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

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Profile and Settings

Affiliation

• Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture

Profile and Settings

• Name (Japanese)

  Hanako

• Name (Kana)

  Bai

• Name

  201601002391312954

Affiliation

• Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture

Achievement

Research Interests

• 反芻動物   子宮   妊娠認識   着床   

Research Areas

• Life sciences / Animal production science

Research Experience

• 2015/12 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
• 2013/04 - 2015/11 明治飼糧株式会社

Awards

• 2015/03 日本畜産学会 奨励賞
   

  受賞者: 唄花子
• 2013/03 日本畜産学会 優秀発表賞
   

  受賞者: 唄花子
• 2012/09 関東畜産学会 優秀発表賞
   

  受賞者: 唄花子
• 2011/09 日本繁殖生物学会 優秀発表賞
   

  受賞者: 唄花子

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 

  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 

  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
• 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]
   

  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]
  
• Analysis of sequential ruminal temperature sensor data from dairy cows to identify cow subgroups by clustering and predict calving through supervised machine learning.

  Eri Furukawa, Yojiro Yanagawa, Akira Matsuzaki, Heejin Kim, Hanako Bai, Masashi Takahashi, Seiji Katagiri, Shogo Higaki

  The Journal of reproduction and development 69 (2) 103 - 108 2023/02/17 [Refereed]
   

  The present study investigated the applicability of a calving prediction model based on supervised machine learning of ruminal temperature (RT) data in dairy cows. The existence of cow subgroups for prepartum RT changes was also examined, and the predictive performance of the model was compared among these subgroups. RT data were collected from 24 Holstein cows at 10 min intervals using an RT sensor system. The average hourly RT was calculated and data were expressed as residual RTs (rRT = actual RT - mean RT for the same time on the previous three days). The mean rRT decreased beginning at approximately 48 h before calving to a low of -0.5°C at 5 h before calving. However, two cow subgroups were identified: cows with a late and small rRT decrease (Cluster 1, n = 9) and those with an early and large rRT decrease (Cluster 2, n = 15). A calving prediction model was developed using five features extracted from the sensor data (indicative of prepartum rRT changes) through a support vector machine. Cross-validation showed that calving within 24 h was predicted with a sensitivity of 87.5% (21/24) and precision of 77.8% (21/27). A significant difference in sensitivity was observed between Clusters 1 and 2 (66.7 vs. 100%, respectively), while none was observed for precision. Therefore, the model based on RT data with supervised machine learning has the potential to efficiently predict calving, although improvements for specific cow subgroups are required.
• Expression of NFIL3 and CEBPA regulated by IFNT induced-PGE2 in bovine endometrial stromal cells during the pre-implantation period.

  Rulan Bai, Kazuya Kusama, Yuta Matsuno, Hanako Bai, Toshihiro Sakurai, Koji Kimura, Kazuhiko Imakawa

  Frontiers in endocrinology 14 1075030 - 1075030 2023 [Refereed]
   

  Prostaglandin E2 (PGE2) is considered as a luteoprotective factor, influencing the corpus luteum during the early pregnant period in the bovine species. Cyclic AMP (cAMP) is activated in response to PGE2 and plays a role in many physiological processes. The maternal recognition signal, interferon τ (IFNT), induces PGE2 secretion from the endometrial epithelial cells, the function of which in stroma cells has not been completely understood. In this study, PGE2 was found to activate cAMP in the bovine endometrial stromal cells (STRs). STRs were then treated with forskolin to activate the cAMP signaling, from which RNA extracted was subjected to global expression analysis. Transcripts related to transcription regulatory region nucleic acid binding of molecular function, nucleus of cellular component, and mitotic spindle organization of biological processes were up-regulated in cAMP-activated bovine STRs. An increase in the transcription factors, NFIL3, CEBPA, and HIF1A via the cAMP/PKA/CREB signaling pathway in the bovine STRs was also found by qPCR. Knockdown of NFIL3, CEBPA, or HIF1A blocked forskolin-induced PTGS1/2 and IGFBP1/3 expression. Moreover, NFIL3 and CEBPA were localized in endometrial stroma on pregnant day 17 (day 0 = estrous cycle), but not on cyclic day 17. These observations indicated that uterine PGE2 induced by conceptus IFNT is involved in the early pregnancy-related gene expression in endometrial stromal cells, which could facilitate pregnancy establishment in the bovine.
• 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][Not invited]
   

  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 2022/04/01 [Refereed]
   

  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.
• Effects of dietary vitamin K3 supplementation on vitamin K1 and K2 (menaquinone) dynamics in dairy cows.

  Hanako Bai, Hikoji Arai, Kentarou Ikuta, Sho Ishikawa, Yoshihisa Ohtani, Kunihiro Iwashita, Nao Okada, Hitoshi Shirakawa, Michio Komai, Fuminori Terada, Yoshiaki Obara

  Animal science journal = Nihon chikusan Gakkaiho 93 (1) e13680  2022/01 [Refereed][Not invited]
   

  The effect of dietary vitamin K3 (VK3) on ruminant animals is not fully investigated. The aim of this study was to examine the effects of dietary VK3 on lactation performance, rumen characteristics, and VK1 and menaquinone (MK, or VK2) dynamics in the rumen, plasma, and milk of dairy cows. Eight Holstein dairy cows in late lactation periods were used in two crossover trials including a control (nontreatment) and a 50 or 200 mg/day (d) VK3 supplementation group. After 14 days, plasma, ruminal fluid, and milk were sampled and their VK1 and MKs contents were measured using fluorescence-high-performance liquid chromatography (HPLC). Milk production was unchanged after feeding 50 mg/day VK3 but marginally decreased after feeding 200 mg/day VK3. The molar ratio of propionate in ruminal fluid was significantly increased on feeding 200 mg/day VK3. Additionally, MK-4 concentrations significantly increased in both plasma and milk after VK3 feeding (50 and 200 mg/day). In ruminal fluid, MK-4 concentrations increased after 200 mg/day VK3 feeding. These results suggest that VK3 may be a good source of MK-4, the biologically active form of VK, in Holstein dairy cows during their late lactation periods. This study provides a basis for understanding the physiological role of VK in dairy cows.
• Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos.

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

  Biochemical and biophysical research communications 584 1 - 6 2021/12/20 [Refereed][Not invited]
   

  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 2021/11/05 [Refereed]
   

  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.
• 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

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology 35 (10) e21904  2021/10 [Refereed]
   

  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.
• 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 2021/09/13 [Refereed]
   

  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.
• 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 2021/09/02 [Refereed]
   

  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.
• 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][Not invited]
   

  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.
• 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 2021/07/09 [Refereed]
   

  An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.
• 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 2021/05/28 [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.
• 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, Hanako Bai, Masashi Takahashi, Manabu Kawahara

  Developmental Biology 468 (1-2) 14 - 25 2020/12/01 [Refereed]
   

  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 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

  Hanako Bai, Hitoshi Hiura, Yoshiaki Obara, Manabu Kawahara, Masashi Takahashi

  Journal of Dairy Science 103 (8) 7531 - 7534 0022-0302 2020/08 [Refereed]
  
• 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.
• 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

  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.
• 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

  Journal of Reproduction and Development 66 (1) 9 - 17 0916-8818 2020/02/14 [Refereed][Not invited]
   

  Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.
• Cluster analysis to evaluate disease risk in periparturient dairy cattle.

  Sho Ishikawa, Kentaro Ikuta, Yoshiaki Obara, Akio Oka, Yoshihisa Otani, Yuji Takahashi, Hanako Bai, Fuminori Terada, Shiro Kushibiki

  Animal Science Journal = Nihon chikusan Gakkaiho 91 (1) e13442  2020/01 [Refereed]
   

  Predicting periparturient disease risk is of immense value to the dairy industry. Periparturient diseases are interrelated with each other; however, predicting the onset risk of these diseases has predominantly been based on a single blood parameter for a single disease. This study examined a new diagnostic method to predict the risk of periparturient diseases. We conducted cluster analysis of multiple blood constituents from 20 Holstein cattle at 1 week post-partum, and the cattle were divided into two groups, A or B. We then compared the periparturient and early-lactation blood constituents of these groups. Group B had significantly higher 3-hydroxybutyric acid concentrations and were suspected to have subclinical ketosis. Group B also had significantly lower calcium concentrations, with a tendency for subclinical hypocalcemia. We also performed discriminant analysis using blood parameters at 1 week post-partum, which grouped the population into the same two groups as the cluster analysis based on three variables: inorganic phosphorus, calcium, and either phospholipids or total cholesterol. We further showed that these discriminant functions could be used to predict the risk of periparturient disease even before parturition. Our results indicate that cluster analysis with multiple blood constituents is useful for predicting periparturient disease risks.
• 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.
• 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

  Journal of Biological Chemistry 294 (50) 19209 - 19223 0021-9258 2019/12/13 [Refereed][Not invited]
   

  Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to a posteriori externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.
• 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 2019/09/01 [Refereed][Not invited]
   

  Conception rate with the use of sexed semen is lower than that with the use of conventional semen, posing a major problem in the dairy industry. The aim of this study was to understand the risk factors that affect the conception rate after artificial insemination (AI) with conventional and sexed semen using field data. The records of the first insemination in Holstein heifers with conventional (n = 41,857) and sexed semen (n = 45,465) in Hokkaido, Japan were analyzed. The mean conception rate after AI from 2012 to 2016 was 56.9% with conventional semen and 47.3% with sexed semen. A multivariable logistic regression model including the effects of year, heifer age, time of the year, semen type, service sire, and their interactions was used to evaluate the interaction effect of heifer age and time of the year by semen type on the conception rate. In the analysis using heifer age, we found that heifers inseminated with sexed semen were approximately 21 days younger than those inseminated with conventional semen. Interestingly, in early, warmer months (Jun, Jul, and Aug), the conception rate after AI with sexed semen significantly decreased compared with that after AI with conventional semen (P < 0.01). Our results showed that more careful implementation of AI is required for a stable conception using sexed semen, particularly during warmer months.
• 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.
• 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 北海道畜産草地学会 7 (1) 7 - 15 2187-5391 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.

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

  Hanako Bai, Talukder Md Abdus Shabur, Hiroki Kunii, Tsukino Itoh, Manabu Kawahara, Masashi Takahashi

  Journal of Reproduction and Development 65 (4) 313 - 318 0916-8818 2019 [Refereed]
  
• 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

  Journal of Reproduction and Development 64 (6) 495 - 502 0916-8818 2018/12/14 [Refereed][Not invited]
   

  Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.
• Significance of CCN2 expression in bovine preimplantation development.

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

  Animal Science Journal = Nihon chikusan Gakkaiho 90 (1) 49 - 54 1344-3941 2018/10 [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.
• 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.
• 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.
• 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

  Journal of Reproduction and Development 64 (1) 57 - 64 0916-8818 2018/02/27 [Refereed][Not invited]
   

  Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5'- and 3'-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses.
• 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.
• Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells

  Kazuya Kusama, Kazuhiro Tamura, Hanako Bai, Toshihiro Sakurai, Hirotaka Nishi, Keiichi Isaka, Kazuhiko Imakawa, Mikihiro Yoshie

  Reproduction, Fertility and Development 30 (11) 1454 - 1454 1031-3613 2018 [Refereed]
   

  Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332 bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2′-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPβ- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPβ and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.
• 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.
• Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling

  Toshihiro Sakurai, So Nakagawa, Hanako Bai, Rulan Bai, Kazuya Kusama, Atsushi Ideta, Yoshito Aoyagi, Kazuyuki Kaneko, Kosuke Iga, Jiro Yasuda, Takayuki Miyazawa, Kazuhiko Imakawa

  Biochemical Journal 474 (20) 3499 - 3512 0264-6021 2017/10 [Refereed][Not invited]
   

  Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.
• 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

  Journal of Reproduction and Development 63 (3) 211 - 220 0916-8818 2017/06/21 [Refereed][Not invited]
   

  Interferon tau (IFN-τ) is a ruminant-specific type I IFN secreted by a conceptus before its attachment to the uterus. IFN-τ induces the expression of IFN-stimulated genes (ISGs) via the type I IFN receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits in the endometrium. However, expression patterns of IFNARs during the estrous cycle have not been reported. We hypothesized that the response to a type I IFN changes along with IFNARs and the IFN-regulatory factors (IRFs) driving transcription of IFN signal-related genes and modulating a type I IFN signal during the estrous cycle. We investigated the estrous cycle stage-dependent type I IFN induction of ISGs and expression patterns of IFN signal-related genes in bovine endometrial tissues. Endometrial tissue pieces collected from bovine uteri at each estrous stage (early, mid, and late) were cultured with or without recombinant bovine IFN-α or concentrated pregnant uterine flushing (PUF) on day 18 after confirming the presence of a conceptus. IFN-α and PUF each significantly increased the expression of ISGs in endometrial tissues. The induction levels of the typical ISGs (MX1-a and ISG15) were significantly higher at the mid stage and correlated with high expression of IRFs at the mid stage. The immunostaining of IFNARs showed strong fluorescence intensities in luminal and glandular epithelia at the early and mid stages. Collectively, these results suggest that the endometrium exhibits estrous cycle stage-dependent responsiveness to type I IFN that may be associated with the expression of IFNARs and IRFs for pregnancy recognition.
• A transcriptional cofactor YAP regulates IFNT expression via transcription factor TEAD in bovine conceptuses

  K. Kusama, R. Bai, T. Sakurai, H. Bai, A. Ideta, Y. Aoyagi, K. Imakawa

  Domestic Animal Endocrinology 57 21 - 30 0739-7240 2016/10 [Refereed][Not invited]
   

  Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the peri-attachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA down regulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNTgene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down regulates IFNT gene transcription after conceptus attachment to the uterine endometrium. (C) 2016 Elsevier Inc. All rights reserved.
• 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.
• The role of endometrial selectins and their ligands on bovine conceptus attachment to the uterine epithelium during peri-implantation period

  Rulan Bai, Kazuya Kusama, Toshihiro Sakurai, Hanako Bai, Changshou Wang, Jinfeng Zhang, Mariko Kuse, Atsushi Ideta, Yoshito Aoyagi, Kiyoshi Okuda, Kazuhiko Imakawa

  Biology of Reproduction 93 (2) 46 - 46 0006-3363 2015/08/01 [Refereed]
   

  A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
• RNA-seq analysis of equine conceptus transcripts during embryo fixation and capsule disappearance

  Yurika Tachibana, Toshihiro Sakurai, Hanako Bai, Kunio Shiota, Yasuo Nambo, Kentaro Nagaoka, Kazuhiko Imakawa

  PLoS ONE 9 (12) e114414 - e114414 1932-6203 2014/12/16 [Refereed]
  
• The localization of GATA2 in the nuclear and cytoplasmic regions of ovine conceptuses

  Hanako Bai, Toshihiro Sakurai, Rulan Bai, James D. Godkin, Kazuhiko Imakawa

  Animal Science Journal 85 (12) 981 - 985 1344-3941 2014/12 [Refereed][Not invited]
   

  GATA transcription factors are emerging as critical regulators in trophoblast development and its gene regulation. The purpose of this study was to examine the expression and cellular localization of GATA2 in ovine conceptuses during the peri-implantation period. In Western blot analyses, GATA2 proteins were found in days 15, 17 and 21 ovine conceptuses (day 0=day of estrus). Using immunohistochemistry and immunofluorescence analyses, we found that GATA2 was localized in days 15, 17 and 21 ovine conceptuses, and more importantly, GATA2 protein was detected in both nuclear and cytoplasmic regions of the trophectoderm. To our knowledge, the present study is the first to demonstrate that GATA2 is localized in two cellular compartments of the trophectoderm in ovine and many other mammalian species, and suggests that the difference in GATA2 location plays a role in the regulation of down-stream genes during the early pregnancy period.
• Establishment and characterization of immortalized bovine endometrial epithelial cells

  Hanako Bai, Toshihiro Sakurai, Rulan Bai, Sachiko Yamakoshi, Etsunari Aoki, Mariko Kuse, Kiyoshi Okuda, Kazuhiko Imakawa

  Animal Science Journal 85 (8) 799 - 804 1344-3941 2014/08 [Refereed]
  
• Involvement of VCAM1 in the bovine conceptus adhesion to the uterine endometrium

  Rulan Bai, Hanako Bai, Mariko Kuse, Atsushi Ideta, Yoshito Aoyagi, Hiroshi Fujiwara, Kiyoshi Okuda, Kazuhiko Imakawa, Toshihiro Sakurai

  Reproduction 148 (2) 119 - 127 1470-1626 2014/08 [Refereed]
   

  Following bidirectional communication, the conceptus and the uterine epithelium must establish a proper cell–cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied whether vascular cell adhesion molecule (VCAM1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM1 expression was minimal in day 17 (day 0=day of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM1 protein was localized to the luminal and glandular epithelia, whereas in the caruncular regions, VCAM1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM1 expression was up-regulated when treated with uterine flushings or growth factor and further increased when EECs were cocultured with bovine trophoblast CT1 cells. VCAM1 expression in CT1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM1 receptor, integrin α 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM1 protein was found in both EECs and conceptuses, but ITGA4 was localized only to trophoblasts. These observations indicate that cell–cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM1 and ITGA4 expression in the bovine species and suggest that uterine VCAM1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.
• Expression and in situ localization of GATA4, 5 and 6 mRNAs in ovine conceptuses and uterine endometria during the peri-implantation period

  Hanako Bai, Toshihiro Sakurai, James D. Godkin, Kazuhiko Imakawa

  Animal Science Journal 85 (4) 388 - 394 1344-3941 2014/04 [Refereed]
  
• Transcriptional regulation of two conceptus interferon tau genes expressed in japanese black cattle during peri-implantation period

  Toshihiro Sakurai, So Nakagawa, Min-Su Kim, Hanako Bai, Rulan Bai, Junyou Li, Kwan-Sik Min, Atsushi Ideta, Yoshito Aoyagi, Kazuhiko Imakawa

  PLoS ONE 8 (11) e80427 - e80427 1932-6203 2013/11/27 [Refereed]
  
• Down-regulation of interferon tau gene transcription with a transcription factor, EOMES

  Toshihiro Sakurai, Hanako Bai, Rulan Bai, Daisuke Sato, Miki Arai, Kiyoshi Okuda, Atsushi Ideta, Yoshito Aoyagi, James D. Godkin, Kazuhiko Imakawa

  Molecular Reproduction and Development 80 (5) 371 - 383 1040-452X 2013/05 [Refereed]
  
• Presence of transcription factor OCT4 limits interferon-tau expression during the pre-attachment period in sheep

  Min-Su Kim, Toshihiro Sakurai, Hanako Bai, Rulan Bai, Daisuke Sato, Kentaro Nagaoka, Kyu-Tae Chang, James D. Godkin, Kwan-Sik Min, Kazuhiko Imakawa

  Asian-Australasian Journal of Animal Sciences 26 (5) 638 - 645 1011-2367 2013/05/01 [Refereed]
  
• Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac)

  K. Kusama, M. Yoshie, K. Tamura, Y. Kodaka, A. Hirata, T. Sakurai, H. Bai, K. Imakawa, H. Nishi, K. Isaka, T. Nagai, T. Nagao, E. Tachikawa

  Placenta 34 (3) 212 - 221 0143-4004 2013/03 [Refereed][Not invited]
  
• Dynamic evolution of endogenous retrovirus-derived genes expressed in bovine conceptuses during the period of placentation

  So Nakagawa, Hanako Bai, Toshihiro Sakurai, Yuki Nakaya, Toshihiro Konno, Takayuki Miyazawa, Takashi Gojobori, Kazuhiko Imakawa

  Genome Biology and Evolution 5 (2) 296 - 306 1759-6653 2013/02 [Refereed]
  
• Expression and potential role of GATA6 in ruminant trophoblasts during peri-Implantation periods

  Hanako Bai, Toshihiro Sakurai, Atsushi Ideta, Yoshito Aoyagi, James D. Godkin, Kazuhiko Imakawa

  Journal of Mammalian Ova Research 29 (3) 135 - 141 1341-7738 2012/10 [Refereed]
   

  マウス胚の発達過程において、転写因子GATA6は原始内胚葉のマーカーとして知られている。しかし、反芻動物では発現や機能の詳細は不明であった。本研究では、反芻動物の着床過程におけるGATA6の発現を検証した。GATA6 mRNA発現は、着床周辺期のウシおよびヒツジ胚で確認され、着床後に発現が上昇した。ウシ栄養膜細胞CT-1、F3、ヒツジ栄養膜細胞oTrでも発現が確認できた。さらに、in situハイブリダイゼーションでも、着床期のヒツジ胚・栄養膜細胞におけるGATA6 mRNA発現を確認した。インターフェロン・タウ(IFNT)遺伝子上流域を挿入したルシフェラーゼレポーター遺伝子アッセイにより、ヒト絨毛性がん細胞株JEG3およびウシ耳由来線維芽細胞EFで、GATA6によりIFNT遺伝子の転写活性が誘導された。本研究の結果、GATA6は反芻動物の胚・栄養膜細胞で発現しているだけでなく、反芻動物に特異的なIFNT遺伝子の転写活性を誘導したことから、動物種特異的な遺伝子発現制御を担うことが示唆される。(著者抄録)
• Coculture system that mimics in vivo attachment processes in bovine trophoblast cells

  Toshihiro Sakurai, Hanako Bai, Rulan Bai, Miki Arai, Makoto Iwazawa, Jinfeng Zhang, Toshihiro Konno, James D Godkin, Kiyoshi Okuda, Kazuhiko Imakawa

  Biology of Reproduction 87 (3) 0006-3363 2012/09/01 [Refereed]
  
• Expression of GATA1 in the ovine conceptus and endometrium during the peri-attachment period

  Hanako Bai, Toshihiro Sakurai, Toshihiro Konno, Atsushi Ideta, Yoshito Aoyagi, James D. Godkin, Kazuhiko Imakawa

  Molecular Reproduction and Development 79 (1) 64 - 73 1040-452X 2012/01 [Refereed]
  
• Expression of GATA1 in Ovine Trophoblast During Implantation Periods

  Hanako Bai, Toshihiro Sakurai, Toshihiro Konno, James D. Godkin, Kazuhiko Imakawa

  BIOLOGY OF REPRODUCTION 85 398  0006-3363 2011/07 [Refereed][Not invited]
  
• Regulation of trophoblast-specific factors by GATA2 and GATA3 in bovine trophoblast CT-1 cells

  Hanako Bai, Toshihiro Sakurai, Yohhei Someya, Toshihiro Konno, Atsushi Ideta, Yoshito Aoyagi, Kazuhiko Imakawa

  Journal of Reproduction and Development THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 57 (4) 518 - 525 0916-8818 2011 [Refereed]
   

  Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.
  
• Function of a transcription factor CDX2 beyond its trophectoderm lineage specification

  Toshihiro Sakurai, Hanako Bai, Toshihiro Konno, Atsushi Ideta, Yoshito Aoyagi, James D. Godkin, Kazuhiko Imakawa

  Endocrinology 151 (12) 5873 - 5881 0013-7227 2010/12/01 [Refereed]
   

  The transcription factor caudal-related homeobox 2 (CDX2) regulates trophectoderm differentiation, but its function beyond trophectoderm differentiation is not well characterized. CDX2 was shown to regulate a trophoblast-specific gene, interferon τ (IFNT), in the ruminants. However, its regulatory mechanism has not been determined. Here, we report a new role of CDX2 in histone modifications of the IFNT gene. Chromatin immunoprecipitation assays using ovine conceptuses obtained from d 14, 16, 16.5, or 20 of pregnancy (d 0, day of mating) revealed that H3K18 acetylation was highly detectable at the upstream and open reading frame regions of the IFNT gene on d 14 and 16, when CDX2 reached its peak expression. From d 16.5, when the conceptus initiates attachment to uterine epithelial cells, histone acetylation along with CDX2 expression declines. Two candidate CDX2 binding sites (−300 to −294 bp and −293 to −287 bp) of the bovine IFNT gene promoter region were detected from chromatin immunoprecipitation and luciferase assay. When Cdx2 constructs were transfected into bovine ear-derived fibroblast cells, histone acetylation was increased, concurrent with the recruitment of cAMP response element binding protein-binding protein, which has histone acetyltransferase activity. H3K18 acetylation was seen in the proximity of the CDX2 binding region located at the IFNT gene’s upstream region in CT-1 cells, but when these cells were treated with specific CDX2 small interfering RNA, H3K18 acetylation was decreased. These findings suggest that CDX2 regulates its targeted gene through cAMP response element binding protein-binding protein recruitment, which correlates with greater histone acetylation.
• Involvement of GATA transcription factors in the regulation of endogenous bovine interferon-tau gene transcription

  Hanako Bai, Toshihiro Sakurai, Min-Su Kim, Yoshikage Muroi, Atsushi Ideta, Yoshito Aoyagi, Hiromi Nakajima, Masashi Takahashi, Kentaro Nagaoka, Kazuhiko Imakawa

  Molecular Reproduction and Development 76 (12) 1143 - 1152 1040-452X 2009/12 [Refereed]
  
• Induction of endogenous interferon tau gene transcription by CDX2 and high acetylation in bovine nontrophoblast cells

  Toshihiro Sakurai, Atsushi Sakamoto, Yoshikage Muroi, Hanako Bai, Kentaro Nagaoka, Kazuhiro Tamura, Toru Takahashi, Kazuyoshi Hashizume, Miki Sakatani, Masashi Takahashi, James D. Godkin, Kazuhiko Imakawa

  Biology of Reproduction 80 (6) 1223 - 1231 0006-3363 2009/06/01 [Refereed]
  

MISC

• 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  56-  (3)  196  -198  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)
• 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  39-  1  -6  2023/02/13  

  Embryo transfer is a widespread assisted reproductive technique for the production of livestock. In general, the blastocyst-stage embryos used for transfer are produced in vivo or in vitro. However, little is known about differences in mitochondrial ultrastructure between in vivo and in vitro blastocysts in mammals. This study aimed to compare the mitochondrial ultrastructure between in vivo and in vitro mouse blastocysts using transmission electron microscopy (TEM). TEM analyses were performed using more than 1,000 mitochondria from blastocyst embryos. No significant difference was observed in mitochondrial roundness. However, the average major axes (nm) and areas (nm2) were significantly greater in the in vitro embryos than in the in vivo embryos (p＜0.01). Furthermore, the average electron density was significantly decreased in the in vitro embryos compared to the in vivo embryos (p＜0.01). Taken together, we conclude that mitochondria in in vitro blastocysts show an increase in size and a decrease in electron density compared with those in in vivo blastocysts. This implies that mitochondrial functions are deteriorated in in vitro blastocysts compared to in vivo blastocysts.
• 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  49-  (2)  88  -90  2023/02  [Refereed][Invited]
   

  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)
• 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  43-  (2)  85  -94  2022/02  [Refereed][Not invited]
  
• 泌乳牛へのビタミンK3の給与効果

  唄花子, 大谷喜永  畜産技術  (806)  24  -28  2022  [Not refereed][Invited]
  
• 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)  269  -271  2022  [Refereed][Invited]
  
• 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  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.
• ウシの繁殖機能とオートファジー

  唄花子, 川原学, 高橋昌志  家畜診療  68-  (12)  695  -702  2021/12  [Not refereed][Invited]
  
• 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
• Maternal recognition and establishment of pregnancy in ruminants

  Bai Hanako  Proceedings of Japanese Society for Animal Nutrition and Metabolism  62-  (1)  19  -25  2018  [Refereed][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。
• 反芻家畜の着床期における転写因子GATAの発現と機能に関する研究

  唄 花子  日本畜産学会大会講演要旨集  119回-  81  -81  2015/03
• Research and issues for pregnancy improvements during the peri-implantation period in ruminants

  Bai H, Sakurai T, Fujiwara H, Ideta A, Aoyagi Y, Imakawa K  Nihon Chikusan Gakkaiho  84-  (3)  301  -308  2013/08  [Refereed][Not invited]
   

  Even after successful fertilization in the mammalian species, nearly 50% of pregnancies fail, 70∼80% of which are lost during the peri-implantation period. It is thought that early embryonic loss results from a lack or shortage of pregnancy recognition. In ruminants, interferon tau (IFNT), an anti-luteolytic substance produced by the embryonic trophectoderm, is a major signal for the maternal recognition of pregnancy. For over 10 years, we have been studying molecular mechanisms associated with IFNT gene transcription for its effective regulation, resulting in the identification many regulatory factors. In addition, intrauterine administration of IFNT has been performed for the means to improve pregnancy rate. However, no effective therapy with IFNT is yet known to exist. For these reasons, future research needs to be directed toward the identification of what conditions that allows the conceptus to express IFNT as well as other factors properly in vivo. More importantly, these findings should be placed into the practical use in which the treatment developed actually improves pregnancy rates.
• Expression and potential role of GATA factors in trophoblast development

  Hanako Bai, Toshihiro Sakurai, James D. Godkin, Kazuhiko Imakawa  Journal of Reproduction and Development  59-  (1)  1  -6  2013/02  [Refereed][Not invited]
   

  Despite exhaustive studies, molecular mechanisms governing blastocyst formation, implantation to the uterine endometrium and placentation have not been definitively characterized. GATA family proteins are a group of zinc finger transcription factors, for which gene ablations eventually result in embryonic death later in pregnancy. These findings suggested that GATA factors are not essential for early embryonic development. However, recent studies from our laboratory and others have revealed that GATA proteins are involved in the regulation of key genes expressed by the trophectoderm that underpin the transition from the morula to trophoblast, and trophectoderm maintenance. Consequently, it is important to consider the current understanding how GATA factors govern early trophectoderm development.
• 栄養膜細胞由来インターフェロン・タウの妊娠認識のための戦略 過去、現在、未来への可能性

  唄 花子, 櫻井 敏博, 染谷 洋平, 今川 和彦  日本受精着床学会雑誌  29-  (2)  275  -282  2012/07  [Refereed]
  
• Functions of interferon tau as an immunological regulator for establishment of pregnancy

  Hanako Bai, Toshihiro Sakurai, Hiroshi Fujiwara, Atsushi Ideta, Yoshito Aoyagi, James D. Godkin, Kazuhiko Imakawa  Reproductive Medicine and Biology  11-  (3)  109  -116  2012  [Refereed][Not invited]
   

  The establishment of a successful pregnancy requires a "fine quality embryo", "maternal recognition of pregnancy", and a "receptive uterus" during the period of conceptus implantation to the uterine endometrium. In ruminants, a conceptus cytokine, interferon tau (IFNT), a major cytokine produced by the peri-implantation trophectoderm, is known as a key factor for maternal recognition of pregnancy. IFNT can be considered one of the main factors in conceptus-uterus cross-talk, resulting in the rescue of ovarian corpus luteum (CL), induction of endometrial gene expressions, activation of residual immune cells, and recruitment of immune cells. Much research on IFNT has focused on the CL life-span (pregnancy recognition) and uterine gene expression through IFNT and related genes however, immunological acceptance of the conceptus by the mother has not been well characterized. In this review, we will discuss the progress in IFNT and implantation research made by us and others for over 10 years, and relate this progress to pregnancy in mammalian species other than ruminants. © Japan Society for Reproductive Medicine 2012.

Presentations

• 妊娠認識と着床過程の研究  [Invited]

  唄 花子

  令和4年度 日本農芸化学会 北海道支部 学術講演会  2022/12
• ルーメンセンサから得られる温度データの機械学習による乳牛の分娩予測

  古川瑛理, 檜垣彰吾, 松崎明, 唄花子, 高橋昌志, 片桐成二, 柳川洋二郎

  日本畜産学会大会講演要旨  2022
• 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  2022
• 第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係

  古川 瑛理, 高橋 昌志, 松崎 明, 唄 花子, 永野 昌志, 片桐 成二, 柳川 洋二郎

  日本畜産学会大会講演要旨集  2021/09  (公社)日本畜産学会
  
• 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  2021  The Society for Reproduction and Development
   

  【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。
• 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  2021  The Society for Reproduction and Development
   

  【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。
• 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  2021
• 夏季および冬季におけるウシ子宮内膜組織の遺伝子発現の検証

  唄 花子, 三谷 朋弘, 川原 学, 高橋 昌志

  The Journal of Reproduction and Development  2020/09  (公社)日本繁殖生物学会
  
• ウシ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

  嶋崎 知哉, 窪 友瑛, 國井 宏樹, 古山 敬祐, 浜口 悠, 浅岡 那月, 唄 花子, 川原 学, 小川 英彦, 高橋 昌志

  The Journal of Reproduction and Development  2020/09  (公社)日本繁殖生物学会
  
• 妊娠ウシ子宮頸管におけるISGs発現誘導へのIFNTの直接関与

  國井 宏樹, 窪 友瑛, 浅岡 那月, 嶋崎 知哉, 古山 敬祐, 木村 康二, 唄 花子, 川原 学, 高橋 昌志

  The Journal of Reproduction and Development  2020/09  (公社)日本繁殖生物学会
  
• 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  2020  THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
   

  [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.

• Species-specific dynamics of mitochondrial content within an embryo during preimplantation development in mice and cattle.

  Komatsu M, Yokoi F, Imai S, Yamane M, Bai H, Takahashi M, Kawahara M

  Fertility, 2020.  2020/01
• 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  2019/08  北海道畜産草地学会
  
• Pregnancy-specific expression of IFIT1 in the bovine external uterine mucosa  [Not invited]

  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  2019  THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
   

  【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。

• Evaluation of cathepsin B in the trophectoderm of vitrified bovine blastocysts  [Not invited]

  NIWA Yuto, BALBOULA Ahmed Zaky, FUJII Takashi, LI Jianye, MORIYASU Satoru, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement  2019  THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
   

  <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>
• Effect of oxidative stress on bovine uterine epithelial cells under heat stress  [Not invited]

  MURATA Hirona, KOMATSU Seimei, BAI Hanako, KAWAHARA Manabu, MASASHI Takahashi

  The Journal of Reproduction and Development Supplement  2019  THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
   

  <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>
• 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

  Journal of Reproduction and Development  2019
• 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  [Not invited]

  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  2018/08
• ウシ胚盤胞期胚におけるTEAD4およびCCN2の発現制御  [Not invited]

  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集  2018/03  (公社)日本畜産学会
  
• Effect of IFN τ on lysosomal functions in bovine leukocytes during early pregnancy  [Not invited]

  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  2018/03  (公社)日本畜産学会
  
• Maternal recognition and establishment of pregnancy in ruminants  [Invited]

  Bai Hanako

  Proceedings of Japanese Society for Animal Nutrition and Metabolism  2018  家畜栄養生理研究会
   

  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  [Not invited]

  OIKAWA Kohei, YAMAZAKI Takeshi, YAMAGUCHI Satoshi, ABE Hayato, BAI Hanako, TAKAHASHI Masashi, KAWAHARA Manabu

  The Journal of Reproduction and Development Supplement  2018  THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT
   

  【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。

• ウシーマウス間ミトコンドリアヘテロプラスミー胚の栄養膜細胞形成能

  詫間光, 小松雅也, 唄花子, 高橋昌志, 小川英彦, 川原学

  日本生殖発生医学会 第13回学術集会  2018
• 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  2017/09  (公社)日本畜産学会
  
• 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集  2017/09  (公社)日本畜産学会
  
• 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  2017/08  北海道畜産草地学会
  
• 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  2017/08  北海道畜産草地学会
  
• 泌乳牛におけるビタミンKの動態に及ぼすビタミンK3添加の影響

  新居 彦治, 生田 健太郎, 石川 翔, 唄 花子, 大谷 喜永, 岩下 有宏, 岡田 菜緒, 白川 仁, 駒井 三千夫, 寺田 文典, 小原 嘉昭

  日本畜産学会大会講演要旨集  2017/03  (公社)日本畜産学会
  
• 着床過程と妊娠認識ーインターフェロン・タウによる反芻動物の妊娠制御ー  [Invited]

  唄花子

  第35回 北海道受精卵移植研究会  2016/08
• 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  2016/04  (一社)日本卵子学会
  
• 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  2016/03  (公社)日本畜産学会
  
• 育成牛への繊維分解酵素の添加が増体と飼料消化率に及ぼす影響

  大谷 喜永, 小川 裕之, 大河原 壮, 永末 将人, 廣瀬 和彦, 吉田 愛美, 唄 花子, 折橋 毅典, 岩下 有宏, 中野 謙一, 小原 嘉昭, 寺田 文典

  日本畜産学会大会講演要旨集  2016/03  (公社)日本畜産学会
  
• Expression dynamics of autophagy-cathepsin-related genes in cyclic and early pregnancy bovine uterine tissues

  SUZUKI Toshiyuki, SHIROZU Takahiro, IWANO Hiroki, OGISO Takatoshi, YAMAUCHI Nobuhiko, YANAGAWA Yojiro, NAGANO Masashi, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

  The Journal of Reproduction and Development Supplement  2016  The Society for Reproduction and Development
   

  【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。
• 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  2016  The Society for Reproduction and Development
   

  【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  2016  The Society for Reproduction and Development
   

  【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。
• 周産期乳牛におけるカルニチンの体内動態と脂肪肝との関連について

  高橋 雄治, 生田 健太郎, 大谷 喜永, 唄 花子, 寺田 文典, 小原 嘉昭

  日本畜産学会大会講演要旨集  2015/09  (公社)日本畜産学会
  
• 血液プロファイルによる周産期乳牛の潜在的リスク評価手法の確立

  大谷 喜永, 生田 健太郎, 高橋 雄治, 唄 花子, 吉田 愛美, 中野 兼一, 小原 嘉昭, 寺田 文典

  日本畜産学会大会講演要旨集  2015/03  (公社)日本畜産学会
  
• 潜在的リスク評価による周産期乳牛の血液および乳中脂肪酸組成の動態解析

  唄 花子, 生田 健太郎, 高橋 雄治, 大谷 喜永, 吉田 愛美, 中野 兼一, 寺田 文典, 小原 嘉昭

  日本畜産学会大会講演要旨集  2015/03  (公社)日本畜産学会
  
• 不死化ウシ子宮内膜上皮細胞の樹立

  櫻井 敏博, 唄 花子, 荒井 未来, 奥田 潔, 今川 和彦

  日本畜産学会大会講演要旨集  2013/03  (公社)日本畜産学会
  
• ウシ特有の内在性レトロウイルス由来配列の探索と発現解析

  唄 花子, 櫻井 敏博, 青木 悦成, 中川 草, 仲屋 友喜, 宮沢 孝幸, 出田 篤司, 青柳 敬人, 五條堀 孝, 今川 和彦

  日本畜産学会大会講演要旨集  2013/03  (公社)日本畜産学会
  
• ウシ着床期周辺期におけるセレクチンの発現と機能解析

  白 汝嵐, 櫻井 敏博, 唄 花子, 荒井 未来, 出田 篤司, 青柳 敬人, 奥田 潔, 今川 和彦

  日本畜産学会大会講演要旨集  2013/03  (公社)日本畜産学会
  
• ウシ胚の子宮内膜への接着はリンパ球ホーミングに類似するか?

  白汝嵐, 櫻井敏博, 唄花子, 久世真理子, 出田篤司, 青柳敬人, 奥田潔, 今川和彦

  関東畜産学会大会講演要旨集  2013
• Regulation of trophoblast factors by GATA factors during bovine implantation periods.

  Bai Hanako, Sakurai Toshihiro, Someya Youhei, Konno Toshihiro, Imakawa Kazuhiko, Ideta Atushi, Aoyagi Yoshito

  The Journal of Reproduction and Development Supplement  2010  The Society for Reproduction and Development
   

  【目的】我々は以前、反芻動物の妊娠認識に必要なサイトカインであるウシ・インターフェロン・タウ(bIFNT)の発現に転写因子GATA2/3が関与していることを示した。転写因子GATAは発現する細胞や組織において特異的な遺伝子発現制御を行うことが知られており、主に血液細胞での発現やその機能について研究されている。しかし、着床期の胚においてGATA2/3が栄養膜細胞において果たす役割についての知見はまだ少ない。本研究では，GATA2/3がbIFNTの発現調節以外にも栄養膜細胞因子の発現に関与するかを検討することを目的とした。 【方法】着床期のウシ胚（妊娠17日、20日、22日、それぞれ着床前・着床時・着床後）を用い，RT-PCR法およびReal time PCR法にてGATA2/3および栄養膜細胞因子のmRNAの発現を確認した。また、ウシ・栄養膜細胞株にGATA2/3のsiRNAを処置し、内在性GATA2/3の発現抑制が栄養膜細胞因子の発現に与える影響をReal time PCR法にて確認した。また、ウシ・栄養膜細胞において発現している内在性GATA2/3の栄養膜細胞因子の上流域への結合状態をクロマチン免疫沈降 (ChIP)法で確認した。 【結果】用いた着床周辺期のウシ胚で、IFNT, CDX2, PL-1 などの栄養膜細胞因子とGATA2/3のmRNAの発現は着床後の妊娠22日には低下するという発現動態を示した。また、ウシ・栄養膜細胞株で内在性GATA2/3発現を抑制すると栄養膜細胞因子の発現も低下するという結果を得た。さらに、ウシ・栄養膜細胞で発現している内在性GATA2/3が栄養膜細胞因子の上流域に結合していることを示した。これらの結果は、転写因子GATA2/3がIFNT以外にも栄養膜細胞因子群の遺伝子発現制御に関与し、栄養膜細胞としての機能（遺伝子発現）を発揮する上でGATAは重要な因子であることを示唆するものである。
• トロホブラスト細胞における転写因子GATAファミリーの発現動態および発現部位

  唄 花子, 櫻井 敏博, 室井 喜景, 永岡 謙太郎, 今川 和彦

  日本畜産学会大会講演要旨集  2009/03  (公社)日本畜産学会
  
• 転写因子CDX2によるクロマチン構造変化

  櫻井 敏博, 唄 花子, 室井 善景, 永岡 謙太郎, 今川 和彦

  日本畜産学会大会講演要旨集  2009/03  (公社)日本畜産学会
  
• IFNT遺伝子発現制御機構へのTEAD4の関与

  山越 祥子, 唄 花子, 櫻井 敏博, 室井 喜景, 永岡 謙太郎, 岡田 幸之助, 友金 弘, 今川 和彦

  日本畜産学会大会講演要旨集  2009/03  (公社)日本畜産学会
  
• Involvement of GATA transcription factor in the IFNT gene

  Bai Hanako, Sakurai Toshihiro, Konno Toshihiro, Takahashi Masashi, Imakawa Kazuhiko

  The Journal of Reproduction and Development Supplement  2009  The Society for Reproduction and Development
   

  09日本繁殖生物学会 Abstruct GATA23 【目的】 インターフェロン・タウ（IFNT）は、反芻動物の着床期に栄養膜細胞から特異的に産生されるタンパクであり、母親の妊娠認識に必須である。しかしながら、IFNT遺伝子の栄養膜細胞特異的な発現制御機構についての知見は乏しい。本研究では、ウシ・栄養膜由来細胞CT-1とウシ・腎臓細胞株MDBK（非栄養膜由来細胞）における遺伝子発現の比較による結果から、遺伝子転写調節因子GATAファミリーに着目し、栄養膜細胞特異的IFNT遺伝子の発現制御への関与を検討した。 【方法】 1．GATA発現増加時のIFNTの発現 ヒト絨毛性がん細胞JEG3、ウシ耳由来繊維芽細胞EF、および卵巣卵丘顆粒膜細胞 oCGにIFNT上流プロモーター領域-631bpの配列を組み込んだレポーターベクター(pGL3-IFNT)と、GATA2, GATA3発現コンストラクトを導入し、ルシフェラーゼアッセイ法によりIFNTの転写活性を測定した。 2．GATA発現抑制時のIFNTの発現 ウシ・栄養膜由来細胞CT-1にGATA2, GATA3のsiRNAを導入し、GATA2,3の発現抑制下でのIFNT遺伝子発現を解析した。 【結果と考察】 1．ルシフェラーゼアッセイの結果、JEG3を用いた場合にはIFNTの転写活性に差は見られなかったが、EF、およびoCG を用いた場合にはGATA2の導入によりIFNT遺伝子の転写活性が上昇した。 2．CT-1細胞では、内在性IFNTの発現におけるGATA3の発現抑制の影響は見られなかったが、GATA2の発現抑制により内在性IFNTの発現が低下した。 今回の結果から、栄養膜細胞特異的に発現するIFNTの発現にGATA2が関与することが明らかになった。
• Role of CDX2 in bovine trophoblast and intestinal epithelium cells

  SAKURAI Toshihiro, BAI Hanako, KONNO Toshihiro, ASO Hisashi, YAMAGUCHI Takahiro, IMAKAWA Kazuhiko

  The Journal of Reproduction and Development Supplement  2009  The Society for Reproduction and Development
   

  【目的】本研究は、トロホブラスト細胞特異的に発現するホメオボックス転写因子CDX2の役割を解明し、妊娠成立機構の新たな手がかりを見出すことを目的とする。CDX2は卵割期から胚全体に発現し始め、胚盤胞形成期にはトロホブラスト特異的に発現する。我々は、反芻動物のトロホブラスト細胞においてCDX2がインターフェロン・タウ（IFNT）遺伝子の転写に関与することを見出している。CDX2は、トロホブラスト細胞のみならず、胃や腸などの消化管にも発現しているが、IFNTは着床前のトロホブラスト細胞でのみ時限的かつ限局的に発現する。【方法】そこで、CDX2を発現するウシ・トロホブラスト細胞株CT-1およびウシ腸管上皮細胞株BIEを用いて、IFNT発現への作用機序を含めたCDX2の役割の相違について検討した。また、CDX2を発現していない耳由来線維芽細胞EFへのCDX2強制発現系を用いても同様に検討した。【結果と考察】EFへのCDX2強制発現系において、CDX2がIFNT遺伝子上流域のCDX2結合コンセンサス配列に結合し、コアクチベーターであるCBPをリクルートすることにより、IFNT遺伝子座のクロマチン構造を弛緩させることを見出した。しかしながら、EFへのCDX2強制発現だけでは、IFNTの発現は見られなかった。また、CT-1、BIE両細胞に対し、抗CDX2抗体を用いたクロマチン免疫沈降(ChlP)スクリーニングを行い、CDX2の標的遺伝子を同定した。その結果、solute carrier familyなどCT-1、BIEに共通したCDX2の標的遺伝子が確認できた。しかしながら、ChlPスクリーニングからは、IFNTのプロモーター領域は含まれていなかったため、現在、ChIPスクリーニングを再検討している。これらの知見は、CDX2は、その標的遺伝子のクロマチン構造を弛緩させることにより、標的遺伝子の発現を制御していること、さらに、CDX2以外のトロホブラスト細胞に特異的に発現する転写因子がIFNTの発現に関与している可能性を示唆した。
• Possible Role of Transcription Factor Cdx2 that Modifies Histone H3 Acetylation in the Interferon-tau Gene

  Sakurai Toshihiro, Bai Hanako, Muroi Yoshikage, Nagaoka Kentaro, Imakawa Kazuhiko

  The Journal of Reproduction and Development Supplement  2008  The Society for Reproduction and Development
   

  インターフェロン・タウ(IFNT)は、反芻動物において胚の栄養膜（トロホブラスト）細胞が妊娠着床期に時期・細胞特異的に分泌するサイトカインであり、母体の妊娠認識に不可欠な因子である。我々はこのIFNT遺伝子の時期・細胞特異的な発現制御機構の解明を目的に解析を行ってきた。その解析の中でトロホブラスト細胞に特異的に発現する転写因子Cdx2がIFNTの発現に大きく関与する因子の１つであることを見出した。トロホブラスト細胞特異的に発現するCdx2は、IFNT発現を制御するだけでなく、トロホブラスト細胞特異的に発現する他の因子についてもクロマチン構造変化を含めた統括的な制御をしており、トロホブラスト細胞がトロホブラスト細胞としての機能を獲得するために必要な因子であると考えられる。ウシ・トロホブラスト細胞株BT-1へのCdx2発現コンストラクト（pSG5-Cdx2）導入によるCdx2の強制発現系において内在性IFNT発現が増強された。また逆にCdx2 siRNAによりその発現をノックダウンすると内在性IFNTの発現が低下した。つまり、このことはCdx2発現レベルとIFNT発現は同調していることを示唆させる。次に、Cdx2およびIFNTを発現していないウシ・腎臓細胞株MDBKにpSG5-Cdx2を導入し、Cdx2の強制発現を行うとIFNT遺伝子領域のヒストンH3リジン18（H3K18）がアセチル化され、IFNT発現が誘導されることを見出した。現在、Cdx2によるH3K18のアセチル化の分子機構の解析を行っている。以上のことから、トロホブラストに発現する転写因子Cdx2は、転写因子としてIFNT発現に関与するだけでなく、IFNT遺伝子座のクロマチン構造を変化させることによってもIFNTの発現に関与する可能性が見出された。
• Changes in Gene Expressions Following Endometrial Cells and Uterine Flushing Treatments in Bovine Trophoblast Cells

  Sato Daisuke, Sakurai Toshihiro, Bai Hanako, Muroi Yoshikage, Okuda Kiyoshi, Nagaoka Kentaro, Imakawa Kazuhiko

  The Journal of Reproduction and Development Supplement  2008  The Society for Reproduction and Development
   

  【目的】着床、胎盤形成の不全は妊娠率、出生率に大きく影響する。一般に着床、初期胎盤形成は胚子側、母体側で様々なホルモン、サイトカインなどにより調節され、進行していく。しかしながら、栄養膜細胞は接着後どのような調節を受け、初期胎盤へと形質転換するのかは未だ明らかにされておらず、またその現象を明らかにするin vitro実験系も確立されていない。本研究では、栄養膜細胞が初期胎盤へと分化していく調節機構を解析するため、ウシ栄養膜細胞株（CT-1）及びウシ子宮内膜上皮細胞（子宮細胞）、ウシ腎臓細胞株（MDBK）を用い、in vitro実験系の確立を試みた。【方法】ウシ子宮細胞上にCT-1細胞を共培養（比較対象として、MDBK細胞上にCT-1細胞を共培養）し、妊娠12日、15日、17日及び性周期15日の子宮灌流液を添加した。また同様に妊娠15日、17日、21日及び性周期19日の子宮内膜上皮細胞（初代培養）の培養上清を添加した。CT-1細胞と子宮細胞を共培養し、子宮灌流液などを添加した時間を0時間として、48時間後に子宮細胞と接着したCT-1細胞を分離回収後、CT-1細胞と子宮細胞それぞれからRNAを回収した。【結果】CT-1細胞における初期胎盤形成に関与する遺伝子群の発現は、子宮灌流液や子宮培養上清を添加しただけでは変化せず、初代培養細胞と共培養し子宮灌流液を添加することにより発現亢進もしくは、発現低下することを見いだした。これは、ウシ栄養膜細胞（in vivo）の妊娠17日、20日の結果と一致した。以上のことから、本実験系は栄養膜細胞の初期胎盤分化調節機構の解析に有効な手段であることが明らかになり、栄養膜細胞の分化はホルモン、サイトカインなどに調節され、接着シグナルが加わることで初期胎盤形成に向けて遺伝子群の発現が切り変わることが示唆された。
• IFNT遺伝子のトロホブラスト細胞特異的な発現制御機構に関与する転写因子の探索

  唄花子, 櫻井敏博, 坂本敦史, 室井喜景, 永岡謙太郎, 中島弘美, 今川和彦

  日本畜産学会大会講演要旨  2008

Association Memberships

• JAPANESE SOCIETY OF ANIMAL SCIENCE   SOCIETY FOR REPRODUCTION AND DEVELOPMENT   

Research Projects

• 子宮環境構築のための新規インターフェロン誘導性遺伝子の機能解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/04 -2025/03 

  Author : 唄 花子
• Analysis of differentiation mechanism of bovine trophoblast cells into placental component cells.

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

  Date (from‐to) : 2022/04 -2025/03 

  Author : 櫻井 敏博, 草間 和哉, 唄 花子
• 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。
• ビタミンが子宮細胞に与える影響の検証

  公益財団法人 三島海雲記念財団：2022 年度【自然科学部門】個人奨励金

  Date (from‐to) : 2022/07 -2023/08
• ウシ子宮内膜上皮細胞における暑熱–酸化ストレス応答経路の解析

  一般財団法人 旗影会：2022年度 旗影会研究助成

  Date (from‐to) : 2022/04 -2023/03
• ウシ子宮内膜上皮細胞における暑熱–酸化ストレス応答経路の解析

  一般財団法人 旗影会：2021年度 旗影会研究助成

  Date (from‐to) : 2021/04 -2022/03
• Identification and analysis of interferon stimulated genes in the uterus

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

  Date (from‐to) : 2020/04 -2022/03 

  Author : Bai Hanako

   

  The objective of this study was to obtain the basic knowledge to elucidate the interaction between the embryo and the maternal uterus in order to improve the pregnancy rate. First, treatment of IFNT were conducted on bovine endometrial cells to identify novel ISGs whose expression is induced in endometrial cells at the protein level. Then, functional analysis of ISGs candidates in uterine endometrial cells was performed. Although there have been some changes from the original plan, the ISGs candidates currently under analysis will continue to be validated.
• Intrauterine environment during implantation in bovine pregnancy.

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : Sakurai Toshihiro

   

  In order to clarify what is different in the intrauterine environment of inseminated cows from that of fertile cows, it is necessary to spatiotemporal analyze the intrauterine environment. Using the in vitro implantation model, we, therefore, identified the factors contributing to implantation to clarify the intrauterine environment. We found Versican, which is an extracellular matrix involved in implantation. We also found IL-6 present in the uterine flushing, that is a cytokine contributes to conceptus elongation. We also found that high expression of SNX5 in serum of ewes on day 22 of gestation could be a marker of embryo loss. It was predicted that the concentration gradient of some cytokine is formed by Versican during the bovine implantation process, thereby contributing to the selective accumulation of immune cells in utero and embryo elongation.
• 北海道における夏季の暑熱ストレスがウシ子宮に及ぼす影響の検証

  公益財団法人 栗林育英財団：令和2年度 研究助成

  Date (from‐to) : 2020/04 -2021/03
• 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.
• 反芻動物の妊娠認識物質産生細胞作製の試み

  公益財団法人 栗林育英財団：令和元年度 研究助成

  Date (from‐to) : 2019/04 -2020/03
• Analysis of fetal-maternal interaction in hypoxic environment

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

  Date (from‐to) : 2017/04 -2019/03 

  Author : Bai Hanako

   

  This study focused on the difference between oxygen concentration in the atmosphere and in utero environment. The objective of this study are: 1. Establishment of hypoxic cell culture system using bovine embryo and endometrial cells, 2. Identification and analysis of important candidate factors of bovine embryo and endometrial cells, and 3. Construction of hypoxic response pathway(s) necessary for pregnancy establishment. 1. I tried culture using bovine embryo and trophoblast cell CT-1 and F3 cells, but we could not stably maintain. Endometrial cells were compared in normal and hypoxic conditions. With regard to 2, and 3, I focused on only endometrial epithelial cells. Now I examine expression analysis of protein levels under hypoxic conditions and are investigating candidate factors.
• ウシ子宮の胚受容能力に及ぼす暑熱－酸化ストレスの影響評価とストレス防除

  一般財団法人 旗影会：2017年度 旗影会研究助成

  Date (from‐to) : 2017/04 -2018/03
• ウシ子宮の胚受容能力に及ぼす暑熱-酸化ストレスの影響評価とストレス防除

  一般財団法人 旗影会：2016年度 旗影会研究助成

  Date (from‐to) : 2016/04 -2017/03
• インターフェロン・タウの自己分泌作用によるウシ胚の遺伝子発現および増殖調節機構の解明

  一般財団法人 畜産ニューテック協会：研究調査助成

  Date (from‐to) : 2016/04 -2017/03
• 妊娠・着床期における栄養膜細胞－子宮細胞相互作用の解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2011 -2012 

  Author : 唄 花子

   

  【研究の内容】 本研究は、着床期の長い反劉動物をモデルとし、胚・栄養膜細胞と子宮細胞の相互作用による妊娠成立機構の解明を目的とした。 初年度には、ウシ栄養膜細胞CT-1と子宮内膜上皮細胞(endometrial epithelial cells, EECs)を共培養することにより、着床周辺期の遺伝子発現を再現するモデルを確立した。しかし、EECsは、数回の継代で性質が変化してしまい、安定性に問題があった。そこで、安定した細胞を得るため、本年度は、不死化子宮内膜上皮細胞(imortalized EECs, imEECs)を作出することを目標として研究を行った。 不死化遺伝子をEEcsに感染させ、薬剤選択を行ない、imEEcsを作出した。得られたimEECsは、60回以上継代した後も増殖能を持ち、形態にも変化はなかった。imEECsは、IFNTおよびホルモン処置への反応性も有しており、子宮上皮細胞としての性質を維持していた。しかし、imEECsは、上皮細胞のマーカー(サイトケラチン)と間質細胞のマーカー(ビメンチン)が共に陽性であり、EECsとしての性質を維持しながらも間質細胞の性質も有することが示唆された。更なる研究に用いるためには、培養基質を変える等の検討が必要である。栄養膜細胞との共培養実験に使用できるか否かも検討している。 また、着床周辺期に重要な胚側の因子として、GATA6がIFNTの遺伝子発現制御に関与することを見出した他、内在性レトロウイルス由来配列に着目し、着床周辺期に発現するいくつかの配列を見出している。 【意義・重要性】 本研究により、長期の継代にも耐えられるimEECsを作出した。課題は残るが、IFNTやホルモンへの応答性を検討するためには研究を行う上での安定性の問題が克服できたといえる。 また、新たにIFNT遺伝子の制御に関与する因子として見出したGATA6は、反芻動物とマウス胚の知見とで違いがある点が興味深い。今後、動物種による違いを探るきっかけとなり得ると考えている。 【研究成果】 本年度の研究成果として投稿した論文は7報が採択された。また、2報は採択決定済である。

Social Contribution

• あぐり大学講座

  Date (from-to) : 2019/04-Today

  Role : Organizing member

  Sponser, Organizer, Publisher  : 北海道大学農学部・北海道新聞社編集局

  農林水産省 第4回 食育活動表彰 消費・安全局長賞 受賞