Researcher Profile and Settings

Master

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

researchmap

Profile and Settings

Profile and Settings

• Name (Japanese)

  Kawahara

• Name (Kana)

  Manabu

• Name

  201301048471341847

Alternate Names

http://kaken.nii.ac.jp/d/r/70468700.ja.html

Achievement

Research Interests

• マウス   家畜   初期胚発生   胎盤形成   核移植   

Research Areas

• Life sciences / Animal production science

Research Experience

• 2010 北海道大学 (連合)農学研究科(研究院) 准教授

Awards

• 2014/07 東北大学 先端農学研究奨励賞受賞
   

  受賞者: 川原学
• 2011/09 日本繁殖生物学会・奨励賞
   

  受賞者: 川原学
• 2009/04 JBC Papers of The Week
   

  受賞者: Manabu Kawahara
• 2005/09 日本畜産学会 優秀発表賞受賞
   

  受賞者: 川原学

Published Papers

• In utero morphological and functional properties of bovine trophoblastic vesicles.

  Shinjiro Kagawa, Yoshihiro Hayashi, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Molecular reproduction and development 91 (8) e23767  2024/08 [Refereed]
   

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

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

  Development (Cambridge, England) 151 (14) 2024/07/15 [Refereed]
   

  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.
• Characterization of conception rate after embryo transfer in comparison with that after artificial insemination in dairy cattle

  Shuhei Fukaya, Takeshi Yamazaki, Hayato Abe, Satoshi Nakagawa, Toshimi Baba, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Journal of Dairy Science 0022-0302 2024/07 [Refereed]
  
• 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)
• Dual barrier system against xenomitochondrial contamination in mouse embryos

  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 [Refereed]
   

  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.
• IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes.

  Shabur Abdus Talukder, Ahmed Z Balboula, Hanako Bai, Manabu Kawahara, Koji Kimura, Masashi Takahashi

  Reproduction (Cambridge, England) 2023/10/01 [Refereed][Not invited]
   

  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.
• Identification of menaquinone-4 (vitamin K2) target genes in bovine endometrial epithelial cells in vitro

  Hanako Bai, Manabu Kawahara, Masashi Takahashi

  Theriogenology 198 183 - 193 0093-691X 2023/03 [Refereed][Not invited]
  
• Differences in Mitochondrial Ultrastructure between In Vivo and In Vitro Mouse Blastocysts

  Hayashi, Yoshihiro, Edure, Taichi, Bai, Hanako, Takahashi, Masashi, Kawahara, Manabu

  Memories of the Research Faculty of Agriculture, Hokkaido University 北海道大学大学院農学研究院 39 1 - 6 1881-8064 2023/02/13 [Refereed][Not invited]
   

  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.
• 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)
• Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells

  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 [Refereed][Not invited]
   

  Progesterone (P4) is a well-known steroid hormone that plays a key role in oocyte growth and the maintenance of pregnancy in mammals, including cattle. Heat stress (HS) has an adverse effect on P4 synthesis through an imbalance in the cellular redox status. We have recently revealed that a standardized extract of Asparagus officinalis stem (EAS) increases P4 through non-HS induction of heat shock protein 70 (HSP70) and a synergistic increase of HSP70 by enhancing the intracellular redox balance, which was adversely affected by HS in bovine granulosa cells (GCs). Bovine GCs collected from bovine ovarian follicles were cultured at 38.5°C and 41°C for 12h with or without 5mg/mL EAS. After treatment, cells and culture suppernatant were collected for the analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect in P4 levels. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to detect expression of steroidogenesis related genes. Fluorescence staining was used to detect mitochondrial activity and lipid droplet. P4 level was increased by EAS treatment in association with increase in steroidogenic acute regulatory protein (STAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), mitochondrial membrane activity and lipid droplet both under non-HS and HS conditions. Notably, synergistic effect of EAS with HS co-treatment was observed to show a greater increase in P4 synthesis when comparison with EAS treatment under non-HS condition. Furthermore, inhibition of HSP70 significantly reduced EAS-induced P4 synthesis, mitochondrial activity and synthesis of lipid droplets. These results suggest that P4 synthesis by EAS is mediated by the steroidogenesis pathway via HSP70-regulated activation of STAR and 3β-HSD, together with improved mitochondrial activity and lipid metabolism in bovine GCs. Moreover, effect of EAS has a synergistic effect of with HSP70-regulated steroidogenesis pathway.
• Zona pellucida removal by acid Tyrode’s solution affects pre- and post-implantation development and gene expression in mouse embryos

  Weihong Fan, Tengda Huang, Tian Wu, Hanako Bai, Manabu Kawahara, Masashi Takahashi

  Biology of Reproduction 0006-3363 2022/08/10 [Refereed]
   

  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.
• Environmental factors affecting the conception rates of nulliparous and primiparous dairy cattle

  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 [Refereed]
   

  Dairy cattle must allocate energy to milk production and reproduction. Therefore, understanding the environmental factors that affect conception rates in nulliparous and primiparous cows is helpful in appropriate feeding management strategies before and after calving. Accordingly, the aim of this study was to investigate the influence of environmental factors before and after the first calving on the conception rate, representing the starting point of milk production. The records of the first artificial insemination (AI) from Holstein nulliparous cows (n = 533,672) and primiparous cows (n = 516,710) in Hokkaido, Japan, were analyzed using separate multivariable logistic regression models. The mean conception rates for nulliparous and primiparous cows from 2012 to 2018 were 55.2 and 39.2%, respectively. In both nulliparous and primiparous cows, the conception rate of crossbreeding using Japanese Black (JB) semen was significantly higher than that for purebred Holstein breeding. The conception rate using sexed semen decreased in the warmer months only in nulliparous cows. Moreover, we grouped primiparous cows according to milk yield during peak lactation (PY; < 25, 25-30, 30-35, ≥35 kg) and the interval from calving to first insemination (CFI; < 60, 60-79, 80-99, ≥100 d), and evaluated their combined effect on the conception rate. Both PY and CFI strongly affected the conception rate in primiparous cows, which decreased with an increase in PY, even for the group with CFI ≥100 d; however, the conception rate increased for a CFI ≥60 d regardless of PY. Taken together, this study demonstrates the long-term effect of PY and an independent effect of CFI on the conception rate of cows. These results provide guidance for management to execute appropriate AI implementation strategies before and after lactation.
• Cattle production by intracytoplasmic sperm injection into oocytes vitrified after ovum pick-up

  Shinjiro Kagawa, Shingo Hiraizumi, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Theriogenology 185 121 - 126 0093-691X 2022/06 [Refereed][Not invited]
   

  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.
• Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes

  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) 324 - 324 2022/02/10 [Refereed]
   

  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.
• Generation of viable calves derived from developmentally mature blastocysts produced by on-gel culture

  Shun SAITO, Hiroki AKIZAWA, Eri FURUKAWA, Yojiro YANAGAWA, Hanako BAI, Masashi TAKAHASHI, Manabu KAWAHARA

  Journal of Reproduction and Development 0916-8818 2022 [Refereed]
   

  Conventional culture systems for bovine embryos are unable to support sustained embryonic development until the developmentally mature blastocyst stage. Although we have previously developed an on-gel culture system that enables bovine blastocysts to complete cell segregation events at day (D) 10 following in vitro culture, the development of D10 blastocysts to term has yet to be achieved. In this study, we attained full-term development of D10 mature blastocysts produced using an on-gel culture system. Two calves derived from on-gel-cultured embryos were vaginally born, showing normal birth and placental weights and no obvious morphological abnormalities. Moreover, we detected no abnormalities in blood metabolic profile analyses. Our findings indicate that on-gel culturing can be used to facilitate the development of developmentally mature blastocysts to term, and produce healthy viable calves. This culture system could make a valuable contribution to cattle production and would enable a range of analyses for characterizing bovine-specific pre-implantation development.
• Recent progress of interferon-tau research and potential direction beyond pregnancy recognition

  Hanako BAI, Manabu KAWAHARA, Masashi TAKAHASHI, Kazuhiko IMAKAWA

  Journal of Reproduction and Development 0916-8818 2022 [Refereed]
   

  Since the discovery of interferon-tau (IFNT) over 30 years ago as the trophectodermal cytokine responsible for the maintenance of the maternal corpus luteum (CL) in ruminants, exhaustive studies have been conducted to identify genes and gene products related to CL maintenance. Recent studies have provided evidence that although CL maintenance, with the up- and down- regulation of IFNT, is important, its regulatory role in the endometrial expression of interferon-stimulated genes (ISGs) is far more important for conditioning the uterine environment for successful conceptus implantation and thereafter. This review initially describes the mammalian implantation process, briefly but focuses on recent findings, as there appears to be a common phenomenon during early to mid-pregnancy among mammalian species.
• Isoflavones and their metabolites influence the milk production ability of bovine mammary epithelial cells in a type‐specific manner

  Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi

  Animal Science Journal 93 (1) e13720  1344-3941 2022/01 [Refereed][Not invited]
   

  Dairy cows feed on isoflavones as physiologically active substances present in legumes. However, the influences of isoflavones (biochanin A, genistein, formononetin, and daidzein) and their metabolites (p-ethylphenol and equol) on milk components production, tight junctions (TJs), and their regulatory pathways are unclear in bovine mammary epithelial cells (BMECs). In this study, we investigated the influences of isoflavones and their metabolites in BMECs using an in vitro culture model. The influences of isoflavones on milk components production, TJ proteins, and STAT5/STAT3 signaling pathways were different in a type-specific manner. Biochanin A decreased the mRNA expression and secretion of both β-casein and lactoferrin while a decrease in activated STAT5 and an increase in activated STAT3. In contrast, equol increased claudin-3, which is the main components for less-permeable TJs in lactation, while an increase in activated STAT5. In addition, a mixture of multiple isoflavones based on the intake of red clover increased secretion of lactoferrin, mRNA expression of β-casein, and amount of claudin-3, but a mixture based on soy did not affect the BMECs. Thus, these results indicate that isoflavones in legumes and the metabolic activity of isoflavones in dairy cows when feeding legumes may affect the milk production ability in BMECs.
• A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells

  Khoi Thieu Ho, Kohei Homma, Jun Takanari, Hanako Bai, Manabu Kawahara, Khang Thi Kim Nguyen, Masashi Takahashi

  Scientific Reports 11 (1) 18175 - 18175 2021/12 [Refereed][Not invited]
   

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

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

  Biochemical and Biophysical Research Communications 584 1 - 6 0006-291X 2021/11 [Refereed]
   

  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.
• The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos

  Weihong Fan, Misato Homma, Renliang Xu, Hiroki Kunii, Hanako Bai, Manabu Kawahara, Toshikazu Kawaguchi, Masashi Takahashi

  Biochemical and Biophysical Research Communications 577 116 - 123 0006-291X 2021/11 [Refereed][Not invited]
   

  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.
• Mitochondrial maturation in the trophectoderm and inner cell mass regions of bovine blastocysts

  Yoshihiro Hayashi, Shun Saito, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Theriogenology 175 69 - 76 0093-691X 2021/11 [Refereed][Not invited]
   

  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.
• Deciphering two rounds of cell lineage segregations during bovine preimplantation development

  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

  The FASEB Journal 35 (10) e21904  0892-6638 2021/10 [Refereed][Not invited]
   

  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.
• Loop-mediated isothermal amplification (LAMP) and machine learning application for early pregnancy detection using bovine vaginal mucosal membrane

  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 0006-291X 2021/09 [Refereed][Not invited]
  
• Heat stress induces oxidative stress and activates the KEAP1-NFE2L2-ARE pathway in bovine endometrial epithelial cells

  Hirona Murata, Hiroki Kunii, Kazuya Kusama, Toshihiro Sakurai, Hanako Bai, Manabu Kawahara, Masashi Takahashi

  Biology of Reproduction 105 (5) 1114 - 1125 0006-3363 2021/07/22 [Refereed]
   

  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.
• Requirement for expression of WW domain containing transcription regulator 1 in bovine trophectoderm development

  Shun Saito, Shota Yamamura, Nanami Kohri, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Biochemical and Biophysical Research Communications 555 140 - 146 0006-291X 2021/05 [Refereed]
   

  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.
• Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells

  Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Atsushi Watanabe, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi

  Cell and Tissue Research 384 (2) 435 - 448 0302-766X 2021/01/12 [Refereed]
   

  Mastitis causes a decrease in milk yield and abnormalities in milk components from dairy cows. Escherichia coli and the E. coli lipopolysaccharide (LPS) cell wall component directly downregulate milk production in bovine mammary epithelial cells (BMECs). However, the detailed mechanism by which this occurs in BMECs remains unclear. Various membrane proteins, such as immune sensors (Toll-like receptors, TLR), nutrient transporters (glucose transporter and aquaporin), and tight junction proteins (claudin and occludin) are involved in the onset of mastitis or milk production in BMECs. In this study, we investigated the influence of LPS on membrane proteins using an in vitro culture model. This mastitis model demonstrated a loss of glucose transporter-1 and aquaporin-3 at lateral membranes and a decrease in milk production in response to LPS treatment. LPS disrupted the tight junction barrier and caused compositional changes in localization of claudin-3 and claudin-4, although tight junctions were maintained to separate the apical membrane domains and the basolateral membrane domains. LPS did not significantly affect the expression level and subcellular localization of epidermal growth factor receptor in lactating BMECs with no detectable changes in MEK1/2-ERK1/2 signaling. In contrast, NFκB was concurrently activated with temporal translocation of TLR-4 in the apical membranes, whereas TLR-2 was not significantly influenced by LPS treatment. These findings indicate the importance of investigating the subcellular localization of membrane proteins to understand the molecular mechanism of LPS in milk production in mastitis.
• Oxidative stress response pathway in bovine endometrial epithelial cells under heat stress culture

  BAI Hanako, KUNII Hiroki, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement 公益社団法人 日本繁殖生物学会 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。
• A Simple and Rapid Bovine Pregnancy Detection Model by RT-LAMP and Machine Learning in the vaginal mucosal membrane

  KUNII Hiroki, KUBO Tomoaki, ASAOKA Natsuki, SHIMASAKI Tomoya, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement 公益社団法人 日本繁殖生物学会 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。
• Lipopolysaccharide and lipoteichoic acid influence milk production ability via different early responses in bovine mammary epithelial cells

  Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi

  Experimental Cell Research 400 (2) 112472 - 112472 0014-4827 2021/01 [Refereed]
   

  Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are cell wall components of Escherichia coli and Staphylococcus aureus, which cause clinical and subclinical mastitis, respectively. However, the reason of the difference in symptoms by pathogen type remains unclear. In this study, the influence of LPS and LTA on early response and milk production in lactating bovine mammary epithelial cells (BMECs) was comparatively investigated. The results showed that LPS decreased the secretion of β-casein, lactose, and triglycerides, whereas LTA decreased the secretion of lactose and triglycerides but increased lactoferrin production without any influence on β-casein secretion. In addition, the influence of milk lipid droplet size in BMECs and gene expression related to milk fat synthesis was different between LPS and LTA. LPS increased the gene expression of interleukin (IL)-1β, tumor necrosis factor-α, and IL-8 through the activation of the nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase pathways, whereas LTA increased IL-1β and CC chemokine ligand 5 expression through the activation of the NF-κB pathway. Moreover, these cytokines and chemokines differently affected the milk production ability of BMECs. These results suggested that the pathogen-specific symptoms may be related to the differences in the early response of BMECs to bacterial toxins.
• Yes-associated protein 1 translocation through actin cytoskeleton organization in trophectoderm cells

  Shota Yamamura, Nanami Goda, Hiroki Akizawa, Nanami Kohri, Ahmed Z. Balboula, Ken Kobayashi, HanakoBai, Masashi Takahashi, Manabu Kawahara

  Developmental Biology 468 (1-2) 14 - 25 0012-1606 2020/09 [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.
• The role of RHOA signaling in trophectoderm cell-fate decision in cattle.

  Nanami Kohri, Hiroki Akizawa, Sakie Iisaka, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Biochemical and biophysical research communications 528 (4) 713 - 718 0006-291X 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.
• Short communication: Menaquinone-4 (vitamin K2) induces proliferation responses in bovine peripheral blood mononuclear cells

  H. Bai, H. Hiura, Y. Obara, M. Kawahara, M. Takahashi

  Journal of Dairy Science 103 (8) 7531 - 7534 0022-0302 2020/08 [Refereed][Not invited]
  
• Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes

  Ahmed Z Balboula, Karen Schindler, Tomoya Kotani, Manabu Kawahara, Masashi Takahashi

  Molecular Human Reproduction 26 689 - 701 1360-9947 2020/07/07 [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 (COCs) 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 spindle assembly checkpoint 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 towards improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.
• Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

  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.
• Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos.

  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 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.
• Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes.

  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.
• Effect of a standardized extract of Asparagus officinalis stem on non-heat shock induction of HSP70 in relation with antioxidant system in bovine cumulus cells

  HO Thieu Khoi, HOMMA Kohei, TAKANARI Jun, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement 日本繁殖生物学会 113 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.

• Examination of gene expression in bovine endometrial tissues collected from summer and winter seasons

  BAI Hanako, MITANI Tomohiro, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement 日本繁殖生物学会 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。

• Detection of Pregnant Down-regulated Gene in Bovine Cervical Mucosa

  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 日本繁殖生物学会 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。

• Direct involvement of IFNT in the stimulation of ISGs in pregnant bovine cervical tissue

  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 日本繁殖生物学会 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。

• Effect of summer heat stress on gene expression in bovine uterine endometrial tissues

  Hanako Bai, Haruka Ukita, Manabu Kawahara, Tomohiro Mitani, Eri Furukawa, Yojiro Yanagawa, Naoto Yabuuchi, Heejin Kim, Masashi Takahashi

  Animal Science Journal 91 (1) e13474  1344-3941 2020/01 [Refereed]
   

  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.
• Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells

  Yusaku Tsugami, Norihiro Suzuki, Manabu Kawahara, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi

  Animal Science Journal 91 (1) :e13355  1344-3941 2020/01 [Refereed][Not invited]
  
• Type-I interferon regulates matrix metalloproteinases clearance of the bovine endometrial spheroid.

  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.
• Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst.

  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 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.
• Effects of use of conventional and sexed semen on the conception rate in heifers: A comparison study

  Kohei Oikawa, Takeshi Yamazaki, Satoshi Yamaguchi, Hayato Abe, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Theriogenology 135 33 - 37 0093-691X 2019/09 [Refereed][Not invited]
  
• 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
• Overgrowth of mice generated from postovulatory-aged oocyte spindles.

  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.
• Significance of CCN2 expression in bovine preimplantation development.

  Akizawa H, Yanagawa Y, Nagano M, Bai H, Takahashi M, Kawahara M

  Animal Science Journal 90 (1) 49 - 54 2019 [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.
• Expression pattern of cathepsin B gene and protein in ICM and TE of bovine blastocyst

  LI Jianye, AHMED Balboula Zaky, FUJII Takashi, MORIYASU Satoru, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement 日本繁殖生物学会 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.

• Evaluation of the immune status of peripheral blood monocytes from dairy cows during the periparturition period.

  Bai H, Talukder M.A.S, Kunii H, Itoh T, Kawahara M, Takahashi M

  Journal of Reproduction and Development 65 (4) 313 - 318 0916-8818 2019 [Refereed][Not invited]
  
• Involvement of interferon-tau in the induction of apoptotic, pyroptotic, and autophagic cell death-related signaling pathways in the bovine uterine endometrium during early pregnancy.

  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 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.
• Analysis of differentially expressed genes and the promoters in bovine endometrium throughout estrus cycle and early pregnancy.

  Sayed A A Musavi, Seiya Yamashita, Taisuke Fujihara, Hironori Masaka, Md Rashedul Islam, Sangwan Kim, Takafumi Gotoh, Manabu Kawahara, Kosuke Tashiro, Nobuhiko Yamauchi

  Animal science journal = Nihon chikusan Gakkaiho 89 (11) 1609 - 1621 2018/11 [Refereed][Not invited]
   

  Endometrial gene expression is primarily regulated by the ovarian steroids and pregnancy recognition factors. This study was aimed to characterize differential expression genes (DEGs) in bovine endometrium together with the analysis of their promoter region. Bovine uteri at follicular stage (FS), luteal stage (LS), and implantation stage (IS) at Day 18 of pregnancy were collected. Total RNA extracted and prepared cDNA were then subjected to high-throughput sequencing. For promoter analysis, 1 kb upstream promoter region of each DEG was analyzed. The numbers of highly expressed DEGs were 496 and 597 at FS and LS, respectively. When compared the gene expression of IS with LS, 383 and 346 DEGs showed higher and lower expression at IS, respectively. It was also observed that 20-30 transcription factors (TFs) were included in each DEGs. In addition, promoter analyses estimated 150-160 TFs for each stage. DLX4 and interferon regulatory factor 4 (IRF4) at FS, and IRF5, IRF9, STAT1, and STAT2 at IS were in common to DEGs and estimated TFs, respectively. This study highlighted potential molecular mechanisms controlling endometrial function during estrus cycle and IS, which will further guide to better understand the endometrial functions in future studies.
• Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes.

  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.
• Time-Lapse Mechanical Characterization of Zona Pellucida Using a Cell Carrier Chip

  Kou Nakahara, Shinya Sakuma, Manabu Kawahara, Masashi Takahashi, Fumihito Arai

  Journal of Microelectromechanical Systems 27 (3) 464 - 471 1057-7157 2018/06/01 [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]
• Reciprocal regulation of TEAD4 and CCN2 for the trophectoderm development of the bovine blastocyst

  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.
• 妊娠初期の牛白血球リソソーム機能に対するIFNτの作用(Effect of IFN τ on lysosomal functions in bovine leukocytes during early pregnancy)

  Talukder Mas, 鈴木 惇文, 白水 貴大, Balboula A.Z., 唄 花子, 川原 学, 高橋 昌志

  日本畜産学会大会講演要旨集 124回 208 - 208 1342-4688 2018/03 [Refereed][Not invited]
  
• Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses.

  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 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.
• Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares

  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.
• Enhancement of sperm motility and viability by turmeric by-product dietary supplementation in roosters

  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
• ウマIGF2遺伝子の全長配列決定と胎子期発現動態

  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 北海道畜産草地学会 5 (2) 21 - 21 2187-5391 2017/08
• Estrous cycle stage-dependent manner of type I interferon-stimulated genes induction in the bovine endometrium.

  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 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.
• Pyridoxine supplementation during oocyte maturation improves the development and quality of bovine preimplantation embryos

  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.
• 発情期及び妊娠認識時のウシ子宮組織におけるオートファジー カテプシン関連因子の発現動態

  鈴木 惇文, 白水 貴大, 岩野 弘暉, 小木曽 貴季, 山内 伸彦, 柳川 洋二郎, 永野 昌志, 唄 花子, 川原 学, 高橋 昌志

  The Journal of Reproduction and Development (公社)日本繁殖生物学会 62 (Suppl.) j64 - j64 0916-8818 2016/09 [Refereed][Not invited]
  
• The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice

  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.
• Conserved Roles of Fibroblast Growth Factor Receptor 2 Signaling in the Regulation of Inner Cell Mass Development in Bovine Blastocysts

  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.
• 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
• Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development

  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.
• Characterization of Toll-Like Receptor 9 expression during Mouse Preimplantation Development

  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]
  
• Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy.

  Takahiro Shirozu, Keisuke Sasaki, Manabu Kawahara, Yojiro Yanagawa, Masashi Nagano, Nobuhiko Yamauchi, Masashi Takahashi

  The Journal of reproduction and development 62 (1) 29 - 35 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.
• Restricted development of mouse triploid fetuses with disorganized expression of imprinted genes

  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.
• Comparing spatial expression dynamics of bovine blastocyst under three different procedures: in-vivo, in-vitro derived, and somatic cell nuclear transfer embryos

  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.
• Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

  Manabu Kawahara, Shiori Koyama, Satomi Iimura, Wataru Yamazaki, Aiko Tanaka, Nanami Kohri, Keisuke Sasaki, Masashi Takahashi

  SCIENTIFIC REPORTS 5 doi: 10.1038/srep14512.  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.
• Status of autophagy, lysosome activity and apoptosis during corpus luteum regression in cattle

  Mansour Aboelenain, Manabu Kawahara, Ahmed Zaky Balboula, Abd El-monem Montasser, Samy Mowaed Zaabel, Kiyoshi Okuda, Masashi Takahashi

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 61 (3) 229 - 236 0916-8818 2015/06 [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 (LC3 alpha, LC beta, 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.
• Characteristics of MX gene that shows resistance to vesicular stomatitis virus from avians to mammals

  Keisuke Sasaki, Manabu Kawahara, Tomomasa Watanabe

  Advances in Genetics Research 11 117 - 130 2014/01/01 

  The antiviral MX gene is an interferon-induced GTPase, which is involved in inhibiting the multiplication of RNA viruses, such as vesicular stomatitis virus (VSV). Due to the severe threats of infectious spread to large numbers of livestock, animal breeding focused on innate immune-associated genes such as MX may play an important role in reducing the incidence of infection. In chicken, Mx gene was thought to lack antiviral activity. However, a specific single nucleotide polymorphism (SNP) in chicken Mx conferred antiviral activity against VSV and influenza virus. This SNP corresponded to amino acid position 631 (631 aa), and resulting in a serine to asparagine change at 631 aa. Therefore, this indicates that it is possible to breed infectious disease-resistant chickens based on the above-mentioned SNP, which could help to prevent the spread of infections. Furthermore, the SNP corresponding to 631 aa was shown to be involved in not only chicken Mx antiviral activity, but also in its intracellular localization. The SNP coincided with the cytoplasmic granular-like protein distribution, suggesting that the intracellular behavior of chicken Mx was likely responsible for its antiviral potential. The intracellular localization and protein behavior of human MxA and mouse MX1 have also indicated a role in viral inhibition. Some MX proteins show granular-like localization, similar to chicken Mx. Therefore, this pattern of chicken Mx localization may also contribute to the antiviral ability against VSV in other livestock animals from avians to mammals. This review focuses on the variety of MX genes among livestock animals and their function in resistance to infectious diseases, especially to VSV
• A single nucleotide polymorphism of porcine MX2 gene provides antiviral activity against vesicular stomatitis virus

  Keisuke Sasaki, Pullop Tungtrakoolsub, Takeya Morozumi, Hirohide Uenishi, Manabu Kawahara, Tomomasa Watanabe

  IMMUNOGENETICS 66 (1) 25 - 32 0093-7711 2014/01 [Refereed][Not invited]
   

  The objective was to determine if single nucleotide polymorphisms (SNPs) in porcine MX2 gene affect its antiviral potential. MX proteins are known to suppress the multiplication of several viruses, including influenza virus and vesicular stomatitis virus (VSV). In domestic animals possessing highly polymorphic genome, our previous research indicated that a specific SNP in chicken Mx gene was responsible for its antiviral function. However, there still has been no information about SNPs in porcine MX2 gene. In this study, we first conducted polymorphism analysis in 17 pigs of MX2 gene derived from seven breeds. Consequently, a total of 30 SNPs, of which 11 were deduced to cause amino acid variations, were detected, suggesting that the porcine MX2 is very polymorphic. Next, we classified MX2 into eight alleles (A1-A8) and subsequently carried out infectious experiments with recombinant VSV Delta G*-G to each allele. In A1-A5 and A8, position 514 amino acid (514 aa) of MX2 was glycine (Gly), which did not inhibit VSV multiplication, whereas in A6 and A7, 514 aa was arginine (Arg), which exhibited the antiviral ability against VSV. These results demonstrate that a SNP at 514 aa (Gly-Arg) of porcine MX2 plays a pivotal role in the antiviral activity as well as that at 631 aa of chicken Mx.
• Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus-oocyte complexes exposed to heat shock during in vitro maturation

  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.
• Transcriptional Wiring for Establishing Cell Lineage Specification at the Blastocyst Stage in Cattle

  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.
• Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle

  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.
• Both antiviral activity and intracellular localization of chicken Mx protein depend on a polymorphism at amino acid position 631

  Keisuke Sasaki, Akihiro Yoneda, Akinori Ninomiya, Manabu Kawahara, Tomomasa Watanabe

  Biochemical and Biophysical Research Communications 430 (1) 161 - 166 0006-291X 2013/01/04 [Refereed][Not invited]
   

  The Mx protein is known to inhibit the multiplication of several RNA viruses. In chickens, a polymorphism at amino acid position 631 (631 aa) of Mx protein has been suggested to be involved in the antiviral ability against vesicular stomatitis virus (VSV) and influenza virus, indicating that a Ser-to-Asn substitution at 631 aa is the source of this antiviral ability. However, how the substitution at 631 aa contributes to the antiviral activity remains to be clarified. In this study, we investigated differences in antiviral activity against VSV and intracellular localization between Ser and Asn types at 631 aa of the chicken Mx protein. The results showed that chicken Mx protein with an Asn at 631 aa inhibited VSV multiplication and Mx distribution in a granular-like pattern in the cytoplasm. However, Mx carrying the Ser type did not inhibit viral growth and homogenous spread throughout the cytoplasm. Furthermore, we found that replacing Ser with Asn at 631 aa provided Mx with antiviral activity against VSV, with Mx showing granular-like distribution in the cytoplasm. These results demonstrated that a single amino acid polymorphism at 631 aa of the chicken Mx protein altered both the antiviral activity and intracellular localization. © 2012 Elsevier Inc.
• Roles of Genes Regulated by Two Paternally Methylated Imprinted Regions on Chromosomes 7 and 12 in Mouse Ontogeny

  Manabu Kawahara, Tomohiro Kono

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 (2) 175 - 179 0916-8818 2012/04 [Refereed][Invited]
   

  We studied the longevity of mice produced without sperm using the genomes of oocytes that are already committed to a germline cell lineage. The first sperm-free mouse "KAGUYA", which we term 'bi-maternal mouse', was born on 3 February, 2003. Bi-maternal embryos were generated using 2 sets of female genomes-one derived from fully grown oocytes from normal adults and the other from non-growing oocytes from newborn pups. These genomes were combined by nuclear transfer. We refined the technique for generating bi-maternal mice and found that genetic manipulations in only 2 regions-the imprinting centres of Igf2-H19 and Dlk1-Gtl2-on chromosomes 7 and 12 of the newborn pups allowed us to generate bi-maternal mice at a high rate. Studying bi-maternal conceptuses and mice provides further insight into the mechanisms by which paternally methylated imprinted genes regulate mammalian ontogenesis.
• Anomalous Oxygen Consumption in Porcine Somatic Cell Nuclear Transfer Embryos

  Satoshi Sugimura, Masaki Yokoo, Ken-ichi Yamanaka, Manabu Kawahara, Satoru Moriyasu, Takuya Wakai, Takashi Nagai, Hiroyuki Abe, Eimei Sato

  CELLULAR REPROGRAMMING 12 (4) 463 - 474 2152-4971 2010/08 [Refereed][Not invited]
   

  Oxygen consumption reflects overall metabolic activity of mammalian embryos. We measured oxygen consumption in individual porcine somatic cell nuclear transfer (SCNT) and in vitro-fertilized (IVF) embryos by modified scanning electrochemical microscopy. Oxygen consumption in IVF embryos rapidly increased at day 5 of the blastocyst stage (D5BL). IVF embryos that consumed >0.81 x 10(14)/mol sec(-1) of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence.
• Longevity in mice without a father

  Manabu Kawahara, Tomohiro Kono

  Human Reproduction 25 (2) 457 - 461 0268-1161 2010/02 [Refereed][Not invited]
   

  Background: Females live longer than males in many mammalian species, including humans. It has been observed that women are at an advantage over men with regard to the lifespan however, the reason for this sex difference in longevity is unclear. Bi-maternal mice (BM), which are produced in a 'sperm-free' manner, could provide an opportunity to analyse the longevity of animals lacking paternal genomes. Methods: AND Results: We studied the longevity of BM, which were generated using two sets of female genomes-one derived from fully grown oocytes from normal adults and the other from non-growing oocytes from newborn pups. These newborn pups were also genetically manipulated in two regions-the imprinting centres of Igf2-H19 and Dlk1-Gtl2-on chromosomes 7 and 12. We determined lifespan of the control (n = 13) and BM (n = 13). Our Results: revealed that the bi-maternal genotype clearly shifted the entire survival curve to the right, suggesting a delay in the expression of all causes of mortality. BM survived 186 days longer than controls. Furthermore, the body weight was significantly lower in the BM as compared with the controls at 20 months after birth (P < 0.05), and leukocyte composition analysis at 8 weeks revealed that the eosinophil count was significantly increased in the BM as compared with the controls (P < 0.05, n = 6). Conclusions: These findings demonstrate that the maternal genome may play a role in ontogenetic longevity. Our Results: further suggested sex differences in longevity, originating at the genome level, implying that the sperm genome has a detrimental effect on longevity in mammals.
• Involvement of Insulin-like Growth Factor 2 in Angiogenic Factor Transcription in Bi-maternal Mouse Conceptuses

  Manabu Kawahara, Qiong Wu, Tomohiro Kono

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 (1) 79 - 85 0916-8818 2010/02 [Refereed][Not invited]
   

  Imprinted genes in which only one of the two parental chromosome copies is expressed have a substantial effect on mammalian ontogenesis. On mouse distal chromosome 7, the paternally expressed gene insulin-like growth factor 2 (Igf2) is separated by approximate 100 kb from the maternally expressed non-coding gene H19. However, there is limited knowledge of the manner in which Igf2 transcription affects the other genes involved in embryonic development. To clarify this, we performed quantitative gene expression analysis for representative angiogenic factors-Vegf, Flt1, Flt4, Flk1, Ang1, Ang2, Tie1, and Tie2-for 3 types of bi-maternal conceptuses containing genomes with non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the rig oocytes were 1) the wild type (ng(wt)), 2) mutant mice carrying a 3-kb deletion of the H19 transcription unit (ng(H19 Delta 3KO)/fg) and 3) mutant mice carrying a 13-kb deletion in the H19 transcription unit, including the germline-derived differentially methylated region on chromosome 7 (ngH(19 Delta 13-KO)/fg)In the ng(wt)/fg and ng(H19 Delta 3KO)/fg placentae, Vegf and Flt1 were upregulated compared with the mean value for the wt placenta, whereas in the ng(H19 Delta 3KO)/fg placenta, these transcriptional levels were restored. In the fetus, however, only 2 genes among the 8 genes analyzed were significantly changed in the bi-maternal fetuses, indicating that the effects of the Igf2 mRNA level on angiogenic factor transcription in the fetus differed from those in the placenta. Our results indicated that the Igf2 mRNA level affects transcription of angiogenic factors in both bi-maternal placentae and fetuses.
• Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

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

  ANIMAL SCIENCE JOURNAL 81 (1) 48 - 57 1344-3941 2010 [Refereed][Not invited]
   

  We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.
• Acetylation Level of Histone H3 in Early Embryonic Stages Affects Subsequent Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

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

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 (6) 638 - 644 0916-8818 2009/12 [Refereed][Not invited]
   

  Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into art embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of historic on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 +/- 2.7 and 56.6 +/- 2.7 vs. 43.5 +/- 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitro-fertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs.
• Defining Contributions of Paternally Methylated Imprinted Genes at the Igf2-H19 and Dlk1-Gtl2 Domains to Mouse Placentation by Transcriptomic Analysis

  Manabu Kawahara, Shinnosuke Morita, Nozomi Takahashi, Tomohiro Kono

  JOURNAL OF BIOLOGICAL CHEMISTRY 284 (26) 17751 - 17765 0021-9258 2009/06 [Refereed][Not invited]
   

  Parental genome functions in ontogeny are determined by interactions among transcripts from the maternal and paternal genomes, which contain many genes whose expression is strictly dependent on their parental origin as a result of genomic imprinting. Comprehensive recognition of the interactions between parental genomes is important for understanding genomic imprinting in mammalian development. The placenta is a key organ for exploring the biological significance of genomic imprinting. To decipher the unknown roles of paternally methylated imprinted genes on chromosomes 7 and 12 in mouse placentation, we performed a transcriptomic analysis on placentae in three types of bimaternal conceptuses that contained genomes derived from both non-growing and fully grown oocytes. Furthermore, we used the Ingenuity pathway analysis software to predict key networks and identify functions specific to paternally methylated imprinted genes regulated by the Igf2-H19 imprinting control region and Dlk1-Dio3 imprinting control region. The data suggested that dynamic conversion of the gene expression profile by restoring the expression of paternally methylated imprinted genes resulted in phenotypic improvements in bimaternal placentae. These results provide a framework to further explore the role of epigenetic modifications in paternal genome during mouse placentation.
• Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs

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

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 55 (3) 299 - 304 0916-8818 2009/06 [Refereed][Not invited]
   

  We evaluated the developmental competence of somatic cell nuclear transfer (SCNT) embryos using in vitro embryo culture systems. Embryos were cultured in NCSU-23, NCSU-23 supplemented with essential and non-essential amino acids (NCSU-23aa) or modified PZM-5 supplemented with BSA instead of PVA (mPZM-5). The rates of blastocyst formation were significantly higher in the mPZM-5 group than in the other groups, regardless of the method of embryo production (38.0 vs. 25.3 or 29.1% for IVF, 18.2 vs. 8.7 or 9.40% for SCNT, respectively). The mean cell numbers of IVF and SCNT blastocysts were also significantly higher in mPZM-5 than in the other groups (62.0 vs. 42.3 or 43.0 for IVF, 46.5 vs. 29.4 or 31.3 for SCNT, respectively). Next, the embryos were cultured in mPZM-5 from days 0 to 4 and then in mPZM-5 (P/P), NCSU-23 (P/N) or NCSU-23aa (P/Naa) until day 6. The rates of blastocyst formation were similar among the 3 two-step culture systems in both embryo groups (36.2, 34.2, and 33.6% for IVF, 20.8, 14.1, and 17.2% for SCNT, respectively). The mean cell number in the IVF and SCNT blastocysts was significantly lower in P/N than in P/P and P/Naa (46.5 vs. 63.5 and 68.7 for IVF, 29.3 vs. 45.5 and 39.7 for SCNT, respectively). Next, we examined the effect of media on apoptosis in IVF and SCNT blastocysts. The apoptosis indices in the blastocysts derived from either NCSU-23 or mPZM-5 were analyzed by TUNEL assay. The apoptosis index of the SCNT blastocysts was significantly lower in mPZM-5 than in NCSU-23 (8.8 vs. 13.6%), whereas no such difference was observed between groups in the IVF embryos (51 vs. 4.4%). These data suggested that SCNT embryos were more easily affected by culture environment compared with IVF embryos, offering the possibility to further enhance the developmental competence of SCNT embryos by developing more appropriate culture conditions in pigs.
• Developmental Ability of Trophoblast Stem Cells in Uniparental Mouse Embryos

  H. Ogawa, N. Shindo, T. Kumagai, Y. Usami, M. Shikanai, K. Jonwn, A. Fukuda, M. Kawahara, Y. Sotomaru, S. Tanaka, T. Arima, T. Kono

  PLACENTA 30 (5) 448 - 456 0143-4004 2009/05 [Refereed][Not invited]
   

  Neither parthenogenetic (PG) nor androgenetic (AG) mouse embryos survive after day 9.5 of pregnancy, owing to the inadequate growth of extraembryonic tissues, including the placenta. At day 9.5 of pregnancy, the placental structures are poorly developed in PC embryos, while trophoblast giant cells are abundant at the implantation site in AG embryos. These findings suggest that both parental genomes are required for placental development. To gain further insight into the trophoblast lineage in PC and AC embryos, we attempted to derive trophoblast stem (TS)-like cell lines from uniparental embryos. Furthermore, we sought to assess their ability to differentiate into cells of the trophoblast lineage by using gene expression analysis. Three cell lines that expressed marker genes for undifferentiated TS cells (Cdx2 and Err beta) were derived from AG embryos. Under differentiation conditions, these cells expressed the trophoblast giant cell-specific genes, but did not express the spongiotrophoblast-specific genes. In contrast, none of the four cell lines from PC embryos expressed marker genes for undifferentiated TS cells, but they expressed Oct3/4, a marker gene for embryonic stem cells. Immunohistochemical analysis indicated that PC blastocysts expressed Oct3/4 and Cdx2 specifically in inner cell mass and the trophectoderm respectively. These results suggest that PC embryos do not possess TS cells, because of the lack of the developmental ability of trophoblast cells. (C) 2009 Elsevier Ltd. All rights reserved.
• Synergistic role of Igf2 and Dlk1 in fetal liver development and Hematopoiesis in bi-maternal mice

  Qiong Wu, Manabu Kawahara, Tomohiro Kono

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 54 (3) 177 - 182 0916-8818 2008/06 [Refereed][Not invited]
   

  Mouse bi-maternal embryos (BMEs) that contain two haploid sets of genomes from non-growing (ng) and fully-grown (fg) oocytes develop to embryonic day (E) 13.5. However, the ng/fg BMEs never develop beyond E13.5 because of repression of the paternally expressed imprinted genes, Igf2 and Dlk1. The present study was conducted to address the issue of whether fetal hematopoietic disorder is involved in the restricted development of BMEs. FACS analysis revealed that the livers of ng(wt)/fg BMEs contained increased numbers of immature c-kit(+)/ter119(-) hematopoietic cells, were while the numbers of mature c-kit(-)/ter119(+) hematopoietic cells were decreased. This finding was supported by histological observations. Quantitative gene expression analysis revealed that Igf2 and Dlk1 expression was repressed in the liver. To understand the role of paternally-methylated imprinted genes on chromosomes 7 and 12, particularly Igf2 and Dlk1, in fetal liver hematopoiesis, we constructed ng(Delta ch7)/fg, ng(Delta ch12)/fg and ng(Delta Double)/fg BMEs using ng oocytes harboring deletion of differentially methylated regions at distal chromosomes 7 and/or 12. The ng(Delta ch7)/fg, ng(Delta ch12)/fg and ng(Delta Double)/fg BMEs, respectively, express Igf2, Dlk1 and both, and these embryos developed to term with specific phenotypes; the ng(Delta ch7)/fg and ng(Delta ch12)/fg BMEs develop to term with severe growth retardation, and the ng(Delta Double)/fg BMEs can survive to become normal female adults. By inducing Igf2 and Dlk1 expression, the proportions of mature and immature hematopoietic cells in the livers of the ng(Delta ch7)/fg, ng(Delta ch12)/fg and ng(Delta Double)/fg BMEs were considerably restored, and particularly in the ng(Delta Double)/fg BMEs, hematopoiesis occurred normally with appropriate expressions of the related genes. These data suggest that inappropriate expression of Igf2 and Dlk1 is involved in impaired fetal hematopoiesis.
• Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries

  Takuya Wakai, Satoshi Sugimura, Ken-Ichi Yamanaka, Manabu Kawahara, Hiroshi Sasada, Hozumi Tanaka, Asako Ando, Eiji Kobayashi, Eimei Sato

  CLONING AND STEM CELLS 10 (2) 249 - 261 1536-2302 2008/06 [Refereed][Not invited]
   

  For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.
• Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

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

  ZYGOTE 16 (2) 153 - 159 0967-1994 2008/05 [Refereed][Not invited]
   

  In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G(0)/G(1) phase derived from miniature pigs. CB (7.5 mu g/ml) and CD (2.5 mu g/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 mu g/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 mu g/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 mu g/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 mu g/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 mu g/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.
• Identification of genes aberrantly expressed in mouse embryonic stem cell-cloned blastocysts

  Yuko Jincho, Yusuke Sotomaru, Manabu Kawahara, Yukiko Ono, Hidehiko Ogawa, Yayoi Obata, Tomohiro Kono

  BIOLOGY OF REPRODUCTION 78 (4) 568 - 576 0006-3363 2008/04 [Refereed][Not invited]
   

  During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psrnc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.
• Protocol for the production of viable bimaternal mouse embryos

  Manabu Kawahara, Yayoi Obata, Yusuke Sotomaru, Nobuhiro Shimozawa, Siqin Bao, Toshitaka Tsukadaira, Atsushi Fukuda, Tomohiro Kono

  NATURE PROTOCOLS 3 (2) 197 - 209 1754-2189 2008 [Refereed][Not invited]
   

  A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.
• Induction of estrus in pubertal miniature gilts

  Takuya Wakai, Hozumi Tanaka, Ken-ichi Yamanaka, Satoshi Sugimura, Hiroshi Sasada, Manabu Kawahara, Eiji Kobayashi, Eimei Sato

  ANIMAL REPRODUCTION SCIENCE 103 (1-2) 193 - 198 0378-4320 2008/01 [Refereed][Not invited]
   

  Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs. (C) 2007 Elsevier B.V. All rights reserved.
• Appropriate expression of imprinted genes on mouse chromosome 12 extends development of bi-maternal embryos to term

  Manabu Kawahara, Qiong Wu, Anne C. Ferguson-Smith, Tomohiro Kono

  FEBS LETTERS 581 (27) 5178 - 5184 0014-5793 2007/11 [Refereed][Not invited]
   

  Recently, we reported that the restored regulation of imprinted gene expression from two regions - H19 differentially methylated region (H19-DMR) and intergenic germline-derived DMR (IG-DMR) - is sufficient for accomplishing full-term development in mice. In the present study, we determined the developmental ability of the bi-maternal embryos (BMEs) containing the non-growing oocyte genome with the IG-DMR deletion (ng(Delta ch12)) and fully-grown (fg) oocyte genome. Foetuses derived from ng(Delta ch12)/fg BMEs were alive at E19.5 but could not survive further. Comparison with BMEs derived from Igf2+/- ng/fg genomes suggests that bi-allelic H19 expression might be involved in foetal development. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
• High-frequency generation of viable mice from engineered bi-maternal embryos

  Manabu Kawahara, Qiong Wu, Nozomi Takahashi, Shinnosuke Morita, Kaori Yamada, Mitsuteru Ito, Anne C. Ferguson-Smith, Tomohiro Kono

  NATURE BIOTECHNOLOGY 25 (9) 1045 - 1050 1087-0156 2007/09 [Refereed][Not invited]
   

  Mammalian development to adulthood typically requires both maternal and paternal genomes, because genomic imprinting places stringent limitations on mammalian development, strictly precluding parthenogenesis(1,2). Here we report the generation of bi-maternal embryos that develop at a high success rate equivalent to the rate obtained with in vitro fertilization of normal embryos. These bi-maternal mice developed into viable and fertile female adults. The bi-maternal embryos, distinct from parthenogenetic or gynogenetic conceptuses, were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. The results provide conclusive evidence that imprinted genes regulated by these two paternally methylated imprinting-control regions are the only paternal barrier that prevents the normal development of bi-maternal mouse fetuses to term.
• Complementary roles of genes regulated by two paternally methylated imprinted regions on chromosomes 7 and 12 in mouse placentation

  Manabu Kawahara, Qiong Wu, Yukio Yaguchi, Anne C. Ferguson-Smith, Tomohiro Kono

  HUMAN MOLECULAR GENETICS 15 (19) 2869 - 2879 0964-6906 2006/10 [Refereed][Not invited]
   

  Imprinted genes have prominent effects on placentation; however, there is limited knowledge about the manner in which the genes controlled by two paternally methylated regions on chromosomes 7 and 12 contribute to placentation. In order to clarify the functions of these genes in mouse placentation, we examined transcription levels of the paternally methylated genes, tissue differentiation and development and the circulatory system in placentae derived from three types of bi-maternal conceptuses that contained genomes of non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were as follows: one was derived from the wild-type (ng(WT)) and another from mutant mice carrying a 13 kb deletion in the H19 transcription unit including the germline-derived differentially methylated region (H19-DMR) on chromosome 7 (ng(Delta ch7)). Another set of oocytes was derived from mutant mice carrying a 4.15 kb deletion in the intergenic germline-derived DMR (IG-DMR) on chromosome 12 (ng(Delta ch12)). Although placental mass was lower in the ng(WT)/fg placentae compared with that in the WT placentae, it was recovered in the ng(Delta ch7)/fg placentae, but not in the ng(Delta ch12)/fg placentae. The ng(Delta ch7)/fg placental growth improvement was associated with severe dysplasia such as an expanded spongiotrophoblast layer and a malformed labyrinthine zone. In contrast, the ng(Delta ch12)/fg placentae retained the layer structures with expanded giant cells, but their total masses were smaller with a normal circulatory system in order. Our findings demonstrate that the genes controlled by the two paternally methylated regions, H19-DMR and IG-DMR, complementarily organize placentation.
• Regulated expression of two sets of paternally imprinted genes is necessary for mouse parthenogenetic development to term

  Q Wu, T Kumagai, M Kawahara, H Ogawa, H Hiura, Y Obata, R Takano, T Kono

  REPRODUCTION 131 (3) 481 - 488 1470-1626 2006/03 [Refereed][Not invited]
   

  Mouse parthenogenetic embryos (PEs) are developmentally arrested until embryo day (E) 9.5 because of genomic imprinting. However, we have shown that embryos containing genomes from non-growing (ng) and fully grown (fg) oocytes, i.e. ng(wt)/fg(wt) PE (wt, wild type), developed to E13.5. Moreover, parthenogenetic development could be extended to term by further regulation of lgf2 and H19 expression using mice with deletion of the H19 transcription unit (H19 Delta 13) together with its differentially unit (DMR). To gain an insight into the extended development of the parthenotes to term, we have here investigated the expression levels of paternally imprinted genes in ngH(19 Delta 13)/fg(wt) PE throughout their development. In ngH(19 Delta 13)/fg(wt) Pes that died soon after recovery, the expression of Igf2 and H19 was restored to the appropriate levels except for low Igf2 expression in the liver after E15.5. Further, the paternally expressed Dlk1 and Dio3 were repressed, while the expression levels of the maternal Gtl2 and Mirg were twice those of the controls. However, the above-mentioned four genes showed almost normal expression in the surviving ngH(19 Delta 13)/fg(wt) PEs. The methylation analysis revealed that the intragenic DMR of the Dlk1-Gtl2 domain was hypermethylated in the ng(H19 Delta 13)/fg(wt) PEs that survived, but not in the PEs that died soon after recovery. The present study suggests that two sets of co-ordinately regulated but oppositely expressed genes, Igf2-H19 and Dlk1-Gtl2, act as a critical barrier to parthenogenetic development in order to render a paternal contribution obligatory for descendants in mammals.
• Paternal dual barrier by lfg2-H19 and Dlk1-Gtl2 to parthenogenesis in mice

  T. Kono, M. Kawahara, Q. Wu, H. Hiura, Y. Obata

  STEM CELLS IN REPRODUCTION AND IN THE BRAIN 60 23 - + 0947-6075 2006 [Refereed][Not invited]
   

  The functional difference between the maternal and paternal genome, which is characterized by epigenetic modifications during gametogenesis, that is genomic imprinting, prevents mammalian embryos from parthenogenesis. Genomic imprinting leads to nonequivalent expression of imprinted genes from the maternal and paternal alleles. However, our research showed that alteration of maternal imprinting by oocyte reconstruction using nongrowing oocytes together with deletion of the H 19 gene, provides appropriate expression of maternally imprinted genes. Here we discuss that further alteration of paternally imprinted gene expressions at chromosomes 7 and 12 allows the ng/fg parthenogenetic embryos to develop to term, suggesting that the paternal contribution is obligatory for the descendant.
• Paternal dual barrier by Ifg2-H19 and Dlk1-Gtl2 to parthenogenesis in mice（マウス単為発生に対するIgf2-H19およびGtl2-Dlk1による父方の二重障壁）

  Kono T, Kawahara M, Wu Q, Hiura H, Obata Y

  Ernst Schering Research Foundation Workshop 60 23 - 33 0947-6075 2006 [Not refereed][Invited]
   

  精子形成過程でメチル化インプリントを受ける遺伝子群がマウス胚の発生に及ぼす影響について報告した。マウスでは単為発生は、妊娠10日前に致死となるが、卵子形成過程でメチル化インプリントを受ける遺伝子の発現を調節すると、その二母性胚は妊娠14日まで発生延長する。加えて、精子形成過程でメチル化インプリントを受けるH19-Igf2領域とGtl2-Dlk1領域をそれぞれ制御すると、これらの二母性胚は妊娠満期まで発生することが示された。これらの結果から、精子形成過程でメチル化インプリントを受ける遺伝子群は、単為発生胚が妊娠後期から妊娠満期へ発生するのを阻止していることが示唆された
• Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer

  M Kawahara, T Wakai, KI Yamanaka, J Kobayashi, S Sugimura, T Shimizu, H Matsumoto, JH Kim, H Sasada, E Sato

  REPRODUCTION 130 (3) 351 - 357 1470-1626 2005/09 [Refereed][Not invited]
   

  When the nucleus in G0/G1 phase is transferred to an enucleated oocyte by nuclear transfer (NT), its nuclear envelope is broken, followed by condensation of chromosome structure by maturation promoting factor (MPF). This morphological remodeling of the transferred interphase nucleus seems to be essential for subsequent development of NT embryos. in this study, we treated porcine NT embryos with caffeine, which has been reported to increase MPF activity, to keep their MPF level high during NIT. When 2.5 mM caffeine was added to the handling medium, the proportion of NT embryos showing condensed chromosome increased significantly (P < 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P < 0.05) higher than in those without caffeine at 3 h post-injection. in addition, the rate of development to the blastocyst stage after activation was significantly (P < 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.
• Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

  T Shinozawa, E Mizutani, Tomioka, I, M Kawahara, H Sasada, H Matsumoto, E Sato

  MOLECULAR REPRODUCTION AND DEVELOPMENT 68 (3) 313 - 318 1040-452X 2004/07 [Refereed][Not invited]
   

  In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage, Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest. (C) 2004 Wiley-Liss, Inc.
• The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes

  M Kawahara, T Mori, H Tanaka, H Shimizu

  THERIOGENOLOGY 58 (6) 1081 - 1095 0093-691X 2002/10 [Refereed][Not invited]
   

  A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated occytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin Filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 It after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation. (c) 2002 Elsevier Science Inc. All rights reserved.

MISC

• いまさら聞けない、インターフェロン・タウと妊娠認識の話

  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
• ウシ卵子成熟および発生における熱ショック非依存的なHSP70発現誘導と酸化ストレスに及ぼすEASの影響

  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
• 胎盤とウイルス 胎盤とウイルスの意外な関係

  唄花子, 川原学, 高橋昌志  化学と生物  60-  (6)  2022
• 牛の繁殖機能とオートファジー

  唄 花子, 川原 学, 高橋 昌志  68-  (12)  695  -702  2021/12
• ホルスタイン種初産牛の人工授精受胎率に及ぼす環境要因の解析

  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
• 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  7-  (2)  2019
• 道内未経産牛における性選別精液の初回授精受胎率は7-8月に低下しやすい

  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
• Effect of oxidative stress on bovine uterine epithelial cells under heat stress

  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>
• Evaluation of cathepsin B in the trophectoderm of vitrified bovine blastocysts

  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>
• Survey of active site of interferon-tau by treating chemically synthetic peptides in endometrium cells

  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.

• Pregnancy-specific expression of IFIT1 in the bovine external uterine mucosa

  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]
   

  【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ISG15, MX1, MX2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（IFIT1）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IFIT1発現を解析した。【結果】AI後18日目に採取したCMにおいては，IFIT1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IFIT1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，IFIT1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IFIT1発現には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。

• 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]
  
• ウシ胚盤胞期胚におけるTEAD4およびCCN2の発現制御

  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]
  
• Maternal recognition and establishment of pregnancy in ruminants

  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。
• Influences of environmental factors on cow conception rate after artificial insemination using sex-sorted semen

  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。

• Effect of heat stress on IFNT responsiveness in bovine uterine epithelial cells

  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。

• Effects of the Hippo pathway effector TAZ gene suppression on bovine preimplantation development

  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。

• Analyses of stress- and immune-related factors in peripheral blood mononuclear cells in dairy cows before and after calving

  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。

• 胚由来IFNTによるウシ子宮外組織での新規応答性検出

  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]
  
• ウシ初期胚発生における結合組織成長因子CTGF遺伝子発現の役割

  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]
  
• ウマIGF2遺伝子の全長配列決定と胎子期発現動態

  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)  2017
• 発情期及び妊娠認識時のウシ子宮組織におけるオートファジー カテプシン関連因子の発現動態

  鈴木 惇文, 白水 貴大, 岩野 弘暉, 小木曽 貴季, 山内 伸彦, 柳川 洋二郎, 永野 昌志, 唄 花子, 川原 学, 高橋 昌志  The Journal of Reproduction and Development  62-  (Suppl.)  j64  -j64  2016/09  [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。
• MII期核置換による再構築卵母細胞由来マウスの出生後の生存性

  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]
  
• Effect of subunit-specific knockdown of IFNAR on expression of IFNτ signal-related genes

  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。

• Influence of polyploid state on genomic imprinting in mice

  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。

• ウシおよびマウスのミトコンドリアヘテロプラスミーが初期胚発生に及ぼす影響

  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]
  
• CIITA遺伝子発現の抑制がウシ初期胚発生に与える影響

  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]
  
• 発情周期におけるウシ生殖器のアクポリン遺伝子群現動態

  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]
  
• 発情周期別ウシ子宮内膜におけるTRPチャネルの発現動態

  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]
  
• 異種間ミトコンドリア混在が個体発生に及ぼす影響

  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]
  
• 発情周期に伴うウマ子宮組織内の粘液分泌・血管新生因子およびホルモン受容体遺伝子発現の変動

  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]
  
• ウシ妊娠子宮におけるMX1遺伝子のスプライシング変異体の発現解析

  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]
  
• マウス初期胚発生過程におけるパターン認識受容体TLR9の発現動態

  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。
• マウス胎子成長に及ぼす倍数性およびインプリント遺伝子発現の影響

  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。
• ウシ子宮内膜組織におけるMX1とMX1B発現に及ぼすI型インターフェロンの応答性およびその受容体の発情周期別比較

  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  J Reprod Dev  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。
• Transcriptional analyses for establishing cell lineage specification at the blastocyst stage in cattle

  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]
  
• Vitrification of mouse blastocysts by hollow fiber device

  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]
  
• ウシ初期胚発生におけるCTGFおよびGATA2遺伝子の発現動態

  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]
  
• 中空糸によるマウス胚盤胞期胚のガラス化凍結保存

  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]
  
• 赤外線サーモグラフィーによる黒毛和種雌牛発情時の体温変動の検出

  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。
• マウス初期胚におけるRNAウイルスレセプターRIG‐1を介した抗ウイルス応答

  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。
• ウシ初期胚発生の進行に伴うCTGFおよびTEAD4遺伝子の発現動態

  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。
• 二母性マウス胚を用いた新規父性メチル化インプリント遺伝子の検証

  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。
• ブタMx2遺伝子の品種間多型における1アミノ酸置換がもたらす抗ウイルス活性の変化

  佐々木恵亮, TUNGTRAKOOLSUB Pullop, 両角岳哉, 上西博英, 川原学, 渡辺智正  日本畜産学会大会講演要旨  115回-  162  -162  2012/03  [Not refereed][Not invited]
  
• A study of TEAD4 and YAP expression in bovine blastocyst.

  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。
• Calves production after vitrification of immature and mature bovine oocytes retrieved by ovum pick up in Japanese Black cows

  TAKUMA Tetsuya, EZOE Daisuke, ICHIMARU Hitoshi, AONO Akira, KAWAHARA Manabu, OTOI Takeshige  農林水産技術研究ジャーナル  34-  (4)  40  -43  2011/04/01  [Not refereed][Not invited]
  
• Gene expression analysis of Inner cell mass snd Trophectoderm in bovine blastocysts

  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。
• Longevity in mice without a father

  M. Kawahara, T. Kono  Journal of Urology  184-  (4)  1457  -1458  2010/10  [Not refereed][Not invited]
  
• Comparative transcriptpmic analysis for inner cell mass and trophectoderm cells in the bovine blastocyst

  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。
• Effect of the expression allele of genes regulated by paternal methylation imprint on development of bi-maternal mouse embryos

  TSUKADAIRA Toshitaka, KAWAHARA Manabu, KONO Tomohiro  Journal of mammalian ova research = 日本哺乳動物卵子学会誌  26-  (2)  S84  2009/04/01
• Birth of somatic cell nuclear transfer(SCNT)-cloned miniature piglet following co-transfer parthenogenetic embryos

  Sugimura,S, Yamanaka,K, Wakai,T, Kawahara,M, Tanaka,H, Kobayashi,J, Kobayashi,E, Sato,E  Tohoku J.Agr.Res.  59-  (3,4)  51  -61  2009  [Not refereed][Not invited]
  
• Production of bi-maternal mice

  河野友宏, 尾畑やよい, 川原学  Cell technology.  27-  (8)  812  -819  2008/08  [Not refereed][Not invited]
   

  哺乳類の個体発生には、父母ゲノム（精子および卵子）の寄与が不可欠である。卵子ゲノムの機能を特徴付けているメチル化インプリントを人為的に操作し，母性ゲノムを父性ゲノム化させることにより、卵子ゲノムのみから確実かつ高率に個体発生（二母性マウス）させることに成功した。本稿では，筆者らが構築してきた二母性マウス作出方法ならびに分子生物学的機構についてあわせて解説した。
• ゲノムインプリンティングと二母性マウスの開発

  川原学, 尾畑やよい  科学と生物  46-  (7)  452  -459  2008/07/01  [Not refereed][Not invited]
   

  pp452-459
• ゲノムインプリンティングと二母性マウスの開発

  川原学, 尾畑やよい, 河野友宏  化学と生物  46-  (7)  452  -459  2008/07  [Not refereed][Not invited]
   

  ほ乳類の胚発生には父母のゲノムが必須であるが、インプリント遺伝子の発現を制御することで、母親の遺伝情報のみをもつ二母性マウスを誕生させることができる。ここでは、その方法と原理を概説し、父母のゲノムが胚発生にどのように寄与するのかを詳述した。
• 二母性胚からの高頻度なマウス発生

  川原学  実験医学  26-  (3)  417  -421  2008/02/01  [Not refereed][Not invited]
  
• Follicular microvasculature in the porcine and bovine ovary visualized by SEM of vascular corrosion casts

  Sato,E, Kawahara,M, Sakurai,M  Morphology  3-  1  -6  2008  [Not refereed][Not invited]
  
• Morphological aspects of porcine embryos resonstructed by somatic cell nuclear transfer

  Wakai,T, Kawahara,M, Yamanaka,K, Hoshino,Y, Tomioka,I, Sugimura,S, Sugawara,A, Sato,E  Morphology  2-  (1)  23  -27  2007  [Not refereed][Not invited]
  
• 卵子形成過程におけるゲノミックインプリンティング

  尾畑やよい, 樋浦仁, 川原学, 河野友宏  蛋白質 核酸 酵素 増刊  52-  (16)  2142  -2148  2007  [Not refereed][Not invited]
   

  卵子形成過程におけるゲノミックインプリンティングが卵母細胞成長過程に生じるこ、ほ乳類の胚発生に必須であることを概説した。
• Birth of parthenogenetic mouse

  Yayoi Obata, Qiong Wu, Takuya Kumagai, Manabu Kawahara, Hidehiko Ogawa, Tomohiro Kono  ZOOLOGICAL SCIENCE  22-  (12)  1381  -1382  2005/12  [Not refereed][Not invited]
  
• 単為発生マウス誕生へのアプローチ

  川原学  産婦人科の世界  57-  (8)  607  -615  2005/08  [Not refereed][Not invited]
  
• Birth of Parthenogenetic Mouse(Symposium of the Process of Fertilization-From Sex Determination to Epigenetics,Symposium,PROCEEDING OF THE 76^ ANNUAL MEETING OF THE ZOOLOGICAL SOCIETY OF JAPAN) :

  Obata Yayoi, Wu Qiong, Kumagai Takuya, Kawahara Manabu, Ogawa Hidehiko, Kono Tomohiro  Zoological science  22-  (12)  1381  -1382  2005
• カフェイン添加によるMPF活性の低下抑制およびドナー細胞の条件がミニブタ再構築はいの発生に及ぼす影響

  山中賢一, 川原学, 若井拓哉, 杉村智史, 佐々田比呂志, 佐藤英明  日本畜産学会大会講演要旨  103rd-  104  2004/03/20  [Not refereed][Not invited]
  
• Follicular microvasculature and angiogenic factors in the ovaries of domestic animals

  T Shimizu, M Kawahara, Y Abe, M Yokoo, H Sasada, E Sato  JOURNAL OF REPRODUCTION AND DEVELOPMENT  49-  (3)  181  -192  2003/06  [Not refereed][Not invited]
   

  The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate the process of thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation. A recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the process of thecal angiogenesis during follicular development. We are developing a novel technology for the induction of follicular development using the technique of in vivo gene administration. Here, we summarize the recent progress of our research.
• Porcine embryonic stem cell lines; Possibility of medical applications and strategy for production

  Osonoi,M, Taguchi,Y, Endo,N, Kawahara,M, Matsumoto,H, Sasada,H, Sato,E, Edited by Sato,E, Miyamoto,H, Manabe,N  Animal Frontier Sciences -Life science update in animal science-,Hokuto Shobo, Kyoto,  219  -223  2003  [Not refereed][Not invited]
  
• Factors affecting the development of the reconstructed embryos produced from in vitro system in pigs.

  Kawahara,M, Mori,T, Tanaka,H, Sasada,H, Sato,E, Edited by Sato,E, Miyamoto,H, Manabe,N  Animal Frontier Sciences -Life science update in animal science-,Hokuto Shobo, Kyoto,  225  -238  2003  [Not refereed][Not invited]
  
• Activation of porcine reconstituted embryos by complex treatment

  FUCHINOUE Kohei, KAWAHARA Manabu, WAKAI Takuya, SASADA Hiroshi, KYONO Koichi, SATO Eimei  Journal of mammalian ova research = 日本哺乳動物卵子学会誌  19-  (2)  S17  2002/04/01  [Not refereed][Not invited]
  
• Combined activation of porcine oocytes injected with nuclei of fetal miniature pig cells and their subsequent development in vitro.

  K Fuchinoue, M Kawahara, T Wakai, H Sasada, K Kyono, E Sato  BIOLOGY OF REPRODUCTION  66-  238  -239  2002  [Not refereed][Not invited]
  
• 遺伝子改変ブタ作出技術-体細胞クローンとその関連技術の最近の進歩

  佐藤英明, 川原 学, 小園井真人  Organ biology  8-  223  -229  2001  [Not refereed][Not invited]
  
• Technologies for the production of gene-modified pigs, Current status of the somatic cell clone and related technologies

  Sato,E, Kawahara,M, Osonoi,M  Organ biology  8-  223  -229  2001  [Not refereed][Not invited]
  

Research Projects

• 物理化学的刺激を感知するTRPチャネルが乳腺組織の形態形成と乳分泌を制御する機構

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2025/03 

  Author : 小林 謙, 川原 学, 磯部 直樹

   

  乳腺上皮細胞は妊娠にともなって増殖・分化し、分娩後に乳分泌を行う細胞である。また、乳腺上皮細胞は温度・伸縮などの物理的刺激やpH変化・生理活性物質などの化学的刺激に曝される細胞でもある近年、温度感受性TRPチャネルが温度のみならず、機械刺激、浸透圧刺激、生理活性物質などの化学刺激をも感知し、細胞の性状を調節することが明らかになりつつある。そこで本研究では物理化学的刺激を感知するTRPチャネルが乳腺上皮組織の形態形成と乳産生を調節すると仮説を立て、その実証を進めている。 研究初年度の今年は、特定のTRPチャネルをノックアウトした細胞において野生型の細胞と乳産生能力に違いがあるかを調べた。その結果、予想に反してノックアウト細胞と野生型細胞の間に違いは認められなかった。しかし、培養温度の変化や生理活性物質に対する応答性に違いがあった。培養液中の浸透圧を調節した実験では、乳腺上皮細胞のカゼイン産生能が浸透圧依存的に増減することがわかった。また、乳腺上皮細胞のスフェロイドを三次元培養した形態形成モデルの実験では、培養温度の上昇が乳腺上皮細胞の増殖を抑制し、乳管伸長や乳腺胞の形成を阻害することがわかった。培養温度を上昇させた場合、細胞増殖や生存性を制御する細胞内シグナル分子のAktとERKの活性化も認められた。同様の結果は、特定のTRPチャネルを活性化する生理活性物質を添加した場合においても起きていた。また、泌乳ヤギを用いた検証を行った。その結果、乳房の加温処理や特定のTRPチャネルのアゴニストを乳房表面に塗布することによって、ヤギ乳汁中の乳質、乳量、および抗菌成分濃度が変化することがわかった。 以上の結果より、乳腺上皮細胞の形態形成や乳産生能力は多様な物理化学的刺激によって調節されていること、その調節にはTRPチャネルが関与していることが示唆された。
• 初期胚分化制御機構の解明に基づくウシガラス化保存胚の発生能改善

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 川原 学, 柳川 洋二郎, 唄 花子

   

  個体発生の第一歩となる胚盤胞期では、胎子側と胎盤側に運命が分かれる時期であるとともに、ウシでは個体生産に重要な受精卵移植に供される発生ステージである。したがって、胚盤胞期胚の胎子側(ICM)および胎盤側(TE)への細胞分化機構を解明することは、生物学的にも動物生産の効率化のためにも不可欠である。本研究では、YAP1タンパク質の核外流出現象、これに及ぼす細胞膜/細胞骨格関連タンパク質NF2の役割をマウスおよびウシ胚を用いて明らかにする。加えて、得られた基礎的知見を栄養外胚葉細胞のYAP1核外流出が問題となるウシガラス化保存胚の発生能改善に結び付ける。 本年度では、まず胚盤胞期胚における細胞分化を制御するYAP1-TEAD4の上流調節因子としての足場タンパク質NF2の役割についてマウス胚およびウシ胚を用いて解析した。マウス胚ではNF2-GFP融合タンパク質を作製して、その挙動を追跡することで初期胚発生間におけるNF2の細胞内局在を精査した。その結果、細胞分化の確立に伴ってNF2の局在が大きく変化することを示唆する結果が得られた。次年度には、これを更に生細胞イメージングで解析し、さらに、同様の実験系をウシ胚でも適用する。また、YAP1局在保持に留意した新たなガラス化保存法の開発に先駆けて、従来の胚盤胞期よりも更に発生ステージを進行させる培養方法を考案した。これにより、より細胞骨格進んだステージまでウシ胚を発育させることが可能になった。体外培養下における発育の正常性の検証のみならず、新規培養系で作出したウシ胚の個体までの発生能を確認するため受精卵移植試験も実施し、複数頭のウシ新生子を作出した。さらに、これらのウシの健常性も確認されたため、新規培養法による個体作出系を確立した。これに基づいて、新規培養法で作出したウシ胚のガラス化保存を試していく方向で研究を進めていく。
• temperature-responsive molecules in mammary epithelial cells regulate the morphogenesis and functional expression of mammary epithelial cells.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2022/03 

  Author : Kobayashi Ken

   

  Mammary epithelial cells (MECs) develop mammary epithelial tissues during pregnancy and produce milk secretion after parturition. MECs are also exposed to active temperature changes due to changes in environmental temperature and metabolic heat associated with milk production. MECs express heat shock proteins (HSPs) and temperature responsive TRP channels. In this study, we investigated the role of HSPs and TRP channels on the MEC functions. First, we confirmed that hormones and inflammatory components changes the expression levels of the thermos responsive molecules. In addition, activation and inactivation of the thermos responsive molecules influenced milk production and tight junction barrier. Furthermore, local heat treatment on goat udder affected milk composition and somatic cell counts. These things suggest that activation and inactivation of HSPs and TRP channels expressed in MECs regulate mammary epithelial development during pregnancy and milk production during lactation.
• Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Kawahara Manabu

   

  The blastocyst consists of two types of cells, the inner cell mass (ICM) and the trophectoderm (TE), which develop into an embryo proper and a large part of the placenta, respectively. We have shown that, since bovine ICM cells can regenerate TE, they are capable of forming both the embryo and placenta. To test developmental ability of TE-regenerated embryos, we then transferred the regenerated blastocysts to recipient females. After the embryo-transfer, to their surprise, one of the four cows became pregnant and a female calf was naturally born with an apparently normal placenta. Our study suggests that we can remove and use a large part of TE for genetic testing to breed cattle with improved qualities. Also, further studies could reveal the mechanism of cell fate decision in mammals and its differences between species.
• The role of nuclear-mitochondrial interaction in mouse embryo implantation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : Kawahara Manabu

   

  Mitochondria are present in every eukaryotic cell, and have unique DNA different from nuclear DNA. Compared with nuclear DNA, it has a close structure between species. However, mitochondrial heteroplasmic embryo (mtB-M embryo) in which heterologous mitochondria derived from cattle has been mixed in mouse embryos shows implantation failure. In this study, we examined the ability of mtB-M embryo to form trophoblast stem cells (TS cells) and investigated the cause of mtB-M embryo implantation failure. Mouse oocytes were subjected to in vitro fertilization, and subsequently, mitochondrial regions derived from bovine pronuclear stage embryos were transplanted into pronuclear stage embryos by the cell fusion. In the establishment test of TS cells, colonies derived from the mtB-M embryos could not be maintained until passage 5. In summary, the function of trophoblast cells cannot be maintained in the mtB-M embryos, which is considered to be one of causes of implantation failure.
• Role of autophagy-lysosomal related responses on uterine modulation at the time of maternal-fetal recogntion

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  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.
• Elucidation of embryonic secretory factors using bovine endometrial spheroids

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Yamauchi Nobuhiko, Md. Rashedul Islam, Mohamed El-Sharawy

   

  The present study aimed to analyze genes whose expression is induced by embryos and to identify embryonic secretory factors that induce gene expression in cattle. Analysis of the embryonic secretory factor of bovine elongated embryo by LC / MS / MS resulted in the identification of 6 cytokines and 2 growth factors. Furthermore, from the results obtained by RNAseq analysis, upstream factors were analyzed for genes whose expression increased in the endometrium of the implantation period. As a result, in addition to IFNT, genes considered to be controlled by MIF was included. These results suggested that MIF might control endometrial function during implantation as an embryonic secretory factor other than IFNT.
• Exploring novel paternally methylated imprinted genes using bi-maternal mouse fetuses

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : KAWAHARA manabu

   

  Parental genome functions in ontogeny are determined by interactions among transcripts from the maternal and paternal genomes. Comprehensive recognition of the interactions between parental genomes is important for understanding genomic imprinting in mammalian development. The bi-maternal (BM) embryos were constructed by only oocyte genomes, and therefore, analyses using those embryos enable us to investigate unknown paternally methylated imprinted genes in mouse genome. Comparing transcriptome between wild type (WT) and BM fetuses revealed that Mbnl1 expression in BM those was significantly reduced. We further examined Mbnl1 expression in uniparental fetuses, in which we found that Mbnl1 gene was downregulated in both parthenogenetic and androgenetic fetuses comparing that in WT fetus. These results suggested that Mbnl1 was non-imprinted gene but regulated by imprinted genes during development.
• Determination of paternally methylated imprinted regions in mouse genome

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2009 

  Author : KAWAHARA Manabu

   

  Parental genome functions in ontogeny are determined by interactions among transcripts from the maternal and paternal genomes, which contain many genes whose expression is strictly dependent on their parental origin as a result of genomic imprinting. Comprehensive recognition of the interactions between parental genomes is important for understanding genomic imprinting in mammalian development. The bi-maternal embryos were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. Through a sequence of studies, we demonstrated that analyses of ng/fg bi-maternal conceptuses helped in elucidating the roles of paternally methylated imprinted genes in mouse ontogeny.
• ブタ核移植法の開発および遺伝子改変ブタ作出への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2004 

  Author : 川原 学

   

  本研究ではブタ核移植法の確立が極めて重要なポイントとなる。 申請書では平成14年度の研究計画において、(1)活性化刺激と(2)卵子の体外成熟培養時間が核移植した胚の発生に及ぼす影響を調べる予定であった。 この研究計画に則り、以下の知見を得た。 活性化刺激法として、卵内カルシウムイオンの一過性の上昇を引き起こすイオノマイシン、電気刺激(150V/mm,60μsec)、また、サイクリンBレベルの低下を誘起しMPF活性を抑制するシクロヘキシミドをそれぞれ単独で適用した場合と、カルシウムイオン系、サイクリンB抑制系各々でより活性可能が高いものを組み合わせた複合活性化法について検討した。実験の結果、活性化刺激を与えた単為発生卵の胚盤胞期への発生率が最も高かったのはイオノマイシン、電気刺激、シクロヘキシミドこれら3者の複合刺激を施した処理区であった。さらに、顕微注入によって核移植した胚に対しても同様に3者の複合処理を施した場合、単独で活性化した場合よりも有意に高い発生率を示した。以上から、イオノマイシン、電気刺激、シクロヘキシミドの3者複合活性化刺激は核移植した胚の発生率を向上させることが示された。 レシピエント卵子の体外成熟培養時間が核移植胚の発生に及ぼす影響を調べた。体外成熟培養36、40、44時間での卵子の成熟率には差がなかった。しかし、36時間体外成熟培養を施した成熟卵子は核移植胚の胚盤胞期までの発生を支持することができなかった。また、40と44時間の体外成熟培養時間では40時間の方が核移植に供した場合有利に高い発生率を引き出すことが明らかになった。 また、研究の進行状況から平成15年度の研究計画の内容にも着手した。 移植後の核の形態変化を調べ、その形態変化が核移植の発生に及ぼす影響を調べた。その結果、卵子内の成熟促進因子MPFによる核の凝縮が核移植した胚の発生に重要であることが明らかになった。(2)の実験結果とも考慮し、核の初期化にはMPFの高い卵細胞質に核を曝すことが重要であると推察された。 以上(1)から(3)の研究成果を踏まえ、平成15年度にはMPF活性と核の初期化との関係を分子レベルで解析し、さらに核移植された胚の個体への発生を確認すべく仮親豚への核移植胚移植を行った。核移植の操作間で、特に核注入操作によってレシピエント卵子内のMPF活性が低下してしまうことをつきとめ、問題解決として操作培地中へのMPF活性維持剤であるカフェインを添加することで活性の低下を防ぐことに成功した。またカフェイン処理によって早期染色体凝縮を起こす核移植胚の割合が高まり、体外での発生率を改善することにも成功した。さらに、リアルタイムPCR法によって未分化特異的遺伝子Oct4の遺伝子発現を解析し、核移植胚のOct4発現が体外受精卵よりも低くなることをつきとめた。しかし、全30回に渡って仮親へ核移植卵を胚移植したが妊娠70日前後で流産してしまい、個体を得ることはできなかった。以上本研究をまとめると、体内での発生能力に課題を残しつつも、体外でのミニブタ体細胞由来核移植卵の発生能力を高めることには成功し、新たな問題を提起するとともに本研究の知見はミニブタクローンの研究領域に貢献するものと考えられる。