Updated on 2024/02/02

写真a

 
KIMURA Koji
 
Organization
Faculty of Environmental, Life, Natural Science and Technology Professor
Position
Professor
External link

Degree

  • PhD ( 2005.11   Kyoto University )

Research Areas

  • Life Science / Animal production science

Professional Memberships

Committee Memberships

  • 日本繁殖生物学会   理事  

    2020.8   

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  • 日本繁殖生物学会   編集委員  

       

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  • 岡山バイオアクティブ研究会   理事  

       

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  • 日本畜産学会   編集委員  

       

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Papers

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

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

    Reproduction (Cambridge, England)   2023.10

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    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.

    DOI: 10.1530/REP-23-0098

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  • Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows. International journal

    Shuichi Matsuyama, Sho Nakamura, Shiori Minabe, Satoshi Ohkura, Koji Kimura

    Biology of reproduction   2023.4

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    Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B, and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. Since neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

    DOI: 10.1093/biolre/ioad039

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  • Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows. International journal

    Shuichi Matsuyama, Sho Nakamura, Shiori Minabe, Miki Sakatani, Naoki Takenouchi, Takuya Sasaki, Yuki Inoue, Hisataka Iwata, Koji Kimura

    Molecular reproduction and development   2023.1

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    To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.

    DOI: 10.1002/mrd.23670

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  • Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro

    Yuki Yamamoto, Maho Kurokawa, Taiji Ogawa, Sayaka Kubota, Koji Kimura

    Reproduction   2023.1

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    Publishing type:Research paper (scientific journal)   Publisher:Bioscientifica  

    Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0–4 days after ovulation), at the mid-late luteal stage (10–17 days), and at the follicular stage (1–3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher at the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

    DOI: 10.1530/rep-22-0174

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    Other Link: https://rep.bioscientifica.com/downloadpdf/journals/rep/aop/rep-22-0174/rep-22-0174.xml

  • Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles

    Ahmad Farid RAWAN, Hikmatullah LANGAR, Maho MUNETOMO, Yuki YAMAMOTO, Kohei KAWANO, Koji KIMURA

    Journal of Reproduction and Development   2023

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    Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.2023-047

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  • Expression of NFIL3 and CEBPA regulated by IFNT induced-PGE2 in bovine endometrial stromal cells during the pre-implantation period. International journal

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

    Frontiers in endocrinology   14   1075030 - 1075030   2023

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    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.

    DOI: 10.3389/fendo.2023.1075030

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  • A Chinese Medicine, Tokishakuyakusan, Increases Bovine Oviductal Tonus <i>via</i> G Protein-Coupled Estrogen Receptor 1

    Sayaka Kubota, Yuki Yamamoto, Koji Kimura

    Biological and Pharmaceutical Bulletin   45 ( 8 )   1133 - 1141   2022.8

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    Authorship:Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Pharmaceutical Society of Japan  

    DOI: 10.1248/bpb.b22-00201

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  • Evaluation of bovine uterine gland functions in 2D and 3D culture system

    Yosuke SUGINO, Taiki SATO, Yuki YAMAMOTO, Koji KIMURA

    Journal of Reproduction and Development   2022

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    Authorship:Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.2022-029

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  • Immunohistochemical identification of epithelial cell types in the isthmus of bovine oviduct: comparison with the ampulla

    Sayaka ITO, Yuna YAMAGUCHI, Sayaka KUBOTA, Yuki YAMAMOTO, Koji KIMURA

    Journal of Reproduction and Development   2022

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    Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Animal Reproduction  

    DOI: 10.1262/jrd.2022-104

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  • Hyperthermia alters interleukin-6 production in response to lipopolysaccharide via endoplasmic reticulum stress in bovine endometrial cells. Reviewed International journal

    Shunsuke Sakai, Yuki Inoue, Keisuke Tanaka, Yuki Yamamoto, Hisataka Iwata, Koji Kimura

    Journal of cellular physiology   2021.10

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    In the postpartum period, cows experience the uterine bacterial infection and develop the endometritis. To eliminate bacteria and recover from endometritis, endometrial epithelial and stromal cells secrete the cytokine and chemokine, such as interleukin 6 (IL-6), IL-8, and monocyte chemotactic protein 1 (MCP1), to recruit immune cells. Moreover, the symptom of endometritis is prolonged in summer and we have recently indicated that hyperthermia suppresses and enhances the IL-6 production in response to lipopolysaccharide (LPS) challenge in endometrial epithelial and stromal cells, respectively. However, the mechanisms for the opposite reaction of IL-6 secretion in response to LPS challenge in both types of endometrial cells under hyperthermia conditions were still unclear. To reveal these mechanisms, both types of endometrial cells were cultured with LPS under the control (38.5°C) or hyperthermia (40.5°C) conditions and comprehensively analyzed differential gene expressions of them by RNA-seq. In addition, based on these results, we examined the effect of endoplasmic reticulum (ER) stress on the IL-6 production in both types of endometrial cells cultured with LPS under hyperthermia conditions. In comprehensive analysis, hyperthermia induced the ER stress in the endometrial stromal cells but not in the endometrial epithelial cells. Actually, we confirmed that hyperthermia increased the gene expression of BiP, ATF4, and sXBP1 and protein expression of BiP and phosphorylated inositol requiring 1, ER stress marker, in the endometrial stromal cells but not in the endometrial epithelial cells. Moreover, in the endometrial stromal cells exposed to LPS, activation and inhibition of ER stress enhanced the IL-6 production under control conditions and suppressed it under hyperthermia conditions, respectively. In this study, we could uncover the one of causes for the disruption of IL-6 production in response to LPS challenge in the endometrial cells under hyperthermia conditions. This finding might be a clue for the improvement of the symptom of endometritis in cows during summer.

    DOI: 10.1002/jcp.30604

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  • Effects of cortisol on prostaglandin F2α secretion and expression of genes involved in the arachidonic acid metabolic pathway in equine endometrium - In vitro study. Reviewed International journal

    Anna Z Szóstek-Mioduchowska, Haruki Shiotani, Yuki Yamamoto, Agnieszka Sadowska, Anna Wójtowicz, Keisuke Kozai, Takuo Hojo, Koji Kimura, Dariusz J Skarzynski, Kiyoshi Okuda

    Theriogenology   173   221 - 229   2021.8

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    Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF2α by affecting the AA cascade in the equine endometrium during the estrous cycle.

    DOI: 10.1016/j.theriogenology.2021.08.009

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  • Heat stress influences the attenuation of prostaglandin synthesis by interferon tau in bovine endometrial cells. Reviewed International journal

    Shunsuke Sakai, Mami Yagi, Nao Fujime, Mariko Kuse, Ryosuke Sakumoto, Yuki Yamamoto, Kiyoshi Okuda, Koji Kimura

    Theriogenology   165   52 - 58   2021.2

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    Heat stress (HS) reduces reproductive performance of cattle, possibly by disrupting endocrine regulation such as prostaglandin (PG) production from uterus and estradiol 17β production from the dominant follicle. Prostaglandin F2α (PGF2α) secretion from endometrium surges during the luteal phase due to tumor necrosis factor (TNF) α stimulation and a positive-feedback loop with oxytocin (OT) from the corpus luteum, ultimately triggering luteolysis, while interferon τ (IFNT) inhibits upregulation of PGF2α production by TNFα and OT, thereby preventing luteolysis and triggering recognition of pregnancy. In the present study, we investigated the effect of OT, TNFα, and IFNT on PGF2α production in both types of endometrial cells under HS conditions. Stimulation of PGF2α production in endometrial epithelial cells by OT was unaffected by HS, while stimulation of PGF2α production in endometrial stromal cells by TNFα was enhanced by HS, and this increased PGF2α production was not significantly suppressed by IFNT. These results suggest that HS disrupted the regulation of PGF2α production by TNFα and IFNT in bovine endometrial stromal cells and it might be one of causes for low conception rate of cattle in summer.

    DOI: 10.1016/j.theriogenology.2021.02.005

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  • Gene-expression profile and postpartum transition of bovine endometrial side population cells†. Reviewed International journal

    Ryoki Tatebayashi, Sho Nakamura, Shiori Minabe, Tadashi Furusawa, Ryoya Abe, Miki Kajisa, Yasuhiro Morita, Satoshi Ohkura, Koji Kimura, Shuichi Matsuyama

    Biology of reproduction   104 ( 4 )   850 - 860   2021.1

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    The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene Ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells.

    DOI: 10.1093/biolre/ioab004

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  • Alteration of chemokine production in bovine endometrial epithelial and stromal cells under heat stress conditions. Reviewed International journal

    Shunsuke Sakai, Toshimitsu Hatabu, Yuki Yamamoto, Koji Kimura

    Physiological reports   8 ( 22 )   e14640   2020.11

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    After parturition, cows frequently develop uterine bacterial infections, resulting in the onset of endometritis. To eliminate the bacteria, bovine endometrial cells secrete chemokines, such as IL-6 and MCP1, which attract macrophages (MΦs) to the subepithelial stroma. These attracted MΦs are not only involved in bacterial elimination but also the orchestration of inflammation and tissue repair. These immune responses aid in the recovery from endometritis; however, the recovery from endometritis takes longer in summer than in any other season. Based on these findings, we hypothesized that heat stress (HS) affects the chemokine production in endometrial cells. To confirm this hypothesis, we compared IL-6 and MCP1 production induced by lipopolysaccharide (LPS) in bovine endometrial epithelial and stromal cells under normal (38.5°C) and HS conditions (40.5°C). In the endometrial epithelial cells, IL-6 production stimulated by LPS was significantly (p < .05) suppressed under HS conditions. MCP1 production in endometrial epithelial cells was not detected under both the control and HS conditions regardless of the presence of LPS. Moreover, LPS significantly (p < .05) stimulated IL-6 and MCP1 production in endometrial stromal cells. Moreover, HS significantly (p < .05) enhanced their production compared to that under the control conditions. In addition, HS did not affect the migration ability of MΦs; however, the supernatant of the endometrial stromal cells cultured under the HS condition significantly (p < .05) attracted the MΦs when compared to the control condition. These results suggest that HS disrupts chemokine production in two types of endometrial cells and alters the distribution of MΦs in the endometrium during the summer.

    DOI: 10.14814/phy2.14640

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  • Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows. Reviewed International journal

    Yuki Yamamoto, Sayaka Ito, Kiyoshi Okuda, Koji Kimura

    Theriogenology   153   143 - 150   2020.9

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    Activin (ACV) A induces various cellular functions via activin receptor type 2 (ACVR2A/2B)-activin receptor-like kinase (ALK) 4 -Smad 2/3 pathway. Although the production of ACVA is indicated in bovine oviducts, its role on the oviduct is unclear. Oviductal isthmus needs to change its function rapidly at peri-fertilization, however, the mechanism is unknown. This study was aimed to clarify the role of ACVA in the morphological changes of oviductal isthmus in cows. First, mRNA expressions of INHBA (ACVA component) and its receptors (ALK4, ACVR2A and ACVR2B) in the isthmic tissues were examined throughout the estrous cycle. INHBA was the highest, however, ACVR2A was the lowest on the day of ovulation, suggesting reduced ACV signal transduction in the isthmus just after ovulation. Proteins of ACVRs and Smad2/3 were clearly detected in the cultured epithelial cells. It is known that ACVA regulates cellular apoptosis. Our data showed that the number of cleaved caspase-3-positive epithelial cells was largest at 2-3 days after ovulation in the isthmus. Interestingly, our study demonstrated that follistatin (ACV/TGFB/BMP inhibitor) significantly decreased the BCL2/BAX ratio in the cultured isthmic epithelial cells. To clarify which ALK pathway is involved in the regulation of BCL2/BAX ratio, the effects of SB431542 (ACV signaling (ALK4) and TGFB signaling (ALK5) inhibitor), SB525334 (ALK5 inhibitor) and LDN193189 (BMP signaling (ALK2/3) inhibitor) were investigated in the next study. The results showed that only SB431542 significantly decreased BCL2/BAX and the others had no effects. These results suggest that decreased ACVA-ACVR2A-ALK4 signal at the post-ovulation induces cyclic apoptosis of isthmic epithelial cells in bovine oviducts.

    DOI: 10.1016/j.theriogenology.2020.05.009

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  • ウシ子宮内膜side population(SP)細胞の遺伝子発現解析と分娩後におけるSP細胞割合の推移

    舘林 亮輝, 中村 翔, 美辺 詩織, 古澤 軌, 阿部 良哉, 加治佐 実希, 森田 康広, 大蔵 聡, 木村 康二, 松山 秀一

    The Journal of Reproduction and Development   66 ( Suppl. )   j41 - j41   2020.9

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    Language:Japanese   Publisher:(公社)日本繁殖生物学会  

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  • Analysis of ciliogenesis process in the bovine oviduct based on immunohistochemical classification. Reviewed International journal

    Sayaka Ito, Yuki Yamamoto, Koji Kimura

    Molecular biology reports   47 ( 2 )   1003 - 1012   2020.2

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    The oviductal epithelium is composed of ciliated and non-ciliated cells. The proportions of these cells change during the estrous cycle. However, the mechanism underlying this cyclic change in the cell proportions remains unclear. Our previous study indicated that ciliated cells are derived from non-ciliated cells. Here, we aimed to investigate the mechanism regulating the changes in the populations of ciliated and non-ciliated cells during the estrous cycle. To this end, we examined the numbers of cells that were positive for acetylated-α-tubulin (cilia marker), Ki67 (proliferation marker), PAX8 (non-ciliated cell marker), and FOXJ1 and MYB (ciliogenesis markers) in the epithelial cells at four different estrous stages (Stage I: days 1-4 after ovulation, Stage II: days 5-10, Stage III: days 11-17, and Stage IV: days 18-20) by immunohistochemistry. The oviductal epithelial cells expressed either FOXJ1 or PAX8. All the acetylated-α-tubulin+ cells were positive for FOXJ1, although there were a few acetylated-α-tubulin-/FOXJ1+ cells. MYB was expressed in both the FOXJ1+ and PAX8+ cells, but it was not expressed in the Ki67+ cells. The numbers of Ki67+ and MYB+ cells were the highest in Stage IV, while the numbers of FOXJ1+ and acetylated-α-tubulin+ cells were the highest in the following Stage I, suggesting that ciliogenesis is associated with the estrous cycle. Thus, based on immunological classification, the oviductal epithelium contains at least seven types of cells at different translational/transcriptional states, and their number is regulated by the estrous cycle. This cyclic event might provide an optimal environment for gamete transport, fertilization, and embryonic development.

    DOI: 10.1007/s11033-019-05192-w

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

    KUNII Hiroki, KUBO Tomoaki, ASAOKA Natsuki, SHIMASAKI Tomoya, KOYAMA Keisuke, KIMURA Koji, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

    The Journal of Reproduction and Development Supplement   113 ( 0 )   P - 65-P-65   2020

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    Language:Japanese   Publisher:THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

    DOI: 10.14882/jrds.113.0_P-65

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  • Effects of miR-98 in intrauterine extracellular vesicles on maternal immune regulation during the peri-implantation period in cattle. International journal

    Keigo Nakamura, Kazuya Kusama, Atsushi Ideta, Koji Kimura, Masatoshi Hori, Kazuhiko Imakawa

    Scientific reports   9 ( 1 )   20330 - 20330   2019.12

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    Evidence accumulated suggests that extracellular vesicles (EVs) present in uterine lumen play a role in conceptus-endometrial cell interactions during peri-implantation periods. However, how intrauterine EVs function on endometrium have not been well characterized. To study how intrauterine EVs affect endometrial milieu in cattle, bovine endometrial epithelial cells (EECs) were treated with EVs isolated from uterine flushing fluids (UFs) on day 17 or 20 pregnancy (P17, P20, respectively; conceptus implantation to endometrium begins on days 19-19.5). RNA extracted from EECs were then subjected to RNA sequence analysis. The analysis revealed that transcripts related to immune system were down-regulated in EECs treated with EVs on P20 compared with those on P17. To investigate whether microRNAs (miRNAs) in EVs regulate maternal immune system in the endometrium during the peri-implantation, microRNA sequence and in silico analyses were performed, identifying bta-miR-98 in EVs as a potential miRNA to regulate maternal immune system. Furthermore, the treatment of EECs with bta-miR-98 negatively regulated several immune system-related genes, CTSC, IL6, CASP4 and IKBKE, in EECs. These results suggest that EVs containing bta-miR-98 is a regulator of maternal immune system, possibly allowing the conceptus attachment to the endometrial epithelium during the peri-implantation period.

    DOI: 10.1038/s41598-019-56879-w

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  • The effects on the endocrine system under hepatotoxicity induction by phenobarbital and di(2-ethylhexyl)phthalate in intact juvenile male rats. Reviewed

    Yamaguchi T, Maeda M, Ogata K, Abe J, Utsumi T, Kimura K

    The Journal of toxicological sciences   44 ( 7 )   459 - 469   2019

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    Phenobarbital (PB) and Di (2-ethylhexyl) phthalate (DEHP), an anti-epileptic drug and a plasticizer used in flexible polyvinylchloride formulations, respectively, are well-known typical hepatotoxicants. This study investigated the effects of PB (100 mg/kg/day) or DEHP (500 mg/kg/day) on the endocrine system in intact juvenile/peripubertal male rats exposed for 31 days beginning on postnatal day 23. Slight hormone level changes, histopathological changes in thyroid gland or induction of UDP-glucuronosyltransferase in liver were observed in both the PB and DEHP groups. One of the assumed mechanisms inducing thyroid effects is predictable to be secondary changes based on the enhancement in thyroid hormone metabolism via the induction of hepatic microsomal enzymes. No reproductive system-related changes in organ weights, histopathology, and sexual maturation were observed in both groups. Lower testosterone level was observed in the PB group. CYP2B and CYP3A, which are involved in testosterone metabolism, were induced in liver of the PB group. There was no change of 17β-hydroxysteroid dehydrogenase activity in testis of both groups. Lower testosterone level in the PB-treated male rats was attributed to an indirect, hepatotoxicity-associated effect on the reproductive system and not to direct effects on testis such as the antiandrogenic activity and the inhibition of steroidogenesis. These results did not indicate that PB or DEHP exposure affects the endocrine system directly.

    DOI: 10.2131/jts.44.459

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  • 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. Reviewed

    Toshiyuki Suzuki, Ryosuke Sakumoto, Ken-Go Hayashi, Takatoshi Ogiso, Hiroki Kunii, Takahiro Shirozu, Sung-Woo Kim, Hanako Bai, Manabu Kawahara, Koji Kimura, Masashi Takahashi

    The Journal of reproduction and development   64 ( 6 )   495 - 502   2018.12

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    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.

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  • Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves. Reviewed International journal

    Nakamura S, Haga S, Kimura K, Matsuyama S

    Journal of animal science   96 ( 11 )   4902 - 4911   2018.9

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    Short-chain fatty acids (SCFAs) are the main source of energy for postweaning ruminants. The monocarboxylic acid transporters, MCT1 and MCT4, are thought to contribute to the absorption of SCFAs from the surface of the rumen following weaning. The present study measured changes in MCT1 and MCT4 expression in ruminal epithelial cells isolated from male preweaning (22 to 34 d old, n = 6) and postweaning (55 to 58 d old, n = 8) calves after euthanasia and sought to examine whether SCFAs stimulate the expression of these transporters. In the current study, cluster of differentiation 147 (CD147) gene expression in the rumen was also investigated since CD147 has been considered to act as ancillary protein for MCT1 and MCT4 to express their correct function. The gene expression levels of MCT1, MCT4, and CD147 in the rumen were found to be significantly higher in postweaning calves than in preweaning calves. Strong MCT1 immunoreactivity was detected in both the stratum basale (SB) and the stratum spinosum (SS) in postweaning ruminal epithelium. Expression of MCT1 in preweaning calves was localized to a specific region of the SB and of the SS. MCT4-immunopositive cells were detected in the stratum corneum (SC) of the ruminal epithelium in postweaning calves. However, only a low level of signal was detected in the SC of preweaning animals. Furthermore, in vitro experiments, ruminal epithelial cells were incubated for 24 h with acetate (0.04, 0.4, and 4 mM), propionate (0.2, 2, and 20 mM), butyrate (0.1, 1, and 10 mM), or β-hydroxybutyrate (BHBA; 0.1, 1, and 10 mM), respectively. Both propionate and butyrate induced an increase in the gene expression levels of MCT4 and CD147, but did not affect MCT1 gene expression. There are no significant effects of acetate and BHBA treatment on these gene expressions. Taken together, these results suggest that an increase in MCT4 and CD147 gene expression in the ruminal epithelium of postweaning calves is likely to be due to the effects of propionate and butyrate derived from a solid-based diet, which may contribute to ruminal development following weaning.

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  • 子宮内膜における免疫系は暑熱ストレスにより変化する

    酒井 駿介, 畑生 俊光, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   64 ( Suppl. )   j83 - j83   2018.9

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  • Heat stress affects prostaglandin synthesis in bovine endometrial cells. Reviewed

    Shunsuke Sakai, Natsumi Hagihara, Mariko Kuse, Koji Kimura, Kiyoshi Okuda

    The Journal of reproduction and development   64 ( 4 )   311 - 317   2018.8

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    Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG) E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.

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  • ウシ黄体における骨形成蛋白質結合蛋白質の役割

    西村 亮, 長谷川 啓喜, 山下 真路, 伊藤 典彦, 岡本 芳晴, 木村 康二, 奥田 潔

    日本獣医学会学術集会講演要旨集   161回   404 - 404   2018.8

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  • Activation of lysosomal cathepsins in pregnant bovine leukocytes. Reviewed International journal

    Md Abdus Shabur Talukder, Ahmed Zaky Balboula, Takahiro Shirozu, Sung Woo Kim, Hiroki Kunii, Toshiyuki Suzuki, Tsukino Ito, Koji Kimura, Masashi Takahashi

    Reproduction (Cambridge, England)   155 ( 6 )   515 - 528   2018.6

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    In ruminants, interferon-tau (IFNT)-mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy.

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  • Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium. Reviewed International journal

    Rulan Bai, Kazuya Kusama, Keigo Nakamura, Toshihiro Sakurai, Koji Kimura, Atsushi Ideta, Yoshito Aoyagi, Kazuhiko Imakawa

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology   32 ( 6 )   3371 - 3384   2018.6

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    Embryo implantation into the uterine endometrium is required for pregnancy establishment in most mammals. By using global expression analysis, we investigated the molecules that are related to epithelial-mesenchymal transition (EMT) in noninvasive bovine trophoblasts and found that the transcription factor, ovo-like zinc finger 2 ( OVOL2), which is essential for mesenchymal-epithelial transition in various cancers, was down-regulated after trophoblast attachment to the endometrial epithelium in utero. In cultured bovine trophoblast cells, OVOL2 down-regulation occurred only when cells were allowed to attach to bovine endometrial epithelial cells via the TEAD3/YAP signaling pathway. This resulted in the up-regulation of the EMT-associated transcription factors, ZEB1 and SNAI2, and the mesenchymal cell markers, N-cadherin ( CDH2) and vimentin ( VIM), whereas epithelial cell marker, E-cadherin ( CDH1), was down-regulated. In contrast, OVOL2 overexpression in bovine trophoblast cells exhibited a decrease in ZEB1 transcripts and an increase in E-cadherin. These observations revealed that ovo-like protein (OVOL)2 down-regulation occurred concurrently with conceptus implantation into the uterine endometrium via the YAP/TEAD3 signaling pathway, and suggest that the down-regulation of OVOL2 expression contributes to the up-regulation of EMT-related transcription factor expression, which enables EMT progression in the noninvasive bovine trophectoderm postimplantation.-Bai, R., Kusama, K., Nakamura, K., Sakurai, T., Kimura, K., Ideta, A., Aoyagi, Y., Imakawa, K. Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.

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  • 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. Reviewed

    Bai R, Kusama K, Nakamura K, Sakurai T, Kimura K, Ideta A, Aoyagi Y, Imakawa K

    FASEB JOURNAL   32 ( 6 )   3371 - 3384   2018.6

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  • Publisher Correction: Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle. Reviewed

    Ikeda M, Matsuyama S, Akagi S, Ohkoshi K, Nakamura S, Minabe S, Kimura K, Hosoe M

    Scientific reports   8 ( 1 )   1470   2018.1

  • ウシ子宮内膜におけるミトコンドリア機能の亢進は受胎率を飛躍的に向上させる

    松山 秀一, 中村 翔, 美辺 詩織, 岩田 尚孝, 木村 康二

    日本胚移植学雑誌   40 ( 1 )   24 - 24   2018.1

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  • Acute stimulation of a smooth muscle constrictor by oestradiol-17β via GPER1 in bovine oviducts Reviewed

    Nishie T, Kobayashi Y, Kimura K, Okuda K

    Reproduction in Domestic Animals   53 ( 2 )   326 - 332   2018

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  • Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle Reviewed

    Mitsumi Ikeda, Shuichi Matsuyama, Satoshi Akagi, Katsuhiro Ohkoshi, Sho Nakamura, Shiori Minabe, Koji Kimura, Misa Hosoe

    SCIENTIFIC REPORTS   7 ( 1 )   17827   2017.12

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    Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

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  • Hypoxia increases glucose transporter 1 expression in bovine corpus luteum at the early luteal stage Reviewed

    Ryo Nishimura, Hiroki Hasegawa, Masamichi Yamashita, Norihiko Ito, Yoshiharu Okamoto, Takashi Takeuchi, Tomoaki Kubo, Kosuke Iga, Koji Kimura, Mitsugu Hishinuma, Kiyoshi Okuda

    JOURNAL OF VETERINARY MEDICAL SCIENCE   79 ( 11 )   1878 - 1883   2017.11

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    A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. Hypoxia-inducible factor-1 (HIF1) induces the expression of glucose transporter 1 (GLUT1). To clarify the physiological roles of GLUT1 in bovine CL, we examined GLUT1 mRNA expression in the CL under hypoxic conditions by quantitative RT-PCR. We also measured the effects of glucose (0-25 mM) and GLUT1 inhibitors (cytochalasin B, STF-31) on P4 production in bovine luteal cells. GLUT1 mRNA expression in bovine CL was higher at the early luteal stage compared to the other later stages. Hypoxia (3% O-2) increased GLUT1 mRNA expression in early luteal cells, but not in mid luteal cells. Glucose (0-25 mM) increased P4 production in early luteal cells, but not in mid luteal cells. Both GLUT1 inhibitors decreased P4 production in early and mid luteal cells. Overall, the results suggest that GLUT1 (possibly induced by hypoxic conditions in the early CL) plays a role in the establishment and development of bovine CL, especially in supporting luteal P4 synthesis at the early luteal stage.

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  • Interferon Tau Regulates Cytokine Production and Cellular Function in Human Trophoblast Cell Line Reviewed

    Nao Tanikawa, Kotomi Seno, Ryouka Kawahara-Miki, Koji Kimura, Shuichi Matsuyama, Hisataka Iwata, Takehito Kuwayama, Koumei Shirasuna

    JOURNAL OF INTERFERON AND CYTOKINE RESEARCH   37 ( 10 )   456 - 466   2017.10

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    Type I interferons (IFN), including IFN-beta (IFNB), activate multiple STAT signaling to drive various biological responses. Another type I IFN, IFN-tau (IFNT), secreted by ruminant embryonic trophoblast cells, has multiple functions with low cytotoxicity. Here, we examined the effects of IFNT on human trophoblast cell functions. First, we performed next-generation sequencing and demonstrated that IFNT-dependent changes in the human Sw.71 trophoblast cell line are partly mediated by proinflammatory as well as IFN signaling. Next, we validated candidate genes, and data confirmed that IFNT stimulated interleukin-6 (IL-6) and IL-8 mRNA expression and secretion. However, human IFNB did not affect IL-6 and IL-8 mRNA expression and secretion. IFNT-induced cytokine secretion was dependent on STAT3 signaling, but not STAT1 signaling. In addition, treatment with IFNT, IL-6, or IL-8 increased cell proliferation, and IFNT also stimulated cell migration in human trophoblast cells. Although IFNT did not affect superoxide dismutase (SOD) 1 mRNA expression, it clearly increased mitochondrial SOD2 mRNA expression, resulting in the acceleration of SOD activity. We demonstrated that in addition to IFN signaling, IFNT also regulated inflammation-related signaling as well as cell proliferation, migration, and redox signaling in human trophoblast cells.

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  • Production of calves by the transfer of cryopreserved bovine elongating conceptuses and possible application for preimplantation genomic selection Reviewed

    Takashi Fujii, Hiroki Hirayama, Akira Naito, Masashi Kashima, Hitomi Sakai, Shigeo Fukuda, Hitomi Yoshino, Satoru Moriyasu, Soichi Kageyama, Yoshikazu Sugimoto, Shuichi Matsuyama, Hiroyuki Hayakawa, Koji Kimura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   63 ( 5 )   497 - 504   2017.10

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    Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.

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  • Age-associated mRNA expression changes in bovine endometrial cells in vitro. Reviewed International journal

    Nao Tanikawa, Ayaka Ohtsu, Ryouka Kawahara-Miki, Koji Kimura, Shuichi Matsuyama, Hisataka Iwata, Takehito Kuwayama, Koumei Shirasuna

    Reproductive biology and endocrinology : RB&E   15 ( 1 )   63 - 63   2017.8

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    BACKGROUND: Endometrial cells secrete various cytokines and the dysfunction of endometrial cells may directly lead to infertility. Interferon tau (IFNT) secreted by trophoblast cells, a well-known pregnancy recognition signal in ruminants, acts on the uterus to prepare for pregnancy. Aging causes cellular and organ dysfunction, and advanced maternal age is associated with reduced fertility. However, few studies have investigated age-dependent changes in the uterus. METHODS: Using next generation sequencing and real-time PCR, we examined mRNA expression in bovine endometrial cells in vitro obtained from young (mean 45.2 months) and aged (mean 173.5 months) animals and the effects of IFNT depending on the age. RESULTS: We showed that inflammation-related (predicted molecules are IL1A, C1Qs, DDX58, NFKB, and CCL5) and interferon-signaling (predicted molecules are IRFs, IFITs, STATs, and IFNs) pathways were activated in endometrial cells obtained from aged compared to young cows. Also, the activation of "DNA damage checkpoint regulation" and the inhibition of "mitotic mechanisms" in endometrial cells obtained from aged cows were evident. Moreover, we showed lower cell viability levels in endometrial cells obtained from aged compared to young cows. Although treatment with IFNT upregulated various types of interferon stimulated genes both in endometrial cells obtained from young and aged cows, the rate of increase by IFNT stimulus was obviously lower in endometrial cells obtained from aged compared to young cows. CONCLUSIONS: Endometrial cells obtained from aged cows exhibited higher levels of inflammatory- and IFN-signaling, and dysfunction of cell division compared with young cows. In addition, a high basal level of IFN-related genes in endometrial cells of aged cows is suggested a concept of "inflammaging".

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  • Adrenomedullin regulates the speed of oviductal fluid flow in cattle Reviewed

    Yuka Yoshimoto, Takumi Nishie, Sayaka Ito, Yoshihiko Kobayashi, Yuki Yamamoto, Kiyoshi Okuda, Koji Kimura

    MOLECULAR REPRODUCTION AND DEVELOPMENT   84 ( 8 )   712 - 718   2017.8

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    Unidirectional flow of oviductal fluid from the ovarian to uterine side of the ampulla plays a significant role in successful pregnancy, and is produced by ciliary beating. Various systems regulate ciliary beating, such as paracrine, autocrine, and endocrine. We hypothesized that Adrenomedullin (ADM)-a peptide hormone that acts via its receptors, which are complexes of Calcitonin receptor-like receptor (CRLR) and Receptor activity-modifying protein (RAMP) 2 or 3-promotes oviductal fluid flow in the ampulla of bovine oviducts. First, we examined the expression of ADM, CRLR, RAMP2, and RAMP3 mRNAs in isolated epithelial cells throughout the estrous cycle, and the localization of ADM receptor protein constituents in the ampulla. RAMP2 expression was significantly higher in the follicular phase. Furthermore, RAMP2 protein was detected only in ciliated cells, whereas CRLR and RAMP3 were detected in all epithelial cells. The effects of ADM and an ADM antagonist on fluid-flow speed were examined using microbeads in ampullary tissue. ADM antagonist decreased bead transport speed, and this decrease was reversed by ADM. In addition, ADM recovered the bead transport speed that decreased in the absence of calcium. Overall, our results suggest that ADM contributes to the regulation of oviductal fluid flow in ampulla.

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  • Estrous cycle stage-dependent manner of type I interferon-stimulated genes induction in the bovine endometrium. Reviewed

    Takahiro Shirozu, Hiroki Iwano, Takatoshi Ogiso, Toshiyuki Suzuki, Ahmed Z Balboula, Hanako Bai, Manabu Kawahara, Koji Kimura, Hitomi Takahashi, Bai Rulan, Sung-Woo Kim, Yojiro Yanagawa, Masashi Nagano, Kazuhiko Imakawa, Masashi Takahashi

    The Journal of reproduction and development   63 ( 3 )   211 - 220   2017.6

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    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.

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  • Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum Reviewed

    Kei Horihata, Shin Yoshioka, Masahiro Sano, Yuki Yamamoto, Koji Kimura, Dariusz J. Skarzynski, Kiyoshi Okuda

    REPRODUCTION FERTILITY AND DEVELOPMENT   29 ( 7 )   1280 - 1286   2017.6

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    The corpus luteum (CL) synthesises and secretes progesterone (P4), which is essential for the establishment and maintenance of pregnancy in mammals. P4 is synthesised from cholesterol. Cholesterol is internalised by low-density lipoprotein receptor (LDLR) and/or scavenger receptor B1 (SR-BI), and is effluxed by ATP-binding cassette (ABC) transporter A1 (ABCA1) and G1 (ABCG1). To test the hypothesis that lipoprotein receptors and ABC transporters are involved in functional luteolysis, we examined the expression of LDLR, SR-BI, ABCA1 and ABCG1 in bovine CL during the luteal stages and after injection of prostaglandin (PG) F(2 alpha)on Day 10 after ovulation. Expression of LDLR and SR-BI mRNA and protein was lower in the regressed luteal than late luteal stage. Injection of cows with a PGF(2 alpha) did not affect LDLR mRNA and protein levels in the CL. Although expression of SR-BI mRNA did not change, SR-BI protein expression decreased 12 and 24 h after PGF(2 alpha) injection. The overall findings of the present study suggest that the decreased expression of SR-BI induced by PGF(2 alpha) is one of the factors responsible for the continuous decrease in P4 production during functional luteolysis.

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  • Local effect of lysophosphatidic acid on prostaglandin production in the bovine oviduct Reviewed

    Yuka Yoshimoto, Yoshihiko Kobayashi, Izabela Woclawek-Potocka, Emilia Sinderewicz, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    REPRODUCTION FERTILITY AND DEVELOPMENT   29 ( 5 )   1021 - 1028   2017.4

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    The mammalian oviduct plays an important role in the fertilisation and transport of gametes and embryo. Prostaglandins (PGs) are local mediators of oviductal functions and are involved in fertilisation and the transport of gametes and embryo. Lysophosphatidic acid (LPA), a kind of phospholipid, is involved in various physiological actions. We hypothesised that LPA regulates PG production in the bovine oviduct. To test this hypothesis, we examined the mRNA expression of LPA receptors (LPAR1-6) and LPA-producing enzymes (ATX, PLA1, PLA1) in ampullary and isthmic tissues and in cultured epithelial and stromal cells isolated from the bovine oviduct. We also investigated the effects of LPA on PG synthase expression and PG production in cultured cells. The mRNA of LPAR1-4, 6, ATX and PLA1 were expressed in cultured epithelial and stromal cells. The expressions of LPAR1-3 were significantly lower and the expression of LPAR4 was significantly higher in the isthmic than in the ampullary tissues. Lysophosphatidic acid significantly stimulated PG production in the cultured isthmic stromal cells. The overall findings suggest that LPA stimulates PG production via LPAR4 in the bovine oviduct. Since PGs are important for fertilisation and the transport of gametes and embryo, these findings show that locally produced LPA regulates oviductal functions.

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  • ウシ初期および中期黄体細胞のプロジェステロン(P4)分泌に及ぼす低酸素環境の影響

    長谷川 啓喜, 西村 亮, 菱沼 貢, 山下 真路, 伊藤 典彦, 岡本 芳晴, 窪 友瑛, 伊賀 浩輔, 木村 康二, 奥田 潔

    日本畜産学会大会講演要旨集   122回   160 - 160   2017.3

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  • Hypoxia-inducible factor 1 mediates hypoxia-enhanced synthesis of progesterone during luteinization of granulosa cells Reviewed

    Fadhillah, Shin Yoshioka, Ryo Nishimura, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   63 ( 1 )   75 - 85   2017.2

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    Hypoxia has been suggested to enhance progesterone (P4) synthesis in luteinizing granulosa cells (GCs), but the mechanism is unclear. The present study was designed to test the hypothesis that the hypoxia-induced increase in P4 synthesis during luteinization in bovine GCs is mediated by hypoxia-inducible factor 1 (HIF-1). GCs obtained from small antral follicles were cultured with 2 mu g/ml insulin in combination with 10 mu M forskolin for 24 h as a model of luteinizing GCs. To examine the influence of HIF-1 on P4 synthesis, we determined the effect of changes in protein expression of the alpha-subunit of HIF-1 (HIF1A) on P4 production and on the expression levels of StAR, P450scc, and 3 beta-HSD. CoCl2 (100 mu M), a hypoxia-mimicking chemical, increased HIF-l alpha protein expression in luteinizing GCs. After the upregulation of HIF-1 alpha, we observed an increase in P4 production and in the gene and protein expression levels of StAR in CoCl2-treated luteinizing GCs. In contrast, CoCl2 did not affect the expression of either P450scc or 3 beta-HSD. Echinomycin, a small-molecule inhibitor of HIF-1's DNA-binding activity, attenuated the effects of CoCl2 and of low oxygen tension (10% O-2) on P4 production and StAR. expression in luteinizing GCs. Overall, these findings suggest that HIF-1 is one of the factors that upregulate P4 in GCs during luteinization.

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  • ウシ過剰排卵処置時におけるニューロキニンB受容体作動薬の静脈内投与が回収胚の発育ステージおよび遺伝子発現に及ぼす影響

    松山 秀一, 中村 翔, 美辺 詩織, 大石 真也, 大蔵 聡, 木村 康二

    日本胚移植学雑誌   39 ( 1 )   61 - 61   2017.1

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  • Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality Reviewed

    Shun Takeo, Koji Kimura, Koumei Shirasuna, Takehito Kuwayama, Hisataka Iwata

    REPRODUCTION FERTILITY AND DEVELOPMENT   29 ( 4 )   759 - 767   2017

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    Maternal age affects the quality of oocytes. The present study examined whether follicular fluid (FF) is a casual factor for age-associated decline in oocyte quality. First, we measured the concentration of advanced glycation end-products (AGE) in FF derived from young (21-45 months; Young-FF) and aged (120 months; Aged-FF) cows and found significantly higher concentrations of AGE in Aged-FF than Young-FF. Second, oocytes were collected from ovaries of young or aged cows and cultured in maturation medium containing 10% FF derived from young or aged cows. Regardless of oocyte origin, Aged-FF accelerated nuclear maturation progression and gap junction closure between oocytes and cumulus cells, increased reactive oxygen species (ROS) content and the rate of abnormal fertilisation of oocytes and decreased blastulation rate compared with Young-FF. Furthermore, supplementation of maturation medium with AGE induced similar age-associated events in oocytes derived from young cows, in that AGE accelerated the progression of nuclear maturation, increased ROS content in oocytes, increased the rate of abnormal fertilisation and decreased blastulation rate. In conclusion, maternal aging increased the concentration of AGE in FF, and both AGE and Aged-FF accelerated nuclear maturation and reduced the developmental competence of oocytes.

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  • Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors Reviewed

    Takamasa Kawaguchi, Dooseon Cho, Masafumi Hayashi, Tomoyuki Tsukiyama, Koji Kimura, Shuichi Matsuyama, Naojiro Minami, Masayasu Yamada, Hiroshi Imai

    PLOS ONE   11 ( 12 )   e0167550   2016.12

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    Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERR/3, and /IFN-tau) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy -related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

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  • Remodeling of bovine oviductal epithelium by mitosis of secretory cells Reviewed

    Sayaka Ito, Yoshihiko Kobayashi, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    CELL AND TISSUE RESEARCH   366 ( 2 )   403 - 410   2016.11

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    Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

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  • Superovulation with a single administration of FSH in aluminum hydroxide gel: a novel superovulation method for cattle Reviewed

    Koji Kimura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   62 ( 5 )   423 - 429   2016.10

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    Superovulation (SOV) is a necessary technique to produce large numbers of embryos for embryo transfer. In the conventional methods, follicular stimulating hormone (FSH) is administered to donor cattle twice daily for 3 to 4 days. As this method is labor intensive and stresses cattle, improving this method has been desired. We previously developed a novel and simple SOV method, in which the intramuscular injection of a single dose of FSH in aluminum hydroxide gel (AH-gel) induced the growth of multiple follicles, ovulation and the production of multiple embryos. Here we show that AH-gel can efficiently adsorb FSH and release it effectively in the presence of BSA, a major interstitial protein. When a single intramuscular administration of the FSH and AH-gel mixture was performed to cattle, multiple follicular growth, ovulation and embryo production were induced. However, the treatments caused indurations at the administration sites in the muscle. To reduce the muscle damage, we investigated alternative administration routes and different amounts of aluminum in the gel. By administering the FSH in AH-gel subcutaneously rather than intramuscularly, the amount of aluminum in the gel could be reduced, thus reducing the size of the induration. Moreover, repeated administrations of FSH with AH-gel did not affect the superovulatory response. These results indicate that a single administration of FSH with AH-gel is an effective, novel and practical method for SOV treatment.

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  • Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers Reviewed

    Ahmed Saad Ahmed Hassaneen, Yousuke Naniwa, Yuta Suetomi, Shuichi Matsuyama, Koji Kimura, Nahoko Ieda, Naoko Inoue, Yoshihisa Uenoyama, Hiroko Tsukamura, Kei-ichiro Maeda, Fuko Matsuda, Satoshi Ohkura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   62 ( 5 )   471 - 477   2016.10

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    Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black x Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

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  • 分娩後のウシ子宮内膜におけるSide population細胞の発現挙動

    松山 秀一, 美辺 詩織, 中村 翔, 古澤 軌, 池田 光美, 木村 康二

    The Journal of Reproduction and Development   62 ( Suppl. )   j143 - j143   2016.9

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  • Possible Contribution of Alpha2,6-Sialylation to Luteolysis in Cows by Inhibiting the Luteotropic Effects of Galectin-1 Reviewed

    Kazuhisa Hashiba, Junko Nio-Kobayashi, Masahiro Sano, Megumi Maeda, Yoshinobu Kimura, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    BIOLOGY OF REPRODUCTION   95 ( 1 )   17   2016.7

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    The corpus luteum (CL) is essential for establishing pregnancy. If pregnancy does not occur during the estrous cycle, luteolysis is induced by prostaglandin (PG) F2alpha secreted from the uterus. Galectin-1, a beta-galactose-binding protein, is expressed in the functional CL of cows and increases the viability of bovine luteal steroidogenic cells (LSCs) by modifying the functions of membrane glycoproteins. The binding of galectin-1 to glycoproteins is blocked by alpha2,6-sialylation of the terminal galactose residues of glycoconjugates, which is catalyzed by a sialyltransferase (ST6Gal-I). However, the physiological role of alpha2,6-sialic acid in bovine CL is unclear. The level of alpha2,6-sialylation of the bovine CL was higher during the regressed-luteal stage than in other luteal stages. Lectin histochemistry revealed that alpha2,6-sialylated glycoconjugates were localized to luteal endothelial cells throughout the estrous cycle. In addition, alpha2,6-sialylated glycoconjugates concentrated to the membrane of LSCs during the regressed-luteal stage. PGF2alpha treatment for 72 h enhanced the expression of ST6Gal-I mRNA and the level of alpha2,6-sialylated glycoproteins in mid-LSCs. The level of alpha2,6-sialylated glycoproteins of late-stage LSCs (Days 15-17 after ovulation) was higher than that of mid-stage LSCs (Days 8-12 after ovulation), and galectin-1 increased the viability of mid-LSCs but not that of late-stage LSCs. Furthermore, galectin-1 increased the viability of late-LSCs when alpha2,6-sialic acid residues were removed by neuraminidase. The overall findings suggest that alpha2,6-sialylation stimulated by PGF2alpha contributes to luteolysis by inhibiting the luteotropic effects of galectin-1 in bovine CL.

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  • Roles of EDNs in regulating oviductal NO synthesis and smooth muscle motility in cows Reviewed

    Yoshihiko Kobayashi, Yuka Yoshimoto, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    REPRODUCTION   151 ( 6 )   615 - 622   2016.6

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    Endothelins (EDNs) participate in various physiological events including smooth muscle contraction, nitric oxide (NO) synthesis, and embryonic development. In this study, we investigated the regional roles of EDNs produced by bovine oviductal epithelial cells in NO synthesis and smooth muscle motility. Quantification of mRNA expressions indicated that expression of EDN receptor B (EDNRB) in the ampullary region was higher after ovulation than before ovulation, whereas expression of FONRA in the isthmic region was higher after ovulation than before ovulation. Immunohistochemistry revealed that the EDN receptors (EDNRA and EDNRB) were expressed in the epithelium, whereas smooth muscle showed positive staining only for EDNRA. The expressions of inducible NO synthase (i NOS) protein and its mRNA (NOS2) in cultured epithelial cells isolated from the ampulla were stimulated by EDN1, but not by EDN2 or EDN3, after 1 h of incubation. In isthmic epithelial cells, none of the EDNs affected the expression of NOS2. Isometric contraction tests indicated that spontaneous waves were strong in the isthmic region but weak in the ampullary region. EDN1 modulated smooth muscle motility in both the regions. The overall findings suggest that EDN1 plays region-specific roles in smooth muscle motility and epithelial NO synthesis, providing an optimal oviductal microenvironment for transport of gametes, fertilization, and development/transport of early embryo.

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  • Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins Reviewed

    Yoshihiko Kobayashi, Yuki Yamamoto, Soichi Kageyama, Hiroki Hirayama, Koji Kimura, Kiyoshi Okuda

    REPRODUCTION   151 ( 6 )   577 - 587   2016.6

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    Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2 alpha. increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradio1-17 beta decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptor a (ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

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  • Evidence for a PGF2α auto-amplification system in the endometrium in mares. Reviewed International journal

    Keisuke Kozai, Shota Tokuyama, Anna Z Szóstek, Yuko Toishi, Nobuo Tsunoda, Kazuyoshi Taya, Miki Sakatani, Masashi Takahashi, Yasuo Nambo, Dariusz J Skarzynski, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

    Reproduction (Cambridge, England)   151 ( 5 )   517 - 26   2016.5

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    In mares, prostaglandin F2α (PGF2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF2α (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF2α significantly stimulated (P < 0.05) PGF2α production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF2α synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF2α auto-amplification system in mares.

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  • ウシ黄体におけるresistin発現制御

    伊藤 さやか, 小林 芳彦, Rak Agnieszka, 山本 ゆき, 美辺 詩織, 松山 秀一, 木村 康二, Skarzynski Dariusz J., 奥田 潔

    日本畜産学会大会講演要旨集   121回   191 - 191   2016.3

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  • The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts Reviewed

    Mark P. Green, Alexandra J. Harvey, Lee D. Spate, Koji Kimura, Jeremy G. Thompson, R. Michael Roberts

    MOLECULAR REPRODUCTION AND DEVELOPMENT   83 ( 1 )   50 - 60   2016.1

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    The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon-tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4mM) with or without exposure to DNP (0, 10, 100M) between Days 5 and 8 post-fertilization. The absence or presence of high (4mM) glucose reduced blastocyst development and favored the development of male embryos (P&lt;0.001). DNP at 10M had no effect, whereas 100M had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10M DNP further skewed the sex ratio toward males (P&lt;0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (&lt;2.5mM) medium to minimize potential embryonic stress, as higher concentrations have sexually dimorphic effects on development and an embryo's ability to signal to the maternal reproductive tract. Mol. Reprod. Dev. 83: 50-60, 2016. (c) 2015 Wiley Periodicals, Inc.

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  • 肉用繁殖牛における分娩前の腟内温度および電気抵抗値の変動

    松山 秀一, 美辺 詩織, 吉岡 耕治, 木村 康二

    日本胚移植学雑誌   38 ( 1 )   45 - 45   2016.1

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  • Regulation of apoptosis by activin signal in the isthmic epithelial cells of the bovine oviduct

    YAMAMOTO Yuki, ITO Sayaka, KOBAYASHI Yoshihiko, KIMURA Koji, OKUDA Kiyoshi

    The Journal of Reproduction and Development Supplement   109 ( 0 )   P - 65-P-65   2016

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  • Regulation of oviductal function by secretory epithelial cells proliferation and differentiation in cattle

    ITO Sayaka, KOBAYASHI Yoshihiko, YAMAMOTO Yuki, KIMURA Koji, OKUDA Kiyoshi

    The Journal of Reproduction and Development Supplement   109 ( 0 )   OR1 - 4-OR1-4   2016

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  • Remodeling of the oviductal epithelium by stem-like cells beneath the basement membrane

    KOBAYASHI Yoshihiko, ITO Sayaka, KIMURA Koji, OKUDA Kiyoshi

    The Journal of Reproduction and Development Supplement   109 ( 0 )   P - 15-P-15   2016

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  • Possible role of interferon tau on the bovine corpus luteum and neutrophils during the early pregnancy Reviewed

    Koumei Shirasuna, Haruka Matsumoto, Shuichi Matsuyama, Koji Kimura, Heinrich Bollwein, Akio Miyamoto

    REPRODUCTION   150 ( 3 )   217 - 225   2015.9

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    When pregnancy is established, interferon tau (IFNT), a well-known pregnancy recognition signal in ruminants, is secreted by embryonic trophoblast cells and acts within the uterus to prepare for pregnancy. IFNT acts as an endocrine factor on the corpus luteum (CL) to induce refractory ability against the luteolytic action of PGF(2 alpha). Hypothesising that IFNT may influence not only the uterine environment but also the CL in cows via local or peripheral circulation, we investigated qualitative changes in the CL of pregnant cows during the maternal recognition period (day 16) and the CL of non-pregnant cows. The CL of pregnant animals had a higher number of neutrophils, and the expression of interleukin 8 (IL8) mRNA and its protein was higher as well as compared with the CL of non-pregnant animals. Although IFNT did not affect progesterone (P-4) secretion and neutrophil migration directly, it stimulated IL8 mRNA expression on luteal cells (LCs), influencing the neutrophils, resulting in the increased migration of IFNT-activated neutrophils. Moreover, both IFNT-activated neutrophils and IL8 increased P-4 secretion from LCs in vitro. Our novel finding was the increase in neutrophils and IL8 within the CL of pregnant cows, suggesting the involvement of IFNT function within the CL toward establishment of pregnancy in cows. The present results suggest that IFNT upregulates neutrophil numbers and function via IL8 on LCs in the CL of early pregnant cows and that both neutrophils and IL8, stimulated by IFNT, are associated with an increase in P-4 concentrations during the maternal recognition period in cows.

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  • ウシ子宮内膜上皮細胞におけるSide population細胞の解析

    松山 秀一, 古澤 軌, 池田 光美, 美辺 詩織, 木村 康二

    The Journal of Reproduction and Development   61 ( Suppl. )   j140 - j140   2015.9

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  • ウシにおける最終糖化産物の蓄積状況の検討

    美辺 詩織, 松山 秀一, 木村 康二

    The Journal of Reproduction and Development   61 ( Suppl. )   j170 - j170   2015.9

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  • Generation of Naive Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors Reviewed

    Takamasa Kawaguchi, Tomoyuki Tsukiyama, Koji Kimura, Shuichi Matsuyama, Naojiro Minami, Masayasu Yamada, Hiroshi Imai

    PLOS ONE   10 ( 8 )   e0135403   2015.8

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    Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naive PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naive-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRR beta, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naive-type biPSCs showed the hallmarks of naive mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naive-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naive mouse PSCs and a demonstrated potential to contribute to chimeras.

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  • Regulation of gonadotropin secretion by monitoring energy availability Reviewed

    Shuichi Matsuyama, Koji Kimura

    Reproductive Medicine and Biology   14 ( 2 )   39 - 47   2015.4

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    Nutrition is a principal environmental factor influencing fertility in animals. Energy deficit causes amenorrhea, delayed puberty, and suppression of copulatory behaviors by inhibiting gonadal activity. When gonadal activity is impaired by malnutrition, the signals originating from an undernourished state are ultimately conveyed to the gonadotropin-releasing hormone (GnRH) pulse generator, leading to suppressed secretion of GnRH and luteinizing hormone (LH). The mechanism responsible for energetic control of gonadotropin release is believed to involve metabolic signals, sensing mechanisms, and neuroendocrine pathways. The availabilities of blood-borne energy substrates such as glucose, fatty acids, and ketone bodies, which fluctuate in parallel with changes in nutritional status, act as metabolic signals that regulate the GnRH pulse generator activity and GnRH/LH release. As components of the specific sensing system, the ependymocytes lining the cerebroventricular wall in the lower brainstem integrate the information derived from metabolic signals to control gonadotropin release. One of the pathways responsible for the energetic control of gonadal activity consists of noradrenergic neurons from the solitary tract nucleus in the lower brainstem, projecting to the paraventricular nucleus of the hypothalamus. Further studies are needed to elucidate the mechanisms underlying energetic control of reproductive function.

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  • Local target cells of activin in the bovine oviduct

    YAMAMOTO Yuki, KOBAYASHI Yoshihiko, KOBAYASHI Hiroshi, KIMURA Koji, OKUDA Kiyoshi

    The Journal of Reproduction and Development Supplement   108 ( 0 )   P - 63-P-63   2015

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    DOI: 10.14882/jrds.108.0_P-63

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  • Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1 beta in Macrophages Reviewed

    Kyoko Hara, Koumei Shirasuna, Fumitake Usul, Tadayoshi Karasawa, Yoshiko Mizushina, Hiroaki Kimura, Akira Kawashima, Akihide Ohkuchi, Shuichi Matsuyama, Koji Kimura, Masafumi Takahashi

    PLOS ONE   9 ( 12 )   e113974   2014.12

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    Background: Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1 beta, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1 beta secretion in human THP-1 macrophages.
    Methods and Results: IFNT dose-dependently inhibited IL-1 beta secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1 beta and mature IL-1 beta. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1 beta mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1 beta secretion was restored by anti-IL-10 neutralizing antibody.
    Conclusions: Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1 beta secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1 beta induction. It may be a therapeutic alternative to IFNA and IFNB.

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  • Effect of estradiol during culture of bovine oocyte-granulosa cell complexes on the mitochondrial DNA copies of oocytes and telomere length of granulosa cells Reviewed

    M. Endo, K. Kimura, T. Kuwayama, Y. Monji, H. Iwata

    ZYGOTE   22 ( 4 )   431 - 439   2014.11

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    During the development of oocytes from early antral follicles (EAFs) to antral follicles (AFs), the mitochondrial DNA copy number (Mt DNA number) increases, and granulosa cells markedly proliferate. This study examined the effect of supplementation of culture medium with estradiol-17 beta(E-2) on the in vitro growth of oocytes, and increases in the Mt DNA number, and telomere length during the in vitro culture of oocytes derived from EAFs (0.4-0.7 mm in diameter). The E-2 supplementation improved antrum formation and the ratio of oocytes reaching the metaphase II (MII) stage, and there was a significant difference in these values between addition E-2 concentrations of 10 mu g/ml and 0.1 mu g/ml. When the oocytes were cultured in the medium containing 10 mu g/ml E-2, the Mt DNA number determined by real-time polymerase chain reaction (PCR) significantly increased, and the ratio of the Mt DNA number at the end of culture to the Mt DNA number at the beginning of the culture was greatly different among cows, and could be predicted by the degree of the difference between the Mt DNA number of oocytes derived from EAFs and that of oocytes derived from AFs (3-6 mm in diameter). When oocytes were cultured for 16 days in a medium containing 10 mu g/ml E-2 or 0.1 mu g/ml E-2, the Mt DNA number of oocytes grown in vitro did not differ, but the telomere length of the granulosa cells was significantly greater in the 10 mu g/ml E-2 group than in the 0.1 mu g/ml group. In conclusion, E-2 supplementation in culture medium improved the growth of oocytes derived from EAFs, and a high E-2 concentration increased the telomere length of the granulosa cells.

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  • Successful Nonsurgical Transfer of Bovine Elongating Conceptuses and Its Application to Sexing Reviewed

    Koji Kimura, Shuichi Matsuyama

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   60 ( 3 )   210 - 215   2014.6

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    The objectives of the present study were to establish a nonsurgical transfer method for elongating bovine conceptuses and to combine this method with biopsy and sexing. Bovine conceptuses were recovered from donor cows on days 13-14 of the estrus cycle. In experiment 1, day 13 conceptuses were transferred to recipient cows using a standard day 7 embryo transfer (ET) method. The pregnancy rate of day 13 conceptus transfer (CT) is comparable to that of day 7 ET. In experiment 2, day 14 conceptuses were transferred using modified methods (balloon catheters or ET guns with modified sheaths). Using the standard ET method, no pregnancies were obtained; however, when balloon catheters or ET guns with modified sheaths were used, the pregnancy rates after CT were 48.0% and 44.8%, respectively. In experiment 3, day 14 conceptuses were biopsied without a micromanipulator, sexed using the loop-mediated isothermal amplification method and transferred to recipient cows. The pregnancy rate of biopsied conceptuses was 46.2% and did not differ significantly from that of unbiopsied conceptuses. Moreover, all pregnant cows transferred conceptuses following biopsy and sexing delivered calves with the expected sexes. These results suggested that the nonsurgical bovine CT method was comparable to day 7 ET and that this technique enables biopsy and sexing without expensive equipment such as a micromanipulator or specialized skills.

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  • Resveratrol improves the mitochondrial function and fertilization outcome of bovine oocytes. Reviewed

    Takeo S, Sato D, Kimura K, Monji Y, Kuwayama T, Kawahara-Miki R, Iwata H

    Journal of Reproduction and Development   60 ( 2 )   92 - 99   2014.4

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    レスベラトロールがウシ卵子のミトコンドリアの品質を改善し発生率を向上させることを示した。

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  • Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes Reviewed

    Shun Takeo, Daichi Sato, Koji Kimura, Yasunori Monji, Takehito Kuwayama, Ryoka Kawahara-Miki, Hisataka Iwata

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   60 ( 2 )   92 - 99   2014.4

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    The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 mu M resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 mu M Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.

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  • Aerococcus vaginais sp nov., isolated from the vaginal mucosa of a beef cow, and emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenil and Aerococcus sanguinicola Reviewed

    Masanori Tohno, Maki Kitahara, Shuichi Matsuyama, Koji Kimura, Moriya Ohkuma, Kiyoshi Tajima

    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY   64 ( Pt 4 )   1229 - 1236   2014.4

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    A gram-stain-positive, facultatively anaerobic, non-spore-forming, catalase-negative, coccoid-shaped bacterial strain, designated BV2(T), was isolated from the vaginal mucosa of a beef cow in Japan. Phylogenetic analysis showed that the isolate shared high 16S rRNA gene sequence similarity (92.9%) with Aerococcus suis 1821102(T) and low similarity (&lt;92.7%) with any other recognized species of the genus Aerococcus. The DNA G+C content was 44.7 mol%, which is within the range observed among species of the genus Aerococcus (37.5-48.4 mol%). The major cellular fatty acid was C-18:1 omega 9c, similar to other type strains of species of the genus Aerococcus. The results of genotypic, phenotypic and chemotaxonomic analyses as well as the low degree of DNA DNA relatedness with all recognized members of the genus Aerococcus indicate that strain BV2(T) represents a novel species of the genus Aerococcus, for which the name Aerococcus vagina/is sp. nov. is proposed. The type strain is BV2(T) (=JCM 19163(T)=DSM 27293(T)). Emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenii and Aerococcus sanguinicola are also presented.

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  • 高濃度エストロジェン投与が去勢雄ウシのLH分泌におよぼす影響

    佐々木 拓弥, 松山 秀一, 木村 康二, 上野山 賀久, 束村 博子, 松田 二子, 大蔵 聡

    日本畜産学会大会講演要旨集   118回   199 - 199   2014.3

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  • ウシDDX4プロモーター制御下でDsRedを発現するトランスジェニックマウスの解析

    古澤 軌, 大越 勝広, 細江 実佐, 池田 光美, 赤木 悟史, 木村 康二, 松山 秀一, 徳永 智之

    日本繁殖生物学会 講演要旨集   107   P - 87-P-87   2014

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    [目的]マウスでは,体外でES細胞やiPS細胞のような多能性幹細胞から機能的な生殖系列細胞を分化誘導する研究が進んでいる。ウシのように世代間隔が長い家畜動物に応用できれば,育種改良にかかる時間とコストを大幅に削減できる。本研究ではウシ生殖系列細胞の分化誘導過程を可視化するため,生殖系列細胞特異的に発現するウシDDX4(VASAホモログ)遺伝子の発現調節領域にDsRed遺伝子を連結したレポーターベクターを構築し,マウスを用いてその発現制御の特異性を検証した。[方法]ウシDDX4遺伝子開始コドンの上流3kbの配列にDsRed遺伝子を連結し,さらに終止コドンの下流1kbの配列を連結した。これを導入細胞選別のためのloxP-CAG-GFP-SVpA-loxPカセットを含むPiggyBacトランスポゾンベクターに組み込み,TT2マウスES細胞株に導入した。遺伝子導入ES細胞を用いて作成されたキメラマウスとその後代について,胎齢13.5日,生後7,11,14及び35日齢の雄生殖腺におけるDsRedの発現を調べた。[結果及び考察]免疫組織学的解析では胎齢13.5日の胎子においても生殖細胞特異的なDdx4の発現が確認されたが,これと一致したDsRedの発現が最初に観察されたのは生後14日齢からであった。蛍光顕微鏡による解析では,11日齢の精細管の一部でDsRed陽性細胞の出現が観察され,フローサイトメーター解析では14日齢で約3%,35日齢で約60%の精巣細胞がDsRed陽性を示すことが明らかになった。定量的PCR解析においても,生殖腺特異的なDsRedの発現が検出されるのは生後14日齢からであり,免疫組織学的解析の結果と一致した。これらの結果から,マウス生殖細胞において本研究で用いたウシDDX4発現制御領域による転写が開始するのは生後7–11日齢の間であり,これは分化型精原細胞と精母細胞の出現時期と一致した。

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  • POU5F1発現誘導システムによる高品質ウシES細胞樹立の試み

    池田 光美, 古澤 軌, 大越 勝広, 細江 実佐, 木村 康二, 松山 秀一, 赤木 悟史, 徳永 智之

    日本繁殖生物学会 講演要旨集   107   P - 96-P-96   2014

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    【目的】我々は第106回本学会において体外受精ウシ胚から樹立したES様細胞がDay 62のキメラウシ胎子組織に存在すること,また,胚体外組織にも寄与する可能性を報告した。しかし生殖系列細胞への分化が確認されておらず,また胚様体形成実験でも,分化誘導時の急速な細胞死が原因で多能性の証明には至っていない。本研究ではウシES様細胞に多能性関連因子の発現誘導システムを導入し,発現量と発現時期を調節することでより高品質なウシES細胞に改変することを試みた。【方法】Tet-On発現誘導システムにPOU5F1(OCT3/4)遺伝子を組み込み,PiggyBacトランスポゾンベクターを用いてウシES様細胞へ導入した。ドキシサイクリン(Dox)存在下でコロニーをピックアップし,遺伝子導入が確認された株について,Doxの有無による細胞の形態及び増殖性の比較と,幹細胞関連遺伝子発現の変化をqRT-PCRにより調べた。さらに,浮遊培養による胚様体形成実験を行った。【結果】POU5F1発現誘導株はコロニー形態がよりドーム状になり,細胞増殖速度は親株と比較して約2.5倍に亢進した。Doxを除去しても形態及び倍化時間が変化しなかったことから,外因性POU5F1の過剰発現により活性化された経路が,外因性POU5F1発現低下後も持続することが示唆された。qRT-PCR解析の結果,Dox添加による外因性POU5F1と幹細胞マーカーNANOG,CDH-1(E-cadherin),MBD3の発現量増加,及びMycの発現量低下を確認した。さらにDoxの有無にかかわらず,親株と比較してCCND1(cyclin D1)の発現量の増加とBCL2の発現量の低下が認められた。胚様体形成実験では,分化培地での培養3週間後に胚様体が形成され,培養系での分化誘導が可能であることが示された。以上の結果により,外因性POU5F1の発現誘導により細胞増殖が亢進し,さらに多能性関連因子の発現誘導を介してES様細胞の性状を大きく変化させることが示唆された。

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  • Characteristics of Bovine Inner Cell Mass-Derived Cell Lines and Their Fate in Chimeric Conceptuses Reviewed

    Tadashi Furusawa, Katsuhiro Ohkoshi, Koji Kimura, Shuichi Matsuyama, Satoshi Akagi, Masahiro Kaneda, Mitsumi Ikeda, Misa Hosoe, Keiichiro Kizaki, Tomoyuki Tokunaga

    BIOLOGY OF REPRODUCTION   89 ( 2 )   28   2013.8

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    Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

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  • Age-Associated Changes in Gene Expression and Developmental Competence of Bovine Oocytes, and a Possible Countermeasure Against Age-Associated Events Reviewed

    S. Takeo, R. Kawahara-Miki, H. Goto, F. Cao, K. Kimura, Y. Monji, T. Kuwayama, H. Iwata

    MOLECULAR REPRODUCTION AND DEVELOPMENT   80 ( 7 )   508 - 521   2013.7

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    In general, maternal age affects the quality of oocytes and embryos. The present study aimed to examine the features and age-associated gene expression profiles of bovine oocytes and embryos as well as to discover possible countermeasures against age-associated events. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and 8- to 16-cell-stage embryos were conducted using next-generation sequencing technology. The gene expression profiles of aged cows showed high expression of genes related to oxidative phosphorylation, eIF4 and p70S6K signaling, and mitochondrial dysfunction in MII-stage oocytes. Oocytes derived from aged cows, compared with those derived from their younger counterparts, exhibited high levels of abnormal fertilization and blastocysts with low total cell numbers. Levels of reactive oxygen species (ROS) and SIRT1 were higher in in vitro-matured oocytes derived from aged cows than in those derived from their younger counterparts. Supplementation of maturation medium with N-acetyl-cysteine (NAC), but not resveratrol, reduced the levels of ROS in the oocytes derived from cows of both age groups; however, resveratrol, but not NAC, improved the fertilization ratio. Conversely, EX 527, an inhibitor of SIRT1, increased the ratio of abnormal fertilization. In conclusion, gene expression profiles of oocytes and embryos derived from aged cows differ from those of oocytes and embryos derived from young cows; in particular, oocytes derived from aged cows show protein and mitochondrial dysfunction. In addition, activation of SIRT1 in oocytes may be a potential countermeasure against age-associated events in oocytes derived from aged cows. Mol. Reprod. Dev. 80: 508-521, 2013. (c) 2013 Wiley Periodicals, Inc.

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  • The Promoter of the Oocyte-Specific Gene, Oog1, Functions in Both Male and Female Meiotic Germ Cells in Transgenic Mice Reviewed

    Miya Ishida, Eriko Okazaki, Satoshi Tsukamoto, Koji Kimura, Akira Aizawa, Seiji Kito, Hiroshi Imai, Naojiro Minami

    PLOS ONE   8 ( 7 )   e68686   2013.7

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    Oog1 is an oocyte-specific gene whose expression is turned on in mouse oocytes at embryonic day (E) 15.5, concomitant with the time when most of the female germ cells stop proliferating and enter meiotic prophase. Here, we characterize the Oog1 promoter, and show that transgenic GFP reporter expression driven by the 2.7 kb and 3.9 kb regions upstream of the Oog1 transcription start site recapitulates the intrinsic Oog1 expression pattern. In addition, the 3.9 kb upstream region exhibits stronger transcriptional activity than does the 2.7 kb region, suggesting that regulatory functions might be conserved in the additional 1.2 kb region found within the 3.9 kb promoter. Interestingly, the longer promoter (3.9 kb) also showed strong activity in male germ cells, from late pachytene spermatocytes to elongated spermatids. This is likely due to the aberrant demethylation of two CpG sites in the proximal promoter region. One was highly methylated in the tissues in which GFP expression was suppressed, and another was completely demethylated only in Oog1pro3.9 male and female germ cells. These results suggest that aberrant demethylation of the proximal promoter region induced ectopic expression in male germ cells under the control of 3.9 kb Oog1 promoter. This is the first report indicating that sex-dependent gene expression is altered according to the length and the methylation status of the promoter region. Additionally, our results show that individual CpG sites are differentially methylated and play different roles in regulating promoter activity and gene transcription.

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  • Age-associated changes in gene expression and developmental competence of bovine oocytes, and a possible countermeasure against age-associated events Reviewed

    S. Takeo, R. Kawahara-Miki, H. Goto, F. Cao, K. Kimura, Y. Monji, T. Kuwayama, H. Iwata

    Molecular Reproduction and Development   80 ( 7 )   508 - 521   2013.7

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    In general, maternal age affects the quality of oocytes and embryos. The present study aimed to examine the features and age-associated gene expression profiles of bovine oocytes and embryos as well as to discover possible countermeasures against age-associated events. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and 8- to 16-cell-stage embryos were conducted using next-generation sequencing technology. The gene expression profiles of aged cows showed high expression of genes related to oxidative phosphorylation, eIF4 and p70S6K signaling, and mitochondrial dysfunction in MII-stage oocytes. Oocytes derived from aged cows, compared with those derived from their younger counterparts, exhibited high levels of abnormal fertilization and blastocysts with low total cell numbers. Levels of reactive oxygen species (ROS) and SIRT1 were higher in in vitro-matured oocytes derived from aged cows than in those derived from their younger counterparts. Supplementation of maturation medium with N-acetyl-cysteine (NAC), but not resveratrol, reduced the levels of ROS in the oocytes derived from cows of both age groups
    however, resveratrol, but not NAC, improved the fertilization ratio. Conversely, EX 527, an inhibitor of SIRT1, increased the ratio of abnormal fertilization. In conclusion, gene expression profiles of oocytes and embryos derived from aged cows differ from those of oocytes and embryos derived from young cows
    in particular, oocytes derived from aged cows show protein and mitochondrial dysfunction. In addition, activation of SIRT1 in oocytes may be a potential countermeasure against age-associated events in oocytes derived from aged cows. © 2013 Wiley Periodicals, Inc.

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  • Estradiol supports in vitro development of bovine early antral follicles Reviewed

    M. Endo, R. Kawahara-Miki, F. Cao, K. Kimura, T. Kuwayama, Y. Monji, H. Iwata

    Reproduction   145 ( 1 )   85 - 96   2013.1

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    Antrum formation and estradiol (E2) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E2 on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E2 or androstenedione (A4) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A4, developmentally competent OGCs secreted more E 2 than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E2 and A4 on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E2 (1 μg/ml
    E2(+)), ii) GCs of OGCs cultured for 4 days without E2 (E2(-)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E2 (1 μg/ml
    AF group). GCs of the E2(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E2 biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E 22 impacts the gene expression profile of GCs to support the in vitro development of OGCs. © 2013 Society for Reproduction and Fertility.

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    Other Link: http://orcid.org/0000-0003-2072-8851

  • piggyBacトランスポゾンを用いたナイーブ型ウシiPS細胞の樹立の試み

    川口 高正, 築山 智之, 南 直治郎, 山田 雅保, 松山 秀一, 木村 康二, 今井 裕

    日本繁殖生物学会 講演要旨集   106   OR1 - 38-OR1-38   2013

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    【目的】人工多能性幹細胞(iPS細胞)などの多能性幹細胞は,高度な育種改良・効率的な家畜生産への利用が期待される。その際,多能性幹細胞が個体形成能を有することが必要となるが,齧歯類以外において個体形成能を持つ多能性幹細胞樹立に関する報告はほとんどない。その理由として,齧歯類以外の種で樹立されている多能性幹細胞の多くは,個体形成能に乏しいヒトES細胞様の細胞株であることによると考えられる。そこで本研究では,ウシにおいて,高い個体形成能を持つことが期待できるマウスES細胞様(ナイーブ型)のiPS細胞(biPS細胞)の樹立を試みた。【方法】妊娠50日齢の胎仔から採取した羊膜細胞に,Oct3/4,Sox2,Klf4およびc−Mycの転写因子を,piggyBacトランスポゾンベクターを用いて導入した。このベクターには,ドキシサイクリン(Dox)添加の有無によって,導入した転写因子の発現が制御できるシステムを組み込んだ。Doxの添加から4日後,遺伝子導入後の細胞をフィーダー細胞上へと移し,幾つかの細胞分化シグナル阻害剤の存在下で培養した。【結果】Doxの添加から2週間後,明確な細胞境界を示すコロニーが出現した。このコロニーを継代すると,マウスES細胞様の球形でコンパクトな形態を示すbiPS様細胞が得られた。得られた細胞は,トリプシンにより単一細胞に分散しても10回以上継代を行うことができた。また,幹細胞マーカーであるアルカリフォスファターゼ活性を示し,多能性関連遺伝子(OCT3/4,NANOG,ESRRβ,REX1)の発現が認められた。biPS様細胞をJAK阻害剤の存在下で培養すると,細胞の増殖率が低下したことから,その細胞増殖はLIF/STAT3シグナルに依存していることが示唆された。さらに,低接着ディッシュ上で数日間浮遊培養し,分化を促すと,胚様体を形成し,三胚葉へと分化した。これらの結果から,得られたbiPS様細胞株はナイーブ型細胞の特徴を示す高度な多能性を有する幹細胞である可能性が示唆された。

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  • 伸長胚移植によるウシES様細胞の多能性解析

    池田 光美, 徳永 智之, 古澤 軌, 大越 勝広, 細江 実佐, 木村 康二, 松山 秀一, 赤木 悟史, 金田 正弘, 木崎 景一郎

    日本繁殖生物学会 講演要旨集   106   P - 115-P-115   2013

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    【目的】我々は第103,104回本学会において体外受精および体細胞核移植ウシ胚からES様細胞(bovine Embryonic Stem-like cells, bESLCs)を樹立したことを報告した。本研究では,bESLCsの多能性を調べるため,伸長胚移植を用いたキメラ形成能実験を行ったので報告する。【方法】蛍光色素(DiI)あるいはGFP遺伝子導入により蛍光標識したbESLCsを8–16細胞期胚へ注入し,in vitroで胚盤胞期胚まで培養して局在を調べた。次に内部細胞塊(inner cell mass, ICM)に蛍光標識細胞が認められたDay 8–9の胚盤胞期胚を受胚ウシ1頭当たり20–30個移植し,6–7日後に非外科的潅流法により伸長胚を回収してbESLCsの局在を調べた。さらにGFP導入bESLCs注入胚由来伸長胚のうち,胚盤及びその近傍にGFP陽性細胞が認められた胚4個を2個ずつ2頭の受胚ウシに再移植し,Day 62で胎子を回収して,蛍光観察及びgenomic PCRによりbESLCsの局在を調べた。【結果】Day 8–9胚盤胞期では,50.7–77.7%の胚でbESLCsがICMあるいはその近傍に局在し,Day 14伸長胚で,18.8–42.9%の胚で胚盤内にbESLCsの局在が観察された。伸長胚再移植実験では,Day 62に1頭の生存胎子とその胎盤及び死亡胎子の残存胎盤1個を得た。回収した胎子の体表及び臓器表面に蛍光顕微鏡下ではGFP陽性細胞は観察されなかったものの,PCR解析において,腸,肝臓,胃,心臓,腎臓,右前足及び頭部(脳を除く)にbESLCsが存在していることが明らかになった。また,羊膜細胞の一部にGFP陽性細胞のクラスターが観察され,PCR解析においても羊膜と絨毛膜,及び死亡胎子の残存胎盤からGFP遺伝子が検出された。以上の結果から,bESLCsは少なくともDay 62の胎子組織に存在すること,また,胚体外組織にも寄与する可能性が示された。

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  • Effect of estradiol during culture of bovine oocyte-granulosa cell complexes on the mitochondrial DNA copies of oocytes and telomere length of granulosa cells. Reviewed

    Endo M, Kimura K, Kuwayama T, Monji Y, Iwata H

    Zygote   12   1 - 9   2012.12

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  • Relationship between Plasma Progesterone Concentration and Number of Conceptuses and Their Growth in Superovulated Cattle Reviewed

    Shuichi Matsuyama, Yosuke Sakaguchi, Koji Kimura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   58 ( 5 )   609 - 614   2012.10

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    Elevated concentrations of circulating progesterone (P-4) in the immediate post-ovulation period are associated with advancement of conceptus elongation in cattle. Superovulated (SOV) cattle have not only elevated plasma P-4 concentrations but also multiple embryos in the uterus because of the formation of multiple corpora lutea. We examined the relationship between plasma P-4 concentration and uterine glucose level in the immediate post-ovulation period and the presence and growth of multiple conceptuses in SOV cattle. SOV cattle were artificially inseminated with frozen-thawed semen at standing estrus (day 0), and the conceptuses were recovered by nonsurgical flushing of the uterus on day 13. In the SOV cattle, there were quadratic relationships between plasma P-4 concentration on days 4, 5 and 7 and conceptus length and between number of conceptuses in the uterus and conceptus length. These results suggest that conceptus growth in SOV cattle is regulated by both systemic P-4 level and number of conceptuses and that there are ranges of plasma P-4 concentrations and numbers of conceptuses in the uterus that are suitable for conceptus growth and development. Plasma P-4 concentrations on days 5 and 7, but not the numbers of conceptuses, were quadratically correlated with uterine glucose levels on day 13 in SOV cattle. In addition, conceptus length was positively correlated with uterine glucose level in SOV cattle. Accordingly, regardless of the number of conceptuses in the uterus, the plasma P-4 concentration was well correlated with the regulation of conceptus growth via changes in uterine glucose levels in SOV cattle.

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  • Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET Reviewed

    Shuichi Matsuyama, Takatoshi Kojima, Satoru Kato, Koji Kimura

    REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY   10   21   2012.3

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    Background: Interferon tau (IFNT), which is secreted into the uterine cavity during the maternal recognition period (MRP), is a key factor for establishment of pregnancy. The present study aims to clarify the relationship between the ability of a bovine conceptus to produce IFNT during the MRP and the conceptus's ability to establish pregnancy.
    Methods: In the first experiment, IFNT (0, 500, or 1000 micrograms) was administered into the uterine horn ipsilateral to the CL 16 or 17 d after standing estrus, and mRNA levels of IFN-stimulated gene 15-kDa protein (ISG15) and Mx2 in peripheral blood mononuclear cells (PBMCs) were determined. In the second experiment, we investigated ISG15 mRNA expression in PBMCs during the MRP in cattle after either artificial insemination (AI) or embryo transfer (ET).
    Results: Intrauterine administration of IFNT stimulated ISG15 and Mx2 gene expressions in PBMCs in cattle, and there was a positive correlation between the expressions of peripheral markers and the quantity of IFNT administered. In pregnant and normal interestrous interval (&lt; 25 d) cattle (nIEI cattle), expression levels of the ISG15 gene showed similar patterns after AI and ET, and ISG15 mRNA expression was increased in pregnant cattle but unchanged in nIEI cattle. In contrast, ISG15 gene expression in extended interestrous interval (greater than or equal to 25 d) cattle (eIEI cattle) differed after ET compared with AI. In eIEI cattle after ET, ISG15 gene expression increased, such that the value on day 18 was intermediate between those of pregnant and nIEI cattle. In eIEI cattle after AI, ISG15 gene expression did not increase throughout the observation period.
    Conclusions: The results of the current study indicate that the quantity of conceptus-derived IFNT can be estimated by measuring ISG15 mRNA levels in PBMCs from cattle. Using this approach, we demonstrate that ISG15 gene expression during the MRP in eIEI cattle differed after ET compared with AI. In addition, the modest increase in ISG15 gene expression in eIEI cattle after ET suggests that late embryo losses were due to delayed or insufficient growth of the conceptus during the MRP in cattle.

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  • Successful spermatogenesis in the xenografted immature bovine testicular tissue.

    TOKUNAGA Tomoyuki, NOGUCHI Junko, KIMURA Koji, MATSUYAMA Shuichi, HOSOE Misa, IKEDA Mitsumi, OHKOSHI Katsuhiro, FURUSAWA Tadashi

    The Journal of Reproduction and Development Supplement   105   1138 - 1138   2012

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  • Effects of purine base metabolism on bovine embryo development

    KIMURA Koji, MATSUYAMA Shuichi

    The Journal of Reproduction and Development Supplement   105   1107 - 1107   2012

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  • Possible involvement of IFNT in lymphangiogenesis in the corpus luteum during the maternal recognition period in the cow Reviewed

    Akane Nitta, Koumei Shirasuna, Shingo Haneda, Motozumi Matsui, Takashi Shimizu, Shuichi Matsuyama, Koji Kimura, Heinrich Bollwein, Akio Miyamoto

    REPRODUCTION   142 ( 6 )   879 - 892   2011.12

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    The corpus luteum (CL), which secretes large amounts of progesterone and is thus essential for establishing pregnancy, contains various types of immune cells that may play essential roles in CL function by generating immune responses. The lymphatic system is the second circulation system and is necessary for immune function, but the lymphatic system of the bovine CL has not been characterized in detail. We collected bovine CLs on days 12 and 16 of the estrous cycle (C12 and C16) and days 16 and 40 of early pregnancy (P16 and P40). Lymphatic endothelial hyaluronan receptor 1 (LYVE1) protein was detected in the CL by immunohistochemistry and western blotting and increased at P40 compared with C16. The mRNA expression levels of lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGFC), VEGFD, and their common receptor VEGFR3, as well as the lymphatic endothelial cell (LyEC) marker podoplanin, increased in P16 and P40 CLs. Thus, it is suggested that the lymphatic system of the bovine CL reconstitutes during early pregnancy. Interferon tau (IFNT) from the conceptus in the uterus is a candidate for activating luteal lymphangiogenesis during the maternal recognition period (MRP). We found that treatment of LyECs isolated from internal iliac lymphatic vessels with IFNT stimulated LyEC proliferation and significantly increased mRNA expression of VEGFC and IFN-stimulated gene 15. Moreover, both IFNT and VEGFC induced LyECs to form capillary-like tubes in vitro. In conclusion, it is suggested that new lymphangiogenesis in the bovine CL begins during the MRP and that IFNT may mediate this novel phenomenon. Reproduction (2011) 142 879-892

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  • Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage Reviewed

    Shogo Shiratsuki, Hisataka Iwata, Koji Kimura, Takeshi Kuge, Yasunori Monji, Takehito Kuwayama

    THERIOGENOLOGY   75 ( 5 )   841 - 848   2011.3

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    The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P &lt; 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P &lt; 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P &lt; 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts. (C) 2011 Elsevier Inc. All rights reserved.

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  • Effect of maternal age on mitochondrial DNA copy number, ATP content and IVF outcome of bovine oocytes Reviewed

    Hisataka Iwata, Hiroya Goto, Hiroshi Tanaka, Yosuke Sakaguchi, Koji Kimura, Takehito Kuwayama, Yashunori Monji

    REPRODUCTION FERTILITY AND DEVELOPMENT   23 ( 3 )   424 - 432   2011

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    The primary aim of the present study was to examine the effect of maternal age (in months) on mitochondrial DNA copy number (Mt number), ATP content and IVF outcome of bovine oocytes. We also compared the Mt number of oocytes with fertilisation outcome and ATP content. Oocytes were collected from cows aged 20-204 months and the Mt number was determined by real-time polymerase chain reaction. The Mt number in immature and mature oocytes was determined to be 368 118 and 807 794, respectively; the ATP content in these oocytes was 1.2 and 2.0 pM, respectively. Both Mt number and ATP content increased during oocyte maturation. However, after 90 months of age, the Mt number of mature oocytes decreased with increasing maternal age, whereas the ATP content of mature oocytes was positively correlated with maternal age (P&lt;0.01); there was no obvious relationship observed between Mt number and ATP content. Furthermore, maternal age was positively correlated with the abnormal fertilisation rate (P&lt;0.01). Mt number and fertilisation outcome were unrelated, but the nature of this relationship differed between young (21-89 months) and old (&gt;89 months) cows. Thus, we conclude that Mt number, the ATP content and fertilisation outcome of bovine oocytes are affected by maternal age.

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  • Expression of bovine interferon-tau variants according to sex and age of conceptuses Reviewed

    Angela M. Walker, Koji Kimura, R. Michael Roberts

    THERIOGENOLOGY   72 ( 1 )   44 - 53   2009.7

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    Interferon-tau (IFNT), which plays a major role in maternal recognition of pregnancy in cattle, is transcribed from multiple genes. Moreover, there are at least 12 cDNA variants, many presumably allelic. Although the IFNT locus is autosomal, Day 8 female blastocysts produced approximately twice as much antiviral activity as males. The questions addressed here are whether male and female blastocysts differed in the kind and number of IFNT they expressed, and whether this pattern changed over development. Day 8, in vitro-produced blastocysts were bisected, and one half of each was sexed by PCR. Demi-embryos (n = 64) were grouped according to whether they were male or female, to provide two pools of each sex. Individual cDNA were sequenced after RT-PCR amplification and shot-gun cloning to provide comparisons between male and female blastocysts, elongating conceptuses of various developmental ages (Days 14-19), and a female trophoblast cell line (CT-1). A total of 376 cDNA clones were sequenced. Six additional cDNA were identified, in addition to the forms described earlier. There were no differences between male and female blastocysts (P = 0.54), and between blastocysts and a trophoblast model system (CT-1 cells; P = 0.24) in the IFNT transcripts expressed, indicating that sexual dimorphism wass not correlated with particular IFNT variants. There were differences in variant frequencies (P &lt; 0.001) among conceptuses of different age, although two, representing boIFN1a and boIFN3c, predominated throughout development. Notably, no alteration in overall IFNT variant diversity was detected in CT-1 cells over time (P = 0.124). (C) 2009 Elsevier Inc. All rights reserved.

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  • Food Deprivation Induces Monocarboxylate Transporter 2 Expression in the Brainstem of Female Rat Reviewed

    Shuichi Matsuyama, Satoshi Ohkura, Kinuyo Iwata, Yoshihisa Uenoyama, Hiroko Tsukamura, Kei-ichiro Maeda, Koji Kimura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   55 ( 3 )   256 - 261   2009.6

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    Ketone bodies are considered to act as a signal to suppress gonadotropin release during negative energy balance. The present study examined the effect of 48-h fasting on the mRNA expressions of monocarboxylate transporter 1 (MCT1) and MCT2, which are involved in ketone body transport, in several brain regions. Quantitative real-time RT-PCR analysis showed that the MCT2 mRNA levels were significantly increased by 48-h fasting in the area postrema-solitary tract nucleus (AP-NTS) region but not the arcuate nucleus-ventromedial hypothalamic nucleus (ARC-VMH) and central gray-supragenual nucleus around the 4th ventricle (CG-SGe) regions. Fasting did not significantly affect MCTI mRNA expression in any of the brain areas examined. Luteinizing hormone (LH) pulse frequency significantly decreased and plasma concentrations of beta-hydroxybutyric acid, a ketone body, significantly increased after 48-h fasting. The present results suggest that increased uptake of ketone bodies via MCT2 in the AP-NTS region is likely involved in the mechanism of fasting-induced suppression of LH secretion in rats.

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  • ラット脳におけるMCT1およびMCT2遺伝子発現に及ぼす絶食の影響

    松山 秀一, 大蔵 聡, 上野山 賀久, 束村 博子, 前多 敬一郎, 木村 康二

    The Journal of Reproduction and Development   54 ( Suppl. )   j107 - j107   2008.8

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  • Nutritional skewing of conceptus sex in sheep: effects of a maternal diet enriched in rumen-protected polyunsaturated fatty acids (PUFA) Reviewed

    Mark P. Green, Lee D. Spate, Tina E. Parks, Koji Kimura, Clifton N. Murphy, Jim E. Williams, Monty S. Kerley, Jonathan A. Green, Duane H. Keisler, R. Michael Roberts

    REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY   6   21   2008.6

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    Background: Evolutionary theory suggests that in polygynous mammalian species females in better body condition should produce more sons than daughters. Few controlled studies have however tested this hypothesis and controversy exists as to whether body condition score or maternal diet is in fact the determining factor of offspring sex. Here, we examined whether maternal diet, specifically increased n-6 polyunsaturated fatty acid (PUFA) intake, of ewes with a constant body condition score around the time of conception influenced sex ratio.
    Methods: Ewes (n = 44) maintained in similar body condition throughout the study were assigned either a control ( C) diet or one ( F) enriched in rumen-protected PUFA, but otherwise essentially equivalent, from four weeks prior to breeding until d13 post-estrus. On d13, conceptuses were recovered, measured, cultured to assess their capacity for interferon-tau (IFNT) production and their sex determined. The experiment was repeated with all ewes being fed the F diet to remove any effects of parity order on sex ratio. Maternal body condition score (BCS), plasma hormone and metabolite concentrations were also assessed throughout the study and related to diet.
    Results: In total 129 conceptuses were recovered. Ewes on the F diet produced significantly more male than female conceptuses ( proportion male = 0.69; deviation from expected ratio of 0.5, P &lt; 0.001). Conceptus IFNT production was unaffected by diet ( P &gt; 0.1), but positively correlated with maternal body condition score ( P &lt; 0.05), and was higher ( P &lt; 0.05) in female than male conceptuses after 4 h culture. Maternal plasma hormone and metabolite concentrations, especially progesterone and fatty acid, were also modulated by diet.
    Conclusion: These results provide evidence that maternal diet, in the form of increased amounts of rumen-protected PUFA fed around conception, rather than maternal body condition, can skew the sex ratio towards males. These observations may have implications to the livestock industry and animal management policies when offspring of one sex may be preferred over the other.

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  • Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

    H. Iwata, H. Shiono, Y. Kon, K. Matsubara, K. Kimura, T. Kuwayama, Y. Monji

    Animal Reproduction Science   105 ( 3-4 )   234 - 244   2008.5

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    The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (&gt
    5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%
    P &lt
    0.05), and the highest rate of normal fertilization was observed for 5 h culture (73.4%
    P &lt
    0.05). The sex ratio was significantly (P &lt
    0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1 h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP
    P &lt
    0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%
    P &lt
    0.05
    experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1 h (treatment 5) or 5 h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively
    P &lt
    0.05), and the 1 h co-incubation significantly skewed (P &lt
    0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5 h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%
    P &lt
    0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9
    1.0 mg/ml treatment 10) significantly (P &lt
    0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes. © 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.anireprosci.2007.03.006

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  • The effect of glucosamine concentration on the development and sex ratio of bovine embros Reviewed

    Koji Kimura, Hisataka Iwata, Jeremy G. Thompson

    ANIMAL REPRODUCTION SCIENCE   103 ( 3-4 )   228 - 238   2008.1

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    Glucosamine is a component of hyaluronic acid and an altenative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP-N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5 mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal-zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-alpha -D-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT. (c) 2006 Elsevier B.V. All rights reserved.

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  • Successful superovulation of cattle by a single administration of FSH in aluminum hydroxide gel Reviewed

    Koji Kimura, Makoto Hirako, Hisataka Iwata, Mari Aoki, Mamoru Kawaguchi, Makoto Seki

    THERIOGENOLOGY   68 ( 4 )   633 - 639   2007.9

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    We investigated whether Al-gel could adsorb and release FSH effectively in vitro and in vivo, and whether a single administration of FSH in Al-gel could successfully induce superovulation (SOV) in cattle. Porcine FSH (pFSH; 30 mg) was mixed with 5 mL of Al-gel; 99.98 +/- 0.01% of pFSH was adsorbed by the gel and 71.6 +/- 1.1% of the adsorbed pFSH was subsequently released in the presence of BSA. In cattle given a single i.m. treatment of 30 mg of pFSH in 5 mL of At-gel, the numbers of CL, total ova recovered, and transferable embryos per cow were not significantly different from conventional (twice daily for 4 d) pFSH treatment (12.3 +/- 1.7 versus 11.7 +/- 1.8, 10.0 +/- 2.5 versus 9.3 +/- 1.7, and 8.6 +/- 2.3 versus 8.0 +/- 1.8, respectively, mean +/- S.E.M.); plasma pFSH concentrations were increased for 4 d, indicating sustained release from the Al-gel. Five cows were given 30 mg pFSH in 5 mL of Al-gel i.m. on five occasions (once every 2-3 months); there was no significant difference among treatments for the number of CL (12.4 +/- 3.8, 13.8 +/- 4.8, 9.0 +/- 1.9, 9.8 +/- 3.0, 12.0 +/- 2.1), total ova recovered (12.0 +/- 3.8, 12.6 +/- 5.1, 6.8 +/- 1.9, 7.6 +/- 1.8, 11.4 +/- 2.5), and transferable embryos (11.4 +/- 3.9, 10.4 +/- 5.8, 6.6 +/- 2.1, 4.8 +/- 1.4, 10.4 +/- 2.6). In conclusion, a single i.m. treatment of 30 mg pFSH in 5 mL Al-gel effectively induced SOV in cattle. (c) 2007 Elsevier Inc. All rights reserved.

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  • Responsiveness of rabbits to superovulation treatment by a single injection of follicle-stimulating hormone with aluminum hydroxide gel Reviewed

    Shu Hashimoto, Koji Kimura, Takashi Kuramochi, Kazuki Aoyagi, Makoto Hirako, Mamoru Kawaguchi, Hisataka Iwata, Masao Hirao, Kensaku Kitada, Kazuo Hirasawa, Masatsugu Ueda

    MOLECULAR REPRODUCTION AND DEVELOPMENT   74 ( 9 )   1208 - 1212   2007.9

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    Aluminum hydroxide gel (Al-gel), which is used as an adjuvant, can absorb macromolecules. We investigated the applicability of Al-gel to the sustained release of follicle-stimulating hormone (FSH) as a simplified method of superovulation (SOV) in rabbits. The responsiveness of rabbits to SOV by a single injection of FSH dissolved in Al-gel suspension (3.2 mg Al/ml) and in 10% (w/v) polyvinylpyrrolidone (PVP), and by multiple injections of FSH in saline was examined. The numbers of total and fertilized eggs recovered from rabbits treated with FSH in Al-gel (40.5 and 26.3, respectively) were similar to multiple injections (47.4 and 28.6, respectively) and were significantly greater (P &lt; 0.05) than single injection of FSH with PVP (17.3 and 11.5, respectively). We also compared the plasma FSH levels of rabbits which were induced SOV by multiple or a single injection of Al-gel. Al-gel provided sustained release of FSH to the blood stream at a high enough dose for SOV. Moreover, the developmental competence of the pups of DNA-injected embryos from rabbits treated with a single injection of FSH mixed with Al-gel (18%) was similar to that of DNA-injected embryos, recovered from rabbits treated with FSH dissolved in saline (21%). Two transgenic pups were obtained from embryos recovered from rabbits by a single injection of FSH with Al-gel. These results indicate that a single injection of FSH with Al-gel is an effective method for SOV of rabbit and that this technique is applicable to research requiring large numbers of rabbit embryos such as the production of transgenic rabbits.

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  • The effect of glucose on the progression of the nuclear maturation of pig oocytes

    H. Sato, H. Iwata, T. Hayashi, K. Kimura, T. Kuwayama, Y. Monji

    Animal Reproduction Science   99 ( 3-4 )   299 - 305   2007.6

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    The progression of the nuclear maturation of oocytes is a useful marker for the estimation of the subsequent developmental competence of oocytes. In this study, we examined the effect of energy substrates in an in vitro maturation medium on the progression of the nuclear maturation of oocytes. In experiment 1, the supplementation of the maturation medium with 0, 5 and 10 mM of glucose lead to increase in the total cell number of the blastocysts. In experiments 2 and 3, the maturation phase was divided into two stages (germinal vesicle (GV) stage: 0-20 h and nuclear maturation stage: 20-44 h), and the effects of glucose or pyruvate added at each stage on the kinetics of nuclear maturation were examined. The addition of glucose at the nuclear maturation stage rather than at the GV stage of maturation effected greater acceleration in the progression of nuclear maturation. However, the addition of pyruvate at both stages had the same effect on the progression of nuclear maturation was the same. In addition, when glucose was added to the medium containing pyruvate, an additive effect on the progression of nuclear maturation was observed (experiment 4). In experiment 5, the inhibitors of glucose-6-phosphate dehydrogenase (G6PD), dehydroepiandrosterone (DHEA) and 6-aminonicotinamide (6-AN) decreased the rate of the final maturation of oocytes and reduced the difference between the rates of the final maturation of oocytes cultured with glucose and those cultured with pyruvate. In the experiment 6, when the activator of G6PD, brilliant cresyle blue (BCB), was added to the maturation medium, the progression of nuclear maturation was significantly accelerated. The results of this study suggested that in addition to the role of an energy substrate, glucose or its metabolites play a role in nuclear maturation. This role was more pronounced at the second stage of maturation (transition from GV breakdown (GVBD) to M2), probably due to the metabolism of glucose via the pentose phosphate pathway (PPP) rather than the glycolysis pathway. © 2006 Elsevier B.V. All rights reserved.

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  • Links between de novo fatty acid synthesis and leptin secretion in bovine adipocytes Reviewed

    Hiroya Kadokawa, Katsuhiro Aikawa, Koji Kimura, Domique Blache, Ian H. Williams, Graeme B. Martin

    JOURNAL OF VETERINARY MEDICAL SCIENCE   69 ( 3 )   225 - 231   2007.3

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    Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.

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  • 黒毛和種牛における卵胞刺激ホルモン皮下1回投与による過剰排卵誘起法 第1報 水酸化アルミニウムゲル吸着卵胞刺激ホルモン投与牛における血漿中卵胞刺激ホルモン濃度の推移および過剰排卵誘起効果 Reviewed

    磯崎良寛, 稲田淳, 浅田研一, 古賀鉄也, 木村康二, 角川博哉, 平子誠

    福岡県農業総合試験場研究報告   26 ( 1 )   61 - 64   2007

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  • Superovulation treatment by a single injection of FSH with aluminum hydroxide gel in Holstein cattle Reviewed

    S. Matoba, M. Tagawa, H. Matsuda, H. Yoshioka, K. Kimura, H. Kadokawa, M. Yonai, K. Imaia

    REPRODUCTION FERTILITY AND DEVELOPMENT   19 ( 1 )   309 - 310   2007

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  • ウシ胚盤胞期胚のcDNAライブラリー構築法 Reviewed

    塚本智史, 今市寿史, 木村康二, 今井 裕, 南 直治郎

    日本胚移植学雑誌 = Japanese journal of embryo transfer   28 ( 3 )   118 - 123   2006.9

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  • Influence of feeding regime on timing of parturition in beef cattle and the relationship of vaginal temperature to parturition Reviewed

    Mari Aoki, Koji Kimura, Osamu Suzuki

    Animal Science Journal   77 ( 3 )   290 - 299   2006.6

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    The timing of parturition was recorded for a total of 56 beef cattle (Japanese Black x Holstein Friesian) on different dietary treatments. The rate of calving during daylight hours in cows night-fed (18.00 hours) with a roughage diet was significantly higher than that in cows night-fed with a high concentrate diet (79.2% vs 38.5%, P &lt
    0.05). Subsequently, the vaginal temperature (VT) of these cows was analyzed using a cosinor method. When the feeding schedule was changed from twice daily (08.30 and 15.30 hours) to night feeding, the periodicity, the acrophase and the bathyphase, which were the parameters of the cosine curve, were unstable from the first day of night feeding until after day 6 (P &lt
    0.05). Prior to parturition, the midline-estimating statistic of rhythm (MESOR) and the amplitude for the cows that were fed a high-roughage diet at night and that calved at night-time were lower and larger, respectively, than that for the other treatments (P &lt
    0.01). Based on these results, the time of parturition in most of the beef cows was influenced by feeding time and diet composition. Those cows that calved at night-time in spite of night feeding had lower vaginal temperatures. © 2006 Japanese Society of Animal Science.

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  • Vascular endothelial growth factor has beneficial effect independent of serum components throughout oocyte maturation and early embryonic development in cattle Reviewed

    HL Luo, K Kimura, M Hirako

    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES   19 ( 4 )   495 - 499   2006.4

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    In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p &lt; 0.05) and FBS+VEGF (42.1%, p &lt; 0.05) groups than in the PVA group (17.1%). Two-factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

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  • Comparison between the characteristics of follicular fluid and the developmental competence of bovine oocytes

    H Iwata, J Inoue, K Kimura, T Kuge, T Kuwayama, Y Monji

    ANIMAL REPRODUCTION SCIENCE   91 ( 3-4 )   215 - 223   2006.2

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    There are great differences in the developmental competence of oocytes collected from individual cows. Oocytes grow and mature in the follicular fluid (FF). In the present study, characteristics of the FF of each ovary and the developmental competence of enclosed oocytes were investigated, and these data were then compared. A total of 37 pairs of ovaries were collected from beef heifers. The concentration of magnesium (Mg), aspirate aminotransferase (AST), nonesterified fatty acids (NEFA), and lactate dehydrogenase (LDH) in the FF were great compared with serum standard. Several significant correlations among these characteristics were detected. Forty-eight hours after fertilization, the stage of embryo development at an advanced developmental stage (&gt;6 cell stage) is related to the rate of blastulation 8 days after fertilization. In addition, a significantly positive or negative correlation was observed between the developmental competence (the rate of cleavage in the embryo and blastulation) and the concentration of the icterus index (ICT) or blood urea nitrogen (BUN) in the FF. In conclusion, the quality of oocytes is affected by the environment in the follicle, and BUN or ICT is a predictable index of the developmental competence of oocytes. (c) 2005 Elsevier B.V. All rights reserved.

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  • Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

    H. Iwata, T. Hayashi, H. Sato, K. Kimura, T. Kuwayama, Y. Monji

    Zygote   13 ( 4 )   303 - 308   2005.11

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    During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35°C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20°C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period. © 2005 Cambridge University Press.

    DOI: 10.1017/S0967199405003412

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  • Effects of D-glucose concentration, D-fructose, and inhibitors of enzymes of the pentose phosphate pathway on the development and sex ratio of bovine blastocysts Reviewed

    K Kimura, LD Spate, MP Green, RM Roberts

    MOLECULAR REPRODUCTION AND DEVELOPMENT   72 ( 2 )   201 - 207   2005.10

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    D-glucose at 5.6 mM reduces the progression of in vitro-produced (IVP) bovine embryos from the morula to the blastocyst stage and skews sex ratio towards males. Possibly, the presence of two transcriptionally active X-chromosomes compromises female embryo development through imbalance in glucose metabolism. Here, we have determined the threshold of embryo sensitivity to glucose, whether substitution Of D-fructose for glucose reduces the selective loss of female embryos, and whether inhibition of an X-linked gene product, glucose 6-phosphate dehydrogenase (G6PD), normalizes sex ratio among bovine blastocysts. IVP zygotes were cultured in glucose-free medium to 72 hr post-insemination, at which time 8-cell embryos were selected for treatment and cultured until 144 hr post-insemination. Addition of 4 mM glucose at the 8-cell stage reduced (P &lt; 0.05) the number of blastocyst that formed, whereas 2.5 and I mM glucose had no effect. Sex ratio in the presence of 4 and 2.5 mM glucose differed significantly from 0.5, while 1 mM glucose had no effect. D-fructose (5.6 mM) did not compromise development to blastocyst and did not alter the sex ratio of blastocysts that formed. When G6PD inhibitors (dehydroepiandrosterone: DHEA and 6-aminonicotinamide: 6-AN), which effectively inhibit the reduction of the NADPH-sensitive dye, brilliant cresyl blue (BCB) in bovine morulae, were added to the culture medium containing 4 mM glucose, embryo development was moderately reduced, but sex ratio skewing was corrected (with 6-AN) or lowered (with DHEA). In conclusion, glucose above 2.5 mM impairs bovine embryo development and increases sex ratio, most likely as a result of increased pentose-phosphate (PP) pathway activity in female embryos.

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  • Comparison of the concentrations of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in maternal and fetal blood, amniotic and allantoic fluids in cattle Reviewed

    M Hirako, M Aoki, K Kimura, Y Hanafusa, H Ishizaki, Y Kariya

    REPRODUCTIVE TOXICOLOGY   20 ( 2 )   247 - 254   2005.7

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    To characterize the maternal-fetal transport of lipophilic endocrine disrupting chemicals, concentrations of polychlorinated (2,3,7,8-substituted) dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) were measured in maternal and fetal blood, and amniotic and allantoic fluids in cattle. Total toxicity equivalent quantity (TEQ) was highest in amniotic fluid on a fat-weight basis, whereas it was highest in maternal blood on a total weight basis. TEQ was lowest in allantoic fluid on either basis; 26 of 29 congeners analyzed in this experiment were detected in one or more samples. The largest number of congeners was detected in amniotic fluid. O8CDD, 2,3,4,7,8-P5CDF and 2,3',4,4',5-P5CB were the major congeners in PCDDs, PCDFs and PCBs, respectively. The O8CDD concentration was higher in fetal blood than in maternal blood on a fat-weight basis, whereas concentrations of other congeners were lower in fetal blood than in maternal blood. Furthermore, on a fat-weight basis, the O8CDD concentration was considerably higher in allantoic fluid compared with other samples. Concentrations of major PCB congeners were higher in amniotic fluid than in maternal and fetal blood on a fat-weight basis. In conclusion, it is suggested that lipophilic endocrine-disrupting chemicals contained in maternal blood are all transferred to the fetal circulation via the placenta in cattle. Furthermore, the results of this experiment imply that O8CDD has different transportation systems from other dioxins in the circulation, and that a considerable amount of PCBs is excreted and accumulated in amniotic fluid during the fetal stage in cattle. (c) 2005 Elsevier Inc. All rights reserved.

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  • Predicting time of parturition from changing vaginal temperature measured by data-logging apparatus in beef cows with twin fetuses Reviewed

    M Aoki, K Kimura, O Suzuki

    ANIMAL REPRODUCTION SCIENCE   86 ( 1-2 )   1 - 12   2005.3

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    Vaginal temperatures (VT) of crossbred (Japanese Black crossed Holstein-Friesian) beef cows (n = 31) were measured by a data-logging apparatus to obtain serial data from days 0 to 6 before parturition. For both single and twin pregnancies, no significant differences were observed in VT during days 3-6 before parturition. Maternal VT was not affected by maternal weight just after parturition, parity, fetal sex, or total fetal litter weight. Average of twin litter weights for two males (MM) and two females (FF) had the strong positive correlations (r = 0.84; P &lt; 0.05) with maternal VT, whereas twin weights of mixed-gender twins (FM) did not correlate with maternal VT (r = -0.26; P = 0.61). Maternal temperature decreased as weights of the female fetus of FM twins became heavier (r = -0.82; P &lt; 0.05). In contrast, maternal VT of FF and MM twins increased as twin weights increased. We defined when the VT began to decrease before parturition by two different methods. One was the "same hours method" where differences in VT between a particular time of day and the corresponding time of the preceding day were compared when the VT was consistently &gt;= 0.3 or &gt;= 0.5 degrees C for more than 3 h. The second method was the "maximum-minimum method" where decreased in the maximum or the minimum values of the day over &gt;= 0.3 and &gt;= 0.5 degrees C were compared to values of preceding day. Onset of decreased VT before expulsion was not different between singletons and twins. In an attempt to define the critical condition in predicting parturition, we estimated assumable predicting probability using the 31 cows that were collected VT. When the parturition occurred within 60 It in the "same hours method" and 72 h since VT was &gt;= 0.3 degrees C and in the "maximum-minimum method", the assumable probability was 100%. In verification experiment under these condition, the "same hours method" had a higher probability of predicting the time of parturition than the "maximum-minimum method", and it was possible to detect the onset of decreased VT at the correct time by the minutes. We concluded that "same hours method" was useful for predicting parturition time in cattle with single and twin pregnancies by the serial measurement of vaginal temperature. (c) 2004 Elsevier B.V. All rights reserved.

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  • 黒毛和種のワンショット過剰排卵処理法の確立に関する研究 Reviewed

    林登, 加藤誠二, 坂口慎一, 澤田幹夫, 関誠, 竹内昭司, 木村康二, 角川博哉

    岐阜県畜産研究所研究報告   5 ( 5 )   30 - 34   2005

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    牛の過剰排卵処理法の簡易化・省力化を図るため、水酸化アルミニウムゲルを卵胞刺激ホルモン(FSH)の溶解剤として用いたFSHの1回投与(ワンショット法)による過剰排卵処理法の有効性を検討した。黒毛和種経産牛9頭を用い、1頭の牛について水酸化アルミニウムゲルを用いたワンショット法(30AU 1回、筋肉内投与)と従来の漸減投与法(5,5,3,3,2,2,計6回、20AU、筋肉内投与)による過剰排卵処理をそれぞれ1回ずつ実施し、発情の状況、採卵時の黄体数、卵胞数および採卵成績を比較した。1.ワンショット区では9頭中8頭に、漸減投与区では9頭中全頭に明瞭な発情が観察された。2.採胚時の平均黄体数、大卵胞数は、ワンショット区で、それぞれ8.7土5.3個、2.9土1.5個、漸減投与区で9.6土5.7個、1.9土1.7個でいずれも有意な差は認められなかった。3.平均回収卵数、正常卵数は、ワンショット区でそれぞれ6.7±6.4個、4.1±3.4個、漸減投与区で8.6土5.8個、4.1±2.8個でいずれも有意な差は認められなかった。以上の結果から、黒毛和種において、水酸化アルミニウムゲルを用いたFSHの1回投与による過剰排卵処理を行うことにより、従来の漸減投与法と比較し遜色ない採卵成績が得られ、この方法が過剰排卵処理法の節易化、省力化を図るための技術として利用可能であることが示唆された。

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  • Effects of oxidative stress and inhibitors of the pentose phosphate pathway on sexually dimorphic production of IFN-tau by bovine blastocysts Reviewed

    K Kimura, LD Spate, MP Green, RM Roberts

    MOLECULAR REPRODUCTION AND DEVELOPMENT   68 ( 1 )   88 - 95   2004.5

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    Bovine interferon-tau (IFN-tau), the anti-luteolytic factor secreted by conceptuses of pecoran ruminants, is a product of autosomal genes, yet in vitro produced (IVP) female expanded blastocysts (EB) secrete about twice as much IFN-tau as males. Two possible explanations have been tested here. One is that embryos of one sex are differentially susceptible to oxidative stress. The second is that female EB produce more IFN-tau because pentose-phosphate pathway (PPP) activity is elevated as a result of delayed X-chromosome inactivation. IVP bovine zygotes were cultured to the 8-cell stage and placed under conditions designed either to promote oxidative stress (+/-H2O2; 20 vs. 5% O-2), or to inhibit glucose 6-phosphate dehydrogenase (G6PDH) activity (addition of dehydroepiandrosterone, DHEA or 6-aminonicotinamide, 6-AN to the medium). At day 8, blastocysts were cultured individually for a further 48 hr to assess IFN-tau production, and embryo sex determined retrospectively. Blastocyst numbers were reduced (P &lt; 0.05) and their continued development impaired (P &lt; 0.05) in presence of H2O2 (200 muM) and 20% O-2, but neither IFN-tau production nor sexually dimorphic expression of IFN-tau were affected. IFN-tau production was reduced, particularly in females (P &lt; 0.05), and sexual dimorphic differences in production were lost in the presence of both DHEA (100 muM) and 6-AN (1 muM). In the case of 6-AN, these effects were achieved without a significant decline in blastocyst developmental progression, quality, or cell number. The data suggest that the higher production of IFN-tau by female EB is an indirect outcome of the increased activity of the oxidative arm of the PPP pathway. (C) 2004 Wiley-Liss, Inc.

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  • Effects of follicle size and electrolytes and glucose in maturation medium on nuclear maturation and developmental competence of bovine oocytes Reviewed

    H Iwata, S Hashimoto, M Ohota, K Kimura, K Shibano, M Miyake

    REPRODUCTION   127 ( 2 )   159 - 164   2004.2

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    The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0-2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5-3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2-3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.

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  • Sexual dimorphism in interferon-tau production by in vivo-derived bovine embryos Reviewed

    K Kimura, LD Spate, MP Green, CN Murphy, GE Seidel, RM Roberts

    MOLECULAR REPRODUCTION AND DEVELOPMENT   67 ( 2 )   193 - 199   2004.2

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    Interferon-tau (IFN-tau) is an antiluteolytic factor responsible for preventing regression of the maternal corpus luteum (CL) during early pregnancy of cattle. In vitro-produced (IVP) bovine embryos first produce IFN-tau as blastocysts. In the present study, we have examined whether sexually dimorphic production of IFN-tau, which is observed among IVP blastocysts, also occurs among in vivo-produced blastocysts, and whether this difference between the sexes persists to day 14 when silencing of one of the X-chromosomes in the trophectoderm is complete. Embryos were flushed from cattle that had been superovulated and bred by AI. Blastocysts (63 male, 62 female) recovered between days 8.5 and 9.5 of pregnancy, were cultured individually. No differences were observed between males and females in either their developmental stage or quality at the beginning, during, and at the end of culture. Female embryos produced more IFN-tau than males by 24 hr (mean values, males: 16.6 +/- 3.7, females: 49.4 +/- 9.0 pg per embryo; P &lt; 0.05) and 48 hr (male: 189.8 +/- 37, 1, female: 410.9 +/- 66.6 pg per embryo; P&lt;0.05). However, the variability in IFN-tau production between individual blastocysts was so great that IFN-tau secretion is unlikely to be of value as a non-invasive means to predict embryo sex. When conceptuses were recovered at day 14, elongating males (n = 25) and females (n = 24) were similar in dimension and did not differ in their IFN-tau production after 4.5 hr (male: 2,550 +/- 607, female: 2,376 +/- 772 ng per conceptus) and 24 hr (male: 12,056 +/- 2,438, female: 8,447 +/- 1,630 ng per conceptus) of culture. Thus, sexual dimorphism in IFN-tau production is observed in both IVP and in vivo-produced expanded blastocysts, but is lost by day 14 of in vivo development. (C) 2004 Wiley-Liss, Inc.

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  • Effect of growth hormone at various periods of development on developmental competence of bovine embryo and its interaction with glucose

    Iwata H, Ohota M, Hashimoto S, Kimura K, Isaji M, Miyake M

    Journal of Reproduction and Development 49 6巻 (2003) 493-499   2003.12

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    胚の体外培養を改善するため、FCSやBSAの血清蛋白を用いた培養条件でGrowth Hormon の添加とグルコースの濃度の胚発生に及ぼす影響について発生ステージごとに検討した。FCS添加条件下でGHは桑実胚以降において発生を促進しすること、またGHの効果は培養条件中のグルコースの濃度と相互効果が見られること、桑実胚以降のグルコース濃度の上昇は胚発生促進には効果がないこと等を示した。

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  • Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro Reviewed

    H Iwata, M Ohota, S Hashimoto, K Kimura, M Isaji, M Miyake

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   49 ( 6 )   493 - 499   2003.12

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    Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.

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  • Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air

    S Hashimoto, K Kimura, H Iwata, R Takakura

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   49 ( 1 )   61 - 66   2003.2

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    The effects of the medium (TCM 199 or SCFaa) and temperature (20 or 39 Q during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 mug/ml CHX in a 50-mul droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P&lt;0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 mu l HSOFaa or as a group (40-50 oocytes) with 170-200 mu l HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-mu l droplet of HSCFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-mu l droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

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  • Vascular endothelial growth factor (VEGF) promotes the early development of bovine embryo in the presence of cumulus cells Reviewed

    HL Luo, K Kimura, M Aoki, M Hirako

    JOURNAL OF VETERINARY MEDICAL SCIENCE   64 ( 11 )   967 - 971   2002.11

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    Three experiments were conducted to determine the effect of Vascular Endothelial Growth Factor (VEGF) on bovine embryonic development in vitro. Human recombinant VEGF(165) was employed at 5 ng/ml in modified synthetic oviduct fluid. In Exp. 1, bovine cumulus oocyte complexes were matured with or without VEGF for 22 hr, inseminated without VEGF for 6 hr, then cultured with or without VEGF for 42 hr. The cleavage rate and the development rate to 4- to 8-cell were higher (P&lt;0.05) in groups with VEGF during in vitro maturation (IVM, 71.4% and 59.6%), in vitro culture (IVC, 70.3% and 62.3%), and both IVM and IVC (75.9% and 67.8%) than in the group cultured thoroughly without VEGF (49.9% and 38.4%, respectively). In Exp. 2, 4- to 8-cell embryos produced in vitro without VEGF were removed from cumulus cells at 48 hr post-insemination (Pi) and cultured with or without VEGF for 144 hr. The development rates to blastocyst at 96 hr (D6), 120 hr (D7) and 144 hr (D8) were similar (P&gt;0.05) in both groups. In Exp. 3, cumulus cells were removed from presumptive embryos produced by IVM and IVF without VEGF at 10 hr Pi. Denuded embryos were cultured with or without VEGF for 38 hr or 182 hr. The cleavage rate and the development rates to 4- to 8-cell at 48 hr Pi and to blastocyst on D6, D7 and D8 were similar (P&gt;0.05) in all groups. These results suggest that VEGF has a beneficial effect on the initial development of bovine embryo through surrounding cumulus cells.

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  • Glucose-6-phosphate dehydrogenase activity of IVM/IVF/IVC bovine embryos is related to the sex and developmental competence under suboptimal gas condition

    Iwata H, Kimura K, Hashimoto S, Oota M, Tominaga T, Minami N

    Journal of Reproduction and Development   5   447 - 453   2002.10

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    Brilliant Cresyl Blueを用いて発生各ステージの体外受精卵の還元力の違いと性比との関係について検討した。その結果桑実胚において還元力の差が雄と雌との間で異なること、8細胞期や胚盤胞期では差がないことを示した。このことより桑実期胚ではX染色体上にあるG6PDの活性に差があり雌雄判別に利用できることやX染色体の不活化が胚盤胞期胚以降で起こっていることを示唆した。

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  • Effect of Vascular Endothelial Growth Factor on maturation, fertilization and developmental competence of bovine oocytes Reviewed

    HL Luo, K Kimura, M Aoki, M Hirako

    JOURNAL OF VETERINARY MEDICAL SCIENCE   64 ( 9 )   803 - 806   2002.9

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    To examine the effect of Vascular Endothelial Growth Factor (VEGF) on the maturation of bovine oocytes, human recombinant VEGF(165) was used in 3 experiments. In Exp. 1, bovine cumulus oocyte complexes (COCs) were matured for 22 hr in modified Synthetic Oviduct Fluid (m-SOF) supplemented with 0 (control) or 5 ng/ml of VEGF. Maturation rate increased (P&lt;0.05) from 78.2% in the control to 90.5% in the VEGF treated group. In Exp. 2, bovine COCs were matured in m-SOF and co-incubated with sperm in modified BO medium, each supplemented with or without 5 ng/ml VEGF. Normal fertilization rate was improved (P&lt;0.05) from 63.0% (control) to 79.8% or 82.3% with VEGF during maturation or both maturation and fertilization. In Exp. 3, bovine COCs were matured the same way as in Exp. 1, then co-incubated with sperm for 6 hr and cultured for 162 hr in m-SOF without VEGF. Cleavage rate and development rate to the 4- to 8-cell stage were examined at 42 hr post-co-incubation and development rate to blastocyst was examined at 162 hr post-co-incubation. Cleavage, the development to the 4- to 8-cell stage and blastocyst rates (82.0%, 70.3% and 45.1%, respectively) were significantly higher (P&lt;0.05) in the VEGF group than those in the control (67.3%, 52.5% and 33.3%, respectively). These results indicate that VEGF has a beneficial effect on the maturation of bovine oocytes.

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  • Role of G6PD activity on sex ratio and developmental competence of bovine bnbryos under oxidative stress Reviewed

    Hisataka Iwata, Koji Kimura, Syu Hashimoto, Maiko Ohta, Keiichirou Tominaga, Naojirou Minami

    Journal of Reproduction and Development   48 ( 5 )   447 - 453   2002

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    Inactivation of the X-linked gene in the female embryo is one of the major events during mammalian early embryogenesis. Before this inactivation, enzyme activity encoded by the X-linked gene is different between male and female embryos. In the present study, we demonstrated a possibility that there may be a different response to specific culture conditions in vitro between male and female early embryo. Glucose-6-phosphate dehydrogenase (G6PD) is one of the X-linked enzymes and its activity is semi-quantitated by brilliant cresyl blue (BCB). In experiment 1, the relationship between semi-quantitated G6PD activity and the sex ratio of the embryos was examined. In experiment 2, the relationship between semi-quantitated G6PD activity and the developmental competence of embryos was examined. No relationship was found between G6PD activity and sex in 8-cell, 16-cell and blastocyst stage embryos. However, G6PD activity was high in female embryos and low in male embryos at the morula stage. When morula stage embryos were categorized by BCB and cultured under high oxygen tension (5% CO2, 95% air), the developmental competence of embryos having high G6PD activity was higher than those having low enzyme activity. However, when the categorized morula stage embryos were cultured under low oxygen tension (5% CO2,5% O2, 90% N2), there was no difference in the developmental rates among the categories. These results indicate that G6PD activity is high in female morula stage embryos compared with that in male embryos, and that the activity of this enzyme strongly affects the developmental competence of morula stage embryo when they are cultured under high oxygen tension.

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  • Sexual dimorphism among bovine embryos in their ability to make the transition to expanded blastocyst and in the expression of the signaling molecule IFN-τ Reviewed

    Melissa A. Larson, Koji Kimura, H. Michael Kubisch, R. Michael Roberts

    Proceedings of the National Academy of Sciences of the United States of America   98 ( 17 )   9677 - 9682   2001.8

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    IFN-τ is a secretory product of trophectoderm of cattle, sheep, and their relatives and is expressed for a few days in early pregnancy after the blastocyst first forms. It serves to alert the mother that she is pregnant. A delayed or less than robust IFN-τ signal is a likely cause of embryonic loss. Here we have determined whether blastocyst production of IFN-τ, which is encoded by a cluster of genes on chromosome 9, differs between the sexes in cattle, as assessed by culture of in vitro-derived embryos on two different media, one complex (tissue culture medium 199 supplemented with serum) with coculture support, the other relatively simple (synthetic oviductal fluid plus albumin). With both media, female blastocysts produced approximately double the amount of IFN-τ as males, regardless of such variables as oocyte batch, blastocyst quality, hatching, and length of time in culture. However, in either tissue culture medium 199, which contains 5.5 mM D-glucose, or in synthetic oviductal fluid, in the presence but not in the absence of added glucose, significantly fewer female than male embryos were able to progress from the morula/early blastocyst stage to more advanced stages of development. It is possible that the differences between male and female embryos both in their production of IFN-τ and in their ability to progress in development in glucose-rich media are manifestations of phenomena that occur in vivo and provide plasticity in embryo selection during early pregnancy.

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  • 放牧における発情牛の歩行数の解析

    植田 直円, 青木 真理, 木村 康二, 加茂 幹男, 伊吹 俊彦, 砂原 弘子

    日本家畜管理学会誌   34   24 - 25   1998

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    DOI: 10.20652/jjlm.34.supplement_24

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  • ポビドンヨード子宮内投与後のウシ子宮内膜における遺伝子発現変化およびside population細胞の変遷

    舘林亮輝, 舘林亮輝, 中村翔, 北川悠梨, 加治佐実希, 市川怜, 森田康広, 森田康広, 大蔵聡, 木村康二, 松山秀一

    日本繁殖生物学会講演要旨集(Web)   114th   2021

  • ウシの子宮内膜における精子走化性因子発現の検討

    宗友 真帆, 酒井 駿介, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   66 ( Suppl. )   j78 - j78   2020.9

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  • 暑熱環境下におけるウシ子宮内膜間質細胞は小胞体ストレス応答経路を介してIL-6発現を増加させる

    酒井 駿介, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   66 ( Suppl. )   j41 - j41   2020.9

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  • ウシ子宮内膜side population(SP)細胞の遺伝子発現解析と分娩後におけるSP細胞割合の推移

    舘林 亮輝, 中村 翔, 美辺 詩織, 古澤 軌, 阿部 良哉, 加治佐 実希, 森田 康広, 大蔵 聡, 木村 康二, 松山 秀一

    The Journal of Reproduction and Development   66 ( Suppl. )   j41 - j41   2020.9

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  • 当帰芍薬散がウシ卵管収縮弛緩運動に与える影響

    窪田 早耶香, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   66 ( Suppl. )   j75 - j75   2020.9

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  • 培養ウシ卵管平滑筋細胞のCa2+変動解析

    山本 ゆき, 石本 健太, 木村 康二

    The Journal of Reproduction and Development   66 ( Suppl. )   j76 - j76   2020.9

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  • ウシ卵管上皮細胞の3次元培養における細胞凝集体およびシスト形成能の評価

    杉野 耀亮, 伊藤 さやか, 佐藤 太紀, 酒井 駿介, 梅原 依吹, 窪田 早耶香, 宗友 真帆, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   66 ( Suppl. )   j76 - j76   2020.9

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  • ウシの発情周期中における卵管自発収縮の制御機構

    山本ゆき, 山本ゆき, 小川泰司, 黒川真帆, 窪田早耶香, 木村康二, 木村康二

    日本分子生物学会年会プログラム・要旨集(Web)   43rd   2020

  • 暑熱ストレスは小胞体ストレスを介して子宮内膜間質細胞のIL6発現を増加させる

    酒井駿介, 山本ゆき, 木村康二

    日本分子生物学会年会プログラム・要旨集(Web)   43rd   2020

  • 【Female genital tractの収縮を再考する】卵管の平滑筋収縮

    木村 康二

    HORMONE FRONTIER IN GYNECOLOGY   26 ( 3 )   185 - 189   2019.9

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    卵管の平滑筋の運動は配偶子や胚の輸送を担う重要な機能の1つである。卵管の平滑筋は自律的な収縮弛緩による蠕動運動を行うことで輸送機能は果たしている。消化管と同じくある種のペースメーカー細胞の存在が示唆されているが、その詳細はいまだ解明されていない。また、卵管の平滑筋による輸送機能は性周期に依存して変化しており、これは卵巣由来の性ステロイドホルモンの直接または間接的な影響によるものと考えられている。(著者抄録)

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  • 排卵前後のウシ卵管上皮における繊毛形成過程の進行

    伊藤 さやか, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   65 ( Suppl. )   j83 - j83   2019.9

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  • ウシ子宮内膜細胞によるマクロファージ誘引に対する暑熱ストレスの影響

    酒井 駿介, 山本 ゆき, 木村 康二

    The Journal of Reproduction and Development   65 ( Suppl. )   j128 - j128   2019.9

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  • ウシ子宮内膜side population細胞における幹細胞マーカーの発現解析

    舘林 亮輝, 中村 翔, 美辺 詩織, 古澤 軌, 阿部 良哉, 森田 康広, 大蔵 聡, 木村 康二, 松山 秀一

    The Journal of Reproduction and Development   65 ( Suppl. )   j133 - j133   2019.9

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  • ウシ卵管培養平滑筋細胞におけるカルシウムオシレーション制御メカニズムの探索

    山本 ゆき, 黒川 真帆, 木村 康二

    The Journal of Reproduction and Development   65 ( Suppl. )   j134 - j134   2019.9

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

    ASAOKA Natsuki, KIMURA Koji, TAKAHASHI Masashi, KUNII Hiroki, KOYAMA Keisuke, KUBO Tomoaki, HAMAGUCHI Yu, OGAWA Hidehiko, KOBAYASHI Hisato, BAI Hanako, KAWAHARA Manabu

    The Journal of Reproduction and Development Supplement   65 ( Suppl. )   j129 - j129   2019.9

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    DOI: 10.14882/jrds.112.0_P-71

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  • ウシ主席卵胞および次席卵胞の顆粒層細胞において発現する遺伝子の網羅的解析

    大下 雪奈, 渡辺 雄貴, 春日 崇, 中平 陽子, 棟朝 亜理紗, 木村 康二, 松山 秀一, 大蔵 聡, 真方 文絵, 松田 二子

    日本獣医学会学術集会講演要旨集   162回   439 - 439   2019.8

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

    SUZUKI Toshiyuki, KIMURA Koji, BAI Hanako, KAWAHARA Manabu, TAKAHASHI Masashi

    Hokkaido Journal of Livestock and Grassland Science   7 ( 1 )   7 - 15   2019

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    <p>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.</p>

    DOI: 10.32253/hlgs.7.1_7

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  • ウマ子宮内膜におけるPGF2α自己増幅機構

    香西 圭輔, 徳山 翔太, Szostek Anna Z, 登石 裕子, 角田 修男, 田谷 一善, 阪谷 美樹, 高橋 昌志, 南保 泰雄, Skarzynski Dariusz J, 山本 ゆき, 木村 康二, 奥田 潔

    馬の科学 = Equine science   56 ( 4 )   282 - 293   2019

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  • 暑熱環境下におけるウシ子宮内膜細胞のマクロファージ走化性物質産生の変化

    酒井駿介, 山本ゆき, 木村康二

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019

  • 卵管自発収縮に関わるイオンチャネルと培養平滑筋細胞内カルシウム変動

    山本ゆき, 山本ゆき, 黒川真帆, 小川泰司, 木村康二, 木村康二

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   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

    国井宏樹, 伊藤月乃, 小木曽貴季, 鈴木惇文, 古山敬祐, Md. Abdus, Shabur Talkder, Balboula Ahmed Zaky, 唄 花子, 川原 学, 永野昌志, 木村康二, 高橋昌志

    北海道牛受精卵移植研究会会報   37   17 - 17   2018.8

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  • ウシ黄体におけるbetaglycanの役割とその発現調節

    長谷川啓喜, 山下真路, 伊藤典彦, 岡本芳晴, 窪友瑛, 伊賀浩輔, 木村康二, 菱沼貢, 奥田潔, 西村亮, 西村亮

    日本畜産学会大会講演要旨   124th   205 - 205   2018.3

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

    KOMATSU Seimei, SUZUKI Toshiyuki, KUNII Hiroki, KAWAHARA Manabu, KIMURA Koji, TAKAHASHI Masashi, BAI Hanako

    The Journal of Reproduction and Development Supplement   111 ( 0 )   P - 38-P-38   2018

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    DOI: 10.14882/jrds.111.0_P-38

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  • ウシ卵管自発収縮に関与するイオンチャネルと平滑筋細胞内カルシウム変動の解析

    山本ゆき, 山本ゆき, 黒川真帆, 小川泰司, 木村康二, 木村康二

    Journal of Reproduction and Development   64 ( Suppl Japanese Issue )   2018

  • ウシ卵管上皮構成細胞のタンパク質発現解析による分化経路の推測

    伊藤さやか, 山本ゆき, 木村康二

    Journal of Reproduction and Development   64 ( Suppl Japanese Issue )   2018

  • ウシ卵管上皮にみられる段階的な繊毛形成過程

    伊藤さやか, 山本ゆき, 木村康二

    日本分子生物学会年会プログラム・要旨集(Web)   41st   2018

  • 暑熱によるウシ子宮内膜のプロスタグランジン分泌増加と温度感受性チャネルの関係

    山田亜依, 高見恵都, 酒井駿介, 山本ゆき, 山本ゆき, 木村康二, 木村康二

    Journal of Reproduction and Development   64 ( Suppl Japanese Issue )   2018

  • ウシ初期黄体における低酸素環境のglucose transporter 1(GLUT1)発現を介した機能亢進

    西村亮, 西村亮, 長谷川啓喜, 山下真路, 伊藤典彦, 岡本芳晴, 竹内崇, 窪友瑛, 伊賀浩輔, 木村康二, 菱沼貢, 奥田潔, 奥田潔

    日本獣医学会学術集会講演要旨集   160th   431 - 431   2017.8

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  • ウシ初期および中期黄体細胞のプロジェステロン(P4)分泌に及ぼす低酸素環境の影響

    長谷川 啓喜, 西村 亮, 菱沼 貢, 山下 真路, 伊藤 典彦, 岡本 芳晴, 窪 友瑛, 伊賀 浩輔, 木村 康二, 奥田 潔

    日本畜産学会大会講演要旨集   122回   160 - 160   2017.3

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  • The effect of interferon tau on human placental cells

    Nao Tanikawa, Yugo Suzue, Schuichi Matsuyama, Koji Kimura, Hisataka Iwata, Takehito Kuwayama, Koumei Shirasuna

    JOURNAL OF REPRODUCTIVE IMMUNOLOGY   118   134 - 134   2016.11

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    DOI: 10.1016/j.jri.2016.10.075

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  • 農家で実施可能なウシ伸長胚を利用した雌雄産み分け技術 (特集 目指せ、確実な子牛生産)

    松山 秀一, 中村 翔, 木村 康二

    養牛の友   ( 488 )   34 - 37   2016.11

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  • ウシ伸長胚を利用した現場実施可能な雌雄産み分け技術

    松山 秀一, 中村 翔, 木村 康二

    畜産技術 = Livestock technology   ( 738 )   8 - 11   2016.11

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

    SHIROZU Takahiro, SUZUKI Toshiyuki, IWANO Hiroki, OGISO Takatoshi, KIM Sung Woo, BAI Hanako, KAWAHARA Manabu, KIMURA Koji, TAKAHASHI Masashi

    The Journal of Reproduction and Development Supplement   109 ( 0 )   P - 14-P-14   2016

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    DOI: 10.14882/jrds.109.0_P-14

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  • 着床前ゲノム選抜技術開発に向けたウシ伸長胚保存法の検討

    藤井貴志, 平山博樹, 陰山聡一, 内藤学, 福田茂夫, 吉野仁美, 松山秀一, 木村康二, 早川宏之, 津田栄, 森安悟

    Journal of Reproduction and Development   62 ( Suppl Japanese Issue )   2016

  • ウシ伸長胚を用いた着床前ゲノム選抜技術開発に向けたSNP解析精度の検討

    平山博樹, 内藤学, 鹿島聖志, 藤井貴志, 陰山聡一, 坂井ひとみ, 杉本喜憲, 松山秀一, 木村康二, 早川宏之, 森安悟

    Journal of Reproduction and Development   62 ( Suppl Japanese Issue )   2016

  • ウシ過剰排卵処置時におけるニューロキニンB受容体作動薬の末梢投与が黄体形成ホルモン分泌および回収胚の品質に及ぼす影響

    中村翔, 木村康二, 美辺詩織, 大石真也, 大蔵聡, 松山秀一

    Journal of Reproduction and Development   62 ( Suppl Japanese Issue )   2016

  • ウシ胚におけるインターフェロンτの局在

    細江実佐, 林憲悟, 作本亮介, 古澤軌, 池田光美, 大越勝広, 徳永智之, 木村康二, 松山秀一, 野口純子, 高橋透

    日本畜産学会大会講演要旨   119th   204   2015.3

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  • ウシ過剰排卵処置時におけるウシ型キスペプチン‐10の静脈内への頻回投与が性腺刺激ホルモン分泌および排卵成績に及ぼす影響

    MATSUYAMA SHUICHI, OKURA SATOSHI, OISHI SHIN'YA, KIMURA KOJI

    日本はい移植学雑誌   37 ( 1 )   34   2015.1

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  • ウシ黄体におけるtestosterone合成酵素の遺伝子発現

    入江結唯, 羽柴一久, 吉岡伸, 木村康二, 山本ゆき, 奥田潔

    Journal of Reproduction and Development   61 ( Suppl Japanese Issue )   2015

  • ウシ卵管における細胞模型エストロジェン受容体(GPER)を介したestradiol-17β(E2)の作用

    西江拓海, 小林芳彦, 山本ゆき, 木村康二, 奥田潔, 奥田潔

    日本畜産学会大会講演要旨   119th   2015

  • piggyBac トランスホソンを用いたリフログラミンク因子導入によるウシ羊膜細胞からの栄養膜細胞株樹立の試み

    川口 高正, 築山 智之, 松山 秀一, 木村 康二, 南 直治郎, 山田 雅保, 今井 裕

    第107回日本繁殖生物学会大会   2014.8

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  • インターフェロン(IFN)τはナノ粒子の取り込みを阻害してインターロイキン(IL)-1β産生を抑制する

    原 教子, 白砂 孔明, 臼井 文武, 唐澤 直義, 木村 博昭, 川島 晃, 大口 昭英, 松山 秀一, 木村 康二, 高橋 将文

    The Journal of Reproduction and Development   60 ( Suppl. )   j137 - j137   2014.8

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

    白水貴大, 川原学, 木村康二, 高橋ひとみ, 柳川洋二郎, 永野昌志, 白汝嵐, 今川和彦, 高橋昌志

    J Reprod Dev   107 ( 0 )   OR2 - 24-OR2-24   2014

  • Generation of murine-ES-cell-like iPS cells in cattle using piggyBac transposition of doxycycline-inducible transcription factor Reviewed

    T. Kawaguchi, T. Tsukiyama, N. Minami, M. Yamada, K. Kimura, H. Imai

    CiRA International Symposium 2013 ~Raising the next generation of stem cell research~, Kyoto, Japan   2013.3

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  • ウシにおけるマウスES細胞様iPS細胞の樹立の試み Reviewed

    川口 高正, 南 直治郎, 山田 雅保, 木村 康二, 今井 裕

    第1回 関西生殖医学集談会   2013.3

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  • ウシ過剰排卵処置時におけるウシ型キスペプチン‐10の単回末梢投与が性腺刺激ホルモン分泌および卵巣機能に及ぼす影響

    MATSUYAMA SHUICHI, OKURA SATOSHI, OISHI SHIN'YA, KIMURA KOJI

    東日本家畜受精卵移植技術研究会大会資料   29th   39   2013

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  • Transdifferentiation of bovine aminion cells into trophoblast-like cells using piggyBac transposition of doxycycline-inducible reprogramming factors Reviewed

    T. Kawaguchi, T. Tsukiyama, N. Minami, M. Yamada, K. Kimura, H. Imai

    Satellite symposium to the International Congress on Animal Reproduction (ICAR)   2012.7

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  • Effect of maternal ageing on the glutathione content and p38 MAPK activity in bovine granulosa cells

    TAKEO Shun, GOTO Hiroya, KIMURA Koji, IWATA Hisataka, MONJI Yasunori, KUWAYAMA Takehito

    29 ( 2 )   S60   2012.4

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  • CHANGES IN PLASMA STEROID CONCENTRATIONS DURING GESTATION IN COWS WITH SPONTANEOUS ABORTION OF SOMATIC CELL CLONED FETUSES

    M. Hirako, H. Takahashi, K. Kimura, N. Adachi, S. Akagi

    REPRODUCTION FERTILITY AND DEVELOPMENT   24 ( 1 )   121 - 121   2012

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  • Oog1 promoter controls the meiosis-specific gene expression in germ cells Reviewed

    Ishida M, Okazaki E, Tsukamoto S, Kimura K, Ohota Y, Kito S, Imai H, Minami N

    The 45th Annual Meeting of the Society for the Study of Reproduction   2012

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  • Sexual dimorphism during early embryonic development in mammals

    Koji Kimura, Shuichi Matsuyama

    Journal of Mammalian Ova Research   29 ( 3 )   103 - 112   2012

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    Although most sexual dimorphism appears after gonadal differentiation, some occur at earlier stages of development. One example of this phenomenon is the skewing of the sex ratio of embryos due to the glucose concentration of the culture medium. This skewing occurs because male and female embryos differ in their abilities to metabolize glucose. Another example is the sexually dimorphic expression of the bovine embryo-derived signal for maternal recognition of pregnancy, IFNT. The difference in expression of X-linked genes between male and female embryos, that results from incomplete X-chromosome inactivation, is considered to be involved, either directly or indirectly, in the sexual dimorphism that appears during early development. © 2012 Japanese Society of Mammalian Ova Research.

    DOI: 10.1274/jmor.29.103

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  • Establishment of induced pluripotent stem cells in cattle using piggyBac transposition of doxycycline-inducible transcription factors

    T. Kawaguchi, T. Tsukiyama, K. Kimura, N. Minami, M. Yamada, H. Imai

    World Congress on Reproductive Biology (WCRB), Cairns, Australia   2011.10

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  • Identification of Oog1 Promoter Regions in Mouse Growing Oocytes

    Miya Ishida, Eriko Okazaki, Satoshi Tsukamoto, Koji Kimura, Yuki Ohta, Seiji Kito, Hiroshi Imai, Naojiro Minami

    BIOLOGY OF REPRODUCTION   85   2011.7

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  • The Possible Involvement of IFN-Tau in Lymphangiogenesis in the Corpus Luteum During Maternal Recognition Period in the Cow

    Akane Nitta, Koumei Shirasuna, Haruka Matsumoto, Shingo Haneda, Motozumi Matsui, Takashi Shimizu, Shuichi Matsuyama, Koji Kimura, Heinrich Bollwein, Akio Miyamoto

    BIOLOGY OF REPRODUCTION   85   2011.7

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  • ウシにおける人工多能性幹細胞の樹立の試み

    川口 高正, 築山 智之, 木村 康二, 南 直治郎, 山田 雅保, 今井 裕

    第136回 日本生殖医学会関西支部集談会、ハービスプラザ、大阪   2011.3

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  • The Relationship Between Pregnancy Status and Production of IFN-Tau of Bovine Conceptus by Measuring Expression of Interferon- Stimulated Gene 15 (ISG15) of Peripheral Leukocyte in Cattle

    Shuichi Matsuyama, Takatoshi Kojima, Satoshi Kato, Shogo Shiratsuki, Hiroshi Tanaka, Keiichi Nagataki, Shoichi Hoshi, Koichi Muroi, Koji Kimura

    BIOLOGY OF REPRODUCTION   152 - 152   2010

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  • RELATIONSHIP BETWEEN FETAL ABNORMALITIES AND PERIPHERAL STEROID CONCENTRATIONS DURING GESTATION IN COWS TRANSFERRED WITH EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

    M. Hirako, H. Takahashi, K. Kimura, N. Adachi, S. Akagi

    REPRODUCTION FERTILITY AND DEVELOPMENT   22 ( 1 )   187 - 187   2010

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  • Identification of Oog1 Promoter Regions which Function During the First Meiosis in Oocytes. Reviewed

    Eriko Okazaki, Satoshi Tsukamoto, Koji Kimura, Yuki Ohta, Seiji Kito, Hiroshi Imai, Naojiro Minami

    BIOLOGY OF REPRODUCTION   170 - 170   2010

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  • Renewal of techniques related to embryo transfer in cattle : a novel superovulation method using aluminum hydroxide gel

    KIMURA Koji, MATSUYAMA Shuichi, IWATA Hisataka, SEKI Makoto, HIRAKO Makoto

    The Journal of reproduction and development   55   j60 - j61   2009.8

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  • ZP3 Promoter Is Not Enough to Analyze the Early Expressing Genes During Oogenesis, Such as Oog1

    Hisashi Imaichi, Rieko Okazaki, Satoshi Tsukamoto, Yuki Ohta, Seiji Kito, Koji Kimura, Hiroshi Imai, Naojiro Minami

    BIOLOGY OF REPRODUCTION   128 - 128   2009

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  • ZP3 promoter is not enough to analyze the early expressing genes during oogenesis, such as Oog1 Analysis of Oog1, an oocyte-specific gene, using transgenic RNAi approach. Reviewed

    Imaichi H, Okazaki E, Tsukamoto S, Ohota Y, Kito S, Kimura K, Imai H, Minami N

    42nd Society for the Study of Reproduction   2009

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  • Oog1 is a maternal effect gene required for the development of mouse preimplantation embryo Reviewed

    N. Minami, H. Imaichi, S. Tsukamoto, Y. Ohta, S. Kito, K. Kimura, H. Imai

    REPRODUCTION IN DOMESTIC ANIMALS   43   149 - 150   2008.7

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  • Effect of FCS and BSA on hatching rate, apoptosis, and stress activated protein kinase activity in bovine blastocysts

    Hisataka Iwata, Yosuke Sakaguchi, Yoshihiro Kon, Koji Kimura, Takehito Kuwayama, Yasuniri Monji

    BIOLOGY OF REPRODUCTION   94 - 94   2008

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  • Analysis of Oog1, an oocyte-specific gene, using transgenic RNAi approach

    Hisashi Imaichi, Satoshi Tsukamoto, Yuki Ohta, Seiji Kito, Koji Kimura, Hiroshi Imai, Naojiro Minami

    BIOLOGY OF REPRODUCTION   133 - 133   2008

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  • A comparison of pregnancy maintenance after embryo transfer of Day 7 or 13 bovine embryos and the relationship between interferon-tau production of day 13 bovine embryos and pregnancy success

    K. Kimura, N. Hayashi, H. Iwata

    REPRODUCTION FERTILITY AND DEVELOPMENT   19 ( 1 )   222 - 223   2007

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  • 第3章 流・死産の発生要因の解明(4)妊娠末期のクローン胎子の発育および分娩発来機構と正常分娩誘起法の開発に関する研究

    平子誠, 平子誠, 青木真理, 青木真理, 木村康二, 木村康二, 花房泰子, 石崎宏, 假屋喜弘

    農林水産省農林水産技術会議事務局研究成果   ( 444 )   2007

  • The effect of glucosamine concentration on the development and sex ratio of bovine embryos.

    Koji Kimura, Hisataka Iwata, Jeremy G. Thompson

    BIOLOGY OF REPRODUCTION   124 - 124   2006

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  • Expression of bovine interferon-tau variants according to sex and age of conceptuses.

    Angela M. Walker, Koji Kimura, R. M. Roberts

    BIOLOGY OF REPRODUCTION   93 - 93   2006

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  • Mechanisms for Establishment of Pregnancy in Mammalian Species

    KIMURA Koji

    Journal of mammalian ova research   22 ( 3 )   101 - 118   2005.10

  • A compariosn of IFN-tau messenger RNA expression between somatic nuclear transfer and in vitro produced female bovine blastocysts.

    K Kimura, S Akagi, S Takahashi, H Iwata, H Kadokawa, M Aoki, M Hirako

    BIOLOGY OF REPRODUCTION   230 - 230   2005

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  • Responsiveness of rabbits to superovulation treatment by single injection of follicle-stimulating hormone mixed with aluminum hydroxide gel.

    S Hashimoto, K Kimura, T Kuramochi, K Aoyagi, M Hirako, M Kawaguchi, H Iwata, H Takahashi, M Hirao, K Kitada, K Hirasawa, M Ueda

    BIOLOGY OF REPRODUCTION   236 - 237   2004

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  • Successful superovulation of cattle using a single intramuscular injection of FSH with aluminum hydroxide gel.

    K Kimura, M Hirako, H Iwata, M Aoki

    BIOLOGY OF REPRODUCTION   237 - 238   2004

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  • The effects of high versus low oxygen and of the presence of glucose and fructose during culture on the sex ratio of bovine blastocysts.

    RM Roberts, MP Green, LD Spate, K Kimura

    BIOLOGY OF REPRODUCTION   68   250 - 251   2003

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  • Effect of oxidative stress and inhibitors of the pentose-phosphate pathway on the sexual dimorphic expression of IFN-tau production by bovine blastocysts

    K Kimura, LD Spate, RM Roberts

    BIOLOGY OF REPRODUCTION   68   158 - 158   2003

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  • Cannulation for a bovine fetus in late gestation under regional anesthesia

    M Aoki, K Kimura, M Hirako, Y Hanafusa, H Ishizaki, Y Kariya

    JOURNAL OF REPRODUCTION AND DEVELOPMENT   48 ( 5 )   455 - 460   2002.10

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    This experiment was carried out to collect fetal blood and fluid via catheters fitted to elucidate interaction between the mother and her fetus during a comparatively long term. Fifteen cows in late gestation were used for this study. Under regional anesthesia, an incision was made in the left paralumbar fossa in the standing position. A catheter for fetal blood was inserted into a vein of the fetus and catheters for fetal fluid were inserted into amniotic and allantoic sacs respectively. Four types of catheters were used for collecting fetal blood. The best of these catheters for fetal vein was the angiographic catheter covered with spring tube. Fetal blood samples were able to be collected for more than seven days constantly from four fetuses until parturition. Fetal fluid could be collected daily from three fetuses until parturition perfectly. Parturition occurred at the end of a normal gestation period. The concentration of fetal and maternal cortisol decreased to the basal level within 24 hours (P&lt;0.05) after operation. The changes in fetal and maternal cortisol during periparturition were the same as previously reported. These results suggested that this method would be beneficial for fetal cannulation and the sample collected by this method was useful for endocrine study during the perinatal period in cattle.

    DOI: 10.1262/jrd.48.455

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  • 交雑種雌牛の親子放牧による双子哺育能力と栄養補給効果

    青木 真理, 木村 康二, 鈴木 修

    畜産草地研究所研究報告   ( 1 )   1 - 6   2002.3

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    交雑種雌牛の親子放牧における哺育能力と母牛あるいはその双子子牛への栄養補給効果を検討した。1996年から1997年にかけて、のべ17組の双子とその母牛をペレニアルライグラス主体草地に分娩約1週間後から輪換放牧し、子牛への別飼い飼料を給与する区(別飼い区)と給与せず母牛へのみ増し飼いする区(増し飼い区)に分け、哺乳量、母牛の栄養状態および子牛の別飼い飼料摂食行動を調査した。別飼い区では、のべ10組の親子を昼夜放牧し、子牛には放牧地内の別飼い施設で別飼い飼料を自由採食させた。増し飼い区では、のべ7組の親子を9:00~16:00の間放牧し、母牛には日量1~2kgの濃厚飼料を給与した。その結果、母牛の泌乳量は日量10.4~14.4kgあり、1頭当たり5.2~7.2kg摂取した。母牛の栄養状態は、両区とも低下傾向を示したが、別飼い区の減少が顕著であった。別飼い区では、別飼い飼料の摂食行動と摂取量が3か月齢以降で顕著に増加した。子牛の平均日増体量は、増し飼い区の方が0-4週齢時で有意に高かったが、その後は別飼い区の方が高く推移した。子牛の体型は、月齢とともに別飼い区が増し飼い区を上回る傾向にあり、6か月齢の胸囲で別飼い区が有意に大きかった。これらから、交雑種雌牛と双子子牛の親子放牧では、生後1か月齢から別飼い飼料の効果が現れることが示された。また、分娩後母牛の体重減少が顕著な草地においては、母牛への補助飼料給与も必要と考えられた。

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    Other Link: http://agriknowledge.affrc.go.jp/RN/2010661547

  • Female, in vivo-produced bovine blastocysts produce over twice the amount of interferon-tau as males at the same stage of development.

    K Kimura, RM Roberts, GE Seidle

    BIOLOGY OF REPRODUCTION   66   154 - 155   2002

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  • 妊娠末期に牛胎子から血液および羊水・尿膜水を連続的に採取できる

    青木真理, 木村康二, 平子誠, 花房泰子, 石崎宏, 仮屋喜弘

    畜産草地研究成果情報   ( 1 )   2002

  • In vitro cultured male bovine embryos make the transition from morula to blastocyst more successfully than females.

    K Kimura, MA Larson, RM Roberts

    BIOLOGY OF REPRODUCTION   64   178 - 178   2001

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  • 娠妊末期における母ウシおよび胎子の血球数および血液生化学値の動態

    花房泰子, 青木真理, 木村康二, 平子誠, 須藤まどか, 石崎宏, 仮屋喜弘

    日本獣医学会学術集会講演要旨集   132nd   2001

  • Effect of Vascular Endothelial Growth Factor on the Early Development of IVM/IVF Bovine Embryos

    LUO Hailing, KIMURA Koji, AOKI Mari, HIRAKO Makoto, ZHAO Youzhang

    17 ( 2 )   S51   2000.4

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  • Effect of bovine interferon tau on the early development of bovine embryos.

    K Kimura, H Iwata, M Aoki, M Hirako

    BIOLOGY OF REPRODUCTION   62   250 - 250   2000

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  • 妊娠末期における母ウシおよび胎子の血球数動態

    花房泰子, 青木真理, 木村康二, 平子誠, 石崎宏, 仮屋喜弘

    関東畜産学会大会講演要旨集   55th   2000

  • 放牧における発情牛の運動量の解析(第2報) : ランダムな間隔で測定した場合の解析方法の検討

    梅田 直円, 加茂 幹男, 青木 真理, 木村 康二, 山田 知哉

    日本家畜管理学会誌 = Japanese journal of livestock management   35   32 - 33   1999.3

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    DOI: 10.20652/jjlm.35.supplement_32

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  • ウシ胎子へのカテーテル装着と胎子血採取の試み

    青木真理, 木村康二, 平子誠, 石崎宏, 仮屋喜弘, 花房泰子

    日本畜産学会大会講演要旨   96th   1999

  • F1雌牛による双子の安定生産技術

    鈴木 修, 青木 真理, 木村 康二

    畜産の研究   50 ( 6 )   675 - 680   1996.6

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Research Projects

  • Three-dimensional construction of artificial seminiferoustubules for in vitro spermatogenesis

    Grant number:20K21656  2020.07 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Yamamoto Yuki

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    Grant amount:\6370000 ( Direct expense: \4900000 、 Indirect expense:\1470000 )

    In this study, we examined the isolation and culture conditions of testicular cells of immature bovine testis and the printing conditions with the aim of constructing seminiferous tubules using a 3D bioprinter. The culture medium containing 10% fetal bovine serum was used to maintain germ cells in the ducts, and the effects of hormones or retinoic acid were examined. Hormones had no effect on cell proliferation or germ cell localization, while the addition of retinoic acid increased the percentage of germ cells expressing Ki67.
    The mixing ratio of commercially available polyethylene glycol diacrylate and gelatin methacrylate as bioink was also examined, but the conditions under which the cells settled and proliferated did not match those maintaining the morphology of the 3D structures, and further verification was required.

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  • Development of technique to decrease voluntary waiting period of cattle using ozone gel

    Grant number:20K21372  2020.07 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)  Grant-in-Aid for Challenging Research (Exploratory)

    Kimura Koji

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    Grant amount:\6370000 ( Direct expense: \4900000 、 Indirect expense:\1470000 )

    In recent years, the voluntary waiting period (the number of days between parturition) in cows has increased by about one month. and no solutions against this issue has not been established, although it would greatly contribute to increased productivity. The uterus is subject to severe physical damage and infection, resulting in inflammation due to detachment of the placenta delivery. For prompt recovery from these injuries, the aims of this study was to investigated the in vitro and in vivo anti-inflammatory effects of ozone gel, which can maintain activity of ozone. In experiments using endometrial stromal cells, the addition of ozone gel to the cell culture medium did not affect IL6 gene expression. Furthermore, intrauterine administration of the gel into the uterus of postpartum cows did not affect the level of infection.

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  • 最終糖化産物がウシ子宮、卵管および胚発育に及ぼす影響とその作用機序の解明

    Grant number:20H03129  2020.04 - 2024.03

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    松山 秀一, 木村 康二

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    Grant amount:\17680000 ( Direct expense: \13600000 、 Indirect expense:\4080000 )

    本研究では、高穀物含量の飼料によって産生されるメチルグリオキサール(MGO)が乳牛における受胎率低下の一因となっていることを検証する。本年度は、牛子宮内膜細胞において、培養液へのMGOの添加により細胞老化のマーカーであるp21遺伝子発現が有意に増加し、子宮内膜間質細胞におけるROS産生量も有意な増加が認められた。以上の結果から、牛子宮内膜間質細胞においてMGOがROS産生を亢進することでDNA損傷を引き起こし、p21などのサイクリン依存性キナーゼ阻害因子の発現を誘導することで細胞周期を停止させ、細胞老化を引き起こす可能性が考えられた。さらに、MGOの9日間にわたる静脈内投与(2時間/日)が牛生体の子宮内膜におけるp21遺伝子発現に及ぼす影響を検討した結果、MGO長期投与後の子宮内膜組織におけるp21 mRNA発現量は投与前と比較して増加する傾向がみられた。このことから、牛へのMGOの長期投与が子宮内膜における細胞老化を誘導することが示唆された。次に、MGOが牛卵管上皮細胞に及ぼす影響を検討した。MGOを添加した卵管上皮細胞におけるROS産生量は有意に増加し、卵管上皮細胞の増殖能は有意に低下した。一方、卵管上皮細胞におけるp21 mRNA発現量についてはMGO添加による変化は認められなかった。これらの結果から、牛卵管上皮細胞において、MGOはROSの発生を促進し、酸化ストレスに伴って生じるアポトーシスが、細胞増殖に影響を与えることが示唆された一方で、細胞老化には関与しない可能性が示された。また、MGOが胚発育に及ぼす時期と影響についても検討を行った。卵子の体外成熟時と比較して体外受精時にMGOを添加した場合の8 cell発生率が著しく低下したことから、MGOは胚の体外受精の段階で作用する可能性が考えられた。

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  • In vitro construction of bovine endometrium including uterine glands and the study on the machanisms of bovine conceptus development

    Grant number:19H03105  2019.04 - 2023.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    木村 康二, 山本 ゆき, 松山 秀一

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    1.3次元培養によって得られた子宮腺シストならびに子宮腺様構造における細胞種を特定するためにサイトケラチン、ビメンチン抗体で染色を行った。両者においてビメンチンポジティブの細胞は存在せずすべてサイトケラチンポジティブであったことから、シスト、子宮腺様構造とも上皮性の細胞のみで構成されていることが示された。2.子宮腺を採取したb所が子宮内膜内のどの部位かによって腺様構造形成に差があるのかを調べるために、子宮内膜浅層ならびに深層のそれぞれから子宮腺を単離し、EGF+WNT存在下でマトリジェル内で培養した。その結果、両者の間でシストならびに子宮腺様構造形成率に差は見られなかった。4.昨年度シストから子宮腺様構造形成の際にEGFは必須であるがWNTは必須でないことが示されたため、単離子宮腺をゲルに包埋直後よりEGFのみで培養したところ、シストの形成は起こらず、直接子宮腺断片から子宮腺様構造が形成することが明らかとなった。またこれはEGF受容体阻害剤によって抑制されることが示された。5.腺様構造、単離子宮腺、シストにおける分泌タンパク質SERPINA14とSPP1遺伝子の発現を比較したところ、単離子宮腺と比較してシストではこれらの発現は有意に上昇したが、腺様構造ではこれらの遺伝子の発現は対照区と比較して有意な差は認められなかった。6.子宮腺ノックアウト牛作出を試みているが、これまでヒツジやマウスで報告された方法に準じて実施した結果、子宮腺のノックアウト個体の作出には至らなかった。当該年度はプロジェステロン量を大幅に増加して投与実験を行い、今年度その個体が性成熟を迎えるので、子宮内膜を採取して子宮腺の存在を確認する予定である。

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  • Culture system of bovine uterine gland for establishment of elongtion of bovine conceptus in vitro

    Grant number:17K19322  2017.06 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)  Grant-in-Aid for Challenging Research (Exploratory)

    KIMURA Koji, YAMAMOTO Yuki

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    Grant amount:\6370000 ( Direct expense: \4900000 、 Indirect expense:\1470000 )

    In this study, we established a method for isolation of bovine endometrial glands enzymatically and its culture system by embedding them in the matrigel. When the isolated uterine glands were cultured by this novel method, the gene expressions of the secretory proteins from bovine endometrium were significantly increased compared to those of glands cultured on plastic dishes (conventional culture method). Moreover, the glands cultured by this novel method had ability to react with sexual steroid hormones such as estradiol 17b and progesterone. The results of the present study are valuable for research on factors which affect the protein secretion ability of the uterine gland and contribute the study on improvement of fertility of cattle.

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  • Role of autophagy-lysosomal related responses on uterine modulation at the time of maternal-fetal recogntion

    Grant number:15H04579  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Takahashi Masashi

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    Grant amount:\16380000 ( Direct expense: \12600000 、 Indirect expense:\3780000 )

    In the present study, we detected the activation of cathepsin (CTS)B in the pregnant bovine uterine tissue. This activation was thought to be stimulated by interferon tau(IFN)T secreted by embryo. Although expression of CTS genes was detected by pregnant and IFNT-treated non pregnant uterine tissue, expression of autophagy related genes was not changed. These results suggest the activation of lysosome and lysosomal CTS is triggered by IFNT for uterine modulation, but contribution of autophagy is low. Further study is needed to clarify the roles of autophagy. RNA interference of each subunit construction of type I IFN receptor subunit (IFNAR1, R1) by siRNA revealed that IFNT stimulated signal transduction is dominantly affected by IFNAR1 for inducing IFN related gene expressions. In addition, high levels of pregnancy specific gene expression were detected in the cervical tissue that is directly connected with uterus for the first time.

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  • Regulatory mechanism of coordinated beating of oviductal cilia

    Grant number:15K14843  2015.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    Kiyoshi OKUDA

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    Grant amount:\3770000 ( Direct expense: \2900000 、 Indirect expense:\870000 )

    Uni-directional ciliary beating is required for successful transport of oocytes and embryos. In some tissues including airway and skin, planar cell polarity (PCP) signaling arranges the direction of cells throughout these tissues. In the present study, we demonstrated that VANGL1, one of PCP core protein, is localized in the ovarian and uterus sides of the cellular membranes of bovine oviductal ciliated cells. Additionally, DVL3 and Inturned, which are the downstream proteins of VANGL1, are also localized in the ciliated cells. These series of VANGL1 signaling may arrange the direction of ciliated cells and cilia throughout the whole tissues from distal to proximal, producing the stream of oviductal fluid toward the uterus to transport the oocytes and embryos.

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  • Activin A as a coordinator of oviductal environment: local action for successful fertilization and establishment of pregnancy in oviducts

    Grant number:15K18771  2015.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)  Grant-in-Aid for Young Scientists (B)

    Yamamoto Yuki, OKUDA Kiyoshi, KIMURA Koji

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    Grant amount:\3250000 ( Direct expense: \2500000 、 Indirect expense:\750000 )

    Environment in mammalian oviducts is highly regulated by many endocrine/paracrine/autocrine factors for fertilization and early embryonic development. The present study has revealed that 1) activin A, a local factor, is constantly present in oviductal lumen, 2) oviductal epithelial cells are target of activin A, and 3) mRNA expression of activin receptor is lowest on a day of ovulation in bovine oviduct. In addition, 4) inhibition of activin signal transduction increased pro-apoptotic gene expression in isthmic epithelial cells of bovine oviduct.

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  • Establishment and evaluation of bovine induced trophoblast cell line and contruction of in vitro implantation model using this cell line.

    Grant number:26292168  2014.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Kimura Koji

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    Grant amount:\16120000 ( Direct expense: \12400000 、 Indirect expense:\3720000 )

    It is necessary for improvement of pregnancy rate in cattle to clarify the mechanisms involved in its embryo development, implantation and establishment of pregnancy. The aims of the present study are to establish the bovine induced pluripotent stem cell and induced trophoblastic cells. Bovine amnion derived cells (bADC) were introduced with transposone expression vectors (piggyBac vectors) containing doxycycline (Dox)-inducible transcription factors. Colonies were selected by morphology criteria and then their characteristics were evaluated by expression of stem cell or trophectoderm markers. We established both of bovine iPS cell lines which can be differentiated to germ cells in vivo and trophoblast cell lines which processed trophoblast stem cell-like characteristics and the potential to differentiate into the extra-embryonic cell lineage.

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  • 伸長胚選別によるウシES細胞キメラ作出の効率化

    Grant number:23380169  2011.04 - 2016.03

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    古澤 軌, 木村 康二, 松山 秀一

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    Grant amount:\18070000 ( Direct expense: \13900000 、 Indirect expense:\4170000 )

    [ウシES細胞の高品質化]
    幹細胞マーカーであるPOU5F1の発現量をTet-onシステムで調節可能なウシES細胞株を樹立した。
    ①キメラ形成能を調べるため、DiIで蛍光標識したES細胞を5個注入した8-16細胞期のマウスあるいはウシ胚をドキシサイクリン(Dox)存在(1μg/ml)、非存在下で培養し、胚盤胞期胚における局在を調べた。8細胞期のマウス胚をホストとした場合、3株中2株において、Dox 存在下の方がICM(inner cell mass)に局在する割合が1.8-3.2倍有意に高かった。Doxの添加時間を0、12、24、及び48時間に設定して局在を比較したところ、24時間ではICMへの局在が2倍以上に増加するものの、それ以上の添加は効果が認められなかった。8-16細胞期のウシ胚をホストにした場合もDox添加24時間でICMへの局在及び取り込みがそれぞれ2倍以上に増加したが、48時間以上の添加は胚発生に悪影響が認められた。TE(trophectoderm)への局在も増加したことから、Dox添加によるPOU5F1の上昇は細胞の未分化性よりも生存性の維持に影響を与え、寄与率が増加したと考えられた。
    ②ウシES細胞とマウスES細胞を2日間単独、あるいは混合培養したマトリゲルビーズをDoxを徐放的に放出する浸透圧ポンプとともにヌードマウスに移植し、3週間後に腫瘍を回収して、蛍光観察及び免疫組織学的解析によりテラトーマ形成能を調べた。ウシES細胞のビーズを単独、あるいはマウスES細胞と隣接して移植した場合、ウシES細胞由来の奇形腫形成が認められたものの、特定の細胞系譜へ分化した像は観察できなかった。一方、両者を混合培養したビーズを移植したところ、マウスES細胞由来のテラトーマ中にウシES細胞由来の上皮、神経叢、血管内皮、及び腺組織が認められたことから、分化誘導をサポートする組織が存在することにより三胚葉に分化し得ることが確認された。

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  • Effects of metabolism of purine derivatives on bovine embryo development

    Grant number:23580398  2011 - 2013

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KIMURA Koji

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    Grant amount:\5460000 ( Direct expense: \4200000 、 Indirect expense:\1260000 )

    The effects of metabolism of purine derivatives on bovine embryo development were investigated. When de novo purine synthesis was inhibited, the embryo development after maternal-to-zygotic transition (MZT) was severely compromised. Moreover, when the purine derivatives (adenine or hypoxanthine) were supplemented in the presence of the inhibitor for de novo purine synthesis, the inhibitory effect disappeared. These results suggested that purines are necessary for bovine embryo development after MZT. However, excess amount of purine derivatives (especially adenine) deteriorated bovine embryo development. In the presence of excess amount of adenine after fertilization, the cleavage of the embryo was arrested and the fragmentation of nuclear was induced.

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  • Mechanisms of sexual dimorphism of IFNτ secretion in bovine blastocyst

    Grant number:20580333  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KIMURA Koji

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    Sexual dimorphic expression of IFNτ of bovine embryo was not caused via the sexual difference of expression of transcription factors, which affect IFNτ gene expression. However, the expression of Dnmt3a, one of DNA methyltransferases, of bovine male blastocysts was significantly higher than that of female embryos. Moreover, addition of 5-azadeoxycytidene, an inhibitor of DNA methyltransferase, to the culture medium of bovine embryo abolished the sexual dimorphic secretion of IFNτ from bovine embryos. These results suggest that the DNA methylation of bovine embryo genome might be involved in the occurrence of sexual dimorphic expression of IFNτ in bovine embryos.

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  • グルコース代謝がウシ受精卵の雌雄比率に及ぼす影響に関する研究

    Grant number:16780208  2004 - 2005

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    木村 康二

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    Grant amount:\3700000 ( Direct expense: \3700000 )

    1.屠場由来卵巣から卵子を吸引し、体外成熟・受精・培養を行い、授精3日目に8細胞期胚を得て以下の実験に供試した。
    (1)3種のグルコース濃度(1、2.5及び4mM)のmSOF培地中で胚を培養し、媒精後8日目に胚盤胞期まで発生した胚を回収し、PCR法によって性を判定した。2.5mMまでの培養液へのグルコース添加は発生率には影響を及ぼさなかった。しかしながら、性比は2.5mMのグルコース添加でも有意に雄に偏ることが示された。
    (2)3種類の培養液(無糖mSOF、5.56mM グルコース添加mSOF、5.56mM フルクトース添加mSOF)で培養し、媒精後8日目に胚盤胞期に発生した胚を回収し、PCR法によって性を判定した。5.6mMのグルコース添加は発生率を有意に低下させた。しかしながら、フルクトースの添加は発生率を低下させることは無かった。性比も同様にグルコースでは有意に雄に偏ったがフルクトースではこの偏りが見られなかった。
    (3)種々の濃度のG6PD阻害剤(DHEAまたは6-AN)を含む4mMグルコース加mSOF培地中で胚を培養し、媒精後8日目に胚盤胞期に発生した胚を回収し、PCR法によって性を判定した。グルコースの添加により生じた性比の偏りは、G6PD阻害剤の添加により消失した。
    以上の結果から、8細胞期胚以降に見られる性比の偏りはグルコースの存在により生じ、これは解糖系ではなくペントースリン酸系へのグルコースの流入によって生じることが明らかとなった。
    2.屠場由来卵巣から卵子を吸引し、体外成熟後、グルコース存在下で授精を行い、媒精後6または18時間後に卵を取り出し、培養に供した。媒精後72時間に8細胞期に発生した胚を回収し、その性をPCRにより決定した。その結果、媒精中のグルコースの添加は、卵の受精に悪影響を及ぼしたが、性比に影響を及ぼすことはなかった。以上の結果から受精および前核形成時のグルコースの存在は性比に影響を及ぼさないことが明らかとなった。

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