研究者詳細 - 木村　康二

論文 - 木村　康二

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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

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