研究者詳細 - 木村　康二

論文 - 木村　康二

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Ovine conceptuses express phospholipase inhibitory genes on days 14-15 of pregnancy, interacting with IFNT pathways. 国際誌

Yuta Matsuno, Kazuya Kusama, Koji Kimura, Kazuhiko Imakawa

Reproduction (Cambridge, England) 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

In mammals, various molecules are involved in biochemical interaction between conceptus and endometrium for pregnancy recognition and establishment. In ruminants, interferon tau (IFNT) is the pregnancy recognition factor; however, IFNT alone does not explain corpus luteum maintenance. Although data on factors expressed during implantation has been accumulated, we hypothesized that the conceptus produces additional, uncharacterized molecules during the period of conceptus attachment. This study aimed to identify new conceptus secretory proteins involved in the biochemical interaction between conceptus and endometrium in sheep. We analyzed RNA-sequence data of ovine conceptuses from pregnant animals on days 12, 14, 15, 16, 17, 19, 20, and 21. To identify novel secretory proteins, we focused on highly expressed but uncharacterized genes and performed in silico protein function analysis, identifying genes encoding phospholipase inhibitory proteins expressed on days 14 and 15. Recombinant proteins from these genes were produced and the effects on cultured bovine endometrial epithelial cells (EECs) and stromal cells (STRs) were analyzed by RNA-sequence analysis. Differentially expressed gene (DEG) analysis demonstrated that the recombinant protein treatment upregulated 31 genes and downregulated 4 genes in EECs; is also upregulated 398 genes and downregulated 66 genes in STRs, including implantation-related genes such as ISG15, OAS1X, OAS1Y, PARP9, PARP14, MX1 and PTGS2. Gene set enrichment analysis revealed that DEGs were enriched in several implantation-related pathways, including ISG15 antivirus mechanisms. These results suggest that, in addition to numerous characterized molecules, phospholipase inhibitory protein is a new candidate molecule in enabling biochemical communication between the conceptus and endometrium.

DOI： 10.1530/REP-24-0286

Effects of intrauterine extracellular vesicle microRNAs on embryonic gene expression in low-fertility cows. 国際誌

Rei Ichikawa, Koji Kimura, Sho Nakamura, Satoshi Ohkura, Shuichi Matsuyama

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 38 ( 20 ) e70116 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Embryo survival and pre-implantation development depend on uterine luminal fluid, which is believed to play a role in early embryonic death and infertility in cows. Extracellular vesicles (EVs) in the uterine luminal fluid contain microRNAs (miRNAs), crucial mediators of intercellular communication. miRNAs regulate conceptus-maternal interactions and participate in embryonic development by suppressing gene expression. Therefore, we hypothesized that miRNAs in the intrauterine EVs of low-fertility cows would hinder embryonic survival and development. EVs were collected from the bovine uterine luminal fluid of both normal- and low-fertility cows 7 days post-estrus. Small RNA-sequencing analysis of miRNAs isolated from these EVs identified eight miRNAs that were highly expressed in normal-fertility cows (normal-fertility miRNAs) and eight with elevated expression in low-fertility cows (low-fertility miRNAs). These two sets of miRNAs were transfected into hatched blastocysts via lipofection. RNA-seq following lipofection with low-fertility miRNAs identified 424 differentially expressed genes (DEGs) relative to the control; in contrast, following lipofection with normal-fertility miRNAs, seven DEGs were identified. Pathway analysis of the DEGs identified following lipofection with low-fertility miRNAs revealed substantial enrichment of mitogen-activated protein kinase (MAPK) signaling. Expression of activator protein 1 (AP1) and interferon-tau (IFNT) mRNA was significantly lower in the low-fertility miRNA transfection group than in the control. IFNT is essential for maternal pregnancy recognition. Therefore, miRNAs in intrauterine EVs from low-fertility cows at 7 days post-estrus may inhibit embryo development and suppress IFNT expression by altering MAPK signaling.

DOI： 10.1096/fj.202401728R

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

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

Reproduction (Cambridge, England) 166 ( 6 ) 411 - 421 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows. 国際誌

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

Biology of reproduction 108 ( 6 ) 936 - 944 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows. 国際誌

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

Molecular reproduction and development 90 ( 3 ) 141 - 152 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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

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

Reproduction 165 ( 3 ) 301 - 312 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioscientifica

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

その他リンク： 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 69 ( 6 ) 337 - 346 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Animal Reproduction

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17β (E2) assumes a central role in follicular development and selection by activating estrogen receptors β (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

DOI： 10.1262/jrd.2023-047

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

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

Frontiers in endocrinology 14 1075030 - 1075030 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pharmaceutical Society of Japan

Early embryo and sperm transport through the oviductal isthmus depends on the contraction and relaxation of the smooth muscle layers. Dysfunction of the oviduct transport is considered to be one of the causes of infertility. For human infertility, Chinese medicine is used in East Asia. Although there are many clinical reports regarding Tokishakuyakusan (TSS), there is little scientific evidence that it affects infertility. In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation via the G protein-coupled estrogen receptor 1 (GPER1). We collected bovine oviductal isthmic tissues at four stages of the estrous cycle, classified based on a macroscopic observation of the ovary. The Magnus method was used to monitor longitudinal contractility (frequency, contraction force, and tonus). The effects of TSS solution, GPER1 agonist (G-1), and antagonist (G-15) on oviductal contractility were examined. The protein expression level of GPER1 in the oviductal isthmic smooth muscle of each estrous stage was assessed by Western blotting. Although TSS did not affect frequency and contraction force, the tonus was significantly increased by TSS or G-1 at all stages (p < 0.05), and the effect was especially highest at days 1-4 after ovulation. The addition of G-15 significantly suppressed the TSS-induced increase of oviductal tonus at all stages (p < 0.05). There was no significant difference in GPER1 protein expression among the estrous stages. TSS affects oviductal contractility by increasing tonus via GPER1, and it may accelerate gamete and early embryo transport by contracting the oviducts longitudinally.

DOI： 10.1248/bpb.b22-00201

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 68 ( 4 ) 254 - 261 2022年

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Animal Reproduction

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in isolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did not affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology between them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

DOI： 10.1262/jrd.2022-029

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 69 ( 1 ) 18 - 24 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Animal Reproduction

The oviductal epithelium consists of ciliated and non-ciliated cells, and their numbers vary depending on the segment of the oviduct and stage of the estrous cycle. Compared with the ampulla, fewer cyclic changes in the number of the two types of cells occur in the isthmus. Recently, we have reported that the epithelium in the ampullary oviduct is composed of many types of cells during different translational/transcriptional states, and their numbers change during the estrous cycle. However, detailed information regarding the epithelial cell subtypes lining the isthmic oviductal epithelium has not yet been reported. In this study, we aimed to identify the epithelial subtypes in the isthmus of the oviduct using immunohistochemistry. Some similarities and differences were observed between the ampulla and isthmus. As observed in the ampulla, epithelial cells of the isthmus expressed either FOXJ1 (ciliogenesis marker) or PAX8 (non-ciliated cell marker). The estrous cycle affected the number of Ki67+ cells but not that of ciliated cells. A relatively high rate of Ki67+ cells (60%) was observed at 1-4 days after the ovulation. Interestingly, unlike the ampulla, Ki67+/FOXJ1+ cells (12.6 ± 1.1%) were discovered in the isthmus. Double staining for Ki67 with FOXJ1, PAX8, or Centrin-1 (a centriole marker) revealed that Centrin-1 was localized on the apical surface of some Ki67+/FOXJ1+ cells. In conclusion, some epithelial cell subtypes exist in the isthmus of the oviduct and isthmus-specific cell subtypes have been identified. These region-specific cells may provide functional and morphological differences between the ampulla and isthmus of the oviduct.

DOI： 10.1262/jrd.2022-104

Hyperthermia alters interleukin-6 production in response to lipopolysaccharide via endoplasmic reticulum stress in bovine endometrial cells. 査読国際誌

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

Journal of cellular physiology 237 ( 1 ) 1087 - 1099 2021年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

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

Effects of cortisol on prostaglandin F2α secretion and expression of genes involved in the arachidonic acid metabolic pathway in equine endometrium - In vitro study. 査読国際誌

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

Heat stress influences the attenuation of prostaglandin synthesis by interferon tau in bovine endometrial cells. 査読国際誌

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

Theriogenology 165 52 - 58 2021年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

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

Gene-expression profile and postpartum transition of bovine endometrial side population cells†. 査読国際誌

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年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

Alteration of chemokine production in bovine endometrial epithelial and stromal cells under heat stress conditions. 査読国際誌

Shunsuke Sakai, Toshimitsu Hatabu, Yuki Yamamoto, Koji Kimura

Physiological reports 8 ( 22 ) e14640 2020年11月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

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

Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows. 査読国際誌

Yuki Yamamoto, Sayaka Ito, Kiyoshi Okuda, Koji Kimura

Theriogenology 153 143 - 150 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

経産牛の受胎性低下は，分娩後の子宮内膜再生時に生じる不具合が一因である可能性が考えられるが，その詳細なメカニズムは未だ明らかでない。ヒトにおいては，月経周期毎に起こる子宮内膜更新に子宮内膜の間葉系幹細胞が関与することが示唆されており，ウシの分娩後の子宮内膜修復においても子宮内膜幹細胞が関与する可能性が考えられる。そこで本研究では，組織幹細胞の集団として考えられているside population（SP）細胞がウシ子宮内膜にも存在することを示すとともに，ウシ子宮内膜SP細胞の特性および分娩後の動態を明らかにすることを目的とした。食肉センター由来のウシ子宮から単離した子宮内膜細胞をHoechst33342で染色し，フローサイトメトリー解析を行った結果，子宮内膜細胞にSP細胞が含まれることが明らかとなった。次に，ウシ子宮内膜SP細胞（n=3）とSP細胞以外の細胞（main population, MP細胞）（n=3）をセルソーターにより分取し，遺伝子発現をRNA-seqにより解析した。その結果，SP細胞とMP細胞では遺伝子発現パターンが異なることが示された。さらに，SP細胞とMP細胞間の発現変動遺伝子（FDR＜0.05）についてGOおよびKEGGパスウェイ解析（p＜0.05）を行ったところ，MP細胞と比較してSP細胞で高発現であった遺伝子には，核酸代謝，血管発達といった機能や幹細胞の自己複製や分化のシグナル経路に関与する遺伝子が多く含まれており，幹細胞マーカーの遺伝子も含まれていた。また，分娩後10，30，50，100日目にバイオプシーによってウシの子宮内膜組織を採取し（n=8），子宮内膜細胞に対するSP細胞の割合を算出した結果，子宮内膜細胞に対するSP細胞の割合は，分娩後10日目で最も低く，その後徐々に増加した。以上の結果より，ウシ子宮内膜に存在するSP細胞は幹細胞/前駆細胞様な遺伝子発現パターンを示し，分娩後に子宮内膜細胞に分化，増殖することで子宮修復に寄与する可能性が示された。

DOI： 10.14882/jrds.113.0_aw1-4

Analysis of ciliogenesis process in the bovine oviduct based on immunohistochemical classification. 査読国際誌

Sayaka Ito, Yuki Yamamoto, Koji Kimura

Molecular biology reports 47 ( 2 ) 1003 - 1012 2020年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

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

妊娠ウシ子宮頸管におけるISGs発現誘導へのIFNTの直接関与

國井宏樹, 窪友瑛, 浅岡那月, 嶋崎知哉, 古山敬祐, 木村康二, 唄花子, 川原学, 高橋昌志

日本繁殖生物学会講演要旨集 113 ( 0 ) P - 65-P-65 2020年

記述言語：日本語出版者・発行元：日本繁殖生物学会

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

DOI： 10.14882/jrds.113.0_P-65

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

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

Scientific reports 9 ( 1 ) 20330 - 20330 2019年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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

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

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

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. 査読

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年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1262/jrd.2018-063

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. 査読国際誌

Nakamura S, Haga S, Kimura K, Matsuyama S

Journal of animal science 96 ( 11 ) 4902 - 4911 2018年9月

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年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】分娩後ほぼすべてのウシは子宮腔への細菌侵入により子宮内膜炎を発症する。これに対し，子宮内膜細胞はIL-6およびIL-8を分泌し，主要感染部位である子宮内膜上皮層へマクロファージ（MΦ）および好中球を誘導することで細菌除去し，子宮内膜炎を治癒する。しかし，夏季に子宮内膜炎を発症した場合，症状が長期化すると報告されており，夏季不妊の一因として考えられる。本研究では，夏季の子宮内膜炎の長期化はHeat Stress（HS）による子宮内膜の免疫機能低下によると仮定し，冬季および夏季の子宮内膜組織におけるMΦおよび好中球の局在解析と，培養子宮内膜細胞を用いた，リポ多糖（LPS）刺激によるIL-6およびIL-8産生へのHSの影響を検討した。【方法】冬季および夏季に採取した子宮内膜においてMΦおよび好中球の局在を免疫組織化学およびギムザ染色により検討した。また，培養子宮内膜上皮および間質細胞にLPS添加後，38.5°Cまたは40.5°Cで培養し，IL-6およびIL-8遺伝子発現および上清中の濃度を定量的RT-PCR法もしくはEIAによりそれぞれ測定した。【結果】MΦは冬季の子宮内膜において上皮層付近に局在するが，夏季には子宮内膜全域に散在した。一方，好中球の局在は冬季と夏季で変化がなかった。また，38.5°Cで培養した上皮細胞においてIL-6およびIL-8遺伝子発現および分泌量はLPSにより有意に増加した。このIL-6増加はHSにより有意に抑制されたが，IL-8増加にHSは影響しなかった。一方，38.5°Cで培養した間質細胞においてIL-6およびIL-8遺伝子発現および産生量はLPS添加により有意に増加し，この増加はHSによって増強された。以上より，HSは子宮内膜細胞による好中球の誘引に影響しない一方，上皮細胞のIL-6産生を抑制し，間質細胞のIL-6産生を刺激することで上皮層へのMΦ誘導を抑制する可能性が考えられた。これにより夏季の子宮内膜炎症状を長期化させ，夏季不妊の一因となることが示唆された。

DOI： 10.14882/jrds.111.0_or2-13

Heat stress affects prostaglandin synthesis in bovine endometrial cells. 査読

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

The Journal of reproduction and development 64 ( 4 ) 311 - 317 2018年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1262/jrd.2018-051

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年8月

Activation of lysosomal cathepsins in pregnant bovine leukocytes. 査読国際誌

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1530/REP-18-0078

Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium. 査読国際誌

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FASEB

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.

DOI： 10.1096/fj.201701131RR

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. 査読

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

FASEB JOURNAL 32 ( 6 ) 3371 - 3384 2018年6月

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

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

Scientific reports 8 ( 1 ) 1470 2018年1月

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年1月

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

Nishie T, Kobayashi Y, Kimura K, Okuda K

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

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

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

SCIENTIFIC REPORTS 7 ( 1 ) 17827 2017年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

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.

DOI： 10.1038/s41598-017-17968-w

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

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年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

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.

DOI： 10.1292/jvms.17-0284

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

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT, INC

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.

DOI： 10.1089/jir.2017.0057

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

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.2017-025

Age-associated mRNA expression changes in bovine endometrial cells in vitro. 査読国際誌

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOMED CENTRAL LTD

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".

DOI： 10.1186/s12958-017-0284-z

Adrenomedullin regulates the speed of oviductal fluid flow in cattle 査読

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

MOLECULAR REPRODUCTION AND DEVELOPMENT 84 ( 8 ) 712 - 718 2017年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

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.

DOI： 10.1002/mrd.22852

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

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1262/jrd.2016-176

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

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

REPRODUCTION FERTILITY AND DEVELOPMENT 29 ( 7 ) 1280 - 1286 2017年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

The 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.

DOI： 10.1071/RD15538

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

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

REPRODUCTION FERTILITY AND DEVELOPMENT 29 ( 5 ) 1021 - 1028 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

The 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.

DOI： 10.1071/RD15409

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年3月

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

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

JOURNAL OF REPRODUCTION AND DEVELOPMENT 63 ( 1 ) 75 - 85 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.2016-068

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年1月

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

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

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

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

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.

DOI： 10.1071/RD15228

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

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

PLOS ONE 11 ( 12 ) e0167550 2016年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

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.

DOI： 10.1371/journal.pone.0167550

Remodeling of bovine oviductal epithelium by mitosis of secretory cells 査読

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

CELL AND TISSUE RESEARCH 366 ( 2 ) 403 - 410 2016年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

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.

DOI： 10.1007/s00441-016-2432-8

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

Koji Kimura

JOURNAL OF REPRODUCTION AND DEVELOPMENT 62 ( 5 ) 423 - 429 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.2016-066

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

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.2016-075

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年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】経産牛における空胎期間延長の一因として子宮回復不全が挙げられる。子宮は分娩時に胎盤が剥離して大きく損傷を受ける一方，その後速やかに子宮内膜が再生することで修復される。したがって経産牛における子宮回復不全には，分娩後の子宮内膜再生の不具合が関与していると考えられるが，ウシの分娩後における子宮内膜再生メカニズムは未だ解明されていない。我々はこれまでに，ウシ子宮内膜においても幹細胞活性を有すると報告されているSide population（SP）細胞が存在することを明らかにしており，子宮内膜幹細胞が子宮内膜の再生に寄与する可能性を示唆している。そこで本研究では，分娩後におけるSP細胞の経日的な発現パターンについて明らかにすることを目的とした。また，これまでの研究で，ウシ子宮内膜上皮細胞を上皮系の幹細胞用無血清培養液で培養することにより，明らかに形態学的に異なる小型の子宮内膜上皮細胞が得られており，これらの小型子宮内膜上皮細胞が前駆細胞として増殖し，分化することで，ウシの子宮内膜再生に寄与する可能性も考えられる。そこで，ウシ小型子宮内膜上皮細胞の分化能についても検討を行った。【方法および結果】分娩後の子宮内膜におけるSP細胞の割合について，分娩後10日，30日，50日，100日で子宮内膜組織をバイオプシーによって採取し，採取した子宮内膜組織から酵素処理によって得られた細胞をフローサイトメトリー解析によって検討した結果，SP細胞はウシ子宮内膜間質に存在し，子宮内膜上皮には含まれないことが示された。さらに，分娩後10日目にはSP細胞の割合が低く，その後徐々に増加することが明らかとなった。また，ウシ小型子宮内膜上皮細胞は，骨，脂肪，軟骨等の中胚葉由来組織に分化しないことが明らかとなった。以上の結果から，分娩後の子宮内膜再生にはウシ子宮内膜間質に存在するSP細胞が関与する可能性が考えられた。

DOI： 10.14882/jrds.109.0_p-87

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

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

BIOLOGY OF REPRODUCTION 95 ( 1 ) 17 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC STUDY REPRODUCTION

The 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.

DOI： 10.1095/biolreprod.116.140194

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

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

REPRODUCTION 151 ( 6 ) 615 - 622 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

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.

DOI： 10.1530/REP-15-0586

Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins 査読

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

REPRODUCTION 151 ( 6 ) 577 - 587 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

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.

DOI： 10.1530/REP-15-0254

Evidence for a PGF2α auto-amplification system in the endometrium in mares. 査読国際誌

Kozai K, Tokuyama S, Szóstek AZ, Toishi Y, Tsunoda N, Taya K, Sakatani M, Takahashi M, Nambo Y, Skarzynski DJ, Yamamoto Y, Kimura K, Okuda K

Reproduction (Cambridge, England) 151 ( 5 ) 517 - 26 2016年5月

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年3月

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

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年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

The 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<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<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 (<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.

DOI： 10.1002/mrd.22590

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年1月

ウシ卵管峡部上皮細胞におけるアクチビンシグナルによるアポトーシス制御

山本ゆき, 伊藤さやか, 小林芳彦, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 109 ( 0 ) P - 65-P-65 2016年

記述言語：日本語出版者・発行元：日本繁殖生物学会

<p>アクチビンA（ACVA）はアポトーシスや細胞分化など様々な生理現象に関与する局所因子で，ウシ卵管上皮でも遺伝子発現が確認されている。哺乳動物の卵管峡部は，受精前は精子の通路であるとともに貯留や受精能獲得の場であり，受精後は初期胚の発育と輸送の場となる。本研究では，卵管峡部におけるACVAの役割を明らかにする目的で以下について検討した。1）ウシ卵管液中ACVAタンパク質をwestern blotによって確認したところ，発情周期を通じて検出された。2） ACV受容体（ActR）1Bおよび2A，シグナル因子Smad2/3のタンパク質発現が培養上皮細胞で確認されたが，培養間質細胞ではActRsタンパク質は検出されなかった。また卵管峡部組織において，排卵日のActR2A遺伝子発現は排卵後8–12日と比較し有意に低かった。3）発情周期中の峡部上皮細胞におけるcleaved caspase-3（CCP3：アポトーシス誘導因子）局在を免疫組織化学で検討したところ，CCP3陽性細胞数は排卵後2–3日で排卵後5–6日に対し有意に多かった。4）アポトーシス関連因子の遺伝子発現に及ぼすACVAの影響を確認するため，培養峡部上皮細胞にACVAおよびフォリスタチン（FST: ACVA作用阻害因子）またはSB431542（SB:ACVシグナル伝達阻害剤）を単独又は組み合わせで添加し，4時間培養した。ACVA単独添加では影響が認められなかったが，FST単独およびACVAとの組み合わせ，ならびにSB単独添加において，Bcl2（抗アポトーシス因子）/Bax （アポトーシス促進因子）の遺伝子発現の比が有意に減少した。以上より，ウシ卵管峡部上皮ではACVA 作用の阻害によりBcl2/Bax比が減少すること，受精前にはActR2A発現の減少に伴いACVA作用が減弱することが示唆されたため，受精後における峡部上皮細胞のアポトーシス増加にACVAシグナルが関与している可能性が示された。ウシ卵管峡部は，受精前後の数日間で精子の輸送・貯留から初期胚輸送・発育のサポートへとその役割を変えることから，ACVAはアポトーシス制御因子として受精前後の峡部上皮機能調節に関与している可能性が考えられる。</p>

DOI： 10.14882/jrds.109.0_P-65

ウシ卵管分泌上皮細胞の増殖と分化による卵管機能の制御

伊藤さやか, 小林芳彦, 山本ゆき, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 109 ( 0 ) OR1 - 4-OR1-4 2016年

記述言語：日本語出版者・発行元：日本繁殖生物学会

<p>【目的】卵管は受精の場，配偶子および初期胚の輸送経路である。膨大部で受精した初期胚は，卵管上皮の繊毛運動により子宮へと輸送される。卵管上皮は卵母細胞および初期胚の輸送を促す繊毛細胞ならびに，初期胚発育に必要な物質を分泌する分泌細胞の主に2種類から構成される。繊毛細胞の数は排卵時に多い一方，分泌細胞の数は黄体の生育に応じて徐々に増加する。この繊毛および分泌細胞数の変化は卵管上皮の機能を制御していると考えられるが，両細胞比の変化が生じるメカニズムは不明である。本研究では卵管の機能制御機構を明らかにするため，排卵周期にともなう細胞分裂の増減とともに，繊毛および分泌細胞の比率を制御するメカニズムをウシをモデルに検討した。【方法】排卵周期を通じたウシ卵管膨大部および峡部組織における1） FOXJ1（繊毛細胞マーカー）および 2） Ki67（細胞分裂マーカー）陽性細胞の割合を検討した。3） Ki67とFOXJ1またはPAX8（分泌細胞マーカー）との共染色により繊毛ならびに分泌細胞における分裂能を評価した。4）分泌細胞における繊毛細胞への分化能を評価するために，分泌細胞の表面抗原であるSSEA1の抗体を結合させた磁気ビーズを用いて単離した細胞をAir-liquid interface（ALI）法で培養後，acetylated tubulin（繊毛マーカー）陽性細胞を調べた。【結果】1）膨大部においてFOXJ1陽性細胞は排卵前後に増加する一方，峡部では変化がなかった。 2） Ki67陽性細胞は膨大部では卵胞期および排卵日に，峡部では排卵前後に多かった。3）膨大部および峡部においてKi67 陽性細胞は全てFOXJ1陰性であった。4） SSEA1陽性細胞をALI法で培養するとacetylated tubulin陽性細胞へと分化した。以上より，排卵周期を通じた繊毛および分泌細胞の比は分泌細胞の増殖・分化によって制御されることが示唆された。また，排卵前には繊毛細胞が増加する一方，分泌細胞が排卵後に増加することで，卵母細胞の輸送，受精，初期胚の輸送および発育などの，卵管内で起こる現象に対応した適切な環境を提供する可能性が示された。</p>

DOI： 10.14882/jrds.109.0_OR1-4

基底膜下 stem-like cells による卵管上皮組織リモデリング

小林芳彦, 伊藤さやか, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 109 ( 0 ) P - 15-P-15 2016年

記述言語：日本語出版者・発行元：日本繁殖生物学会

<p>【目的】哺乳動物の卵管上皮組織は，排卵周期の進行に伴い変化することで卵管内において起こる様々な生理現象に最適な環境を生み出す。上皮層には既知の卵管上皮細胞（分泌細胞および繊毛細胞）に加え，少数のstem-like cells（CD44陽性細胞）の存在することが近年明らかとなったが，その役割は不明である。本研究はCD44陽性細胞の特性を明らかにすることを目的とした。【方法】24–72カ月齢のウシ卵管において，1） CD44陽性細胞の局在およびCD44と種々のマーカータンパク質の共局在を免疫組織化学的に調べることで同細胞の性質を推定した。2）卵管間質層から磁気ビーズ分離法により単離したCD44陽性細胞の基底膜通過能を，基底膜マトリックスMatrigelをコートしたTranswellを用いて調べた。3） Air-liquid interface（ALI）培養した間質層由来CD44陽性細胞に種々の物質を添加し，上皮/間葉マーカー発現の比率（Cytokeratin-7（CK7）：Vimentin（VIM）およびE-cadherin（CDH1）：VIM）を算出するとともに免疫細胞染色により間葉上皮転換能を評価した。【結果】1） CD44陽性細胞は上皮層に加えて間質層にも存在した。上皮層のCD44陽性細胞にはCK7陽性かつVIM陰性のもの，CK7陰性かつVIM陽性のものならびにCK7陽性かつVIM陽性のものが混在する一方，間質層のCD44陽性細胞は全てCK7陰性かつVIM陽性であった。卵管上皮層におけるVIM陽性のCD44陽性細胞は排卵前と比べ排卵後に減少する一方，CK7陽性のCD44陽性細胞は増加した。2）間質層由来CD44陽性細胞はMatrigel層を通過し，この通過は基底膜成分分解酵素matrix metalloproteinase-2の中和抗体により抑制された。3） Wnt4およびcAMP活性化剤Forskolinの共添加がVIM発現に対するCK7およびCDH1発現の比率を上昇させ，上皮様の形態を持つ細胞への分化を誘導した。以上より，新たに存在の示された間質層由来CD44陽性細胞は基底膜を通過でき，上皮細胞へ分化可能であること，さらに基底膜下のより未分化な細胞が，周期的にリモデリングされる卵管上皮組織を構築する細胞の供給源である可能性が示された。</p>

DOI： 10.14882/jrds.109.0_P-15

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

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

REPRODUCTION 150 ( 3 ) 217 - 225 2015年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

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.

DOI： 10.1530/REP-15-0085

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年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】ウシにおいては分娩を機に受胎率が低下することが知られている。このような受胎率低下は分娩後の子宮内膜再生の不具合が一因と考えられるが，ウシの分娩後における子宮内膜再生メカニズムは未だ解明されていない。ウシとは異なり，月経周期ごとに脱落と増殖を繰り返し絶えず更新されるヒトの子宮内膜においては子宮内膜幹細胞が子宮内膜の更新に寄与することが示唆されている。我々はこれまでに，ウシ子宮内膜上皮細胞においても幹細胞活性を有すると報告されているSide population （SP）細胞が存在することを明らかにしており，本研究では，ウシ子宮内膜上皮培養細胞におけるSP細胞の出現パターンおよびSP細胞の分化能について明らかにすることを目的とした。【方法および結果】ウシ子宮からトリプシン処理により単離し，培養した子宮内膜上皮細胞を用いてHoechst33342にて染色後，フローサイトメトリー解析に供し，SP細胞の存在を確認した。さらにセルソーターによって，ウシの子宮内膜上皮培養細胞からSP細胞を分取し，骨芽細胞および脂肪細胞分化用培地を用いて培養したが，それぞれ骨芽細胞および脂肪細胞への分化は認められなかった。また，マトリゲルと混合したウシ子宮内膜上皮SP細胞を免疫不全マウスの皮下に注入し，9週間後に移植部位を確認したところ，テラトーマの形成は認められなかった。このように，ウシの子宮内膜上皮SP細胞はヒト子宮内膜SP細胞で示されているような多分化能を示さないことから，ウシの子宮内膜再生メカニズムは幹細胞が寄与するヒトの子宮内膜更新とは大きく異なる可能性が示唆された。また，子宮内膜上皮細胞におけるSP分画の割合は一定でなく個体間で大きく変動していたことから，ウシ子宮内膜上皮SP細胞は子宮内膜更新の段階に応じて増殖するような前駆細胞として機能することで子宮内膜の更新に寄与している可能性がある。

DOI： 10.14882/jrds.108.0_p-60

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年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】乳牛では，高泌乳化に伴い穀類依存比率を高めた飼養体系に移行している一方で，受胎率は減少の一途を辿っている。近年，体内に過剰に摂取された余剰の糖などのエネルギー物質は非酵素的にタンパク質と結合して最終糖化産物（Advanced Glycation End Products：AGEs）を生み出し，このAGEsが様々な疾患や臓器・組織の老化を引き起こすことが明らかとなってきた。乳牛の低受胎には高エネルギー飼養体系により蓄積したAGEsが関与している可能性も考えられるが，これまで家畜における血中AGEs濃度を測定した報告はない。本研究ではウシの体内におけるAGEsの蓄積状況を把握するとともに，飼養形態や栄養状態との相関を明らかにすることを目的とした。【方法】日本飼養標準に示された養分要求量を充足するようにTMR給与が行われているホルスタイン経産牛（n=20）と粗飼料主体で飼養されているホルスタイン未経産牛（n=20）および黒毛和種（経産牛n=20，未経牛産n=19）から採血し，AGEsの一種であるメチルグリオキサール（MG）誘導体，グルコース，非エステル化脂肪酸（NEFA）濃度を測定した。【結果および考察】ホルスタイン経産牛（平均日齢 1878日，TMR給与）における血中MG誘導体濃度は，加齢による影響を排除するために日齢差がない黒毛和種経産牛（平均日齢 1858日，粗飼料給与）と比較した結果，有意に高かった。一方，血中グルコースおよびNEFA濃度には差が認められなかった。また，粗飼料主体で飼養されている日齢差がないホルスタイン種（平均日齢 517日）および黒毛和種（平均日齢 505日）の未経産牛の間には血中MG誘導体濃度に差が見られなかった。以上のことから，ホルスタイン経産牛におけるAGEsの蓄積は穀類依存比率を高めた飼養体系が一因である可能性が示唆された。

DOI： 10.14882/jrds.108.0_p-119

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors. 査読

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

PloS one 10 ( 8 ) e0135403 2015年8月

Regulation of gonadotropin secretion by monitoring energy availability 査読

Shuichi Matsuyama, Koji Kimura

Reproductive Medicine and Biology 14 ( 2 ) 39 - 47 2015年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：John Wiley and Sons Ltd

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.

DOI： 10.1007/s12522-014-0194-0

ウシ卵管内における局所調節因子アクチビンの標的細胞

山本ゆき, 小林芳彦, 小林宙, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 108 ( 0 ) P - 63-P-63 2015年

記述言語：日本語出版者・発行元：日本繁殖生物学会

【目的】アクチビン（ACV）Aは局所調節因子として卵胞発育や着床に関与する糖タンパク質である。ACV受容体（ActR）は，プロテインキナーゼとして機能する1型（ActR1）と，リガンド結合部を有する2型（ActR2）からなるヘテロ4量体である。ActRにACVが結合すると，シグナルタンパク質Smad2/3がリン酸化されシグナルが伝達される。我々は以前，ウシ卵管由来の活性型ACVAが排卵前後に増加する可能性を示した。本研究では，卵管におけるACVAの役割を解明することを目的に，以下の4点について検討した。【検討項目】1)ウシ卵管組織におけるActRs遺伝子発現，2)培養卵管上皮および間質細胞におけるActRsの発現，3)培養卵管上皮および間質細胞におけるSmad2/3発現，4)ウシ凍結精子（8個体）のActRsならびにSmad2/3発現。1)，2)，3) については卵管膨大部および峡部のそれぞれで検討した。【結果】1) 膨大部および峡部の卵管組織では ActR1BおよびActR2A の遺伝子発現が確認され，各遺伝子発現量は排卵前後で低かった。2) ActR1BおよびActR2A遺伝子は培養上皮および間質細胞の両方に発現していたが，タンパク質は上皮細胞においてのみ発現していた。 3)培養上皮および間質細胞の両方においてSmad2/3遺伝子発現が確認されたが，タンパク質は上皮細胞でのみ発現していた。4)精子において，ActR1BおよびActR2A の遺伝子およびタンパク質発現とともにSmad2/3遺伝子の発現が確認された。【考察】卵管上皮細胞および精子にActRsおよび Smad2/3が発現していたことから，両者はACVAの標的であることが示唆された。ウシ卵母細胞や初期胚にはすでにActRs の発現が確認されている。しかし卵管組織のActR遺伝子発現は，活性型ACVAが増加する排卵前後に低かった。以上より，排卵前後のウシ卵管内ACVAは卵母細胞のみならず精子にも作用している可能性が示された。

DOI： 10.14882/jrds.108.0_P-63

Interferon-tau attenuates uptake of nanoparticles and secretion of interleukin-1β in macrophages. 査読

Hara K, Shirasuna K, Usui F, Karasawa T, Mizushina Y, Kimura H, Kawashima A, Ohkuchi A, Matsuyama S, Kimura K, Takahashi M

PloS one 9 ( 12 ) e113974 2014年12月

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

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

ZYGOTE 22 ( 4 ) 431 - 439 2014年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CAMBRIDGE UNIV PRESS

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.

DOI： 10.1017/S0967199412000603

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

Koji Kimura, Shuichi Matsuyama

JOURNAL OF REPRODUCTION AND DEVELOPMENT 60 ( 3 ) 210 - 215 2014年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The 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.

DOI： 10.1262/jrd.2013-137

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes 査読

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年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The 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.

DOI： 10.1262/jrd.2013-102

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 査読

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年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC GENERAL MICROBIOLOGY

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 (<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.

DOI： 10.1099/ijs.0.058081-0

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

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年4月

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年3月

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年

記述言語：日本語出版者・発行元：日本繁殖生物学会

［目的］マウスでは，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。

DOI： 10.14882/jrds.107.0_P-87

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年

記述言語：日本語出版者・発行元：日本繁殖生物学会

【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。

DOI： 10.14882/jrds.107.0_P-96

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

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC STUDY REPRODUCTION

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.

DOI： 10.1095/biolreprod.112.106641

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

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年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

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.

DOI： 10.1002/mrd.22187

その他リンク： http://orcid.org/0000-0003-2072-8851

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

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

PLOS ONE 8 ( 7 ) e68686 2013年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

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.

DOI： 10.1371/journal.pone.0068686

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

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年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1002/mrd.22187

Estradiol supports in vitro development of bovine early antral follicles 査読

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

Reproduction 145 ( 1 ) 85 - 96 2013年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：1

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.

DOI： 10.1530/REP-12-0319

その他リンク： 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年

記述言語：日本語出版者・発行元：日本繁殖生物学会

【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。

DOI： 10.14882/jrds.106.0.OR1-38.0

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年

記述言語：日本語出版者・発行元：日本繁殖生物学会

【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。

DOI： 10.14882/jrds.106.0.P-115.0

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

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

Zygote 12 1 - 9 2012年12月

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

Shuichi Matsuyama, Yosuke Sakaguchi, Koji Kimura

JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 ( 5 ) 609 - 614 2012年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.2012-079

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

Shuichi Matsuyama, Takatoshi Kojima, Satoru Kato, Koji Kimura

REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY 10 21 2012年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOMED CENTRAL LTD

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 (< 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.

DOI： 10.1186/1477-7827-10-21

異種間移植した子ウシ精巣組織における伸長精子細胞及び成熟精子の形成

徳永智之, 野口純子, 木村康二, 松山秀一, 細江実佐, 池田光美, 大越勝広, 古澤軌

日本繁殖生物学会講演要旨集 105 1138 - 1138 2012年

出版者・発行元：日本繁殖生物学会

【目的】ブタでは、子ブタの未成熟な精巣組織をヌードマウスに異種間移植することにより成熟精子を形成させ、それを顕微授精に供することにより産子が得られている(Nakai et al., 2010)。本手法をウシに適用できれば、リスクを抱えたクローン及び組換えクローンウシ等の産子や胎子から正常な生殖細胞をレスキューし、直接的に後代を得ることが可能になると考えられる。しかし、ウシでは同様の方法により伸長精子細胞の形成は認められているが、成熟精子を得たとする報告は見あたらない。本研究では、月齢の異なる黒毛和種子ウシ精巣組織の異種間移植を試み、伸長精子細胞及び成熟精子の形成の有無を調べた。【方法】出生後、2週齢、1ヶ月、2ヶ月、3ヶ月、4ヶ月及び6ヶ月齢の黒毛和種子ウシより精巣を摘出し、1～2mm角に細切した組織片を、去勢したICR-nu/nu雄マウス（チャールスリバー）の背部皮下に4カ所移植した。移植後、出生から6ヶ月、8ヶ月及び9ヶ月目に精巣組織を回収し、固定後、組織切片を作製してHE染色を施し、生殖細胞の成熟状態等を観察した。【結果】現在のところ、1ヶ月齢子ウシ由来精巣組織片を移植した例で、移植後7ヶ月（出生後8ヶ月）目で精細管の一部に伸長精子細胞の形成が観察され、さらに1ヶ月後（出生後9ヶ月）では成熟精子が確認された。対照として1ヶ月から8ヶ月齢の子ウシ精巣組織を観察したところでは、8ヶ月齢で成熟精子が観察されることから、移植精巣組織においても、若干遅れるものの生体内とほぼ同じ期間で精子が形成されることが判明した。

DOI： 10.14882/jrds.105.0_1138

プリン塩基代謝のウシ胚発生に及ぼす影響

木村康二, 松山秀一

日本繁殖生物学会講演要旨集 105 1107 - 1107 2012年

出版者・発行元：日本繁殖生物学会

【目的】ウシ初期発生胚は成体細胞と比べて特異な代謝系を備えている。そのためウシ胚は他の細胞と比べて特殊な環境で培養されている。体外成熟・受精・培養に供される胚のすべてが胚盤胞期にまで発育することはなく、その原因の一つは現在用いられてる培養系が健全な胚発生に適切でないことが示されており、さらなるウシ胚培養系の改良が必要である。このためにはウシ胚における物質代謝を詳細に解明することが必須であり、グルコースを中心にさまざまな物質のウシ胚における代謝が明らかとなっている。グルコース代謝の一部はペントースリン酸経路に流入し、その後プリン塩基合成へとつながるが、ウシ胚の核酸の主要構成成分であるプリン塩基代謝については未解明な点が多い。本研究ではプリン塩基代謝のウシ胚発生に及ぼす影響について検討した。【方法】食肉センター由来卵巣から卵子を吸引し体外成熟および体外受精により受精卵を得た。受精後18時間から66時間(1-8細胞期)または66時間から186時間（８細胞期-胚盤胞期）にプリンde novo合成阻害剤であるアミノプテリンおよびでプリン塩基（アデニンおよびヒポキサンチン）を胚培養液に添加し、胚発生率を検討した。【結果】アミノプテリンによってプリン塩基のde novo生合成を抑制したところ、1-8細胞期胚では発生率の低下は見られなかったが、8細胞期-胚盤胞期では濃度依存的に有意に胚発生率を低下させた。次にこのプリン塩基de novo生合成抑制下でプリン塩基を添加したところ、アミノプテリンによるde novo生合成阻害効果は有意に改善された。また、プリン塩基を胚培養液に添加したところ、1-8細胞期では1mMまでの添加でヒポキサンチンは胚発生に影響を与えなかったが、アデニンでは有意に胚発生を抑制し、8細胞期-胚盤胞期ではアデニンおよびヒポキサンチンとも有意に胚発生を抑制したが、その効果はアデニンでより大きいものであった。以上の結果から、プリン塩基は胚発生に必要である一方でその過剰添加は胚発生に負に作用することが明らかとなった。

DOI： 10.14882/jrds.105.0_1107

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

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

REPRODUCTION 142 ( 6 ) 879 - 892 2011年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

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

DOI： 10.1530/REP-11-0157

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

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

THERIOGENOLOGY 75 ( 5 ) 841 - 848 2011年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

The 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 < 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 < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 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.

DOI： 10.1016/j.theriogenology.2010.10.026

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

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

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

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

The 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<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<0.01). Mt number and fertilisation outcome were unrelated, but the nature of this relationship differed between young (21-89 months) and old (>89 months) cows. Thus, we conclude that Mt number, the ATP content and fertilisation outcome of bovine oocytes are affected by maternal age.

DOI： 10.1071/RD10133

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

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

THERIOGENOLOGY 72 ( 1 ) 44 - 53 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

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 < 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.

DOI： 10.1016/j.theriogenology.2009.01.017

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

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.20214

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年8月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】低栄養時にはケトン体の脳での取り込みが増加し、脳内のケトン体濃度上昇が生殖機能を抑制する因子として作用することが示唆されている。しかし、ケトン体の脳での取り込み調節機序については未だ明らかでない。ケトン体の細胞内外への輸送はモノカルボン酸トランスポーター（MCT）を介して行われ、脳ではMCT1が血管内皮細胞、第4脳室上衣細胞、アストロサイトで、また、MCT2が神経細胞で主に発現していることから、脳でのケトン体取り込み調節にMCT1およびMCT2が関与する可能性が考えられる。そこで、本研究では、絶食が脳におけるMCT1およびMCT2遺伝子発現に及ぼす影響について検討した。【方法】卵巣摘出後エストラジオールを代償投与した成熟雌ラット（9-10週齢）を用いた。48時間の絶食を負荷し、血中LH濃度および代謝基質濃度を測定するとともに、視床下部、橋、延髄からtotal RNAを抽出し、リアルタイムRT-PCR法によりMCT1およびMCT2遺伝子発現量を定量した。【結果および考察】絶食により延髄におけるMCT2遺伝子発現量が増加した。視床下部および橋におけるMCT2遺伝子発現量および脳の各部位におけるMCT1遺伝子発現量は絶食による変化はなかった。また、絶食によりLHパルス頻度が減少し、生殖機能の抑制が示された。血中グルコース濃度は低下した一方、血中非エステル化脂肪酸およびケトン体濃度は上昇した。以上の結果から、ラットにおいて、生殖機能の抑制が生じる絶食状態では、延髄におけるMCT2発現の増加が、ケトン体の脳での取り込み増加に関与する可能性が示唆された。

DOI： 10.14882/jrds.101.0.530.0

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

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOMED CENTRAL LTD

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 < 0.001). Conceptus IFNT production was unaffected by diet ( P > 0.1), but positively correlated with maternal body condition score ( P < 0.05), and was higher ( P < 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.

DOI： 10.1186/1477-7827-6-21

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年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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

Koji Kimura, Hisataka Iwata, Jeremy G. Thompson

ANIMAL REPRODUCTION SCIENCE 103 ( 3-4 ) 228 - 238 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

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.

DOI： 10.1016/j.anireprosci.2006.12.014

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

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

THERIOGENOLOGY 68 ( 4 ) 633 - 639 2007年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

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.

DOI： 10.1016/j.theriogenology.2007.02.016

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

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年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

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 < 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.

DOI： 10.1002/mrd.20672

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年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1016/j.anireprosci.2006.05.008

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

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

JOURNAL OF VETERINARY MEDICAL SCIENCE 69 ( 3 ) 225 - 231 2007年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

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.

DOI： 10.1292/jvms.69.225

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査読

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年

記述言語：日本語掲載種別：研究論文（学術雑誌）

現行の牛過剰排卵誘起（SOV）においては、豚由来卵胞刺激ホルモン（FSH-P）を数日間にわたり多回投与する必要があり、作業者や牛の負担が大きい。そこで、高分子吸着・徐放能を有する水酸化アルミニウムゲル（AG）に吸着させたFSH-P1回投与（ワンショット）によるSOV効果について検討した。30AU（アーマー単位）のFSH-PをAGに吸着（最終アルミニウム濃度：1.3mg／ml）させた後に黒毛和種雌牛の皮下に投与し、その2日後にプロスタグランジンF2α類縁体製剤を投与することによりワンショットSOVを実施した。血漿中FSH-P濃度の推移および胚採取成績について、20AUのFSH-Pを3日間朝夕漸減投与した群と比較した。FSH-Pをワンショットすることにより、漸減投与の場合と同様に72時間以上血漿中FSH-P濃度が高値を持続し、複数の卵胞が発育した。ワンショットおよび漸減投与によりSOVを実施した場合の胚採取総数は、12.0および8.2個／頭とワンショットの方が有意（P＜0.05）に多く、正常胚採取数については、7.8および5.8個／頭とワンショットの方が多い傾向にあった。本研究の結果より、FSH-PをAGに吸着させて牛皮下に1回投与することにより、3日間朝夕漸減投与と同等の過剰排卵誘起効果が得られることが明らかとなった。

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

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年

ウシ胚盤胞期胚のcDNAライブラリー構築法査読

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年9月

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

Mari Aoki, Koji Kimura, Osamu Suzuki

Animal Science Journal 77 ( 3 ) 290 - 299 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1111/j.1740-0929.2006.00351.x

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

HL Luo, K Kimura, M Hirako

ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 19 ( 4 ) 495 - 499 2006年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOC

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 < 0.05) and FBS+VEGF (42.1%, p < 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.

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年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

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 (>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.

DOI： 10.1016/j.anireprosci.2005.04.006

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年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

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 査読

K Kimura, LD Spate, MP Green, RM Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 72 ( 2 ) 201 - 207 2005年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

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 < 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.

DOI： 10.1002/mrd.20342

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 査読

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

REPRODUCTIVE TOXICOLOGY 20 ( 2 ) 247 - 254 2005年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

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.

DOI： 10.1016/j.reprotox.2005.01.011

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

M Aoki, K Kimura, O Suzuki

ANIMAL REPRODUCTION SCIENCE 86 ( 1-2 ) 1 - 12 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

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 < 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 < 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 >= 0.3 or >= 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 >= 0.3 and >= 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 >= 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.

DOI： 10.1016/j.anireprosci.2004.04.046

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査読

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年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：岐阜県畜産研究所

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。

その他リンク： http://agriknowledge.affrc.go.jp/RN/2010722694

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

K Kimura, LD Spate, MP Green, RM Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 68 ( 1 ) 88 - 95 2004年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

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 < 0.05) and their continued development impaired (P < 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 < 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.

DOI： 10.1002/mrd.20053

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

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

REPRODUCTION 127 ( 2 ) 159 - 164 2004年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIO SCIENTIFICA LTD

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.

DOI： 10.1530/rep.1.00084

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

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

MOLECULAR REPRODUCTION AND DEVELOPMENT 67 ( 2 ) 193 - 199 2004年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

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 < 0.05) and 48 hr (male: 189.8 +/- 37, 1, female: 410.9 +/- 66.6 pg per embryo; P<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.

DOI： 10.1002/mrd.10389

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年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

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。

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

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

JOURNAL OF REPRODUCTION AND DEVELOPMENT 49 ( 6 ) 493 - 499 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

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.

DOI： 10.1262/jrd.49.493

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年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The 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<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.

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

HL Luo, K Kimura, M Aoki, M Hirako

JOURNAL OF VETERINARY MEDICAL SCIENCE 64 ( 11 ) 967 - 971 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

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<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>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>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.

DOI： 10.1292/jvms.64.967

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

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。

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

HL Luo, K Kimura, M Aoki, M Hirako

JOURNAL OF VETERINARY MEDICAL SCIENCE 64 ( 9 ) 803 - 806 2002年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC VET SCI

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<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<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<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.

DOI： 10.1292/jvms.64.803

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

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

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

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

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.

DOI： 10.1262/jrd.48.447

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 査読

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

DOI： 10.1073/pnas.171305398

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 ( supplement ) 24 - 25 1998年

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