した結果、胎仔への発生率は非ガラス化胚のそれと同等であった(33.3%(40⁄120)vs 29.2%(35⁄120))。また、製品化用試作品を用いてガラス化したBDF1胚およびSAMP1胚の融解後の胚盤胞形成率は、それぞれ非ガラス化胚のそれと同等であった(85.9%(231⁄269)vs 90.0%(9⁄10)、79.4%(27⁄34)vs 86.2%(26⁄29))。【考察】中空糸ガラス化法により、胚の生存性を損なうことなく、マウス胚を多数同時にガラス化保存し得ることが示された。
  • YAMANAKA Ken-ichi, KUBOTA Kaiyu, BALBOULA Ahmed, SAKATANI Miki, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  103-  (0)  148  -148  2010  [Not refereed][Not invited]
     
    【目的】我々は日本繁殖生物学会第101回大会において、RNA干渉によるDNMT1遺伝子のノックダウンが胚盤胞期のDNAメチル化レベルを低下させ、体外発生率を向上させることを報告した。本研究では、DNMT1遺伝子をノックダウンされたNT胚の各発生ステージにおけるDNAメチル化動態および胚盤胞期における胚発生関連遺伝子の発現を調べた。【材料及び方法】子牛雄線維芽細胞をドナー細胞として常法によりNT胚を作製した。RNA干渉はDNMT1に対して設計した二本鎖RNA (siRNA)を活性化処理後のNT胚の細胞質内に顕微注入することにより行った。実験区はIVF区、NT区およびsiRNA区を設け、各発生ステージ(培養開始後24、48、72、120、192時間)の胚からゲノムDNAを抽出した。bisulfite処理後、satellite I領域を増幅し、メチル化感受性制限酵素Aci Iを用いたCOBRA法によりDNAメチル化状態を評価した。さらに、胚盤胞期胚における胚発生関連遺伝子(GLUT1、IGF2R、INFτ、OCT4およびHSP70)の発現をreal-time PCR法により調べた。【結果】IVF区と比べてNT区ではすべてのステージで一貫して高いDNAメチル化レベルであった。これに対して、siRNA区ではNT区と比べるとすべての発生ステージにおいてDNAメチル化レベルの有意な低下が確認された。DNAメチル化レベルの動態では、IVF区では培養開始後24時間から一貫して低く維持されていたのに対して、NT区では192時間までステージ間での有意な低下は見られなかった。一方、siRNA区では培養開始後72時間で急激なDNAメチル化の低下が見られた。また、胚盤胞期のINFτの発現量はNT区ではIVF区と比べて有意に低いのに対して、siRNA区ではIVF区と同等レベルの発現が確認された。以上の結果から、エピジェネティクス制御に関わる遺伝子の調節によりNT胚の発生能を改善できる可能性が示唆された。
  • 吉ざわ努, 平子誠, 下司雅也, 高橋昌志, 永井卓  肉用牛研究会大会講演要旨集  47th-  39  -42  2009/10/06  [Not refereed][Not invited]
  • 宮崎史彦, 中野俊樹, 山口敏康, 佐藤実, 高橋昌志, 尾形信二  日本水産学会大会講演要旨集  2009-  118  2009/09/30  [Not refereed][Not invited]
  • 阪谷美樹, AHMED Balboula, 山中賢一, 高橋昌志  日本畜産学会大会講演要旨  111th-  79  2009/09/28  [Not refereed][Not invited]
  • 山中賢一, 金田正弘, 稲葉泰志, 齋藤公治, 阪谷美樹, 今井敬, 渡邊伸也, 永井卓, 高橋昌志  日本畜産学会大会講演要旨  111th-  75  2009/09/28  [Not refereed][Not invited]
  • 松本光史, 阪谷美樹, 村上斉, 井上寛暁, 高橋昌志, 梶雄次  九州沖縄農業研究成果情報  (24)  113  -114  2009/09/18  [Not refereed][Not invited]
  • 阪谷美樹, 山中賢一, BALBOULA Ahmed Z, 高橋昌志  九州農業研究発表会専門部会発表要旨集  72nd-  100  2009/08  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹  家畜診療  (554)  451  -465  2009/08/01  [Not refereed][Not invited]
  • 高橋 昌志, 阪谷 美樹  家畜診療  56-  (8)  451  -465  2009/08  [Not refereed][Not invited]
  • 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer  31-  (2)  127  -133  2009/05/19  [Not refereed][Not invited]
  • 阪谷美樹, BALBOULLA Ahmed, 山中賢一, 高橋昌志  日本畜産学会大会講演要旨  110th-  96  2009/03/27  [Not refereed][Not invited]
  • 高橋昌志, 山中賢一, 阪谷美樹  畜産技術  (645)  2  -9  2009/02/01  [Not refereed][Not invited]
  • 高橋昌志, 山中賢一, 阪谷美樹  日本胚移植学雑誌  31-  (1)  9  -17  2009/01/28  [Not refereed][Not invited]
  • 齋藤公治, 森将臣, 住尾善彦, 山口大輔, 億正樹, 笹木教隆, 高橋ひとみ, 橋谷田豊, 高橋昌志, 下司雅也  日本胚移植学雑誌  31-  (1)  59  2009/01/28  [Not refereed][Not invited]
  • 高橋昌志, 松成ひとみ, 阪谷美樹, 山中賢一, 笹山典久, 吉川義洋, 白数昭雄, 長嶋比呂志  日本胚移植学雑誌  31-  (1)  54  2009/01/28  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 山中賢一, BALBOULA Ahmed Z  農研機構畜産草地研究所成果情報(Web)  2009-  2009
  • BALBOULA Ahmed, YAMANAKA Ken-ichi, SAKATANI Miki, TAKAHASHI Masashi  The Journal of Reproduction and Development Supplement  102-  (0)  111  -111  2009  [Not refereed][Not invited]
     
    Recently, the amount of cathepsins transcripts in cumulus cells is known to be correlated inversely with the developmental competence of bovine oocytes. Moreover, the Inhibition of cathepsin B during in vitro maturation (IVM) of bovine COCs was found to be associated with high developmental rate. In the present study, we investigated the impact of cathepsin B inhibitor (E-64) addition to in vitro culture (IVC) medium on development and quality of bovine preimplantation embryos. After IVM of cumulus oocyte complexes (COCs), followed by in vitro fertilization, zygotes were cultured with or without E-64 for 7 days. Cleavage and blastocyst rates were evaluated on days 2 and 7, respectively. Embryonic quality was evaluated by both total cell number and TUNEL of day 7 blastocysts. Addition of E-64 during IVC significantly increased both the blastocyst rate and the total cell number. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in day 7 blastocysts. These results indicate that inhibition of cathepsin B during IVC greatly improves the developmental competence of preimplantation embryos and increases the number of high quality embryos.
  • M. Sakatani, K. Yamanaka, M. Takahashi  REPRODUCTION FERTILITY AND DEVELOPMENT  21-  (1)  207  -207  2009  [Not refereed][Not invited]
  • K. Yamanaka, M. Sakatani, M. Takahashi  REPRODUCTION FERTILITY AND DEVELOPMENT  21-  (1)  128  -128  2009  [Not refereed][Not invited]
  • TAKAHASHI Masashi, YAMANAKA Ken-ichi, Ahmed Balboula, SAKATANI Miki, NAGAI Takashi  The Journal of Reproduction and Development Supplement  102-  (0)  109  -109  2009  [Not refereed][Not invited]
     
    ［目的］初期胚を含む細胞内の還元環境維持グルタチオン（GSH）は重要な役割を持つことが知られている。非必須アミノ酸であるシステイン(Cys)はGSH合成、代謝に必須であり、かつ合成反応の律速を果たすが、その供給源としてのシスチン(CyssCy)の外部からの取り込みが重要な因子であることが知られている。我々は、牛胚では各ステージでCyssCy取り込み能が低く、チオール添加による還元作用により胚内に取り込まれGSH合成に使われることを既に報告している。CyssCyの細胞内取り込みにはNa非依存性・酸性アミノ酸輸送系xC-に属するxCTと4F2hcの二両体が主要な輸送体であり、特にxCTがCyssCy取り込みに関わることが報告されているが、牛生殖細胞や初期胚での発現動態は不明である。本研究では牛生殖細胞における両サブユニットの検出を行った。［方法］個体耳より採取、培養した牛線維芽細胞、と場より採取した卵丘細胞、成熟卵子、day1 、day2、 day4、day6およびday8胚からRNAを抽出し、RT-PCRによってxCTと4F2hc遺伝子の発現動態を検出した。併せて、xCTの抗体を用いてタンパク質の蛍光検出を試みた。［結果及び考察］線維芽細胞および卵丘細胞を用いた検出では、xCTと4F2hc遺伝子の両者が検出された、また、細胞表明におけるxCTタンパク質の局在も併せて検出された。初期胚では、各ステージで4F2hc遺伝子の一定な発現が検出されたが、xCT遺伝子についてはday2からday4の時期の周辺で発現の低下が見られた。各ステージの胚におけるxCTタンパク質の局在については明確な変動は確認されなかった。以上の結果より、牛体細胞ならびに初期胚におけるxCTと4F2hc遺伝子の発現が初めて確認された。また、初期胚においては、発生ステージに伴う4F2hc とxCT遺伝子発現のパターンに違いが見られたが、胚で既に観察されているシスチン低利用性と関連については明確ではなかった。このため、xC-アミノ酸輸送系の遺伝子発現とタンパク質機能との相違も示唆された。
  • KUBOTA Kaiyu, YAMAGAMI Kazuki, YAMANAKA Ken-ichi, TAKAHASHI Masashi, SOH Tomoki, YAMAUCHI Nobuhiko, HATTORI Masa-aki  The Journal of Reproduction and Development Supplement  102-  (0)  1083  -1083  2009  [Not refereed][Not invited]
     
    【目的】着床はプロゲステロン（P4）とエストラジオール（E2）の二つのステロイドホルモンにより厳密に制御されている。P4シグナルの仲介因子としてIndian Hedgehog (Ihh)が報告され、上皮―間質間の相互作用を通じ、子宮内膜の受容能獲得に重要な役割を果たすことが示唆されている。我々は妊娠ラット子宮においても着床前の妊娠3.5日目の上皮において、Ihhの一過性の発現上昇が認められることを報告した。一方、その発現調節機構についての詳細は不明である。本研究では、卵巣除去ラット、着床遅延モデルを用い、ステロイドホルモンによるIhhの発現調節機構の解析を目的とした。
    【方法】7週齢Wistarラットから卵巣を切除し、2週間後、P4およびE2を投与し、Ihh mRNAの発現に対する影響をリアルタイムRT-PCRにより解析した。次に妊娠3.5、4.5日目にERアンタゴニスト（ICI182.780）を投与し、Ihh mRNAの発現を解析した。また、常法に従い着床遅延モデルラットを作製し、E2投与の有無および前後におけるIhh mRNAの発現を解析した。
    【結果】卵巣除去ラットによる解析の結果、Ihh mRNAの発現はP4処理により急性に誘導されることが明らかとなった。一方、E2処理では発現が誘導されないのみならず、P4の発現誘導を抑制することが示唆された。そこで着床前期に上昇する一過性のE2産生がIhh mRNAの発現を抑制し着床へ関与している可能性を考慮し、ICI処理によりその発現量を解析した。その結果、着床後の妊娠5.5日目には対象群、ICI処理郡でその発現量に差が認められなかった。一方、妊娠初期に一過性に上昇するIhhの発現が着床前であるのか、着床期（着床ウィンドウ成立時）であるのか、その結論には至っていない。そのため、現在着床遅延モデルにより人為的に着床を制御することで、着床の前後のIhh mRNAの発現量を解析しており、こちらも併せて報告する。
  • Bai Hanako, Sakurai Toshihiro, Konno Toshihiro, Takahashi Masashi, Imakawa Kazuhiko  The Journal of Reproduction and Development Supplement  102-  (0)  236  -236  2009  [Not refereed][Not invited]
     
    09日本繁殖生物学会 AbstructGATA23【目的】インターフェロン・タウ（IFNT）は、反芻動物の着床期に栄養膜細胞から特異的に産生されるタンパクであり、母親の妊娠認識に必須である。しかしながら、IFNT遺伝子の栄養膜細胞特異的な発現制御機構についての知見は乏しい。本研究では、ウシ・栄養膜由来細胞CT-1とウシ・腎臓細胞株MDBK（非栄養膜由来細胞）における遺伝子発現の比較による結果から、遺伝子転写調節因子GATAファミリーに着目し、栄養膜細胞特異的IFNT遺伝子の発現制御への関与を検討した。【方法】1．GATA発現増加時のIFNTの発現ヒト絨毛性がん細胞JEG3、ウシ耳由来繊維芽細胞EF、および卵巣卵丘顆粒膜細胞 oCGにIFNT上流プロモーター領域-631bpの配列を組み込んだレポーターベクター(pGL3-IFNT)と、GATA2, GATA3発現コンストラクトを導入し、ルシフェラーゼアッセイ法によりIFNTの転写活性を測定した。2．GATA発現抑制時のIFNTの発現ウシ・栄養膜由来細胞CT-1にGATA2, GATA3のsiRNAを導入し、GATA2,3の発現抑制下でのIFNT遺伝子発現を解析した。【結果と考察】1．ルシフェラーゼアッセイの結果、JEG3を用いた場合にはIFNTの転写活性に差は見られなかったが、EF、およびoCG を用いた場合にはGATA2の導入によりIFNT遺伝子の転写活性が上昇した。2．CT-1細胞では、内在性IFNTの発現におけるGATA3の発現抑制の影響は見られなかったが、GATA2の発現抑制により内在性IFNTの発現が低下した。今回の結果から、栄養膜細胞特異的に発現するIFNTの発現にGATA2が関与することが明らかになった。
  • KANEDA Masahiro, NAGAI Takashi, AKAGI Satoshi, WATANABE Shinya, GESHI Masaya, HASHOYADA Yutaka, TAKAHASHI Masashi, YAMANAKA Kenichi, SAITO Kimiharu, MORI Masaomi  The Journal of Reproduction and Development Supplement  102-  (0)  1097  -1097  2009  [Not refereed][Not invited]
     
    【目的】インプリント遺伝子とは、父由来あるいは母由来の対立遺伝子のうち一方だけが特異的に発現する哺乳類特有の遺伝子であり、これまでにヒト・マウスでは100個近く見つかっている。これらの遺伝子の働きは様々であるが、胎子の成長をコントロールしたり、胎盤形成に必須であったり、また糖質・脂質代謝を司るものもある。さらに、体細胞クローン動物ではインプリント遺伝子を含む多くの遺伝子に発現異常が見られ、低発生率や発生異常の一因と考えられている。しかし、ウシを含む家畜においては、10個程度のインプリント遺伝子しか同定されておらず、それもヒト・マウスのホモログ遺伝子の解析で終わっている。そこで、母由来対立遺伝子しか持たないウシ単為発生胚由来線維芽細胞を用いて、ウシ新規インプリント遺伝子の探索を行った。
    【方法】ウシ単為発生胚由来線維芽細胞および正常胚由来線維芽細胞（いずれも妊娠40日齢の胎子から採取）よりRNAを調整し、RT-PCR法によりマウスインプリント遺伝子ホモログ遺伝子の発現解析を行った。また、DNAマイクロアレイ（NimbleGen）を用いて、父性発現インプリント遺伝子候補を網羅的に解析した。
    【結果】マウスで父性発現するインプリント遺伝子IGF2、PEG3、 ZAC1、 NDN、DLK1は正常胚でのみ発現していることから、これらの遺伝子はウシでもマウス同様に父性発現インプリント遺伝子であることが示唆された。一方で、SGCE、IMPACT、MAGEL2、SNRPN、PEG1/MESTは正常胚のみならず単為発生胚でも発現が見られたことから、これらの遺伝子はウシではインプリントされていない可能性が考えられる。また、マウスで母性発現するインプリント遺伝子のうち、CDKN1Cは単為発生胚で発現していなかったことから、この遺伝子はウシではマウスとは逆に父性発現するようにインプリントされている可能性が示唆された。DNAマイクロアレイの結果から、正常胚由来線維芽細胞でのみ発現している遺伝子（＝父性発現インプリント遺伝子の候補）を同定し、現在、解析を進めている。
  • 松本光史, 阪谷美樹, 井上寛暁, 村上斉, 高橋昌志, 梶雄次  日本養豚学会誌  45-  (4)  271  2008/12/25  [Not refereed][Not invited]
  • 松本光史, 阪谷美樹, 井上寛暁, 村上斉, 高橋昌志, 梶雄次  日本養豚学会大会講演要旨  90th-  41  2008/10/23  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹  九州沖縄農業研究成果情報  (23)  123  -124  2008/09/08  [Not refereed][Not invited]
  • 谷峰人, 友川浩一郎, 水戸康明, 船越大資, 坂谷美樹, 高橋昌志, 上村俊一  日本獣医学会学術集会講演要旨集  146th-  231  2008/09/05  [Not refereed][Not invited]
  • 高橋昌志  Journal of Reproduction and Development  54-  (Supplement)  j37  2008/08/01  [Not refereed][Not invited]
  • 高橋 昌志, 三石 洋之, 渡辺 正五  JARI research journal  30-  (6)  263  -266  2008/06  [Not refereed][Not invited]
  • 山中賢一, 高橋昌志  日本胚移植学雑誌  30-  (2)  99  -105  2008/05/16  [Not refereed][Not invited]
  • 山中 賢一, 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer  30-  (2)  99  -105  2008/05/16  [Not refereed][Not invited]
  • 高橋昌志, 山中賢一, 阪谷美樹  日本畜産学会大会講演要旨  109th-  109  2008/03/27  [Not refereed][Not invited]
  • 阪谷美樹, 浜野晴三, 山中賢一, 高橋昌志  日本畜産学会大会講演要旨  109th-  109  2008/03/27  [Not refereed][Not invited]
  • 井上寛暁, 松本光央, 高橋昌志, 村上斉, 阪谷美樹, 梶雄次  日本畜産学会大会講演要旨  109th-  138  2008/03/27  [Not refereed][Not invited]
  • 高橋昌志, 山中賢一, 阪谷美樹  日本胚移植学雑誌  30-  (1)  61  2008/01/28  [Not refereed][Not invited]
  • 小林修司, 稲葉泰志, 阪谷美樹, 小林修一, 相川芳雄, 大竹正樹, 今井敬, 高橋昌志  日本胚移植学雑誌  30-  (1)  59  2008/01/28  [Not refereed][Not invited]
  • Yutaka Hashiyada, Masanori Taniguchi, Yoichi Fujii, Hitomi Takahashi, Masaya Geshi, Masashi Takahashi  BIOLOGY OF REPRODUCTION  74  -74  2008  [Not refereed][Not invited]
  • Nakano Kazuaki, Nagashima Hiroshi, Matsunari Hitomi, Ogawa Buko, Fujiwara Tsukasa, Nakayama Naoki, Sasayama Norihisa, Yoshikawa Yoshihiro, Shirasu Akio, Takahashi Masashi  The Journal of Reproduction and Development Supplement  101-  (0)  121  -121  2008  [Not refereed][Not invited]
     
    【目的】我々が新規に開発した中空糸膜法（高橋ら，2008）によりガラス化保存された，ブタ体外生産胚からの産仔作出の可能性を検証することを目的とした。【方法】NCSU23を基本とする培地でブタ卵丘卵子複合体を培養しMII期卵を得た。これらに電気的活性化（直流150V/mm，100μsec，1回）を加え，単為発生を誘導した。また、一部の卵には既法（Kurome et al.，2006）に従ってICSIを行い受精卵を作製した。これらの胚をPZM-5培地で4日間培養し，得られた桑実胚に非侵襲的方法による細胞質内脂肪顆粒除去処置（Esaki et al.，2004 を改良）を施した。すなわち，胚を38.5°Cの4% trypsinで1-4分間処理して透明帯を膨化させた後，7.5μg/ml cytochalasin B存在下で遠心処理（12000g，23min，38°C）し，細胞質内脂肪顆粒を囲卵腔内に偏在させた。胚を1hr培養した後，ガラスキャピラリーに装着したトリアセテート製の中空糸膜(ポアサイズ78Å（半径），内径185μm，膜厚15μm，長さ3-4 cm)に胚を吸入し，ガラス化・融解を行った。ガラス化には20%仔ウシ血清を添加した20mM Hepes緩衝TCM199を基本培地とし、15％ ethylene glycol，15％ DMSO，0.5M sucroseを含むガラス化液を用いた。融解後の培養には10%ウシ胎仔血清を添加したPZM-5培地を使用した。単為発生由来のガラス化胚を融解後3日間培養し，胚盤胞への発達能を調べた。また，ICSI由来のガラス化胚は融解後2日間培養し，発情同期化したレシピエントブタの子宮角に移植した。【結果】単為発生由来胚のガラス化後の胚盤胞形成率は非ガラス化胚のそれと同等であった（80.9%，38/48 vs. 95.1%，39/41）。また，ガラス化されたICSI由来胚盤胞27個を2頭のレシピエントブタに移植した結果，１頭が妊娠した（現在妊娠91日目）。以上より，ブタ胚のガラス化保存における中空糸膜法の有効性が胚移植試験によって実証された。
  • Yamanaka Ken-ichi, Sakatani Miki, Takahashi Masashi  The Journal of Reproduction and Development Supplement  101-  (0)  519  -519  2008  [Not refereed][Not invited]
     
    【目的】体細胞クローン (SCNT) 胚では、通常の受精胚と比較して多くの異常が認められている。その代表として挙げられるのが遺伝子発現の制御に関わるゲノムDNAのメチル化修飾である。このDNAメチル化を制御しているのはDNA methyltransferase (DNMT)であり、クローン胚の発育能との関係も報告されている。そこで、本研究では、ウシSCNT胚におけるDNAのメチル化を制御する手段として目的の遺伝子のみを特異的に抑制することができるRNA干渉に着目し、胚におけるDNMT 1発現抑制の影響を調べた。【方法】DNMT1に対して設計した 21塩基対の二本鎖RNA (siRNA)を合成し、siRNAを体外受精およびSCNT胚の細胞質内に顕微注入した。実験区は無処理区、siRNA区およびSHAM区をそれぞれ体外受精胚およびSCNT胚で設け、体外培養後の発生率、胚盤胞期胚の細胞数およびICM/TE比を調べた。さらに、体外発生培養後8日の胚盤胞期胚をICMおよびTEに分離し、ゲノムDNAを抽出した。bisulfite処理後、satellite I領域を増幅し、メチル化感受性制限酵素Aci Iを用いたCOBRA法によりメチル化状態を評価した。【結果】体外受精胚では、siRNA区で胚盤胞期への発生率が無処理区およびSHAM区と比較して有意に低下した(P<0.05)。一方、SCNT胚ではsiRNA区およびSHAM区で分割率が有意に増加した(P<0.01)。また、胚盤胞期への発生率はsiRNA区で無処理区と比較して有意に増加した(P<0.05)。細胞数およびICM/TE比には有意な差は見られなかった。さらに、胚盤胞期胚のsatellite Iのメチル化状態を調べた結果、SCNT胚のsiRNA区ではICMおよびTEどちらも未処理区およびSHAM区と比較して有意に低下した(P<0.01)。よって、DNMT1のRNA干渉による胚のメチル化状態制御の可能性が示唆された。
  • 松本光史, 阪谷美樹, 井上寛暁, 村上斉, 高橋昌志, 梶雄次  日本養豚学会誌  44-  (4)  224  2007/12/25  [Not refereed][Not invited]
  • 松本光史, 阪谷美樹, 井上寛暁, 村上斉, 高橋昌志, 梶雄次  日本養豚学会大会講演要旨  88th-  17  2007/10/25  [Not refereed][Not invited]
  • 山中賢一, 阪谷美樹, 高橋昌志  西日本畜産学会報  25  2007/09/30  [Not refereed][Not invited]
  • HASHIYADA Yutaka, FUJII Yoichi, TANIGUCHI Masanori, MIYACHI Rie, WATANABE Akiyuki, URATA Hirofumi, SUGAWARA Toru, YOKOTA Masami, KOZAI Chiaki, FUJII Mitutaka, TAKAHASHI Masashi, TAKAHASHI Hitomi  日本胚移植学雑誌  29-  (3)  106  -113  2007/09/21  [Not refereed][Not invited]
  • HASHIYADA Yutaka, TANIGUCHI Masanori, FUJII Yoichi, MIYACHI Rie, WATANABE Akiyuki, KOZAI Chiaki, TAKAHASHI Hitomi, OKADA Masato, SUGAWARA Toru, FUJII Mitutaka, YOKOTA Masami, URATA Hirofumi, TAKAHASHI Masashi, IMAI Kei  日本胚移植学雑誌  29-  (3)  114  -124  2007/09/21  [Not refereed][Not invited]
  • 高橋昌志, 小林修一, 荒木武紀, 山中賢一, 阪谷美樹  九州農業研究発表会専門部会発表要旨集  70th-  92  2007/08  [Not refereed][Not invited]
  • 阪谷美樹, 小林修司, 山中賢一, 高橋昌志  九州農業研究発表会専門部会発表要旨集  70th-  93  2007/08  [Not refereed][Not invited]
  • 高橋 昌志, 三石 洋之, 渡辺 正五  JARI research journal  29-  (7)  307  -310  2007/07  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹  臨床獣医  25-  (4)  19  -23  2007/04/01  [Not refereed][Not invited]
  • 岡野彰, 高橋ひとみ, 高橋昌志, 下司雅也  農業・食品産業技術総合研究機構畜産草地研究所研究報告  (7)  1  -7  2007/03/16  [Not refereed][Not invited]
  • S. Kobayashi, M. Sakatani, Y. Inaba, S. Kobayashi, K. Imai, M. Takahashi  REPRODUCTION FERTILITY AND DEVELOPMENT  19-  (1)  266  -266  2007  [Not refereed][Not invited]
  • Y. Hashiyada, H. Takahashi, M. Asada, N. Sakuta, S. Furuyashiki, S. Yamaya, T. Oike, K. Konishi, M. Takahashi  REPRODUCTION FERTILITY AND DEVELOPMENT  19-  (1)  198  -199  2007  [Not refereed][Not invited]
  • M. Takahashi, K. Nagayama, M. Sakatani, S. Kobayashi, K. Morishita, M. Asakawa  REPRODUCTION FERTILITY AND DEVELOPMENT  19-  (1)  213  -214  2007  [Not refereed][Not invited]
  • M. Sakatani, K. Nagayama, K. Kobayashi, K. Kobayashi, K. Morishita, M. Asakawa, M. Takahashi  REPRODUCTION FERTILITY AND DEVELOPMENT  19-  (1)  271  -271  2007  [Not refereed][Not invited]
  • Takahashi Masashi, Yamanaka Kenichi, Sakatani Miki  The Journal of Reproduction and Development Supplement  100-  (0)  12033  -12033  2007  [Not refereed][Not invited]
     
    【目的】標的遺伝子に対する二本鎖RNAを作成し、細胞内に導入することで遺伝子特異的にmRNAを分解する手法であるRNA干渉法は、多岐にわたる培養細胞や一部の生体組織に加えて初期胚においてもその作用が認められている。組織細胞と異なり、初期胚では１細胞期の胚内に二本鎖RNAを顕微注入する手法が主であり、細胞分裂が進み、構成割球数が多くなった時期の胚への顕微注入は困難であることから、分割胚でのRNA干渉については検討されていない。そこで、本研究ではRNA干渉作用を持たない蛍光標識siRNAを用いて、リポフェクションにより発生の進んだ胚への導入条件と胚発生への影響について検討した。【方法】常法による体外受精後、透明帯除去・非除去１細胞期胚に蛍光標識siRNAをリポフェクション法により導入した。その際に、導入操作条件についても併せて検討した。２－３時間の導入処理後、落射蛍光顕微鏡により導入siRNAの蛍光を測定した。その後、継続して培養し、８日目の胚盤胞形成への影響を観察した。体外受精後２日目の８－１６細胞胚、５日目の桑実期胚および８日目の透明帯脱出胚についても蛍光標識siRNAによる導入を行い、処理後の蛍光量および胚盤胞への発生を観察した。【結果及び考察】透明帯非除去の１細胞期胚では、siRNAの蛍光強度が増加したが、透明帯除去後の胚内での蛍光は観察されなかった。一方、透明帯除去後にリポフェクションを行うことで、胚の蛍光増加が観察された。siRNA導入胚を継続して培養したところ、胚盤胞の形成が見られた。透明帯を除去した８－１６細胞胚、桑実期胚および透明帯脱出胚においても蛍光標識siRNA導入を行った結果、割球における蛍光の増加が観察された。また、８－１６細胞胚、桑実期胚の継続培養後、胚盤胞の形成が観察された。透明帯脱出胚においても導入後の胚の退行は観察されなかった。以上のことから、リポフェクションによるsiRNA導入には透明帯を除去する必要があり、透明帯除去後には割球への導入は可能であることが示唆された。併せて本導入処理による胚発生への毒性は低いと考えられた。
  • Sakatani Miki, Nagayama Koki, Morishita Koremoto, Yamanaka Kenichi, Asakawa Makio, Takahashi Masashi  The Journal of Reproduction and Development Supplement  100-  (0)  20017  -20017  2007  [Not refereed][Not invited]
     
    【目的】暑熱ストレスに起因する酸化ストレスは受精時や胚発生初期に悪影響を及ぼし受胎率を低下させると考えられる。体外培養系では暑熱ストレスにより、胚の活性酸素類が増加し発生率が低下するが、培養液中に抗酸化物質を添加することにより発生率が改善する。そこで本研究では強力な抗酸化能を持つ褐藻類由来ポリフェノールであるフロロタンニン類(PHL)を培養液に添加が暑熱ストレス下での受精、胚発生に及ぼす影響を検討した。【方法】実験１：融解後、洗浄希釈した精子を38.5℃、41℃、41℃＋100ng/ml PHLの各条件下で4時間処理しPI/FDA染色による精子の膜損傷程度並びにコメットアッセイ法による精子のDNA損傷程度を評価した。処理終了後、体外受精を行い発生培養を行った。実験2：38.5℃、41℃、41℃＋100ng/ml PHLの各条件下で6時間の媒精後、発生培養を行った。実験3：体外受精胚を用い、受精後2日目に38.5℃、41℃、41℃＋10ng/ml PHL の条件で6時間の処理を行い、その後38.5Cの温度条件にて発生培養を行った。各実験区とも発生培養開始2日目に分割率、8日目に分割胚数に占める胚盤胞発生率を判定した。【結果・考察】実験1：DNA損傷程度は処理区間の差は認められなかった。精子の膜損傷割合は41℃区で増加する傾向が認められたが、PHL添加により有意に低下した。胚盤胞発生率は41℃区で有意に低下した。しかしPHLの添加により胚盤胞発生率は有意に改善した。実験2：受精中の暑熱ストレスは胚分割率を有意に低下させたが、PHLの添加により暑熱ストレス下での胚盤胞発生率は若干増加した。実験3：受精後2日目の暑熱ストレスは胚盤胞発生率を有意に低下させたが、PHLの添加により胚の発生率は有意に改善された。以上の結果から、褐藻類由来のポリフェノールであるPHLは、暑熱ストレス下における精子の膜損傷を軽減し、受精及び体外培養時の発生低下を防止することが示唆された。
  • HASHIYADA Yutaka, WATANABE Akiyuki, TANIGUCHI Masanori, FUJII Yoichi, MIYACHI Rie, URATA Hirofumi, FUJII Mitsutaka, YOKOTA Masami, TANIMURA Hidetoshi, TAKAHASHI Masashi, TAKAHASHI Hitomi  Nihon Chikusan Gakkaiho  77-  (4)  471  -478  2006/11/25  [Not refereed][Not invited]
     
    Co-transfer of bovine embryos with trophoblastic vesicles (TVs) is a useful method to improve pregnancy rate. However, efficiency of TVs production by recovering the elongating blastocysts derived from superovulation varies depending on the condition of donor cows such as response to hormone treatment. In the present study, we examined the efficacy of TVs production derived from elongating blastocysts which were produced by transfer of in vitro-produced blastocysts (ET) or superovulation-artificial insemination (SOV-AI). On day 14, ET-derived elongating blastocysts were recovered 7 days after transfer of 5-10 and 11-20 blastocysts. On day 17, ET-derived elongating blastocysts were recovered 10 days after transfer of 10 blastocysts. SOV-AI-derived elongating blastocysts were recovered 14 and 17 days after artificial insemination. TVs were generated from culture of trophoblastic fragments, which had been dissected from morphologically normal elongating blastocysts of longer than 3 mm in length. Numbers of recovered elongating blastocysts and TVs were compared among treatments. Numbers of total and intact elongating blastocysts embryos were significantly lower (P<0.05) when elongating blastocysts were recovered on day 14 after transfer of 5-10 blastocysts (2.3 and 0.9) than those from ET of 11-20 (5.7 and 2.3) embryos or SOV-AI (8.3 and 2.7). On day 17, total and intact elongating blastocysts embryos were not different between ET (4.2 and 3.2) and SOV-AI (9.9 and 3.4). The rate of TVs formation and number of TVs from dissected trophoblastic fragments were higher when TVs were produced from elongating blastocysts recovered on day 17 after ET (93.5% and 31.6) than other groups (60.8-88.6% and 2.1-20.1). These results indicate that TVs production can be improved and widened more in a field by the method of embryo transfer of in vitro-produced blastocysts, which have developed to elongating blastocysts in vivo.
  • 小林修司, 阪谷美樹, 高橋昌志  九州沖縄農業研究成果情報  (21)  155  -156  2006/10/16  [Not refereed][Not invited]
  • 小林修一, 高橋ひとみ, 奥田潔, 阪谷美樹, 小林修司, 高橋昌志  西日本畜産学会報  17  2006/09/29  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 小林修一, 澤井健, 志賀一穂  日本胚移植学雑誌  28-  (3)  112  -117  2006/09/22  [Not refereed][Not invited]
  • 高橋ひとみ, 高橋昌志, 平子誠, 下司雅也, 岡野彰, 犬丸茂樹  畜産草地研究成果情報  (5)  15  -16  2006/08/25  [Not refereed][Not invited]
  • 小林修司, 阪谷美樹, 高橋昌志  畜産草地研究成果情報  (5)  189  -190  2006/08/25  [Not refereed][Not invited]
  • 小林修一, 高橋ひとみ, 奥田潔, 阪谷美樹, 小林修司, 高橋昌志  日本畜産学会大会講演要旨  106th-  96  2006/03/20  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 小林修一  日本畜産学会大会講演要旨  106th-  81  2006/03/20  [Not refereed][Not invited]
  • 阪谷美樹, 小林修一, 小林修司, 高橋昌志  日本畜産学会大会講演要旨  106th-  81  2006/03/20  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹  東日本家畜受精卵移植技術研究会大会資料  21st-  8  -11  2006  [Not refereed][Not invited]
  • 高橋ひとみ, 高橋昌志, 下司雅也, 岡野彰, 犬丸茂樹, 奥田潔  東日本家畜受精卵移植技術研究会大会資料  21st-  16  -17  2006  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 小林修一, 濱崎直孝, 入江徹美  東日本家畜受精卵移植技術研究会大会資料  22nd-  42  -43  2006  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 斎藤仁, 崎村武司, 家入誠二, 長嶋比呂志  東日本家畜受精卵移植技術研究会大会資料  22nd-  54  -55  2006  [Not refereed][Not invited]
  • KOBAYASHI SHUJI, SAKATANI MIKI, KOBAYASHI SHUICHI, INABA YASUSHI, IMAI KEI, TAKAHASHI MASASHI  The Journal of Reproduction and Development Supplement  99-  (0)  171  -171  2006  [Not refereed][Not invited]
     
    バイオプシーした性判別胚の凍結融解後の生存性は低く，この技術の普及の大きな課題となっている。その理由の一つとして，透明帯の切開による影響が考えられ，防除策としてマイクロカプセルを用いた人工透明帯の利用が挙げられる。本試験は，胚の凍結保存におけるマイクロカプセルの有効性を検討した。体外受精後７日目の胚盤胞を，透明帯およびマイクロカプセルの有無により，4区（Zona(+)・Cap(-)区、Zona(+)・Cap(+)区、Zona(-)・Cap(-)区およびZona(-)・Cap(+)）に分類し，試験に供した。透明帯は，0.25%アクチナーゼを添加した生理食塩水を用いて除去した。マイクロカプセルは，１％アルギン酸ナトリウム加リンゲル溶液に導入した胚を溶液とともにキャピラリーピペットに吸引し，0.1％塩化カルシウム加リンゲル溶液中に静かに吹き出すことにより作製した。凍結は0.1M シュークロース，1.5M エチレングリコール，20%ウシ胎子血清（FCS）を添加した乳酸加リンゲル液を用い，プログラムフリーザーで凍結保存した。融解後の胚の生存性は，0.1mM βメルカプトエタノール，20%FCSを添加したTCM199で培養し，24，48，72および96時間後に観察して判定した。透明帯を有する胚ではマイクロカプセルの有無により凍結融解後の生存性に差は認められなかったが，透明帯を除去した胚では48時間目以降の生存率に有意差が認められ（Zona(-)・Cap(-)区：65.7%，Zona(-)・Cap(+)区：81.5%，p<0.05），透明帯除去時におけるマイクロカプセルの有効性が示唆された。
  • Sawai Ken, Takahashi Masashi, Moriyasu Satoru, Hirayama Hiroki, Minamihashi Akira, Onoe Sadao  The Journal of Reproduction and Development Supplement  99-  (0)  61  -61  2006  [Not refereed][Not invited]
     
    【目的】近年, 体細胞クローンにおけるエピジェネティクスの異常が明らかになりつつある。前大会で我々はウシ体細胞核移植(NT-SC)胚の胚盤胞期(BC)におけるSatellite I領域のDNAメチル化レベルが体内受精体内発生(Vivo)胚などと比較して高いことを報告した。今回はウシNT-SC胚の発生に伴うDNAメチル化レベルの変化を明らかにする目的で, BCと伸長期(EL)におけるDNAメチル化およびDNAメチルトランスフェラーゼ(Dnmt)遺伝子発現の解析を行った。【方法】NT-SCおよびVivo由来のBC胚を雌ウシに移植し, 発情後16日目にEL胚を回収した。BC胚は胚全体を, EL胚は胚盤(ED)部位と栄養膜(TE)部位をそれぞれ採取した。メチル化DNA割合はBisulfite処理DNAのSatellite I領域を増幅し, 制限酵素消化後の画像解析により測定した。また, Dnmt-1, -3aおよび-3bの発現量をRT-リアルタイムPCR法により測定した。【結果】BCのメチル化DNA割合はNT-SC胚においてVivo胚と比べて有意に高い値を示した。Vivo胚のメチル化DNA割合はBCと比較してEL-ED, -TEにおいて有意に増加し, EDはTEよりも高い値を示した。NT-SC胚のメチル化DNA割合はEL-TEにおいてBCおよびEL-EDと比べて有意に低い値を示し, BCとEL-EDに差は認められなかった。EL各部位のDNAメチル化レベルはNT-SC胚とVivo胚に差は認められなかった。Dnmt遺伝子発現では, BCのNT-SC胚でDnmt-1発現量が有意に低い値を示したが, Dnmt-3a, -3b発現量に差は認められなかった。EL各部位におけるDnmt遺伝子発現量はNT-SC胚とVivo胚に差は認められなかった。本結果から, Satellite I領域のDNAメチル化レベルはNT-SC胚とVivo胚では異なる変化を示し, NT-SC胚のDNAメチル化は発生に伴い正常なレベルに近づくことが示唆された。
  • 久保田健嗣, 長山公紀, 阪谷美樹, 小林修司, 小林修一, 森下惟一, 浅川牧夫, 高橋昌志  西日本畜産学会報  57  2005/10/15  [Not refereed][Not invited]
  • 小林修司, 阪谷美樹, 小林修一, 高橋昌志  西日本畜産学会報  72  2005/10/15  [Not refereed][Not invited]
  • 高橋昌志, 長山公紀, 阪谷美樹, 小林修一, 小林修司, 森下惟一, 浅川牧夫  日本畜産学会大会講演要旨  105th-  70  2005/08/25  [Not refereed][Not invited]
  • 高橋昌志, 大山さやか, 阪谷美樹, 小林修司, 小林修一, 長嶋比呂志  日本畜産学会大会講演要旨  104th-  139  2005/03/20  [Not refereed][Not invited]
  • 渡辺伸也, 当真裕美子, 木村由紀子, 高橋清也, 赤木悟史, 志賀一穂, 藤田達男, 宇津宮恭子, 高橋昌志  日本畜産学会大会講演要旨  104th-  118  2005/03/20  [Not refereed][Not invited]
  • 小林修司, 阪谷美樹, 小林修一, 高橋昌志  日本畜産学会大会講演要旨  104th-  131  2005/03/20  [Not refereed][Not invited]
  • 阪谷美樹, 田川真人, 小林修一, 小林修司, 高橋昌志  日本畜産学会大会講演要旨  104th-  132  2005/03/20  [Not refereed][Not invited]
  • 小林修一, 阪谷美樹, 小林修司, 高橋昌志  日本畜産学会大会講演要旨  104th-  131  2005/03/20  [Not refereed][Not invited]
  • Sawai Ken, Takahashi Masashi, Kageyama Soichi, Moriyasu Satoru, Hirayama Hiroki, Minamihashi Akira, Onoe Sadao  The Journal of Reproduction and Development Supplement  98-  (0)  30  -30  2005  [Not refereed][Not invited]
     
    【目的】マウスやウシ, ブタなどの体細胞核移植胚においては様々な遺伝子の発現異常が報告されており, 核移植産子の作出率を低下させる原因となっていることが考えられる。遺伝子発現を調節する機構の一つとしてDNAのメチル化による転写制御が知られており, 今回我々はウシ体細胞核移植胚のSatellite I領域のメチル化状態を様々な方法により作出された胚と比較するとともに, 体細胞核移植胚の産子作出率と胚盤胞期胚のDNAメチル化状態の関係について検討した。【方法】胎子(NT-FE), 新生子牛(NT-CA)由来線維芽細胞および桑実期胚の割球細胞(NT-EM)を用いた核移植, 体内受精体内発生 (Vivo), 体外受精(IVF)および単為発生(PA)により得られた胚盤胞期胚を1胚づつ採取し, DNAを抽出した。得られたDNAはBisulfite処理後, Satellite I領域をPCR法により増幅した。制限酵素(Aci I)処理したPCR産物を電気泳動し, 得られた消化産物のバンドを画像解析することによりメチル化DNAの割合を測定した。また, NT-CA胚の割球細胞をドナー細胞に用いて核移植した胚のメチル化DNAの割合を測定した。さらに, 核移植産子の作出率が異なる複数のドナー細胞を用いて作出したNT-CA胚におけるメチル化DNAの割合についても測定を行った。【結果】NT-FEおよびNT-CA胚のメチル化DNAの割合はNT-EM, Vivo, IVFおよびPA胚と比較して有意に高い値を示した。NT-EM胚のメチル化DNAの割合はVivo, IVFおよびPAと比較して差は認められなかった。NT-FEおよびNT-CA胚のメチル化DNAの割合はそれぞれのドナー細胞よりも有意に減少していたが, NT-CA割球細胞由来の核移植胚はドナー細胞に用いたNT-CA胚と同程度のメチル化状態にあった。また, 産子の作出率が異なるドナー細胞を用いたNT-CA胚のメチル化DNAの割合に有意な差は認められなかった。
  • NAGAYA Hidekazu, KANAYA Toshimichi, KAKI Hiroki, TOBITA Yoneko, TAKAHASHI Masashi, TAKAHASHI Hitomi, YOKOMIZO Yuichi, INUMARU Shigeki  Journal of Veterinary Medical Science  66-  (11)  1395  -1401  2004/11/25  [Not refereed][Not invited]
     
    We developed a procedure for the large-scale purification of bovine interferon-τ (boIFN-τ) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-τ (rboIFN-τ) was efficiently produced in the silkworm infected with boIFN-τ cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 × 108U/mg) of 91% pure rboIFN-τ by means of a combination of the ANT, followed by QSC and CSCC.
  • 小林修司, 阪谷美樹, 小林修一, 高橋昌志  西日本畜産学会報  14  2004/10/15  [Not refereed][Not invited]
  • 高橋昌志, 小林修一, 小林修司, 阪谷美樹  Journal of Reproduction and Development  50-  (Supplement)  J56  2004/08/25  [Not refereed][Not invited]
  • 阪谷美樹, 須田郁夫, 沖智之, 小林修一, 小林修司, 高橋昌志  Journal of Reproduction and Development  50-  (Supplement)  J56  2004/08/25  [Not refereed][Not invited]
  • 阪谷美樹, 小林修司, 高橋昌志  九州沖縄農業研究成果情報  (19)  251  -252  2004/08/13  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 細江実佐, 古沢軌, 徳永智之  九州沖縄農業研究成果情報  (19)  255  -256  2004/08/13  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 志村英明, 志賀一穂, 谷口雅律  九州沖縄農業研究成果情報  (19)  253  -254  2004/08/13  [Not refereed][Not invited]
  • 岡野彰, 高橋ひとみ, 高橋昌志, 伊賀浩輔, 下司雅也  畜産草地研究成果情報  (3)  3  -4  2004/08/10  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 細江実佐, 古沢軌, 徳永智之  畜産草地研究成果情報  (3)  203  -204  2004/08/10  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司, 志村英明, 志賀一穂, 谷口雅律  畜産草地研究成果情報  (3)  201  -202  2004/08/10  [Not refereed][Not invited]
  • 阪谷美樹, 小林修司, 高橋昌志  畜産草地研究成果情報  (3)  205  -206  2004/08/10  [Not refereed][Not invited]
  • 小林修司, 今井敬, 後藤裕司, 金山佳奈子, 阪谷美樹, 高橋昌志, 小島敏之  九州農業研究  (66)  (109)  -109  2004/05/20  [Not refereed][Not invited]
  • 高橋昌志, 阪谷美樹, 小林修司  日本畜産学会大会講演要旨  103rd-  110  2004/03/20  [Not refereed][Not invited]
  • 淵本大一郎, 本間大輔, 鈴木俊一, 渡辺聡, 岩元正樹, 矢崎智子, 大西彰, 阪谷美樹, 高橋昌志  日本畜産学会大会講演要旨  103rd-  103  -103  2004/03/20  [Not refereed][Not invited]
  • GESHI Masaya, TAKAHASHI Masashi, TAKAHASHI Hitomi, OKANO Akira, TAKAHASHI Hiroshi, MATSUBARA Kazuei, TAKAHASHI Jutaro, NAGAI Takashi  日本胚移植学雑誌 = Japanese journal of embryo transfer  26-  (1)  1  -6  2004/01/16  [Not refereed][Not invited]
  • TAKAHASHI Hitomi, INUMARU Shigeki, TAKAHASHI Masashi, WATANABE Satoko, IGA Kosuke, YOKOMIZO Yuichi, GESHI Masaya, OKANO Akira, OKUDA Kiyoshi  Journal of Reproduction and Development  49-  (6)  433  -440  2003/12/01  [Not refereed][Not invited]
     
    Bovine interferon (bIFN) τ, which plays a key role in maternal-fetal recognition of pregnancy, was expressed by an Autographa californica nuclear polyhedrosis virus expression system. cDNA coding bIFNτ was derived from cultured trophoblast cells. The recombinant (r) bIFNτ had high antiviral activity (1 × 10 8 IU/mg) and the molecular weight of rbIFNτ was estimated to be 23 kDa by Western blotting analysis. We investigated the biological effect of rbIFNτ on prostaglandin (PG) F2α synthesis in cultured bovine endometrial epithelial cells in the presence or absence of oxytocin (OT, 100 nM). rbIFNτ suppressed basal and OT-induced PGF2α production in a dose-dependent manner (1-1,000 ng/ml). These results showed that biologically active rbIFNτ was produced in the baculovirus expression system, and that rbIFNτ had the ability to suppress the synthesis of PGF2α from bovine endometrial epithelial cells.
  • 谷口雅律, 松本道夫, 平田慎一郎, 阪谷美樹, 高橋昌志  熊本県農業研究センター畜産研究所試験成績書  2002-  133  -134  2003/11  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志  九州沖縄農業研究成果情報  (18)  235  -236  2003/11/01  [Not refereed][Not invited]
  • HOSOE Misa, FURUSAWA Tadashi, INOUE Fukashi, SAKATANI Miki, TOKUNAGA Tomoyuki, SCHULTZ Richard. M., TAKAHASHI Masashi  Journal of Mammalian Ova Research  20-  (3)  99  -105  2003/10/01  [Not refereed][Not invited]
     
    Double stranded RNA (dsRNA) interference is a useful tool for interfering with gene function by promoting the sequence-dependent degradation of targeted mRNA in several organisms. In the present study, in order to confirm and improve the effect of dsRNA, we investigated an inhibitory effect of dsRNA on both transient and stable gene expression of enhanced green fluorescent protein (EGFP) in mouse preimplantation embryos. In the transient expression system, the rates of fluorescent embryos were significantly decreased by co-injection of EGFP dsRNA and EGFP expression vector fragment into the pronucleus of zygotes. In the stable expression system, EGFP expression in transgenic embryos was significantly decreased by injection of EGFP dsRNA into both the pronucleus and cytoplasm of zygotes, but, cytoplasmic injection caused a more significant EGFP inhibition than pronuclear injection. In quantitative PCR analysis, the expression of the EGFP gene was also inhibited by dsRNA injection, whereas the endogenous gene expression was not affected. These data suggest that dsRNA can inhibit the specific gene expression without affecting the development and expression of other genes.
  • 高橋ひとみ, 高橋昌志, 下司雅也, 岡野彰  畜産草地研究成果情報  (2)  11  -12  2003/08/30  [Not refereed][Not invited]
  • 阪谷美樹, 高橋昌志  畜産草地研究成果情報  (2)  221  -222  2003/08/30  [Not refereed][Not invited]
  • 高橋ひとみ, 高橋昌志, 下司雅也, 岡野彰  畜産草地研究成果情報  (2)  13  -14  2003/08/30  [Not refereed][Not invited]
  • 阪谷美樹, 小林修一, 高橋昌志  日本胚移植学雑誌  25-  (2)  75  -81  2003/05/16  [Not refereed][Not invited]
  • 阪谷 美樹, 小林 修一, 高橋 昌志  日本胚移植学雑誌 = Japanese journal of embryo transfer  25-  (2)  75  -81  2003/05/16  [Not refereed][Not invited]
  • 高橋昌志, 古沢軌, 阪谷美樹, 徳永智之, 細江実佐  日本畜産学会大会講演要旨  101st-  60  2003/03/20  [Not refereed][Not invited]
  • 伊賀浩輔, 高橋ひとみ, 高橋昌志, 岡野彰  日本胚移植学雑誌  24-  (3)  83  -89  2002/09/17  [Not refereed][Not invited]
  • 下司雅也, 志水学, スクマー サハ, 高橋ひとみ, 高橋昌志, 岡野彰, 渡辺聡子, 犬丸茂樹, 横溝祐一  畜産草地研究成果情報  (1)  9  -10  2002/08/30  [Not refereed][Not invited]
  • 渡辺晃行, 戸塚豊, 近藤守人, 宮地利江, 小財千明, 藤井満貴, 谷口雅律, 橋谷田豊, 高橋昌志  関東東海北陸農業研究成果情報  2001-  (1)  78  -79  2002/07/31  [Not refereed][Not invited]
  • 高橋昌志  日本胚移植学雑誌  24-  (2)  49  -57  2002/05/16  [Not refereed][Not invited]
  • HOSOE Misa, FURUSAWA Tadashi, SAKATANI Miki, TOKUNAGA Tomoyuki, TAKAHASHI Masashi  Journal of mammalian ova research = 日本哺乳動物卵子学会誌  19-  (2)  S52  2002/04/01  [Not refereed][Not invited]
  • 高橋昌志, 永井卓, 岡村直道, 高橋ひとみ, 阪谷美樹, 岡野彰  日本畜産学会大会講演要旨  100th-  122  2002/03/20  [Not refereed][Not invited]
  • 淵本大一郎, 菊地和弘, 岩元正樹, SOMFAI T, 永井卓, 大西彰, 渡部聡, 阪谷美樹, 高橋昌志  日本畜産学会大会講演要旨  100th-  124  2002/03/20  [Not refereed][Not invited]
  • 下司雅也, 志水学, 高橋ひとみ, 高橋昌志, 渡辺聡子, 犬丸茂樹, 横溝祐一  東日本家畜受精卵移植技術研究会大会資料  17th-  32  -33  2002/01/24  [Not refereed][Not invited]
  • TAKAHASHI Hitomi, IGA Kosuke, SATO Taro, TAKAHASHI Masashi, OKANO Akira  Journal of Reproduction and Development  47-  (3)  181  -187  2001/06/01  [Not refereed][Not invited]
  • 小川英彦, 高橋昌志, 高橋ひとみ, 岡野彰  畜産研究成果情報  (15)  33  -34  2001/03/24  [Not refereed][Not invited]
  • 高橋昌志, 高橋ひとみ, 岡野彰, 永井卓  畜産研究成果情報  (15)  31  -32  2001/03/24  [Not refereed][Not invited]
  • 高橋昌志, 高橋ひとみ, 岡野彰, 永井卓  畜産研究成果情報  (15)  35  -36  2001/03/24  [Not refereed][Not invited]
  • 小川英彦, 高橋昌志, 高橋ひとみ, 永井卓, 岡野彰  日本畜産学会大会講演要旨  98th-  126  2001/03/20  [Not refereed][Not invited]
  • 高橋ひとみ, 高橋昌志, 浜野晴三, 渡辺聡子, 犬丸茂樹, 永井卓, 岡野彰  日本畜産学会大会講演要旨  98th-  122  2001/03/20  [Not refereed][Not invited]
  • 岸久司, 小川英彦, 高橋昌志, 高橋ひとみ, 永井卓, 岡野彰  日本畜産学会大会講演要旨  98th-  125  2001/03/20  [Not refereed][Not invited]
  • 加藤(森)ゆうこ, 高橋昌志, 森匡, 福永重治, 竹之内一昭, 中村富美男, 近藤敬治  日本畜産学会大会講演要旨  98th-  149  2001/03/20  [Not refereed][Not invited]
  • 菅原徹, 近藤守人, 小財千明, 藤井満貴, 谷口雅律, 橋谷田豊, 高橋昌志  東日本家畜受精卵移植技術研究会大会資料  16th-  52  -53  2001/02/01  [Not refereed][Not invited]
  • H Takahashi, M Takahashi, S Hamano, S Watanabe, S Inumaru, Y Yokomizo, T Nagai, A Okano  BIOLOGY OF REPRODUCTION  64-  183  -183  2001  [Not refereed][Not invited]
  • 高橋昌志  Journal of Reproduction and Development  46-  (Supplement)  J79-J86  -86  2000/12/25  [Not refereed][Not invited]
  • 岡野彰, 小川英彦, 岸久司, 高橋ひとみ, 高橋昌志  日本畜産学会大会講演要旨  97th-  122  2000/03/20  [Not refereed][Not invited]
  • 小川英彦, 岸久司, 高橋昌志, 伊賀浩輔, 高橋ひとみ, 岡野彰  日本畜産学会大会講演要旨  97th-  180  2000/03/20  [Not refereed][Not invited]
  • 高橋昌志, 慶長久美子, 高橋ひとみ, 小川英彦, 岡野彰  日本畜産学会大会講演要旨  97th-  104  2000/03/20  [Not refereed][Not invited]
  • 伊賀浩輔, 高橋ひとみ, 高橋昌志, 岡野彰  日本畜産学会大会講演要旨  97th-  180  2000/03/20  [Not refereed][Not invited]
  • 太田土美, 高橋ひとみ, 高橋昌志, 伊賀浩輔, 菅原徹, 宇田三男, 岡野彰  東日本家畜受精卵移植技術研究会大会資料  15th-  20  -21  2000/01/27  [Not refereed][Not invited]
  • 高橋ひとみ, 渡辺聡子, 高橋昌志, 伊賀浩輔, 国保健浩, 犬丸茂樹, 岡野彰  日本獣医学会学術集会講演要旨集  128th-  256  1999/09/01  [Not refereed][Not invited]
  • 小川英彦, 高橋昌志, 高橋ひとみ, 岡野彰  日本畜産学会大会講演要旨  95th-  80  1999/03/10  [Not refereed][Not invited]
  • 渡辺聡子, 高橋ひとみ, 国保健浩, 内村昭彦, 高橋昌志, 岡野彰, 横溝祐一, 犬丸茂樹  日本獣医学会学術集会講演要旨集  127th-  134  1999/03/01  [Not refereed][Not invited]
  • M Takahashi, K Keicho, M Hosoe, H Ogawa, H Takahashi, A Okano  THERIOGENOLOGY  51-  (1)  256  -256  1999/01  [Not refereed][Not invited]
  • 高橋昌志, 慶長久美子, 高橋ひとみ, 小川英彦, 岡野彰  日本繁殖生物学会講演要旨  91st-  38  1998/08  [Not refereed][Not invited]
  • 小川英彦, 高橋昌志, 高橋ひとみ, 岡野彰  日本繁殖生物学会講演要旨  91st-  95  1998/08  [Not refereed][Not invited]
  • 高橋ひとみ, 高橋昌志, 小川英彦, 岡野彰  日本繁殖生物学会講演要旨  91st-  97  1998/08  [Not refereed][Not invited]
  • 佐藤太郎, 高橋ひとみ, 高橋昌志, 小川英彦, 岡野彰  日本畜産学会大会講演要旨  94th-  135  1998/03  [Not refereed][Not invited]
  • 慶長久美子, 高橋昌志, 細江実佐, 高橋ひとみ, 吉田光敏, 岡野彰  日本畜産学会大会講演要旨  94th-  165  1998/03  [Not refereed][Not invited]
  • 小川英彦, 高橋昌志, 高橋ひとみ, 岡野彰  日本畜産学会大会講演要旨  94th-  135  1998/03  [Not refereed][Not invited]
  • 高橋昌志, 高橋ひとみ, 犬丸茂樹, 橋爪一善, 今川和彦, 岡野彰  日本畜産学会大会講演要旨  93rd-  139  1997/08  [Not refereed][Not invited]
  • 高橋昌志, 浜野晴三, 細江実佐, 高橋ひとみ, 岡野彰  日本畜産学会大会講演要旨  92nd-  205  1997/03  [Not refereed][Not invited]
  • 高橋昌志  畜産の研究  51-  (2)  270  -274  1997/02  [Not refereed][Not invited]
  • 高橋昌志  ブレインテクノニュース  (58)  16  -18  1996/11  [Not refereed][Not invited]
  • TAKAHASHI Masashi, NAGAI Takashi, OKANO Akira  Animal Science Technology  67-  (8)  740  -741  1996/08/25  [Not refereed][Not invited]
  • 高橋昌志, 岡野彰  日本畜産学会大会講演要旨  91st-  232  1996/03  [Not refereed][Not invited]
  • 岡野彰, 作本亮介, 高橋昌志, 奥田潔  日本畜産学会大会講演要旨  91st-  206  1996/03  [Not refereed][Not invited]
  • 坂代江, 高橋昌志, 金井幸雄, 岡野彰  日本畜産学会大会講演要旨  91st-  216  1996/03  [Not refereed][Not invited]
  • 八木隆史, 染井英夫, 高橋昌志, 奥田潔, 岡野彰  日本畜産学会大会講演要旨  91st-  209  1996/03  [Not refereed][Not invited]
  • 坂代江, 高橋昌志, 金井幸雄, 岡野彰  日本畜産学会大会講演要旨  90th-  127  1995/03  [Not refereed][Not invited]
  • 岡野彰, 奥田潔, 高橋昌志  家畜繁殖学会講演要旨  86th-  70  1994/09  [Not refereed][Not invited]
  • OKANO Akira, TAKAHASHI Masashi  Nihon Chikusan Gakkaiho  65-  (10)  923  -927  1994  [Not refereed][Not invited]
     
    This study was conducted to determine the effect of bovine, porcine and avian amniotic fluid (AmF) on the in vitro development of mouse embryos. The growth rates from 2-cell embryo through to hatched blastocyst (HBlast) were examined in whole AmF or in culture media supplemented with and without AmF for 96h. Whole bovine, porcine and avian AmF were not effective for the development of mouse embryos cultured in vitro. In a BSA-free M16 medium supplemented with 10% bovine AmF, the development of 2-cell embryos was equal to that in M16 supplemented with BSA. The effective factor in bovine AmF was destroyed by heat treatment at 100°C for 5min, and was not absorbed with Dextran-charcoal treatment. The moleculsr weight (MW) of this fraction was less than 104. Therefore, this factor is not asteroid or a lipid but presumably a polypeptide of MW less than 104.
  • M TAKAHASHI, A OKANO  BIOLOGY OF REPRODUCTION  50-  124  -124  1994  [Not refereed][Not invited]
  • 高橋昌志, 永井卓, 岡村直道, 岡野彰  家畜繁殖学会講演要旨  84th-  50  1993/10  [Not refereed][Not invited]
  • 岡野彰, 奥田潔, 高橋昌志  日本畜産学会大会講演要旨  87th-  251  1993/03  [Not refereed][Not invited]
  • 高橋昌志, 永井卓, 浜野晴三, 桑山正成, 岡村直道, 岡野彰  日本畜産学会大会講演要旨  87th-  283  1993/03  [Not refereed][Not invited]
  • 桑山正成, 吉兼由美, 浜野晴三, 岩崎説雄, 高橋昌志, 岡野彰, 中原達夫, 永井卓  日本畜産学会大会講演要旨  87th-  283  1993/03  [Not refereed][Not invited]
  • 高橋昌志, 永井卓, 浜野晴三, 桑山正成, 岡村直道, 岡野彰  家畜繁殖学会講演要旨  82nd-  9  1992/09  [Not refereed][Not invited]
  • 高橋昌志, 永井卓, 岡村直道, 浜野晴三, 桑山正成, 吉田光敏, 岡野彰  日本畜産学会大会講演要旨  85th-  223  1992/03  [Not refereed][Not invited]
  • 浜野晴三, 桑山正成, 高橋昌志, 永井卓, 岡野彰  日本畜産学会大会講演要旨  85th-  223  1992/03  [Not refereed][Not invited]
  • 高橋ひとみ, 桑山正成, 浜野晴三, 高橋昌志, 永井卓, 岡野彰, 角川博哉, 仮屋尭由  日本畜産学会大会講演要旨  85th-  222  1992/03  [Not refereed][Not invited]
  • 鎌田八郎, 高橋昌志, 岡野彰, 浜田龍夫  日本畜産学会大会講演要旨  85th-  205  1992/03  [Not refereed][Not invited]
  • 辻典子, 高橋昌志, 岡野彰, 栗崎純一, 荒井綜一  日本畜産学会大会講演要旨  84th-  60  1991/03  [Not refereed][Not invited]
  • 岡野彰, 高橋昌志  日本畜産学会大会講演要旨  84th-  31  1991/03  [Not refereed][Not invited]
  • OKANO Akira, TAKAHASHI Masashi  The Japanese journal of animal reproduction  37-  (2)  121  -125  1991  [Not refereed][Not invited]
     
    Prostaglandin (PG) E2 at the final concentrations between 2010000 ng/ml, stimulated progesterone (P4) output from the porcine corpus luteal cells in culture. It is suggested that PGE2 has a luteotropic effect to porcine corpus lutea in vivo. PGF2α did not have any clear effect on P4 output from luteal cells. On the contrary, oxytocin and platelet activating factor have the suppressive effect on P4 output from luteal cells in culture. These results mean that they have luteolytic effect to porcine corpus lutea.
  • 高橋昌志, 岡野彰  日本畜産学会大会講演要旨  83rd-  5  1990/03  [Not refereed][Not invited]

Association Memberships

  • 北海道受精卵移植研究会   東日本受精卵移植研究会   JAPAN EMBRYO TRANSFER SOCIETY   北海道畜産草地学会   THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT   JAPANESE SOCIETY OF ANIMAL SCIENCE   

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 高橋 昌志, 窪 友瑛
  • Production of molecular chaperone-enhanced bovine embryos by utilizing heat-independent HSP70 inducer
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
    Date (from‐to) : 2022/06 -2024/03 
    Author : 高橋 昌志
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2019/04 -2022/03 
    Author : Masashi Takahashi
     
    The purpose of this study was to clarify the bovine pregnancy response in extrauterine tissues temporally and spatially and to elucidate the kinetics of the involvement of interferon (IFN)τ produced by the embryo. We first detected IFNτ in the cervical mucus as well as the pregnancy-dependent increase in IFN-induced gene expression in the cervix around 18 days of pregnancy, when preimplantation bovine embryos are at their peak production of IFNτ in the uterus. In addition, RNA sequence analysis of cervical tissue has revealed a marked increase in IGFBP3 expression just before ovulation. These results suggest the possibility of detecting pregnancy- and estrus-related factors in cervical tissues that can be collected less-invasively, and the development of early pregnancy and estrus detection technology is expected.
  • リソソームシステインプロテアーゼ制御による牛卵巣の長期保存技術の開発
    一般 財団法人旗影会 財団法人旗影会：研究助成事業
    Date (from‐to) : 2018/04 -2019/03 
    Author : 高橋昌志
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    Date (from‐to) : 2015/11 -2018/03 
    Author : 高橋 昌志, BALBOULA AHMED
     
    本年度は、特別研究員Ahmed Z Balboura博士が別ポストへの採用のため、研究機期間が短縮たため、昨年度の研究の継続に注力した。特に、本研究で期待される凍結保存後のウシ胚の生存性向上への効果に関しては、前年度に明らかとなったガラス化保存胚の生存性向上へのカテプシン阻害剤の効果の検証を行い、カテプシンB阻害剤であるe-64および、カテプシンK阻害剤であるOdanactib、それぞれが凍結後の生存性への向上効果が確認された。実際にガラス化処理後の胚盤胞において、カテプシンBの活性を詳細に観察したところ、無処理胚と比べてガラス化処理胚では、胚盤胞外周を構成し、胎盤に分化する栄養膜細胞(TE)において、胚の内部に位置し、将来胎児に分化する内部細胞塊(ICM)と比べてカテプシン活性の増加が検出された。同時に、TUNEL染色によるDNA損傷の検出では、TE細胞でのDNA損傷の増加が確認された。また、同時にアポトーシス関連遺伝子であるcaspase9の有意な増加が確認された。これらのことから、ガラス化処理を含む凍結処理によって保存液に直接接する栄養膜細胞への物理的な障害が細胞への損傷を直接的に引き起こし、それを受けた細胞内のカテプシンの活性化－その下流のシグナルとして知られているアポトーシスの経路が活性化することが示唆された。今後は、ICMとTEの異なった細胞における応答性への際の研究が今後、必要である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2015/04 -2018/03 
    Author : Takahashi Masashi
     
    In the present study, we detected the activation of cathepsin (CTS)B in the pregnant bovine uterine tissue. This activation was thought to be stimulated by interferon tau(IFN)T secreted by embryo. Although expression of CTS genes was detected by pregnant and IFNT-treated non pregnant uterine tissue, expression of autophagy related genes was not changed. These results suggest the activation of lysosome and lysosomal CTS is triggered by IFNT for uterine modulation, but contribution of autophagy is low. Further study is needed to clarify the roles of autophagy. RNA interference of each subunit construction of type I IFN receptor subunit (IFNAR1, R1) by siRNA revealed that IFNT stimulated signal transduction is dominantly affected by IFNAR1 for inducing IFN related gene expressions. In addition, high levels of pregnancy specific gene expression were detected in the cervical tissue that is directly connected with uterus for the first time.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2015/04 -2017/03 
    Author : Masashi Takahashi
     
    The purpose of the present research is to search the activities of synthetic peptides constituting IFN-τ for a possible substitution for recombinant IFN-τ. Eleven peptides (long chain: 27-28 aa, short chain: 7-17 aa) have been chemically synthesized from amino acid sequence and structure of IFN-τ. Each or mixed peptides or recombinant IFN-τ were added to cultured bovine endometrium stromal cells to detect the expression of MX1, 2 and ISG15 that belong to interferon-stimulated genes (ISGs). Next, each or mixed peptides were added to the cells with recombinant IFN-τ and expression of ISGs was evaluated.IFN-τ significantly stimulated ISGs expression, whereas all single or mixed peptides did not. Addition of single or mixed peptides did not show the inhibitory effect of IFN-τ.These result suggest that synthetic peptides would not have agonist and antagonist ability or binding to IFN receptor by the possible structural change.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2014/04 -2017/03 
    Author : EDASHIGE Keisuke
     
    In this study, we examined whether a low-temperature sensitive transient receptor potential (TRP) channel was involved in the chilling injury of pig oocytes/embryos. The mRNA of TRPA1, which is sensitive to the temperature at lower than 17oC, was expressed in pig oocytes and embryos at various stages. Exposure of oocytes/embryos to 15oC for 15 min markedly decreased their viability. Exposure of oocytes to 15oC increased intracellular Ca2+ immediately. The pretreatment of oocytes with AP-18, a specific inhibitor for TRPA1, suppressed the increase in their intracellular Ca2+ and the decrease in their viability by the exposure to 15oC. These results indicate that TRPA1 is involved in the chilling injury of pig oocytes/embryos, and that the injury can be circumvented by suppressing TRPA1 activity. Our findings would be useful for developing a cryopreservation method for pig oocytes/embryos.
  • Detection of type I interferon-mediated catabolic activity in blood and somatic cells for early pregnancy diagnosis
    公益財団法人伊藤記念財団：研究助成事業
    Date (from‐to) : 2014/04 -2015/03 
    Author : Masashi Takahashi
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012 -2012 
    Author : TAKAHASHI MASASHI
     
    This study investigated 1)the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus oocyte complexes (COCs) and cathepsin(CTS) B activity in relation to apoptosis and 2) the effect of supplementation of CTSB inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development were investigated. Heat stress exposure during maturation significantly decreased the cleavage and blastocyst rate as well as increase in lysosome and CTSB activities. Besides, increase of caspase-3 activities in HS-treated oocytes showed significant increase of TUNEL-positive cells in blastocysts.Addition of CTDB inhibitor E-64 to maturation medium significantly increased the developmental rate and total cell number of blastocyst as well as decreasing the activities of lysosome and caspase-3. These results indicate that the regulation of lysosomal CTS by specific inhibitors improve the developmental competence of normal and heat-shocked COCs.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2005 -2007 
    Author : Masashi TAKAHASHI
     
    RNA interference (RNAi) is a strong tool to interfere a specific mRNA and widely applied for basic research for cell and tissue functions as well as medical treatment. Since the first report of RNAi in mouse preimplantation embryos (1999), RNAi research of gene regulation in mammalian embryos has started. However, RNAi in livestock animals such as bows and pigs has not been fully investigated. Therefore, in the present project, we investigated the efficiency of RNA interference on early development and tissue cell functions of bovine preimplantation embryos and cells. In the first experimen...

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