研究者詳細 - 舟橋　弘晃

豚液状精液人工授精におけるカフェイン添加による暑熱期の分娩率低下の改善

山口昇一郎, 金子和典, 金子和典, 川島幸子, 川島幸子, 竹村陽, 竹村陽, 増本憲考, 笠正二郎, 笠正二郎, 舟橋弘晃, 吉岡耕治

日本養豚学会誌 54 ( 4 ) 2017年

ウシ精巣からの精原幹細胞採取の試み

永原知樹, 若井拓哉, 舟橋弘晃

岡山実験動物研究会報 ( 33 ) 50 - 50 2017年

クワの実は上皮細胞死を抑制することでインドメタシン誘発性胃潰瘍発症を阻止する

定兼侑里, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃, 畑生俊光

日本農芸化学会大会講演要旨集(Web) 2016 2016年

クワの実凍結乾燥粉末摂取による肥満抑制効果の検討

畑生俊光, 渡邉智, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃

日本農芸化学会大会講演要旨集(Web) 2016 2016年

生殖補助医療技術者の養成とスキルアップに高等教育機関が果たすべき役割

舟橋弘晃, 高山修, 本橋秀之, 中塚幹也

日本卵子学会誌 1 ( 1 ) S12 - S12 2016年

生殖補助医療現場で使用されている手技等に関する全国調査結果

高山修, 倉田裕子, 小倉初音, 坂東裕香里, 大和礼, 本橋秀之, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃, 舟橋弘晃

日本卵子学会誌 1 ( 1 ) S46 - S46 2016年

IVM中のブタ小中卵胞由来COC,裸化卵母細胞,卵丘細胞内のcAMPおよびcGMP量

奥平裕一, 若井拓哉, 舟橋弘晃

Journal of Reproduction and Development 61 ( Suppl Japanese Issue ) J91 - j91 2015年9月

記述言語：日本語出版者・発行元：(一社)日本繁殖生物学会

【目的】体外成熟（IVM）中の細胞内cAMPとcGMP量の劇的な変化は，卵母細胞の減数分裂再開に大きく関与している。本研究は，卵丘細胞-卵母細胞複合体（COC），裸化卵母細胞（DO）および卵丘細胞塊（CC）内のcAMPとcGMP量のIVM中の変動を，小卵胞（SF：直径1–3 mm）および中卵胞（MF：3–6 mm）間で比較した。【方法】SFまたはMFからCOCsを採取し，24時間IVMを行った（eCG，hCG，dibutyryl cAMP添加mPOM中で20時間培養，その後4時間はそれらを不含のmPOM中で培養）。IVM培養開始0，10，20，24時間に，COCsを回収し，COC，DO，CCそれぞれのcAMPとcGMP量をELISA法で測定した。また，IVM培養開始0, 10, 20時間に，COCあたりの卵丘細胞数を測定し，卵丘細胞あたりのcAMPとcGMP量を推定した。【結果】IVM中のSFおよびMF由来DOのcAMPとcGMP量に差は無かった。MF由来のCOCとCCのcAMP量は，IVM培養開始後20時間にSF由来のそれらより有意に高かった（P < 0.05）。また，IVM培養開始後20時間のSF由来CCのcAMP量は，IVM培養開始後10時間のそれより，有意に低下した（P < 0.01）。MF由来のCOCとCCのcGMP量は，IVM培養開始後0および10時間にSF由来のそれらより有意に高かった（P < 0.05）。IVM中のMF由来のCOC中の卵丘細胞数は，SF由来のそれより有意に多かった（P < 0.01）。IVM培養開始後10時間のSF由来卵丘細胞の推定cAMP量は，MF由来のそれより有意に高かった（P < 0.01）。IVM培養開始0時間のMF由来卵丘細胞の推定cGMP量は，SF由来のそれより有意に高かった（P < 0.01）。以上の結果から，IVM時のSFおよびMF由来COC中の卵丘細胞内cAMPとcGMP量の変動パターンは大きく異なっていることが明らかになった。

J-GLOBAL

Methods for improving in vitro and in vivo boar sperm fertility 国際誌

H. Funahashi

Reproduction in domestic animals = Zuchthygiene 50 40 - 47 2015年7月

記述言語：英語

Fertility of boar spermatozoa is changed after ejaculation in vivo and in vitro. During processing for in vitro fertilization (IVF), although spermatozoa are induced capacitation, resulting in a high penetration rate, persistent obstacle of polyspermic penetration is still observed with a high incidence. For artificial insemination (AI), we still need a large number of spermatozoa and lose a majority of those in the female reproductive tract. Fertility of cryopreserved boar spermatozoa is still injured through freezing and thawing process. In the present brief review, factors affecting fertility of boar sperm during IVF, AI and cryopreservation are discussed in the context of discovering methodologies to improve it.

DOI： 10.1111/rda.12568

完全合成溶液中での未成熟ブタ卵母細胞のガラス化保存加温温度の検討

クワの実凍結乾燥粉末投与によるインドメタシン誘発性胃潰瘍形成抑制効果の検討

畑生俊光, 定兼侑里, 藤原崇子, 大西直規, 村田芳行, 舟橋弘晃

日本農芸化学会大会講演要旨集(Web) 2015 2015年

ヒト初期卵胞体外培養時の調整培地の効果

高山修, 本橋秀之, 奥平裕一, FERRE Pillar, ATHURUPANA Rukmali, MI Bui Thi Tra, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

日本IVF学会雑誌 18 ( 2 ) 79 - 79 2015年

ヒト小卵胞および中卵胞由来卵丘細胞-卵母細胞複合体の体外成熟能

奥平裕一, 高山修, BUI Thi Tra Mi, FERRE Pilar, 本橋秀之, 若井拓哉, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

日本IVF学会雑誌 18 ( 2 ) 81 - 81 2015年

段階的な精子の融解はイノシシの精子の融解後の質を改善する(Stepwise thawing enhanced post-thaw quality of boar sperms)

Athurupana Rukmali, 舟橋弘晃

日本畜産学会大会講演要旨集 118回 201 - 201 2014年3月

ヒト中卵胞および小卵胞由来裸化卵母細胞の体外成熟能

奥平裕一, 李楊, RUKMALI Athurupana, PILAR Ferre, 高山修, 本橋秀之, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

日本生殖医学会雑誌 59 ( 4 ) 391 - 391 2014年

ヒト初期卵胞の体外培養(IVG)における培養条件の検討

高山修, 本橋秀之, 奥平裕一, 李楊, PILAR Ferre, RUKMALI Athurupana, 中塚幹也, 中塚幹也, 舟橋弘晃, 舟橋弘晃

日本生殖医学会雑誌 59 ( 4 ) 317 - 317 2014年

高橋大山, 舟橋弘晃

Journal of mammalian ova research 31 ( 2 ) S43 - S43 2014年

現役の胚培養士におけるリカレント教育士への意識

本橋秀之, 高山修, 沖津摂, 沖津摂, 舟橋弘晃, 舟橋弘晃, 中塚幹也, 中塚幹也

Journal of mammalian ova research 31 ( 2 ) S97 - S97 2014年

中卵胞由来卵丘細胞塊分泌因子は小卵胞由来ブタ卵母細胞の体外成熟能を改善する

清水舞, 舟橋弘晃, 船橋弘晃

Journal of mammalian ova research 31 ( 2 ) S103 - S103 2014年

ヒト卵巣組織ガラス化保存融解後の生存性はFreshと同等か?

本橋秀之, 高山修, RUKMALI Athurupana, 奥平裕一, PILAR Ferre, 李楊, 中塚幹也, 舟橋弘晃

日本生殖医学会雑誌 59 ( 4 ) 411 - 411 2014年

完全合成溶液による未成熟ブタ卵丘細胞-卵母細胞複合体のガラス化保存

高橋大山, 舟橋弘晃

Journal of reproduction and development 59 ( Suppl Japanese Issue ) j165 - j165 2013年

豚の非外科的胚移植と新しい人工授精技術の実用化・産業化のための研究開発

吉岡耕治, 舟橋弘晃, 三角浩司, 星宏良, 阿部宏之, 中根崇, 坂上信忠, 柴田貴子, 中野貞雄

農林水産省農林水産技術会議事務局研究成果 ( 481 ) 2013年

タンパク質合成阻害剤クロラムフェニコールおよびシクロヘキシミド存在下でのブタ精子の受精能獲得および体外受精能

奥平裕一, 舟橋弘晃

Journal of reproduction and development 59 ( Suppl Japanese Issue ) j136 - j136 2013年

子宮内免疫反応制御による豚人工授精技術

山口昇一郎, 鈴木千恵, 野口倫子, 笠正二郎, 森美幸, 磯崎良寛, 舟橋弘晃, 吉岡耕治

Journal of reproduction and development 58 ( Suppl Japanese Issue ) j77 - j77 2012年

ブタ受精卵の体外生産技術

舟橋弘晃

Journal of reproduction and development 58 ( Suppl Japanese Issue ) j76 - j76 2012年

中小卵胞由来ブタ卵丘細胞卵母細胞複合体のヒアルロナン産生能

中小路宗洋, 舟橋弘晃

Journal of reproduction and development 58 ( Suppl Japanese Issue ) j97 - j97 2012年

グリセリンおよびトレハロースがブタ精液の凍結融解後の精子機能の正常性に及ぼす影響

廣瀬悠人, 舟橋弘晃, 舟橋弘晃

Journal of reproduction and development 58 ( Suppl Japanese Issue ) j205 - j205 2012年

豚液状精液へのカフェイン添加が人工授精後の繁殖成績に及ぼす影響

増本憲考, 山口昇一郎, 笠正二郎, 舟橋弘晃, 吉岡耕治

日本養豚学会大会講演要旨 97th 2012年

精子内RNA量を指標としたブタ精液の評価の可能性

奥平裕一, 佐々木真也, 舟橋弘晃

Journal of reproduction and development 57 ( Suppl Japanese Issue ) j118 - j118 2011年

津高牧場への発情発見システム「牛歩」の導入と今後の課題

舟橋弘晃, 舟橋弘晃, 野久保隆, 片山佳司

岡山大学農学部センター報告 ( 33 ) 2011年

Brilliant cresyl blueで判別された中小卵胞由来ブタ卵母細胞の体外成熟能とRNA量

THANH Lam Thi Ngoc, 舟橋弘晃

Journal of reproduction and development 57 ( Suppl Japanese Issue ) 2011年

豚凍結精液人工授精における精液希釈液へのカフェイン添加が融解精子の膜正常性,子宮内白血球数および炎症性サイトカインmRNA発現に及ぼす影響

山口昇一郎, 鈴木千恵, 野口倫子, 笠正二郎, 森美幸, 磯崎良寛, 舟橋弘晃, 吉岡耕治

Journal of reproduction and development 57 ( Suppl Japanese Issue ) j138 - j138 2011年

ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

R. Romar, M. J. Izquierdo-Rico, H. Funahashi

REPRODUCTION FERTILITY AND DEVELOPMENT 23 ( 1 ) 221 - 221 2011年

REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

M. J. Izquierdo-Rico, R. Romar, C. Kohata, H. Funahashi

REPRODUCTION FERTILITY AND DEVELOPMENT 23 ( 1 ) 229 - 230 2011年

小卵胞由来体外成熟ブタ卵母細胞の体外受精および前核形成能

小畑千由貴, 舟橋弘晃

日本畜産学会大会講演要旨 112th 85 - 85 2010年

タンパク質合成阻害剤がブタ精子内RNA量および運動性に及ぼす影響

山下佳佑, 舟橋弘晃

Journal of reproduction and development 56 ( Suppl Japanese Issue ) j79 - j79 2010年

分化誘導したマウスES細胞及びiPS細胞におけるCy3標識REIC/Dkk-3タンパク質のエンドサートーシスによる取り込み

片岡健, 阪口政清, LI Kun Peng, DU Gang, 武田千佳, 舟橋弘晃, 村田等, HUH Nam-Ho

組織培養研究 29 ( 1 ) 74 - 74 2010年

ラット胚盤胞の非外科的受精卵移植による産子の獲得

金時宇, 柏崎直巳, 舟橋弘晃

Journal of reproduction and development 56 ( Suppl Japanese Issue ) j70 - j70 2010年

動的環境における胚培養成績上昇機構に関する考察

松浦宏治, 黒田ユカ, 舟橋弘晃, 成瀬恵治

日本生殖医学会雑誌 55 ( 3 ) 97 - 97 2010年

繁殖技術に関する最新動向グローバルな視点での人工授精の技術開発動向

舟橋弘晃

養豚の友 ( 494 ) 2010年

マイクロ空圧駆動型人工卵管で負荷される力学的刺激

李井春, 松浦宏治, 兒玉美恵子, 黒田ユカ, 舟橋弘晃, 成瀬恵治

化学とマイクロ・ナノシステム研究会講演要旨集 21st 2010年

卵黄がウシ及びブタ多核白血球の貪食性と走化性に及ぼす影響

李井春, 舟橋弘晃

日本畜産学会大会講演要旨 112th 86 - 86 2010年

Reduction of immunological sperm loss following artificial insemination of boar semen

FUNAHASHI Hiroaki, LI Jing-Chun, YAMAGUCHI Shoichiro, MURAKAMI Tetsuya

日本養豚学会誌 = The Japanese journal of swine science 46 ( 4 ) 211 - 212 2009年12月

授精のベストタイミングとTop Quality Embryoの獲得 IVM成熟卵子の最適受精条件とは何か?

舟橋弘晃

日本IVF学会誌 12 51 - 53 2009年9月

卵黄含有希釈液の注入がラット子宮内多核白血球数に及ぼす影響

藪崎雅紀, 舟橋弘晃

Journal of reproduction and development 55 ( Supplement ) j158 - j158 2009年

PKC活性化剤によるゴナドトロピン・フリー完全合成培地中でのブタ卵母細胞の体外成熟

中務結貴, 舟橋弘晃, 舟橋弘晃

Journal of reproduction and development 55 ( Supplement ) j139 - j139 2009年

傾斜培養によるマウス胚の培養成績とそのマイクロ空間制御

黒田ユカ, 黒田ユカ, 松浦宏治, 松浦宏治, 舟橋弘晃, 成瀬恵治

化学とマイクロ・ナノシステム研究会講演要旨集 20th 2009年

PDMSの物性を利用した生殖細胞操作デバイスの作製

松浦宏治, 松浦宏治, 黒田ユカ, 黒田ユカ, 舟橋弘晃, 成瀬恵治

化学とマイクロ・ナノシステム研究会講演要旨集 20th 2009年

小卵胞由来および中卵胞由来ブタ卵母細胞に付随する卵丘細胞の生存性およびアポトーシス頻度の比較

松永利恵, 舟橋弘晃

日本畜産学会大会講演要旨 110th 89 - 89 2009年

カフェイン及びヘパリンが豚白血球の走化性と貪食性に及ぼす影響

李井春, 舟橋弘晃

Journal of reproduction and development 55 ( Supplement ) j158 - j158 2009年

シリコンエラストマー製チャンバーによるガラス吸着性精子の運動軌跡測定

松浦宏治, 山下佳佑, 黒田ユカ, 舟橋弘晃, 舟橋弘晃

Journal of reproduction and development 55 ( Supplement ) j75 - j75 2009年

卵丘細胞の共培養が小卵胞由来ブタ卵母細胞の成熟率に与える影響

松永利恵, 舟橋弘晃

Journal of reproduction and development 55 ( Supplement ) j137 - j137 2009年

カフェイン添加希釈液への抗酸化剤の添加が豚凍結融解精子の先体反応及びAI後の繁殖成績に及ぼす影響

山口昇一郎, 舟橋弘晃, 村上徹哉, 徳満茂

日本養豚学会大会講演要旨 92nd 2009年

DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

Y. Akaki, K. Yoshioka, H. Funahashi

REPRODUCTION FERTILITY AND DEVELOPMENT 21 ( 1 ) 218 - 219 2009年

子宮内免疫制御による豚凍結精液人工授精方法の検討

山口昇一郎, 舟橋弘晃, 村上徹哉

日本畜産学会大会講演要旨 109th 2008年

ゴナドトロピン・フリー完全合成培地中でのブタ卵母細胞の体外成熟

赤木友香, 舟橋弘晃

日本畜産学会大会講演要旨 109th 2008年

揺動培養によるマウス胚盤胞到達率の上昇

黒田ユカ, 松浦宏治, 片野坂友紀, 毛利聡, 舟橋弘晃, 成瀬恵治

日本生体医工学会大会プログラム・論文集(CD-ROM) 47th 2008年

牛多核白血球の精子および血清に対する走化性

李井春, 薮崎雅紀, 舟橋弘晃, 舟橋弘晃

Journal of reproduction and development 54 ( Supplement ) j137 - j137 2008年

ラットおよびマウス透明帯除去初期胚の体外培養とヒアルロン酸による卵割促進効果

奥山正信, 舟橋弘晃

Journal of reproduction and development 54 ( Supplement ) j123 - j123 2008年

ラット子宮内多核白血球の性周期および交配後の変動

薮崎雅紀, 李井春, 舟橋弘晃, 舟橋弘晃

Journal of reproduction and development 54 ( Supplement ) j137 - j137 2008年

体外成熟時のシェアストレスがブタ卵母細胞の初期発生能に及ぼす影響

小池孝幸, 成瀬恵治, 舟橋弘晃

日本畜産学会大会講演要旨 109th 2008年

老齢種雄豚精子希釈液へのカルニチン添加が精子ミトコンドリア活性に及ぼす影響

佐野光, 舟橋弘晃

日本畜産学会大会講演要旨 109th 2008年

揺動培養器を用いたブタ卵母細胞の体外成熟と単為発生後の初期発生

舟橋弘晃, 小池孝幸, 成瀬恵治

日本生殖医学会雑誌 53 ( 1/2 ) 44 - 44 2008年

哺乳動物卵母細胞の体外成熟の現況

舟橋弘晃

Journal of clinical embryologist 9 4 - 8 2007年6月

カルニチンがブタ卵母細胞の体外成熟に及ぼす影響

赤木友香, 舟橋弘晃

日本畜産学会大会講演要旨 108th 67 - 67 2007年

限定培地へのPyruvate添加がブタ卵母細胞の核・細胞質成熟に及ぼす影響

小池孝幸, 坂井涼子, 舟橋弘晃

日本畜産学会大会講演要旨 108th 67 - 67 2007年

グルコースおよびピルビン酸がブタ卵母細胞の核成熟およびグルタチオン濃度に及ぼす影響

小池孝幸, 舟橋弘晃

日本畜産学会大会講演要旨 107th 74 - 74 2007年

小中卵胞由来ブタ卵母細胞-卵丘細胞塊におけるグルコース6リン酸脱水素酵素活性の所在

舟橋弘晃, 池原あかり, 石田有希

日本畜産学会大会講演要旨 107th 74 - 74 2007年

Nucleosideが成熟過程のマウス卵母細胞内ミトコンドリア膜電位活性に及ぼす影響

藤井渉, 舟橋弘晃

Journal of reproduction and development 53 ( Supplement ) j177 - j177 2007年

豚凍結精液の人工授精で用いる融解液の違いが人工授精後の繁殖成績および豚凍結精子の先体反応に及ぼす影響

山口昇一郎, 舟橋弘晃, 村上徹哉, 伊東正吾

日本畜産学会大会講演要旨 106th 101 - 101 2006年

豚精液の深部注入器によるAI技術の現状海外および国内の事例から

舟橋弘晃

養豚の友 ( 444 ) 2006年

体細胞を導入したラット卵母細胞の活性化後の発生能

藤井渉, 舟橋弘晃

Journal of reproduction and development 52 ( Supplement ) j77 - j77 2006年

Select antioxidants improve the function of extended boar semen stored at 10 degrees C 国際誌

H Funahashi, T Sano

THERIOGENOLOGY 63 ( 6 ) 1605 - 1616 2005年4月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE INC

The objective was to determine the effects of antioxidant addition to extender on viability, acrosome integrity and penetrability in vitro of boar spermatozoa preserved at 10 degrees C. Washed spennatozoa were resuspended at 1 x 10(8) cells/mL in modified Modena solution containing 20% (v/ v) boar seminal plasma and 5 mM antioxidant (glutathione, cysteine or hypotaurine). Control aliquots were the same suspension without added antioxidants. Sperm suspensions were then chilled to 10 degrees C with a computerized cooling program. Sperm viability after 7 and 14 d was higher in the presence of glutathione or cysteine, whereas hypotaurine did not improve the survival rate. Percentage of chlortetracycline (CTC) fluorescence pattern as intact live cells was higher in spermatozoa preserved with glutathione or cysteine at 7 and 14 d of preservation. When the preservation period was prolonged until 57 d, survival rate was higher with cysteine than controls. When spermatozoa were preserved with cysteine and then inseminated in an IVF system, penetration rate was not different until 15 d of preservation and higher than controls at 15-29 d, whereas no sows became pregnant after AI with spermatozoa preserved for 21-23 d. Therefore, glutathione and cysteine can improve the viability and functional status of boar spermatozoa during liquid preservation and boar spermatozoa penetrated in vitro even after preservation in the presence of cysteine at 10 degrees C for 29 d. (c) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2004.06.016

豚人工授精における技術的課題と技術開発方向２．精液注入器の改良．

舟橋弘晃

畜産技術第599号(2005年4月号) pp13-18 ( 599 ) 2005年

プロテアーゼ阻害剤による体外成熟豚卵子の前処理が体外受精系での精子侵入に及ぼす影響

舟橋弘晃

日本畜産学会大会講演要旨 104th 129 - 129 2005年

Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine 国際誌

H Funahashi, R Romar

REPRODUCTION 128 ( 6 ) 789 - 800 2004年12月

記述言語：英語出版者・発行元：BIO SCIENTIFICA LTD

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 mul modified Medium 199-suspended spermatozoa at 2.5 x 10(5) sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 +/- 2.2) than 8 h (207.6 +/- 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 mumol adenosine/l for 0 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

DOI： 10.1530/rep.1.00295

Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine 国際誌

H Funahashi, R Romar

REPRODUCTION 128 ( 6 ) 789 - 800 2004年12月

記述言語：英語出版者・発行元：BIO SCIENTIFICA LTD

DOI： 10.1530/rep.1.00295

人工授精や体外受精に用いる豚精子受精能の人為制御システムの開発．

舟橋弘晃

平成15年度食肉に関する助成研究調査成果報告書(財団法人伊藤記念財団) Vol.22, p55-61. 2004年

瞬時共培養法による豚卵子への多精子侵入頻度の軽減

舟橋弘晃, ROMAR R

日本畜産学会第103回大会講演要旨 p108 108 - 108 2004年

In vitro fertility of boar spermatozoa preserved at 10oC for 22 days.

Reproduction Fertility and development 16(1,2):255 2004年

IVM-IVF of porcine oocytes treated with roscovitine、

日本畜産学会第103回大会講演要旨 p107 2004年

10℃保存された豚精子の体外受精能と人工授精の成績

豚の繁殖衛生セミナー通信 30, 7-12 2003年

Polyspermic penetration in porcine IVM-IVF systems 国際誌

Hiroaki Funahashi

Reprodution, Fertility and Development 15 ( 3 ) 167 - 177 2003年

記述言語：英語

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm-zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

豚卵母細胞の体外成熟と体外受精

舟橋弘晃

日本不妊学会雑誌 48, 274-275 ( 3/4 ) 274 - 275 2003年

体外培養システムにおけるブタ卵子の細胞質成熟

IVF Conference 2003 68 - 71 2003年

哺乳動物初期胚の対外培養および操作が着床後の成長・分化に残す課題

舟橋弘晃

Journal of Chinical Embryologist 6, 15-22 15 - 22 2003年

Polyspermic penetration in porcine IVM-IVF systems

H Funahashi

REPRODUCTION FERTILITY AND DEVELOPMENT 15 ( 3 ) 167 - 177 2003年

記述言語：英語掲載種別：書評論文，書評，文献紹介等出版者・発行元：C S I R O PUBLISHING

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization ( IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm - zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

DOI： 10.1071/RD02076

同種および異種精しょうが豚凍結精子の生存性および受精能に及ぼす効果

舟橋弘晃, 佐野通

日本養豚学会大会講演要旨 79th 2003年

Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system 国際誌

H Funahashi

REPRODUCTION 124 ( 6 ) 857 - 864 2002年12月

記述言語：英語出版者・発行元：SOC REPRODUCTION FERTILITY

The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO2-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N-omega-vitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N-omega-vitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

DOI： 10.1530/rep.0.1240857

Effect of methyl-beta-cyclodextrin and fertilization promoting peptide on capacitation of boar spermatozoa in a protein-free medium

H Funahashi

JOURNAL OF REPRODUCTION AND DEVELOPMENT 48 ( 1 ) 57 - 63 2002年2月

記述言語：英語出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The present study was undertaken to examine the effect of methyl-beta-cyclodextrin (MBCD) and fertilization promoting peptide (FPP; pGlu-Glu-ProNH(2)) on the capacitation and acrosome reaction of boar spermatozoa in a protein-free medium. After washing with a protein-free medium, ejaculated spermatozoa (resuspended at 1 x 10(6) cells/ml) were cultured in 2 ml of modified Medium-199 containing polyvinylalcohol (mM199-PVA) supplemented or not supplemented with MBCD and/or FPP for 2 h in an atmosphere of 5% CO2 in air at 39 C. Effects of MBCD and FPP on boar spermatozoa were determined by chlortetracycline fluorescence assessment. When spermatozoa were cultured in various MBCD concentrations, capacitation was induced in a concentration-dependent manner, but spontaneous acrosome reaction was not affected. When the effect of FPP in mM199-PVA was determined, sperm capacitation was induced and spontaneous acrosome reaction was inhibited in 100 nM FPP, regardless of the absence or presence of BSA. In the last experiment, spermatozoa were cultured in mM199-PVA supplemented with various concentrations of MBCD and/or FPP. An accelerative effect of MBCD on sperm capacitation was observed in a concentration-dependent manner even in the presence of FPP, whereas 1-2 mM of MBCD neutralized the stimulatory effect of FPP on capacitation. Furthermore, FPP inhibited spontaneous acrosome reaction even in the presence of MBCD. These results demonstrate that MBCD and PFF can induce capacitation of boar spermatozoa in a protein-free medium and that FPP alone also inhibits spontaneous acrosome reaction.

DOI： 10.1262/jrd.48.57

Effect of methyl-beta-cyclodextrin and fertilization promoting peptide on sperm capacitation of boar spermatozoa in a protein-free medium.

Induction of capacitation and the acroxome reaction of boar spermatozoa by 1-arginine and nitric oxide synthesis associated with the anion transport system.

Reproduction 124 ( 6 ) 857 - 864 2002年

カフェインおよび受精促進ペプチド存在下での豚新鮮射出精子のNO産生

第95回日本繁殖生物学会講演要旨集 p161 2002年

豚凍結精液融解後のグリセリン濃度が精子の受精能に及ぼす影響

佐野通, 荒金知宏, 舟橋弘晃

第78回日本養豚学会大会講演要旨集 p11 2002年

チオール類が豚液状保存精子の生存性および自発的先体反応に及ぼす影響

舟橋弘晃, 原田護, 佐野通, 伊藤述史

第77回日本養豚学会大会講演要旨集 p23 2002年

豚精子のHCO3-/C1-輸送がアルギニンによる受精態獲得・先体反応促進効果に及ぼす影響

日本畜産学会第100回大会講演要旨集 p115 2002年

Hiroaki FUNAHASHI

Journal of Reproduction and Development 48 ( 1 ) 57 - 63 2002年

豚精子のHCO₃^-/Cl^-輸送がアルギニンによる受精能獲得・先体反応促進効果に及ぼす影響

舟橋弘晃

日本畜産学会大会講演要旨 100th 2002年

Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro 国際誌

H Funahashi, H Ekwall, K Kikuchi, H Rodriguez-Martinez

REPRODUCTION 122 ( 3 ) 443 - 452 2001年9月

記述言語：英語出版者・発行元：SOC REPRODUCTION FERTILITY

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 mum) and in vitro (5.95 +/- 0.51 mum) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

DOI： 10.1530/rep.0.1220443

Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro

H Funahashi, H Ekwall, K Kikuchi, H Rodriguez-Martinez

REPRODUCTION 122 ( 3 ) 443 - 452 2001年9月

記述言語：英語出版者・発行元：SOC REPRODUCTION FERTILITY

Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine 国際誌

H Funahashi, T Nagai

MOLECULAR REPRODUCTION AND DEVELOPMENT 58 ( 4 ) 424 - 431 2001年4月

記述言語：英語出版者・発行元：WILEY-LISS

Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/1098-2795(20010401)58:4<424::AID-MRD10>3.0.CO;2-1

Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine

H Funahashi, T Nagai

MOLECULAR REPRODUCTION AND DEVELOPMENT 58 ( 4 ) 424 - 431 2001年4月

記述言語：英語出版者・発行元：WILEY-LISS

DOI： 10.1002/1098-2795(20010401)58:4<424::AID-MRD10>3.0.CO;2-1

アルギニンが豚精子の受精能獲得・先体反応に及ぼす影響

舟橋弘晃

日本畜産学会第98回大会講演要旨 98th 110 2001年

緑色蛍光蛋白質の顕微注入法と核移植技術を組み合わせた形質転換牛胚の効率的生産手法の開発

第12回西日本胚移植研究会・第20回北海道牛受精卵移植研究会合同研究発表大会講演要旨集 36 2001年

Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

Sixth International Conference on Pig Reproduction 76 2001年

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

Cloning and Stem Cells 3 ( 4 ) 183 - 190 2001年

記述言語：英語出版者・発行元：Mary Ann Liebert Inc.

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

DOI： 10.1089/15362300152725891

Oviduct involvement in sperm capacitation and oocyte development

Sixth International Conference on Pig Reproduction 62 2001年

An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

Theriogenology 55 ( 1 ) 522 2001年

豚の新しい繁殖技術とその展望

舟橋弘晃

豚の繁殖衛生セミナー通信 27 ( 28 ) 79 - 88 2001年

Effect of fertilization promoting peptide and methyl-beta-cyclodextrin on sperm capacitation of boar spermatozoa in a protein-free medium.

Sixth International Conference on Pig Reproduction 76 2001年

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

Cloning and Stem Cells 3 ( 4 ) 183 - 190 2001年

記述言語：英語出版者・発行元：Mary Ann Liebert Inc.

DOI： 10.1089/15362300152725891

An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer.

Theriogenology 55 ( 1 ) 522 2001年

Involvement of oviduct in sperm capacitation and oocyte development in pigs. 国際誌

H. Rodriguez-Martinez, P. Tienthai, K. Suzuki, H. Funahashi, H. Ekwall, A. Johannisson

Reproduction (cambridge, england) supplement 58 129 - 145 2001年

記述言語：英語

An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for < 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy 国際誌

H Funahashi, H Ekwall, H Rodriguez-Martinez

BIOLOGY OF REPRODUCTION 63 ( 5 ) 1437 - 1442 2000年11月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 mum(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 mum(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

DOI： 10.1095/biolreprod63.5.1437

Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy

H Funahashi, H Ekwall, H Rodriguez-Martinez

BIOLOGY OF REPRODUCTION 63 ( 5 ) 1437 - 1442 2000年11月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

H Funahashi, T Fujiwara, T Nagai

BIOLOGY OF REPRODUCTION 63 ( 4 ) 1157 - 1163 2000年10月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes, These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.

越壁IVF法による精子選別は豚卵子への多精子受精頻度を減少させる

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes 国際誌

H Funahashi, T Fujiwara, T Nagai

BIOLOGY OF REPRODUCTION 63 ( 4 ) 1157 - 1163 2000年10月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

DOI： 10.1095/biolreprod63.4.1157

舟橋弘晃, 永井卓

The journal of reproduction and development 46 ( 5 ) 319 - 324 2000年10月

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro 国際誌

H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

MOLECULAR REPRODUCTION AND DEVELOPMENT 55 ( 1 ) 117 - 124 2000年1月

記述言語：英語出版者・発行元：WILEY-LISS

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome toss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these re sponses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln+FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1098-2795(200001)55:1<117::AID-MRD16>3.0.CO;2-7

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

H Funahashi, A Asano, T Fujiwara, T Nagai, K Niwa, LR Fraser

MOLECULAR REPRODUCTION AND DEVELOPMENT 55 ( 1 ) 117 - 124 2000年1月

記述言語：英語出版者・発行元：WILEY-LISS

DOI： 10.1002/(SICI)1098-2795(200001)55:1<117::AID-MRD16>3.0.CO;2-7

Sperm selection by a climbing-over-a-wall method reduces the incidence of polyspermic penetration of porcine oocytes.

Hiroaki FUNAHASHI, Takashi NAGAI

Journal of Reproduction and Development 46 ( 5 ) 319 - 324 2000年

Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes

Hiroaki Funahashi

Journal of Reproduction and Development 46 ( 5 ) 319 - 324 2000年

記述言語：英語出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

Effect of sperm selection according to the degree of motility after insemination on in-vitro penetration was examined by using a new in-vitro fertilization system designated as a climbing-overa-wall (COW) IVF method. When the sperm penetration rate in the COW-IVF method was compared with a standard method at the same sperm concentration (5 X 10s cells/ml), the rates (95.1 ±1.9 and 98.2 ±1.0%, respectively) were similar, but the incidence of monospermic penetration was higher in the COW-IVF (25.5 ±4.5%) than the standard method (10.4 ±2.5%). When sperm concentration was changed from 0.5 × 105 to 10 × 105 cells/ml in the COW-IVF method, sperm penetration rate was higher at a higher concentration, whereas monospermic penetration rate was increased at a lower concentration. The proportion of monospermic oocytes in matured oocytes was similar among sperm concentrations, 0.5 × 105 to 5 × 105 cells/ml, at fertilization in the COW method. These results demonstrate that the COW-IVF method, selection according to the degree of sperm motility after insemination, can increase the normal penetration of frozen-thawed boar spermatozoa into IVM oocytes without any reduction in the sperm penetration rate.

DOI： 10.1262/jrd.46.319

Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes. (Other,)

The 14th International Congress on Animal Reproduction 2000年

Ultrastructure of the zona reaction of in vivo and in vitro fertilized pig oocytes.

The 14th International Congress on Animal Reproduction 2000年

豚精子の受精能獲得を調節するアデノシン・レセプターの役割

舟橋弘晃, 藤原俊満

日本畜産学会第97回大会講演要旨 97th 124 2000年

The zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy.

Biology of Reproduction 63 ( 5 ) 1447 - 1452 2000年

The zona reaction in porcine oocytes fertilized in vivo and vitro as seen with scanning electron microscopy.

Theriogenology 53 ( 1 ) 420 2000年

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

H Funahashi, N Bandoh, S Nakahira, SH Oh, S Tsuboi

ZYGOTE 7 ( 4 ) 301 - 305 1999年11月

記述言語：英語出版者・発行元：CAMBRIDGE UNIV PRESS

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilised eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilised oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation of rat oocytes. 国際誌

Zygote 7 ( 4 ) 301 - 305 1999年11月

Recent Development in Embryo Technology in Pigs. (共著)

Asian-Australian Journal of Animal Science 12 ( 6 ) 966 - 975 1999年9月

Recent development in embryo technology in pigs - Review

K Niwa, H Funahashi

ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 12 ( 6 ) 966 - 975 1999年9月

記述言語：英語掲載種別：書評論文，書評，文献紹介等出版者・発行元：ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOCIETIES

Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage, has made it possible to utilize follicular oocytes for in, vitro production of embryos and thus stimulated research on various, embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality, Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

DOI： 10.5713/ajas.1999.966

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes

S Said, H Funahashi, K Niwa

ZYGOTE 7 ( 3 ) 249 - 254 1999年8月

記述言語：英語出版者・発行元：CAMBRIDGE UNIV PRESS

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

卵胞膜細胞との共培養による卵丘付着牛卵母細胞の卵核胞発育阻害を伴わない減数分裂の休止

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes 国際誌

S Said, H Funahashi, K Niwa

ZYGOTE 7 ( 3 ) 249 - 254 1999年8月

記述言語：英語出版者・発行元：CAMBRIDGE UNIV PRESS

DOI： 10.1017/S0967199499000635

Production of plasminogen activators(PAs)in bovine cummulus-oocyte complexes during maturation in vitro : effects of epidermal growth factor on production of Pas in oocytes and cumulus cells. (共著)

Biology of Reproduction 61 ( 1 ) 298 - 304 1999年7月

Production of plasminogen activators (PAs) in bovine cumulus-oocyte complexes during maturation in vitro: Effects of epidermal growth factor on production of PAs in oocytes and cumulus cells 国際誌

KW Park, SH Choi, XX Song, H Funahashi, K Niwa

BIOLOGY OF REPRODUCTION 61 ( 1 ) 298 - 304 1999年7月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed, Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex, In COCs before culture, however, no activity of PAs was detected. Al 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

DOI： 10.1095/biolreprod61.1.298

朴光旭, 舟橋弘晃, 丹羽晧二

The journal of reproduction and development 45 ( 3 ) 223 - 231 1999年6月

Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development

Kwang Wook Park, Hiroaki Funahashi, Koji Niwa

Journal of Reproduction and Development 45 ( 3 ) 223 - 231 1999年

記述言語：英語出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

The effects of maturation inhibitors and follicular cells on germinal vesicle (GV) development and meiotic resumption in bovine oocytes were examined. Evaluation of GV development of immature oocytes revealed that oocytes from larger diameter follicles were at more advanced stages. Although culture of cumulus-oocyte complexes (COCs) in the presence of dibutyryl-cAMP, 8-bromo-cAMP or N6-monobutyryl-cAMP did not inhibit meiotic maturation, culture in the presence of cycloheximide (CX) did. Cycloheximide also completely inhibited development of the GV as assessed by changes in morphology. Co-culture of COCs with theca cells (TH) or theca and granulosa cells (TG) also inhibited meiotic resumption but allowed GV development to the most advanced stage, GV-V. Thus co-culture with TH was able to synchronize oocytes at the stage just prior to GV breakdown. When removed from co-culture, the majority of oocytes were able to resume meiosis if co-cultured in a sufficient large volume of medium. The ability of TH to synchronize GV development could be used in a two-step protocol for oocyte maturation, the first step promoting GV development and the second inducing resumption of meiosis. Such an approach might improve the developmental potential of embryos obtained following in vitro fertilization of in vitro matured oocytes.

DOI： 10.1262/jrd.45.223

The presence of Tissue Inhibitor of Matrix Metalloproteinase-1(TIMP-U)During Meiosis Improves Porcine'Oocyte Competence'as Determined by the Early Embryonic Development After In-vitro Fertilization(共著)

FUNAHASHI H, MCINTUSH E W, SMITH M F, DAY B N

Journal of Reproduction and Development 45 ( 4 ) 265 - 271 1999年

卵子への侵入過程におけるラット精子核内チオール類の酸化還元状態の観察と変化(共著)

SAID S, 舟橋弘晃, 丹羽こう二

第95回日本畜産学会大会講演要旨 95th 79 - 79 1999年

Co-culture of cumulus-enclosed bovine oocytes with theca cells induces the meiotic arrest but does not inhibit germinal vesicle development. (共著)

Journal of Reproduction and Development 45 ( 3 ) 223 - 231 1999年

The presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis improves porcine 'oocyte competence' as determined by early embryonic development after in-vitro fertilization

Hiroaki Funahashi, Eric W. McIntush, Michael F. Smith, Billy N. Day

Journal of Reproduction and Development 45 ( 4 ) 265 - 271 1999年

記述言語：英語出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

The present studies were designed to determine the effect of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during meiosis or after in vitro fertilization on early embryonic development of porcine embryos. The presence of purified ovine TIMP-1 in culture medium after in vitro fertilization did not affect the incidence of embryo cleavage or development to the blastocyst stage at 48 h or 6 days after insemination, respectively. In contrast, when various concentrations (1.25, 2.50 and 5.00 μg/ml) of purified ovine TIMP-1 were added to the culture medium during the second half (24 h) of oocyte maturation, normal cleavage rates (64 ± 3% at 1.25 μg/ml, 57 ± 5% at 2.50 μg/ml and 56 ± 1% at 5.00 μg/ml) were higher (p&lt
0.05) as compared to controls (43 ± 2%). In addition, the incidence of embryos that developed to the blastocyst stage was increased (p&lt
0.05) when TIMP-1 was added (34 ± 3% at 1.25 Mg/ml, 32 ± 1% at 2.50 μg/ml and 28 ± 1% at 5.00 μg/ml) as compared to controls (22 ± 3%). There was no difference in the incidence (53 ± 1%) of oocytes fertilized normally. These results indicate that the presence of TIMP-1 during the second half of in vitro maturation enhanced the competence of porcine oocytes to develop to the 2- to 4-cell stages after in vitro fertilization, consequently increasing the incidence of blastocysts.

DOI： 10.1262/jrd.45.265

Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage 国際誌

SH Oh, K Miyoshi, H Funahashi

BIOLOGY OF REPRODUCTION 59 ( 4 ) 884 - 889 1998年10月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

A suitable chemically defined culture medium far 1-cell rat embryos (mR1ECM) was modified to obtain sperm penetration, and the developmental competence of oocytes fertilized in the medium was compared to that of oocytes fertilized in a traditional medium, modified Krebs-Ringer bicarbonate medium (mKRB), Sperm penetration was not observed when polyvinyl alcohol was replaced with ESA in mR1ECM (mR1ECM-BSA); the incidence was improved only when the osmolarity in mR1ECM-BSA was increased to that in mKRB (310 mOsm) by addition of NaCl, The proportion of oocytes penetrated in mR1ECM-BSA with NaCl increased (71.6 +/- 6.9%), which was not different compared to that in mKRB (76.7 +/- 13.7%). High incidences of sperm penetration (88.8 +/- 4.1% to 93.1 +/- 5.1%) were also observed when NaCl concentration in mR1ECM-BSA was increased from 76.7 mM to 100-130 mM. The incidence of embryos developing to the morula and blastocyst stages was higher when fertilized in mR1ECM-BSA containing 110-130 mM NaCl (91-94%) than in mKRB (70%), A total of 5 offspring were obtained after transfer of the morulae and blastocysts (69 embryos/ 7 females), These results demonstrate that a high developmental ability of rat embryos to the blastocyst stage is attained when the embryos have been fertilized in mR1ECM-BSA containing 110-130 mM NaCl and then cultured in mR1ECM.

DOI： 10.1095/biolreprod59.4.884

Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high Nacl concentration and bovine serum albumin maintain developmental ability to the blastocyst stage(共著)

Biology of Reproduction 59 ( 4 ) 884 - 889 1998年10月

Both bovine oocytes and cumulus cells produce plasminogen activator during in vitro maturation

SH Choi, H Funahashi, K Niwa

THERIOGENOLOGY 49 ( 1 ) 309 - 309 1998年1月

Both bovine oocytes and cumulus cells produce plasminogen activator during in vitro maturation(共著)

Theriogenology 49 ( 1 ) 309 - 309 1998年1月

EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

Journal of Reproduction and Fertility, Abstract Series 21 10 1998年

Co-culture of bovine cumulus-oocyte complexes with thece cells inhibits the meiotic resumption at a specific phase of germinal vesicle(共著)

Biology of Reproduction 58 ( Suppl.1 ) 219 - 219 1998年

Viability of boar spermatozoa frozen in 0.5ml-plastic straw by 2-step freezing method(共著)

Proceedings of the 8th World Conference on Animal Production, Contributed Papers 2 252 - 253 1998年

Recent Development in Embryo Technology in Pigs. (共著)

Proceedings of the 8th World Conference on Animal Production, Symposium Series 2 240 - 251 1998年

Cytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic development

87 205 - 214 1998年

EPP and adenosine inhibit spontaneous acrosome loss in boar sperm.

Journal of Reproduction and Fertility, Abstract Series 21 10 1998年

Intracellular calcium release and cortical reaction after thimerosal-treatment of porcine oocytes matured in vitro or ovulated(共著)

Biology of Reproduction 58 ( Suppl.1 ) 219 - 219 1998年

Inhibiting effect of theca cells on meiotic resumption of bovine oocytes may not be via cumulus cells(共著)

Proceedings of the 8th World Conference on Animal Production, Contributed PApers 2 920 - 921 1998年

Effect of cumulus cells on the ability of pig oocytes to utilize cysteine or cystine during maturation in vitro(共著)

Journal of Reproduction and Development 44 ( 2 ) 161 - 168 1998年

Effects of cumulus cells on the ability of pig oocytes to utilize cysteine or cystine during maturation in vitro

Ken Sawai, Hiroaki Funahashi, Koji Niwa

Journal of Reproduction and Development 44 ( 2 ) 161 - 168 1998年

記述言語：英語出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

The present study examined the effect of cysteine and cystine associated with cumulus cells in maturation medium on oocyte glutathione (GSH) concentration at the end of maturation culture (for 48 h) and male pronuclear (MPN) formation following in vitro fertilization of pig oocytes. When cumulus-enclosed oocytes were cultured in a serum-free medium containing 0.57 mM cysteine or cystine, their GSH concentration and incidence of MPN formation were higher than when they were cultured without cysteine and cystine. Supplementation with cysteine during the final 24 h of culture resulted in an increased GSH concentration of oocytes and a higher incidence of MPN formation, regardless of the presence of cumulus cells, than in those cultured without cysteine or cystine. The same high incidence of MPN formation was observed even when oocytes were denuded after 36 h of culture and exposed to cysteine for only 12 h. In contrast, when oocytes were denuded after 24 h of culture and exposed to cystine, neither GSH synthesis nor MPN formation was improved, but both parameters did improve when oocytes were not denuded. Exposure of cumulus-enclosed oocytes to cystine from 36 h of culture did not promote MPN formation. These results indicate that cumulus-free pig oocytes can synthesize GSH in the presence of cysteine and that the presence of cumulus cells is essential for maintaining a high concentration of GSH in oocytes in the presence of cystine.

DOI： 10.1262/jrd.44.161

豚卵胞卵子の体外成熟・受精:とくに初期発生能獲得に関する研究(共著)

Recent Development in Embryo Technology in Pigs. (共著)

Proceedings of the 8th World Conference on Animal Production, Symposium Series 2 240 - 251 1998年

Cytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic developmentCytoplasmic maturation of porcine oocytes for successful male pronuclear formation and early embryonic development

87 205 - 214 1998年

Viability of boar spermatozoa frozen in 0.5ml-plastic straw by 2-step freezing method(共著)

Proceedings of the 8th World Conference on Animal Production, Contributed Papers 2 252 - 253 1998年

舟橋弘晃

Journal of reproduction and development 44 ( 6 ) j47-j52 - j52 1998年

受精促進ペプチド(FPP)およびアデノシンの豚射出精液に及ぼす自発的先体反応阻止作用における競合(共著)

浅野敦之, 舟橋弘晃, 丹羽こう二, FRASER L R

第91回日本繁殖生物学会講演要旨集 91st ( 6 ) 45 - a24 1998年

リン酸によるラット初期胚の発生阻害時期とマイクロフィラメント構造への影響(共著)

呉世薫, 舟橋弘晃, 丹羽こう二

第91回日本繁殖生物学会講演要旨集 91st ( 6 ) 43 - a22 1998年

Co-culture of bovine cumulus-oocyte complexes with theca cells inhibits the meiotic resumption at a specific phase of germinal vesicle.

KW Park, H Funahashi, K Niwa

BIOLOGY OF REPRODUCTION 58 ( Suppl.1 ) 219 - 219 1998年

Intracellular calcium release and cortical reaction after thimerosal-treatment of porcine oocytes matured in vitro or ovulated.

H Funahashi, Z Machaty, WH Wang, TC Cantley, RS Prather, BN Day

BIOLOGY OF REPRODUCTION 58 ( Suppl.1 ) 219 - 219 1998年

Inhibiting effect of theca cells on meiotic resumption of bovine oocytes may not be via cumulus cells(共著)

Proceedings of the 8th World Conference on Animal Production, Contributed PApers 2 920 - 921 1998年

受精促進ペプチド(FPP)およびアデノシンが豚精子の自発的先体反応に及ぼす阻止効果(共著)

15 ( 2 ) S26 1998年

豚受精卵の体外生産と人為操作

舟橋弘晃

家畜人工授精 ( 185 ) 14 - 22 1998年

豚受精卵の体外生産技術の現状

日本畜産学会関西支部報 ( 138 ) 18 - 20 1998年

Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization

H Funahashi, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 57 ( 1 ) 49 - 53 1997年7月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (fetal mean, 82.1 +/- 2.1%) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p < 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p < 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

DOI： 10.1095/biolreprod57.1.49

Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation 国際誌

Ken Sawai, Hiroaki Funahashi, Koji Niwa

Biology of Reproduction 57 ( 1 ) 1 - 6 1997年7月

記述言語：英語

The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-I) to metaphase II (M-II) and M-II at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-I to M-II stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

DOI： 10.1095/biolreprod57.1.1

Stage-specific requirement of oysteine during in-vitro maturation of porcine oocytes for glutathione synthesis ossociated with male pronuclear formation(共著)

Biology of Reproduction 57 ( 1 ) 1 - 6 1997年7月

Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic AMP improves developmental competence following in vitro fertilization(共著) 国際誌

Biology of Reproduction 57 ( 1 ) 49 - 53 1997年7月

Chlortetracycline fluorescence patterns and in vitro fertilization of frozen-thawed boar spermatozoa incuboted under various bicarbonate concentrations(共著) 国際誌

Zygote 5 ( 2 ) 117 - 125 1997年5月

Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations

LR Abeydeera, H Funahashi, NH Kim, BN Day

ZYGOTE 5 ( 2 ) 117 - 125 1997年5月

記述言語：英語出版者・発行元：CAMBRIDGE UNIV PRESS

Porcine oocyte-cumulus complexes were cultured in bovine serum albumin (BSA)-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM) 199 containing caffeine (5 mM), fetal calf serum (FCS; 10%) and varying concentrations (26-56 mM) of NaHCO3 for 9 h (experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%) and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p < 0.05) at 36-56 mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46 mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p < 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56 mM NaHCO3, respectively. The fertilisation medium containing 46 mM NaHCO3 and 1% FCS showed a higher penetration rate (84%) with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes(共著)

56 ( 4 ) 921 - 930 1997年4月

Developmental changes in the intracellular Ca2+ release mechanisms in porcine oocytes 国際誌

Z Machaty, H Funahashi, BN Day, RS Prather

BIOLOGY OF REPRODUCTION 56 ( 4 ) 921 - 930 1997年4月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP(3)), the InsP(3) receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP(3)-induced Ca2+ release was inhibited by heparin, the InsP(3) receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP(3) and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.

DOI： 10.1095/biolreprod56.4.921

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro. 国際誌

H Funahashi, T C Cantley, B N Day

Theriogenology 47 ( 3 ) 679 - 86 1997年2月

記述言語：英語

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

DOI： 10.1016/S0093-691X(97)00026-5

Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

Fifth International Conference on Pig Reproduction 142 1997年

Production of hyaluronic acid by porcine oocyte-cumulus cell complexes during in vitro maturation.

Fifth International Conference on Pig Reproduction 139 1997年

Tris-buffered medium (TBM) is better for in vitro fertilization of porcine oocytes than mM199 which has been traditionally used.

H Funahashi, Y Kajiwara, K Sawai, K Niwa

BIOLOGY OF REPRODUCTION 56 ( Supplementl ) 514 - 514 1997年

Effect of tissue inhibitor of metalloproteinase(TIMP-1)on early development of seine oocytes matured and fertilized in vitro(共著)

Theriogenology 46 ( 1 ) 277 1997年

豚透明帯糖残基の局在性および精子結合・侵入に及ぼす影響(共著)

宋学雄, 楊信志, 舟橋弘晃, 丹羽こう二

第90回日本繁殖生物学会講演要旨集 ( 90th ) 56 1997年

クローン動物とコピー動物

岡山地区高分子懇話会平成9年度第2回高分子講演会 11 - 18 1997年

Advances in in vitro production of pig embryos

H Funahashi, BN Day

JOURNAL OF REPRODUCTION AND FERTILITY Supplement52 271 - 283 1997年

記述言語：英語出版者・発行元：J REPROD FERTIL INC

A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

Additive effects of sodium hyaluronate as inducer of capacitation of boar spermatozoa in vitro.

Fifth International Conference on Pig Reproduction 142 1997年

Production of hyaluronic acid by porcine oocyte-cumulus cell complexes during in vitro maturation.

Fifth International Conference on Pig Reproduction 139 1997年

Effect of tissue inhibitor of metalloproteinase (TIMP-1) on early development of swine oocytes matured and fertilized in vitro

FUNAHASHI H

Theriogenology 47 ( 1 ) 277 - 277 1997年

Tris-buffered medium(TBM)is better for in vitro fertilization of porcine oocytes than mM199 which has been traditionally used(共著)

Biology of Reproduction 56 ( Supplementl ) 514 - 514 1997年

Advances in in-vitro production of porcine embryos(共著)

Journal of Reproduction and Fertility Supplement52 271 - 283 1997年

Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocyte penetrated in vitro(共著)Journal of Reproduction and Development

Journal of Reproduction and Development 43 ( 1 ) 73 - 80 1997年

Effects of cysteine in serum-free maturation medium on male pronuclear formation of maturing pig oocytes penetrated in vitro

Ken Sawai, Hiroaki Funahashi, Wei Hua Wang, Koji Niwa

Journal of Reproduction and Development 43 ( 1 ) 73 - 80 1997年

記述言語：英語出版者・発行元：The Japanese Society of Animal Reproduction (JSAR)

Pig immature oocytes were cultured for 24, 36 and 48 h in serum-free maturation medium with or without 0.57 mM cysteine. The addition of cysteine to the medium was associated with increased glutathione synthesis by oocytes but did not promote nor inhibit nuclear maturation, sperm penetration in vitro, and decondensation of sperm nuclei in penetrated oocytes. The incidence of activation of penetrated oocytes 14 h after insemination in vitro was lower in those cultured both in the presence and absence of cysteine for 24 h than 36 and 48 h. The lower ability of oocytes cultured for 24 h to be activated was not improved by neither the addition of cysteine (0.57 mM) in fertilization medium nor prolonged culture time after insemination. However, male pronuclear formation in activated oocytes after sperm penetration at any stages of maturation was largely accelerated when cysteine was added to maturation medium. An increased concentration of glutathione may induce full decondensation of sperm nuclei in immature pig oocytes penetrated in vitro ensuring transformation of the decondensed sperm nuclei to male pronuclei only in synchronization with oocyte activation.

DOI： 10.1262/jrd.43.73

Preincubation of oocyte-cumulus complexes before exposing to gonadotropins enhanced the developmental ability of porcine embryos maturad and fertilized in vitro(共著)

Theriogenology 47 ( 3 ) 679 - 686 1997年

牛精液用0.5mlストローを用いた豚精子の凍結保存

野上与志郎, 原田護, 関哲生, 舟橋弘晃, 丹羽こう二

日本畜産学会大会講演要旨 93rd 1997年

受精培地中のNaCl濃度がラット卵子への精子侵入およびその後の初期発生におよぼす影響

OH S-H, 舟橋弘晃, 丹羽こう二

Journal of mammalian ova research 14 ( 1 ) 1997年

Advances in in vitro production of pig embryos. 国際誌

H. Funahashi, B. N. Day

Journal of reproduction and fertility. Supplement 52 271 - 283 1997年

記述言語：英語

Gamma-glutamyltranspeptidase of spermatozoa may reduce oocyte glutathione content at sperm penetration(共著) 国際誌

Molecular Reproduction and Development 45 ( 4 ) 485 - 90 1996年12月

Microtubule organization in porcine oocytes during fertilization and parthenogenesis 国際誌

NH Kim, C Simerly, H Funahashi, G Schatten, BN Day

BIOLOGY OF REPRODUCTION 54 ( 6 ) 1397 - 1404 1996年6月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

Microtubule configurations in porcine oocytes after sperm penetration or after artificial activation by electrical stimulation were imaged by immunocytochemistry and laser scanning confocal microscopy. Soon after sperm penetration, an aster was seen adjacent to the incorporated sperm head. Polyspermic penetrations led to the presence of multiple sperm asters in association with each sperm. The sperm aster enlarged and, at the time of pronuclear apposition, filled the cytoplasm. After male and female gamete union, the microtubule matrix was reduced. At the mitotic metaphase stage, microtubules were detected in the spindle, which was anastral and fusiform. At anaphase, asters assembled at each spindle pole, and at telophase, large asters filled the cytoplasm. Artificial activation by electrical stimulation induced in the cytoplasm a dense network of microtubules, which seem to be involved in proper positioning of the female pronucleus. At mitotic metaphase, microtubules were concentrated around the chromatin. The results of experiments using taxol, a microtubule stabilizing agent, suggest that maternal centrosomal material is present in the mature porcine oocyte as dispersed undetectable material that can form a microtubule network after parthenogenetic activation. However, at fertilization, the paternal centrosome collects centrosomal material to form a sperm aster. These results suggest that the functional centrosome that forms during fertilization is a result of the blending of paternal and maternal centrosomal components.

DOI： 10.1095/biolreprod54.6.1397

Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes.(共著)

Biology of Reproduction 54 ( 6 ) 1412 - 1419 1996年6月

Microtuble organization in porcine oocytes during fertilization and parthenogenesis.(共著)

Biology of Reproduction 54 ( 6 ) 1397 - 1404 1996年6月

Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes 国際誌

H Funahashi, NH Kim, TT Stumpf, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 54 ( 6 ) 1412 - 1419 1996年6月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

The effects of organic osmolytes on cytoplasmic maturation of porcine oocytes were examined in maturation medium (modified Whitten's medium) containing various NaCl concentrations. The presence of organic osmolytes, such as taurine and sorbitol, at 6 and 12 mM in maturation medium containing 68.49 or 92.40 mM NaCl increased oocyte glutathione content. Microfilament organization in oocytes was disrupted in maturation medium containing the higher level of NaCl (92.40 mM). However, supplementation with 12 mM sorbitol to the medium reduced the severity of the abnormality. Early embryonic development in vitro to the blastocyst stage was 8.3 +/- 0.9% for oocytes matured in modified Whitten's medium (68.49 mM NaCl) supplemented with 12 mM sorbitol, and 7.9 +/- 0.8% in modified NCSU23 medium (containing 108.73 mM NaCl, 7 mM taurine, 5 mM hypotaurine, and 1 mM glutamine), compared to 4.7 +/- 0.6% in modified Whitten's medium (68.49 mM NaCl), which did not contain organic osmolytes. These results indicate that the presence of organic osmolytes, such as sorbitol and taurine, reduces the detrimental effects of high NaCl concentration in media used for the maturation of porcine oocytes. This effect is reflected by oocyte glutathione content and microfilament organization at the end of maturation and early development following in vitro maturation and in vitro fertilization.

DOI： 10.1095/biolreprod54.6.1412

Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro 国際誌

NH Kim, H Funahashi, LR Abeydeera, SJ Moon, RS Prather, BN Day

JOURNAL OF REPRODUCTION AND FERTILITY 107 ( 1 ) 79 - 86 1996年5月

記述言語：英語出版者・発行元：J REPROD FERTIL INC

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

DOI： 10.1530/jrf.0.1070079

Effects of injecting calcium chloride into in vitromatured porcine oocytes.(共著) 国際誌

Biology of Reproduction 54 ( 2 ) 316 - 322 1996年2月

Effects of injecting calcium chloride into in vitro-matured porcine oocytes

Z Machaty, H Funahashi, MA Mayes, BN Day, RS Prather

BIOLOGY OF REPRODUCTION 54 ( 2 ) 316 - 322 1996年2月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

In vitro-matured porcine oocytes were given injections of 0.1 M CaCl2 and after 6 h evaluated for signs of early and late activation events. CaCl2 injection caused cortical granule exocytosis in 75% (3 of 4) of the oocytes tested. It also induced cell cycle resumption as monitored by the histone H1 kinase assay: the phosphorylation rate of histone H1 decreased to 36.7% of the original value. Treated oocytes completed meiosis, extruded the second polar body, and progressed to first interphase: 79.4% of them formed one or more pronuclei. The elevated intracellular Ca2+ level resulted in activation-related changes in the protein synthetic profile in 90% (9 of 10) of the oocytes. Furthermore, 14.7% (9 of 61) of the treated oocytes developed to the compact morula/early blastocyst stage after a 7-day culture in ligated porcine oviduct, and one blastocyst hatched from the zona pellucida. Control oocytes given injections of 0.1 M MgCl2 or carrier medium (10 mM Hepes) did not show the changes mentioned. The results strengthen the idea that Ca2+ is a cell messenger that plays a central part in oocyte activation; it is concluded that elevated intracellular Ca2+ level caused by a single injection of CaCl2 leads to both early and late events of porcine oocyte activation.

DOI： 10.1095/biolreprod54.2.316

Co-culture with follicular shell pieces(FSP)increases the development al competence of pig oocytes matured in vitro.(共著)

Biology of Reproduction 54 ( Supplement 1 ) 100 - 100 1996年

Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

Hiroaki FUNAHASHI, Todd T. STUMPF, Nam-Hyung KIM, Billy N. DAY

Journal of Reproduction and Development 42 ( 2 ) 109 - 115 1996年

The effect of NaCl in maturation media on histone H1 kinase activity of porcine oocytes was examined at the end of culture for maturation. Oocyte-cumulus complexes were cultured in modified Whitten's medium containing different concentrations of NaCl (44.50, 68.49, 92.40, 116.40 or 140.35 mM). The media were supplemented with 10% porcine follicular fluid and hormones for 20 h and then without hormonal supplements for an additional 20 h. At the end of culture, oocytes were sampled for morphological observation, for glutathione assay and for histone H1 kinase assay. At the end of culture, the incidence of oocytes at each meiotic stage was not different among groups. However, histone H1 kinase activity and glutathione content were higher (P < 0.01) in oocytes cultured in media with lower NaCl concentrations. Histone H1 kinase activity in the oocytes at the end of maturation culture was correlated with intracellular glutathione content (r = 0.899, P < 0.01). These data indicate that histone H1 kinase activity and glutathione content of porcine oocytes at the end of culture for maturation are reduced when a high salt medium is used for maturation.

DOI： 10.1262/jrd.42.109

糖類が豚卵子への精子侵入におよぼす影響(共著)

宋学雄, 沢井健, 許東けい, 舟橋弘晃, 丹羽こう二

第89回日本繁殖生物学会講演要旨 89th 57 1996年

システインおよびシスチンが豚卵胞卵子の応性前核形成能に及ぼす影響(共著)

沢井健, 舟橋弘晃, 丹羽こう二

第89回日本繁殖生物学会講演要旨 89th 44 1996年

成長ホルモンが牛卵胞卵子の成熟分裂の進行速度におよぼす影響(共著)

伊賀浩輔, 舟橋弘晃, 丹羽こう二

第89回日本繁殖生物学会講演要旨 89th 41 1996年

Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.(共著)

Biology of Reproduction 54 ( Supplement 1 ) 408 - 408 1996年

Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

Congress Programme of the 13th International Congress on Animal Reproduction 2 10 - 4 1996年

In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

Theriogenology 45 ( 1 ) 265 1996年

Low salt maturation medium enhances the histone H1 kinase activity of porcine oocytes at the end of in vitro maturation.(共著)

Journal of Reproduction and Development 42 ( 2 ) 109 - 115 1996年

Factors affecting nuclear and cytoplasmic maturation in pig oocytes

P Coy, S Ruiz, N Ouhibi, H Funahashi, BN Day, RM Moor

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 412 - 412 1996年

Co-culture with follicular shell pieces (FSP) increases the developmental competence of pig oocytes matured in vitro.

LR Abeydeera, H Funahashi, TC Cantley, A Rieke, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 100 - 100 1996年

Nuclear morphology of swine oocytes during follicular development following stimulation by eCG injection.

H Funahashi, H Tatemoto, TC Cantley, BN Day

BIOLOGY OF REPRODUCTION 54 ( Supplement 1 ) 408 - 408 1996年

Exposure of swine oocyte-cumulus complexes to dibutyryl cyclic AMP for the first 20 hours of maturation increases the homogeneity of oocyte unclear maturation.(共著)

Congress Programme of the 13th International Congress on Animal Reproduction 2 10 - 4 1996年

In vitro penetration of pig oocytes commercially prepared frozen-thawed boar spermatozoa in a chemically semi-defined bicarbonatefree medium.(共著)

Theriogenology 45 ( 1 ) 265 1996年

Gamma-glutamyltranspeptidase of spermatozoa may reduce oocyte glutathione content at sperm penetration(共著)

Hiroaki Funahashi, Zoltan Machaty, Randall S. Prather, Billy N. Day

Molecular Reproduction and Development 45 ( 4 ) 485 - 490 1996年

The presence of γ-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine 5'-O-(3'-thiotriphosphate) (GTP-γ-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 ± 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 ± 1.3%) at 6 h after injection. Injected GTP-γ-S, however, activated 76.0 ± 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 ± 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-γ-S injected oocytes (4.2 ± 0.7 pmol/oocyte) and noninjected oocytes (4.0 ± 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 ± 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 ± 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP- γ-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes.

DOI： 10.1002/(SICI)1098-2795(199612)45:4<485::AID-MRD11>3.0.CO;2-W

Factors affecting development in vitro of borine and rat 1-cell embryos(共著)

Journal of Mammalian Ova Research 13 ( 2 ) 71 - 80 1996年

Factors Affecting Development in Vitro of Bovine and Rat 1-Cell Embryos

Kazuchika Miyoshi, Hiroaki Funahashi, Koji Niwa

Journal of Mammalian Ova Research 13 ( 2 ) 71 - 80 1996年

Factors affecting nuclear and cytoplasmic maturation in pig oocytes.(共著)

Biology of Reproduction 54 ( Supplement 1 ) 412 - 412 1996年

Pronuclear formation and intracellular glutathione content of in vitro matured porcine oocytes following in vitro fertilization and/or electrical activation.(共著)

Zygote 3 ( 3 ) 273 - 281 1995年8月

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos.(共著)

Theriogenology 44 ( 3 ) 391 - 401 1995年8月

PRONUCLEAR FORMATION AND INTRACELLULAR GLUTATHIONE CONTENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING IN-VITRO FERTILIZATION AND/OR ELECTRICAL ACTIVATION 国際誌

H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

ZYGOTE 3 ( 3 ) 273 - 281 1995年8月

記述言語：英語出版者・発行元：CAMBRIDGE UNIV PRESS

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

DOI： 10.1017/S0967199400002677

PRONUCLEAR VISIBILITY, DEVELOPMENT AND TRANSGENE EXPRESSION IN IVM/IVF-DERIVED PORCINE EMBRYOS

HM KUBISCH, MA LARSON, H FUNAHASHI, BN DAY, RM ROBERTS

THERIOGENOLOGY 44 ( 3 ) 391 - 401 1995年8月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). Alter 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

DOI： 10.1016/0093-691X(95)00193-C

γ-glutamyl-transpeptidase(GGT)of spermatozoa may reduce oocyte glutathione(GSH)contest at sperm penetration.(共著)

Biology of Reproduction 52 ( Supplement1 ) 140 - 140 1995年

Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著) Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's

Role in the Genetic Improvement of Farm Animals 19 1995年

Microtuble and microfilament dynamics in porcine oocytes during meiotic maturation.(共著) 国際誌

Molecular Reproduction and Development 43 ( 2 ) 248 - 255 1995年

Microtuble and microfilament dynamics in porcine oocytes during meiotic maturation.(共著)

Molecular Reproduction and Development 43 ( 2 ) 248 - 255 1995年

EFFECT OF TAXOL ON MICROTUBULE ORGANIZATION IN PORCINE OOCYTES AFTER FERTILIZATION AND PARTHENOGENETIC ACTIVATION

NH KIM, LR ABEYDEERA, RE EVERTS, H FUNAHASHI, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 139 - 139 1995年

EFFECT OF BICARBONATE ON IN-VITRO PENETRATION OF PIG OOCYTES BY COMMERCIALLY PREPARED FROZEN-EJACULATED BOAR SEMEN

LR ABEYDEERA, H FUNAHASHI, NH KIM, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement 1 ) 122 - 122 1995年

Effects of cumulus cells on glutathione content of porcine oocyte during in vitro maturation

FUNAHASHI H

J Anim Sci 73 ( 1 ) 90 - 90 1995年

Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995年

Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

Theriogenology 43 ( 1 ) 262 1995年

Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

Theriogenology 43 ( 1 ) 213 1995年

GAMMA-GLUTAMYL-TRANSPEPTIDASE (GGT) OF SPERMATOZOA MAY REDUCE OOCYTE GLUTATHIONE (GSH) CONTENT AT SPERM PENETRATION

H FUNAHASHI, Z MACHATY, RS PRATHER, BN DAY

BIOLOGY OF REPRODUCTION 52 ( Supplement1 ) 140 - 140 1995年

Effect of exposure of porcine oocyte-cumuluscomplexes to gonadotropins.n.(共著)

Beltsville Symposium (]G0010[)(]G0010[) : Biotechnology's Role in the Genetic Improvement of Farm Animals 19 1995年

Effects of cumulus cells on glutathione content of porcine oocytes during in vitro maturation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995年

Cytoskeletal reorganization in porcine oocytes after chemical activation.(共著)

Journal of Animal Science 73 ( Supplement 1 ) 90 1995年

Comparative expression patterns of two transgenes in murine,porcine and bovine embryos.(共著)

Theriogenology 43 ( 1 ) 262 1995年

Effects of organic osmolytes in maturation medium on intracellular glutathione content of porcine oocytes.(共著)

Theriogenology 43 ( 1 ) 213 1995年

Effect of taxol on microtuble organization in porcine oocytes after fertilization and parthenogenetic activation.(共著)

Biology of Reproduction 52 ( Supplement 1 ) 139 - 139 1995年

Effect of bicarbonate on in vitro penetration of pig oocytes by commercially prepared frozen-ejaculated boar semen.(共著)

Biology of Reproduction 52 ( Supplement 1 ) 122 - 122 1995年

Use of low-salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization.(共著)

Biology of Reproduction 51 ( 4 ) 633 - 639 1994年10月

USE OF LOW-SALT CULTURE-MEDIUM FOR IN-VITRO MATURATION OF PORCINE OOCYTES IS ASSOCIATED WITH ELEVATED OOCYTE GLUTATHIONE LEVELS AND ENHANCED MALE PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION 国際誌

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION 51 ( 4 ) 633 - 639 1994年10月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supple ments; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl(59.3 +/- 8.1 and 70.9 +/- 2.0%,respectively) than in medium containing 68.49 mM NaCl (33.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

DOI： 10.1095/biolreprod51.4.633

Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

Theriogenology 41 ( 7 ) 1425 - 1433 1994年5月

Different hormonal requirements of pig oocyte-cumulus complexes during maturation in vitro.(共著)

Journal of Reproduction and Fertility 101 ( 1 ) 159 - 165 1994年5月

IN-VITRO DEVELOPMENT OF IN VITRO-MATURED PORCINE OOCYTES FOLLOWING CHEMICAL ACTIVATION OR IN-VITRO FERTILIZATION 国際誌

H FUNAHASHI, TC CANTLEY, TT STUMPF, SL TERLOUW, BN DAY

BIOLOGY OF REPRODUCTION 50 ( 5 ) 1072 - 1077 1994年5月

記述言語：英語出版者・発行元：SOC STUDY REPRODUCTION

Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p < 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p < 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (P < 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p < 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p < 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (P < 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes) as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

DOI： 10.1095/biolreprod50.5.1072

In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization.(共著)

Biology of Reproduction 50 ( 5 ) 1072 - 1077 1994年5月

DIFFERENT HORMONAL REQUIREMENTS OF PIG OOCYTE-CUMULUS COMPLEXES DURING MATURATION IN-VITRO 国際誌

H FUNAHASHI, T CANTLEY, BN DAY

JOURNAL OF REPRODUCTION AND FERTILITY 101 ( 1 ) 159 - 165 1994年5月

記述言語：英語出版者・発行元：J REPROD FERTIL INC

Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml(-1), hCG (10 iu ml(-1)) and oestradiol (1 mu g ml(-1)) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte-cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68-70% and 45-49%, respectively) than in oocytes previously exposed to PMSG or hCG (89-99% and 71-89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86-99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63-72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 +/- 72.6 ng ml(-1)) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 +/- 13.3 ng ml(-1)), during the first 20 h of culture (42.8 +/- 19.4 ng ml(-1)) and also during the second 20 h period of culture without hormonal supplements (0.925 +/- 0.544 ng ml(-1)). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 +/- 0.2 ng ml(-1) at 0 h to 6.9 +/- 1.5 ng ml(-1) after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte-cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.

DOI： 10.1530/jrf.0.1010159

DEVELOPMENTAL ABILITY OF PORCINE OOCYTES MATURED AND FERTILIZED IN-VITRO 国際誌

H FUNAHASHI, TT STUMPF, SL TERLOUW, TC CANTLEY, A RIEKE, BN DAY

THERIOGENOLOGY 41 ( 7 ) 1425 - 1433 1994年5月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17 beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42/71) and with monospermic penetration and male and female pronuclei (32%, 23/71) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18/71 and 8%, 6/71, respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28/288 and 13%, 41/318, respectively) were also higher (P < 0.05) than in controls (2%, 6/284 and 6%, 16/248, respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9/288 in PFF-treated group and 2%, 6/284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3/100) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

DOI： 10.1016/0093-691X(94)90193-M

DEVELOPMENT OF RAT ONE-CELL EMBRYOS IN A CHEMICALLY-DEFINED MEDIUM - EFFECTS OF GLUCOSE, PHOSPHATE AND OSMOLARITY

K MIYOSHI, H FUNAHASHI, K OKUDA, K NIWA

JOURNAL OF REPRODUCTION AND FERTILITY 100 ( 1 ) 21 - 26 1994年1月

記述言語：英語出版者・発行元：J REPROD FERTIL INC

Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l(-1) (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-(1). This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l(-1) and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mu mol phosphate l(-1), but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mu mol phosphate l(-1). In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l(-1) (67%) and 10.0 mmol glucose l(-1) (60%) were not statistically different from the percentage at 5.0 mmol glucose l(-1) (46%), but was significantly greater than the percentage at 2.5 mmol l(-1) (33%). When osmolarity of the medium with 5.0 mmol glucose l(-1) was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the few-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

Development of rat one-cell embryos in a chemically defined medium : effects of glucose, phosphate and osmolarity.(共著)

Journal of Reproduction and Fertility 100 ( 1 ) 21 - 26 1994年1月

Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 71 1994年

Histone H1 kinase(H1K)activity and glutathione(GSH)content of porcine oocytes during pronuclear formation after in vitro fertilization.(共著)

Biology of Reproduction 50 ( Supplement 1 ) 128 - 128 1994年

Histone H1 kinase activity after electrical activation of in vitro matured porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 74 1994年

Effects of extracellular potassium on the meiotic and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 71 1994年

Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 75 1994年

Microtubule organization of porcine oocytes during in vitro fertilization.(共著)

Biology of Reproduction 50 ( Supplement 1 ) 146 - 146 1994年

受精卵あるいは卵子輸送装置.(共著)

特許公報特公平6-94401 1994年

MICROTUBULE ORGANIZATION OF PORCINE OOCYTES DURING IN-VITRO FERTILIZATION

NH KIM, H FUNAHASHI, TT STUMPF, BN DAY

BIOLOGY OF REPRODUCTION 50 ( Supplement 1 ) 146 - 146 1994年

Effects of NaCl in maturation media on intracellular glutathione concentration and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 75 1994年

In vitro maturation/in vitro fertilization of porcine oocytes.(共著)

Proceeding for Annual Meeting of the Society for Theriogenology 206 - 214 1994年

HISTONE-H1 KINASE (H1K) ACTIVITY AND GLUTATHIONE (GSH) CONTENT OF PORCINE OOCYTES DURING PRONUCLEAR FORMATION AFTER IN-VITRO FERTILIZATION

H FUNAHASHI, TT STUMPF, TC CANTLEY, NH KIM, BN DAY

BIOLOGY OF REPRODUCTION 50 ( Supplement 1 ) 128 - 128 1994年

Effects of sodium chloride concentration in maturation media on histone H1 kinase(H1K)activity and cytoplasmic maturation of porcine oocytes.(共著)

Journal of Reproduction and Fertility 72 ( Abstract Series 13 ) 38 1994年

Histone H1 kinase activity after electrical activation of in vitro matured porcine oocytes.(共著)

Journal of Animal Science 72 ( Supplement 1 ) 74 1994年

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes.(共著)

Molecular Reproduction and Development 36 ( 3 ) 361 - 367 1993年11月

EFFECTS OF ELECTRICAL-STIMULATION BEFORE OR AFTER IN-VITRO FERTILIZATION ON SPERM PENETRATION AND PRONUCLEAR FORMATION OF PIG OOCYTES 国際誌

H FUNAHASHI, TT STUMPF, SL TERLOUW, BN DAY

MOLECULAR REPRODUCTION AND DEVELOPMENT 36 ( 3 ) 361 - 367 1993年11月

記述言語：英語出版者・発行元：WILEY-LISS

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm-1 AC followed by a 30 musec pulse at 120 V mm-1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88-100%) and polyspermic rates (79-100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 +/- 0.5 vs. 4.6 +/- 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. (C) 1993 Wiley-Liss, Inc.

DOI： 10.1002/mrd.1080360312

EFFECTS OF FOLLICULAR-FLUID AT FERTILIZATION IN-VITRO ON SPERM PENETRATION IN PIG OOCYTES 国際誌

H FUNAHASHI, BN DAY

JOURNAL OF REPRODUCTION AND FERTILITY 99 ( 1 ) 97 - 103 1993年9月

記述言語：英語出版者・発行元：J REPROD FERTIL INC

The effects of porcine follicular fluid (PFF) on sperm penetration of pig oocytes and on prevention of polyspermy were examined and characteristics of spermatozoa exposed to PFF were determined. The addition of PFF at the level of 1 and 10% to the prefertilization and fertilization media decreased penetration rates and the mean number of spermatozoa in penetrated eggs regardless of the origin of PFF. In the presence of BSA, supplementation of 0.1% PFF to prefertilization and fertilization media and 1% PFF to prefertilization media did not decrease the penetration rates but did increase monospermic penetration to 54 and 68%, respectively. When PFF was added to prefertilization media, the number of spermatozoa binding to the zona and the percentage of acrosome-intact spermatozoa decreased with increased PFF concentration (from 43.1 +/- 2.8 and 73.1 +/- 4.9% to 7.2 +/- 1.3 and 15.7 +/- 15.4%, respectively). At the end of prefertilization incubation, sperm agglutination was observed and the degree depended on PFF concentration. Supplementation of fetal calf serum to prefertilization and fertilization media blocked the effects of PFF on sperm penetration and binding of spermatozoa to the zona. These results indicate that the prefertilization incubation of porcine spermatozoa in suitable concentrations of porcine follicular fluid will effectively reduce both the number of spermatozoa that attach to the surface of pig eggs and the incidence of polyspermy.

DOI： 10.1530/jrf.0.0990097

Effects of follicular fluid at fertilization in vitro on sperm penetration of pig oocytes.(共著)

Journal of Reproduction and Fertility 99 ( 1 ) 97 - 103 1993年9月

EFFECTS OF THE DURATION OF EXPOSURE TO HORMONE SUPPLEMENTS ON CYTOPLASMIC MATURATION OF PIG OOCYTES INVITRO 国際誌

H FUNAHASHI, BN DAY

JOURNAL OF REPRODUCTION AND FERTILITY 98 ( 1 ) 179 - 185 1993年5月

記述言語：英語出版者・発行元：J REPROD FERTIL INC

The effect of hormone supplements on cytoplasmic maturation in vitro was examined by incubating oocyte-cumulus complexes in a medium with PMSG (10 iu ml-1), hCG (10 iu ml-1) and oestradiol (1 mug ml-1) for various periods and then transferring them to medium without added hormones for the remainder of the maturation period. Exposure of oocyte-cumulus complexes to hormone supplements for 2 h improved only germinal vesicle breakdown and maturation rates compared with complexes not exposed to added hormones. The removal of hormone supplements at 20 h after the start of culture enhanced the ability of oocytes to form male pronuclei 10 to 12 h after insemination. Further, the effects of transfer of intact, and oocytectomized oocyte-cumulus complexes to hormone-free medium at 20 h on cumulus expansion were examined. The diameter and morphology of the intact oocyte-cumulus complexes were improved after the removal of oocyte-cumulus cell complexes from hormonal exposure. The responses of oocytectomized oocyte-cumulus complexes to hormone were similar to those of intact oocyte-cumulus complexes with the exception of corona radiata expansion. The results suggest that the removal of hormone supplements from maturation media at 20 h after culture enhanced cytoplasmic maturation and cumulus expansion. Further, cumulus expansion does not appear to depend on intercellular communication between cumulus cells and oocytes. Oocytectomy did influence expansion of the corona radiata during culture.

DOI： 10.1530/jrf.0.0980179

Effects of the duration of exposure to hormone supplements on cytoplasmic maturation of pig oocytes in vitro.(共著)

Journal of Reproduction and Fertility 98 ( 1 ) 179 - 185 1993年5月

EFFECTS OF DIFFERENT SERUM SUPPLEMENTS IN MATURATION MEDIUM ON MEIOTIC CYTOPLASMIC MATURATION OF PIG OOCYTES 国際誌

H FUNAHASHI, BN DAY

THERIOGENOLOGY 39 ( 4 ) 965 - 973 1993年4月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P < 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.

DOI： 10.1016/0093-691X(93)90433-6

Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes.(共著)

Theriogenology 39 ( 4 ) 965 - 973 1993年4月

Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium.(共著)

Theriogenology 39 ( 4 ) 875 - 886 1993年4月

GLUCOSE REQUIREMENT AT DIFFERENT DEVELOPMENTAL STAGES OF INVITRO FERTILIZED BOVINE EMBRYOS CULTURED IN SEMI-DEFINED MEDIUM 国際誌

JH KIM, H FUNAHASHI, K NIWA, K OKUDA

THERIOGENOLOGY 39 ( 4 ) 875 - 886 1993年4月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).

Assessment of porcine oocytes using brilliant cresyl blue.(共著)

Theriogenology 39 ( 1 ) 214 1993年

Cytoplasmic maturation and in vitro development of porcine oocytes matured in Whitten's medium and fertilized in vitro.(共著)

Fourth International Conference on Pig Reproduction 36 1993年

Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

Fourth International Conference on Pig Reproduction 23 1993年

Effects of hormonal conditions during last 20 h of in vitro maturation on cytoplasmic maturation and vitro fertilization of pig oocytes.(共著)

Journal of Animal Science 71 ( Supplement 1 ) 70 1993年

Pig oocyte activation and processing of transplanted nuclei.(共著)

39 ( 1 ) 329 1993年

Electrical stimulation of pig pig oocytes in the presence or absence of calcium and magnesium.(共著)

Fourth International Conference on Pig Reproduction 42 1993年

Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

Theriogenology 39 ( 1 ) 322 1993年

Effects of artificial activation before in vitro fertilization on sperm penetration and pronuclear formation in pig oocytes.(共著)

Theriogenology 39 ( 1 ) 222 1993年

ラット前核期胚の体外発生におよぼすリン酸およびグルコ-スの影響.(共著)

三好和睦, 丹羽こう二, 奥田潔, 舟橋弘晃

家畜繁殖学会講演要旨 39 ( 5 ) a13 1993年

Electrical stimulation of pig pig oocytes in the presence or absence of calcium and magnesium.(共著)

Fourth International Conference on Pig Reproduction 42 1993年

Cytoplasmic maturation and in vitro development of porcine oocytes matured in Whitten's medium and fertilized in vitro.(共著)

Fourth International Conference on Pig Reproduction 36 1993年

Developmental ability of porcine oocytes matured and fertilized in vitro.(共著)

Fourth International Conference on Pig Reproduction 23 1993年

Premature chromosomal condensation of nuclei transplanted into in vitro matured porcine oocytes.(共著)

Biology of Reproduction 48 ( Supplement 1 ) 175 1993年

Pronuclear formation and in vitro development folowing artificial activation of pig oocytes matured different culture media.(共著)

Biology of Reproduction 48 ( Supplement 1 ) 171 1993年

Effects of hormonal conditions during last 20 h of in vitro maturation on cytoplasmic maturation and vitro fertilization of pig oocytes.(共著)

Journal of Animal Science 71 ( Supplement 1 ) 70 1993年

Pig oocyte activation and processing of transplanted nuclei.(共著)

39 ( 1 ) 329 1993年

Processing of nuclei transplanted into in vitro matured porcine oocytes.(共著)

Theriogenology 39 ( 1 ) 322 1993年

Effects of artificial activation before in vitro fertilization on sperm penetration and pronuclear formation in pig oocytes.(共著)

Theriogenology 39 ( 1 ) 222 1993年

Premature chromosomal condensation of nuclei transplanted into in vitro matured porcine oocytes.(共著)

Biology of Reproduction 48 ( Supplement 1 ) 175 1993年

Pronuclear formation and in vitro development folowing artificial activation of pig oocytes matured different culture media.(共著)

Biology of Reproduction 48 ( Supplement 1 ) 171 1993年

Assessment of porcine oocytes using brilliant cresyl blue

ERICSSON S

Theriogenology 39 ( 1 ) 214 - 214 1993年

FERTILIZATION AND EARLY CLEAVAGE INVITRO OF AGING BOVINE OOCYTES AFTER MATURATION IN CULTURE 国際誌

RC CHIAN, H NAKAHARA, K NIWA, H FUNAHASHI

THERIOGENOLOGY 37 ( 3 ) 665 - 672 1992年3月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

Bovine follicular oocytes cultured for 20 to 48 hours were inseminated with frozen thawed spermatozoa. Significantly higher penetration rates (94 to 100% vs 86 to 94%: P < 0.05) and proportions of polyspermy (35 to 78% vs 22 to 45%: P < 0.01) were obtained for oocytes cultured continuously for 24 hours with spermatozoa than for those separated from spermatozoa 8 hours after insemination. The most prominent effect of ageing of oocytes on early events of penetration was observed in the incidence of polyspermy rather than in the penetration rate and the proportion of pronuclear plus cleaved oocytes: the proportion of polyspermic oocytes significantly increased (P < 0.05) in oocytes inseminated after 28 to 48 hours of culture (36 to 78%) compared with those cultured for 20 to 24 hours (22 to 35%) for maturation. Culture experiments for early development of penetrated oocytes indicated that no significant differences were observed in the proportions of oocytes cleaved to the two- to four- cell stage 48 hours after insemination among those cultured for 20 to 40 hours for maturation. However, further cleavage to the four- to sixteen-cell stage 72 to 96 hours after insemination was greatly inhibited as ageing of oocytes proceeded from 28 hours in culture for maturation.

DOI： 10.1016/0093-691X(92)90146-I

Fertilization and early cleavage in vitro of ageing bovine oocytes after maturation in culture.(共著)

Theriogenology 37 ( 3 ) 665 - 672 1992年3月

体外受精由来和牛胚の凍結融解後の生存性ならびに移植・分娩成績について.(共著)

北海道牛受精卵移植研究会会報 ( 11 ) 19 - 21 1992年

Developmental abilities of mouse and rat pronuclear eggs following interspecific pronuclear and cytoplasmic transplantation.(共著)

Journal of Reproduction and Development 38 ( 3 ) 179 - 183 1992年

Developmental Abilities of Rat and Mouse Pronuclear Eggs Following Interspecific Nuclear or Cytoplasmic Transplantation

Hiroaki Funahashp, Koji Niwa

Journal of Reproduction and Development 38 ( 3 ) 179 - 183 1992年

記述言語：英語

Homologous and heterologous replacement of a small amount of cytoplasm or both pronuclei were performed in rat and mouse pronuclear eggs. Rat eggs after receiving rat or mouse cytoplast never developed beyond the 2-cell stage in vitro. On the other hand, the in-vitro development of mouse eggs that received rat cytoplasm was possible, but it was greatly inhibited compared with those that received mouse cytoplasm. None of enucleated rat eggs that received rat or mouse pronuclei also developed beyond the 2-cell stage. Although few enucleated mouse eggs after receiving rat pronuclei developed beyond the 2-cell stage, high proportion (64%) of those that received mouse pronuclei could develop to blastocysts in vitro. When 2-cell embryos obtained 18 h after fusing with donor karyoplast were transferred to pseudopregnant rats, 2 (9%) of 23 enucleated rat embryos and 1 (7%) of 15 enucleated mouse embryos each fused with heterologous karyoplast developed to early blastocyst 48-55 h after transfer. These results may indicate that the embryonic genome can be activated in the heterospecific cytoplasm. © 1992, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.

DOI： 10.1262/jrd.38.179

牛体外受精卵の初期発生におよぼすグルコースの影響

金宗興, 舟橋弘晃, 伊賀浩輔, 丹羽こう二

日本畜産学会大会講演要旨 85th 1992年

完全体外培養系で得た牛体外受精凍結卵の移植試験

青柳敬人, 武富敏郎, 舟橋弘晃

JA全農飼料畜産中央研究所試験研究報告 ( 20(1991) ) 1992年

牛における個体別体外受精卵の作出試験

青柳敬人, 舟橋弘晃, 武田哲男

JA全農飼料畜産中央研究所試験研究報告 ( 20(1991) ) 1992年

黄体ホルモン製剤利用による牛の反復過排卵処置に関する検討

青柳敬人, 武富敏郎, 板倉はつえ, 亀山賢次, 舟橋弘晃, 武田哲男, 鬼原辰夫

JA全農飼料畜産中央研究所試験研究報告 ( 20(1991) ) 1992年

First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6℃ for various durations.(共著)

Theriogenology 36 ( 3 ) 411 - 417 1991年9月

1ST CLEAVAGE OF ENUCLEATED RAT EGGS FOLLOWING TRANSPLANTATION OF KARYOPLAST REMOVED FROM PRONUCLEAR EGGS STORED AT 2-DEGREES-C TO 6-DEGREES-C FOR VARIOUS DURATIONS

K NIWA, H FUNAHASHI, K OHMAE, M KATTOH

THERIOGENOLOGY 36 ( 3 ) 411 - 417 1991年9月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

Pronuclear rat eggs were cultured for 24 to 48 hours at 37-degrees-C after storage at 2 to 6-degrees-C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6-degrees-C).

Developmental capacity of bovine oocytes collected from ovaries of individual heifers and fertilized in vitro.(共著)

Theriogenology 36 ( 3 ) 427 - 434 1991年9月

DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES COLLECTED FROM OVARIES OF INDIVIDUAL HEIFERS AND FERTILIZED INVITRO 国際誌

H FUNAHASHI, Y AOYAGI, T TAKEDA, T ONIHARA

THERIOGENOLOGY 36 ( 3 ) 427 - 434 1991年9月

記述言語：英語出版者・発行元：BUTTERWORTH-HEINEMANN

Bovine follicular oocytes from individual heifers (n = 49) were separately matured, fertilized with frozen-thawed spermatozoa and cultured with cumulus cells. Although there were great variations in the number (mean +/- SD = 19.1 +/- 11.9) of oocytes collected from individual heifers and the percentages of the oocytes cleaved 48 hours after insemination (mean +/- SD = 69.5 +/- 18.4) and developed to the morula stage 7 days after insemination (mean +/- SD = 10.9 +/- 10.9), there were significant correlations between the numbers of oocytes collected and cleaved (the correlation coefficient: r = 0.9336) or developed to morula stage (r = 0.6633), indicating that oocytes from different heifers have the same developmental ability after in vitro fertilization. Ten morulae and 12 blastocysts which were obtained 7 and 8 days after insemination were transferred, one by one, to each uterine horn of 11 recipients. At Day 60 of pregnancy, 8 (80%) fetuses were identified in 4 (80%) of 5 recipients into which 10 embryos were transferred at Day -1 of synchrony. However, only 3 (25%) fetuses were identified in 2 (40%) of 6 recipients into which 12 embryos were transferred at Day 0 or +1 of synchrony.

Electric fusion of 2-cell rat embryo balastomeres.

黄体ホルモン製剤を用いた黒毛和種牛の反復過剰排卵処理に関する検討.(共著)

北海道牛受精卵移植研究会会報 ( 10 ) 19 - 21 1991年

ラット前核期胚および桑実期胚の低温保存におよぼすショ糖の影響.(共著)

家畜繁殖学雑誌 37 ( 4 ) a21 1991年

ラットおよびマウスの初期はいにおける属間核移植試験

舟橋弘晃, 武田哲男, 丹羽こう二

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

牛における核移植に関する検討

亀山賢次, 青柳敬人, 舟橋弘晃, 武田哲男, 武富敏郎, 板倉はつえ

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

豚における低コスト麻酔法の検討

武富敏郎, 亀山賢次, 舟橋弘晃, 板倉はつえ

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

ラットおよびマウスの属間核移植卵の体内発育試験

舟橋弘晃, 武田哲男, 丹羽こう二

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

人工透明帯封入牛分割凍結卵の移植試験

武田哲男, 青柳敬人, 亀山賢次, 武富敏郎, 舟橋弘晃, 板倉はつえ, 鬼原辰夫

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

完全体外培養系で得た牛体外受精卵の移植試験

舟橋弘晃, 武富敏郎

JA全農飼料畜産中央研究所試験研究報告 ( 19(1990) ) 1991年

ラット2-細胞期胚における電気的割球融合

舟橋弘晃, 丹羽皓二

家畜繁殖学雑誌 36 ( 2 ) 114 - 119 1990年6月

舟橋弘晃, 丹羽晧二

家畜繁殖学雑誌 36 ( 2 ) 114 - 119 1990年

記述言語：英語出版者・発行元：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

ラット2-胞期胚に1回あるいは連続した2回の直流パルス(1.0~2.0kV/cm)を50~200μs通電し,割球の融合率と融合に要する時間を調べた。1.0kV/cmで50μsのパルスでは融合は困難であり,1あるいは2回付与のいずれにおいても15個の処理胚のうち割球の融合したものはわずか1個(7%)であった。融合率は1.5~2.OkV/cmの電圧および100~200μsの通電において最も高くなった(80~100%)。1.0kV/cmで100~200μsの条件下では1回よりも2回の連続通電により融合率は高くなったが,1.5および2.0kV/cmでは2回通電の効果はほとんど認められなかった。通電後1時間以内に融合した胚について融合に要した時間を測定した結果,1.0および1.5kV/cmでは50~200μsのパルスを2回通電することによって融合時間が短縮されたが,2.0kV/cmでは殆ど短縮されなかった。1.0,1.5および2.0kV/cmで150μs通電された後割球融合の生じた胚を偽妊娠雌ラットの卵管に移植し,3日後に回収した結果,回収卵のうちそれぞれ15(83%),12(80%)および9個(64%)が桑実期あるいは胚盤胞にまで発生していたが,これら3者間に有意差は認められなかった。

DOI： 10.1262/jrd1977.36.114