記述言語：英語   掲載種別：研究論文（学術雑誌）  

Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprogramming research. However, the standard mouse SCNT protocol remains expensive, labor-intensive, and requires hard work for many hours. Therefore, we have been trying to reduce the cost and simplify the mouse SCNT protocol. This chapter describes the methods to use low-cost mouse strains and steps from the mouse cloning procedure. Although this modified SCNT protocol will not improve the success rate of mouse cloning, it is a cheaper, simpler, and less tiring method that allows us to perform more experiments and obtain more offspring with the same working time as the standard SCNT protocol.

DOI： 10.1007/978-1-0716-3064-8_7

PubMed

記述言語：日本語  

J-GLOBAL

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-022-22850-5

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0270781

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s42003-022-03623-2

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-022-31216-4

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1101/gr.276363.121

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2021-115

PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science (PLoS)  

Conventional in vitro culture and manipulation of mouse embryos require a CO₂ incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO₂ incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO₂ gas-generating agent, to increase the CO₂ partial pressure of CZB medium to 4%–5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO₂ incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO₂ incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO₂ incubator: 34.3%). This study demonstrates that CO₂ incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

DOI： 10.1371/journal.pone.0260645

PubMed

記述言語：英語   出版者・発行元：Research Square Platform LLC  

<title>Abstract</title>
Parental pronuclei (PN) are asymmetrical in several points but the underlying mechanism for this is still unclear. Recently, a theory has been become broadly accepted that sperm are more than mere vehicles to carry the paternal haploid genome into oocytes. Here, in order to reveal the formation mechanisms for parental asymmetrically relaxed chromatin structure in zygotes, we investigated histone mobility in parthenogenetic-, androgenic-, ROSI-, ELSI-, tICSI-, and ICSI-zygotes with several numbers of PNs with the use of zygotic fluorescence recovery after photobleaching, a method previous established by our group. The results showed that sperm played a role to cause chromatin compaction in both parental PNs. Interestingly, during spermiogenesis, male germ cells acquired this ability and its resistance. On the other hand, oocytes harbored chromatin relaxation ability. Furthermore, the chromatin relaxation factor was competed for between PNs. Thus, these results indicated that the parental asymmetrically relaxed chromatin structure was established as a result of a competition between the PNs for the chromatin relaxation factor that opposed the chromatin compaction effect by sperm. Together, it was suggested that parental germ cells cooperated for their just arisen newborn zygotes by playing a distinct role in the regulation of chromatin structure.

DOI： 10.21203/rs.3.rs-806059/v1

その他リンク： https://www.researchsquare.com/article/rs-806059/v1.html

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.isci.

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

Freeze-drying techniques allow the preservation of mammalian spermatozoa without using liquid nitrogen. However, the current method requires the use of glass ampoules, which are breakable, expensive, and bulky to store or transport. In this study, we evaluated whether mouse freeze-dried (FD) spermatozoa can be preserved and transported on thin materials. In this study, we demonstrated that FD sperm can be preserved in thin plastic sheets. Its DNA integrity was comparable to that of glass ampoule spermatozoa, and healthy offspring were obtained after preservation at -30°C for more than 3 months. We attached preserved FD sperm to postcards, and transported these to other laboratory inexpensively at room temperatures without any protection. This method will facilitate the preservation of thousands of mouse strains in a single card holder, promote collaboration between laboratories, conservation of genetic resources, and assisted reproductive technology.

DOI： 10.1016/j.isci.2021.102815

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/sciadv.abg5554.

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Association for the Advancement of Science (AAAS)  

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

DOI： 10.1126/sciadv.abg5554

PubMed

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】凍結乾燥精子を用いた顕微授精胚は，新鮮精子を用いた場合と比べて産仔率が大きく低下してしまう。この原因として最初の凍結で生じるとされる氷晶形成に着目し，凍結処理を行わずに乾燥させた非凍結真空乾燥精子から子供が産まれるのか検討した。また凍結保護剤の添加や乾燥時の周囲温度が産仔率へ与える影響についても検証した。【方法】ICR系統のマウス精子と卵子を用いた。精子は前培養後，アンプル瓶に分注しEYELA FDU2200を用いて6時間の乾燥処理を行った。試料は冷凍庫で3日以上保存し，加水して実験に使用した。実験1では非凍結の効果，実験2では精子懸濁液量の影響，実験3では乾燥保護剤（PVP）の効果，実験4では冷却により乾燥速度を低下させた場合の影響について，精子の形態，DNA損傷度（コメットアッセイ），ICSI後の発生率，胚盤胞の細胞数，および産仔率で評価した。【結果】実験1: 非凍結真空乾燥精子は従来の凍結乾燥精子に比べ，精子の頭部と尾部が分離している精子の割合が低下したが，胚盤胞率に差はなかった。実験2: 精子懸濁液を50 µLから5 µLまで少なくすると，DNA損傷度が増加し胚盤胞率が低下した。実験3・4: PVPの添加と乾燥時の周囲温度の低下（約0.4℃）による乾燥速度の低下で，精子のDNA損傷と形態的損傷を抑え，胚盤胞率が向上し胚盤胞の細胞数が増加した。胚移植により非凍結真空乾燥精子から産仔を得ることに成功したが産仔率は改善されなかった。【考察】本研究により，精子を乾燥保存するために凍結処理は必須ではないこと，および凍結乾燥精子の低産仔率の原因は，凍結処理よりも乾燥処理で生じるダメージによるものである可能性が示された。今後，乾燥条件に的を絞り乾燥精子の産仔率向上を目指すのと同時に，ACS率やγ-H2AX解析を用いて，コメットアッセイでは得られなかったより詳細な精子のDNA損傷を解析し，乾燥保護剤や冷却処理が精子に対してどのような作用をもたらして発生率を改善したか明らかにしたい。

DOI： 10.14882/jrds.114.0_p-87

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】円形精子細胞を用いた顕微注入（ROSI）胚は，精子注入（ICSI）胚と比較し産仔率は低い。その原因として，RNAおよび，精子完成プロセスでのヒストン-プロタミン置換により本来除去されるべきエピゲノムが持込まれ，胚性ゲノム活性化時のトランスクリプトームに異常が生じていることが考えられる。そこで本研究ではROSI/ICSI胚のRNA-seqを行い，まずはICSI胚との比較により，ROSI胚特有なトランスクリプトームの全容を把握し，その制御機構を解明する。【方法】ROSIおよびICSIにより作出した1および2細胞期胚のRNA-seqデータからEdgeRを用いて発現変動遺伝子（DEG）を同定した。また，雄性生殖細胞からのRNAの持込を検証するため，同定したDEGについて，既存の円形精子細胞ならびに精子のRNA-seqデータからその発現量を調べた。【結果】ICSI胚と比較しROSI胚におけるDEGを1細胞期胚に406個（1c-DEG），2細胞期胚に3,434個（2c-DEG）を同定した（FDR<0.05）。これらDEGの内，1細胞期においてROSI胚で高い発現を示す1c-DEGの321個は，円形精子細胞で精子よりも有意に発現が高かった。一方，2c-DEGにおいて，1細胞期から2細胞期への変動を調べると，ICSI胚では発現が下がるがROSI胚では上がる遺伝子群が79個，その逆を示す遺伝子群が131個同定された。これらはいずれも円形精子細胞と精子との間で発現量の差が少ない遺伝子であった。【考察】1c-DEGは，主に円形精子細胞由来のRNA持込に起因することが示唆された。また2c-DEGの要因は，1細胞期のDEGにより誘発された可能性の他，円形精子細胞由来のエピゲノムが原因である可能性が考えられた。現在ROSI/ICSI胚のATAC-seqや既存の雄性生殖細胞のChIP-seqデータの統合的な解析により，円形精子細胞由来エピゲノムの持ち込みが，2c-DEGを生じる機構へ関与するのかを検証している。

DOI： 10.14882/jrds.114.0_p-39

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】我々は1細胞期胚の雌雄前核における，ヒストンH2Bの流動性を指標としたクロマチン構造の“緩さ”について解析を行い，精子に由来する雄性前核（mPN）が極端に緩んだ構造をとり，同じ細胞質に共存する雌性前核（fPN)が，mPNよりも締まった構造をとる“雌雄差”を報告している（Ooga et al. 2016）。今回この雌雄差の形成に，精子が果たす役割を明らかにすることを試みた。【方法】eGFP-H2BのmRNA注入未受精卵を用いて，各種1細胞期胚を作製し，受精後8–10時間でzygotic Fluorescence Recovery After Photobleaching（zFRAP; Ooga et al. 2018）を用い，H2Bの流動性を各種胚について解析した。【結果】(1）顕微授精胚と2倍体雌性単為発生胚を作成し，精子がfPNを締めるのかを調べると，精子と共存するfPNは単為発生胚のfPNよりも締まっていた。(2)精子がmPNも締めるのかを単一の前核にして調べるため，除核した卵子に1匹または2匹の精子を顕微授精し作成した雄性単為発生胚，卵子の紡錘体を移植した半数体雌性単為発生胚を調べた。それぞれ単一前核にすると，mPNはfPNよりも締まり，精子数に比例してより締まった。(1)-(2)より，精子が両前核クロマチンへの凝集作用を持つことがわかった。(3)未受精卵に1匹または2匹の精子を注入して，精子による凝集作用を増強した顕微授精胚を作成し，fPNとmPNの凝集作用への感受性を比べた。fPNは1匹の精子で限界まで締まり，2匹の精子を注入してもより締まることはなかった。一方mPNがfPN並に締まるには，2匹の精子が必要で，凝集作用に抵抗性を示した。(4)円形精子細胞，伸長精子細胞，および精子注入胚を作製し，凝集能は精子形成過程のどのステージで獲得されるかを調べると，精子形成が進むに連れ，fPNへの凝集能は強くなった。【結論】精子形成過程で獲得されるクロマチン凝集作用とその感受性の差によって，雌雄差は形成される。

DOI： 10.14882/jrds.114.0_p-69

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】精子の凍結乾燥保存には，これまでガラスバイアルやガラスアンプル瓶が使用されてきたが，消耗品として比較的に高価であり，ガラスのため破損の危険性及び熱封入操作の必要性などの欠点が普及を妨げる原因となっている。そこで本研究では，安い汎用品であるプラスチック製の1.5 mL チューブに着目し，高真空度での凍結乾燥精子の保存が可能か，保存後に産仔の作出は可能か検討した。【方法】採取した精子はHTF 培地にて30 分培養後に1.5 mL のチューブに50 μL ずつ分注した。チューブの蓋をテープで鋭角に固定し，液体窒素で凍結後密栓式真空乾燥機において3–24 時間乾燥した。その後，真空状態のチャンバー内でのチューブの蓋を閉め密栓した。チューブ内の真空度については，テスラコイル検出器による非破壊検査で作製直後及び保存中の真空度を測定した。一部のチューブは水中で開き空気混入量を測定した。チューブは室温，4℃，及び –30℃ で最長一ヶ月間保存した。顕微授精は加水直後に行い，胚盤胞への発生率及び卵管移植による産仔率を調べた。【結果】作製直後のチューブは高真空だったが，保存期間及び保存温度の上昇に連れて真空保持率は低下する傾向が見られた。乾燥は3 時間以降は重量が減少しなかったことから3時間で十分なことが分かった。チューブ内の凍結乾燥精子を用いた顕微授精はどの区でも可能であり，体外発生率に差は見られなかった。室温，4℃ 及び –30℃で3 日間保存した後に作製した顕微授精胚を2 細胞期に移植した結果，いずれの区からも産仔を得ることができた（18% vs. 11% vs. 15%）。さらに，–30℃ で一ヶ月間保存したチューブからも正常な産仔を得ることができた。【考察】本研究により，凍結乾燥精子が安価な1.5 mL チューブでも保存できることが初めて明らかとなった。現時点では室温保存は3 日間が限界であるが，国内輸送なら可能である。長期保存には冷凍庫が必要だが，膨大な量のマウス系統を保存するためには安価なチューブは効果的である。

DOI： 10.14882/jrds.114.0_p-24

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】一般に精子の前培養は，精子に受精能を獲得させ体外受精の成功率を高めるために行われている。しかし凍結乾燥精子は顕微授精で受精させるため，精子の前培養は必要ないと考えられてきた。だが，受精能を獲得した精子を凍結乾燥に用いることで，凍結乾燥精子の低い産仔率を改善できるかもしれない。そこで本研究では，凍結乾燥精子を作製する前にさまざまな時間で精子の前培養処理を行い，前培養の効果について検討した。【方法】ICR系統の雄マウスの精巣上体尾部から精子を採取し，0，1，6，24時間の精子前培養を行った後，液体窒素で凍結し，6時間の真空乾燥処理によって凍結乾燥精子を作製した。作製した凍結乾燥精子は使用するまで–30℃で保存し，実験当日に加水し，常法に従って顕微授精を行った。顕微授精にはICR系統の卵子を使用した。顕微授精胚は120時間培養後，胚盤胞までの発生率，胚盤胞のICM/TE 細胞数，およびTUNEL法によるDNA損傷率を調べた。また，2細胞期胚で偽妊娠雌マウスへ移植を行い，産仔率の比較も行った。加えて，それぞれの前培養時間ごとにコメット法による精子DNAの損傷率も比較を行った。【結果】胚盤胞への発生率は，前培養0時間と比較して前培養1時間で明らかな向上がみられた（0時間:39％ vs.1時間:64％）。しかし，さらに長い前培養を行うと前培養時間の増加に伴って発生率は低下した。胚盤胞での細胞数やDNA損傷率に関しては，前培養時間ごとに大きな差はみられなかった。また，産仔率に関しては，胚盤胞までの発生率の結果と同様に前培養1時間で改善した（0時間:14％ vs.1時間:24％）。一方，精子DNAの損傷率は前培養時間の長さに比例して増加した。【考察】本研究によって，凍結乾燥精子作製においても，精子の前培養による受精能獲得が重要であることが示唆されたが，前培養時間が増えるにつれて精子DNAのダメージが増加するため，最適な前培養時間の決定が必要である。

DOI： 10.14882/jrds.114.0_p-86

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】「哺乳類の胚発生に地球の重力は必要か」この問いの解明に向け，我々は国際宇宙ステーション（ISS）にマウス凍結2細胞期胚を運び，微小重力環境での融解，培養を計画している。この計画には大量の胚を用意し，–80℃で保存する制約のもと必ず成功させなければならない。胚の回収から凍結までの最も効率的な凍結胚の作製方法を決定するため，–80℃で2細胞期胚の保存が可能なHOV法（Mochida et al., 2013）を用いて，Vivo胚，IVF胚及びICSI胚の凍結耐性と発生能を比較した。更にVivo胚の回収時期と凍結の最適タイミングについても検討した。【方法】ICR系統マウスを用いて以下の実験を行った。実験1ではVivo胚，IVF胚，ICSI胚の2細胞期胚をそれぞれ凍結し，–80℃で3日間以上保存後に融解し，生存率，発生率及び産仔率を比較した。実験2ではVivo胚を卵管から回収する時期と凍結までの体外培養時間について最適条件を検討した。【結果・考察】実験1：融解後の生存率はVivo胚がIVF胚及びICSI胚よりも有意に高かった（62％ vs. 35–40%）。胚盤胞率はVivo胚とIVF胚がICSI胚よりも有意に高かった（50–68％ vs. 10%）。胚盤胞率の高かったVivo胚とIVF胚を移植した結果，両者に有意な差はなかった（66%及び55%）。実験2：2細胞期のVivo胚を17時に回収し，18時に凍結した区の生存率，発生率，産仔率は9時回収–10時凍結区，及び9時回収–18時凍結区と比較して最も高いことが分かった（産仔率：59％ vs. 43–44%）。またDNA合成阻害剤を用いた実験から，9時回収胚はG2期（84％）とS期（16％）の胚が存在していたのに対し，17時回収胚は全てG2期の胚だった。この結果から細胞周期の違いが凍結耐性に影響していることが示唆された。本研究によって，ISSへ運ぶ凍結胚は17時に回収した後期2細胞期Vivo胚を1時間休ませてから凍結した作製方法が最適であることが分かった。

DOI： 10.14882/jrds.114.0_p-95

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】クロマチンの緩さはES細胞の分化能の指標として知られている。そこで，我々は胚においてもクロマチンの緩さが個体発生能の高低に関わっているのではないかと考えた。本研究ではFRAP（Fluorescence recovery after photobleaching）法により，胚を生かしたままクロマチンの緩さを測定することで，個体発生能を評価する方法の確立を目指す。【方法】過排卵処理を施したBDF1マウスから採卵を行い，ICRマウスから採取した精子を用いてIVFを行った。媒精から2–3時間後にmRNAを注入し，蛍光標識ヒストン（eGFP-H2B）を発現させた。その後，1細胞期または2細胞期にてFRAP解析を行い，ヒストンの置換速度を測定した。このヒストンの置換速度からMobile Fraction; MF（%）を算出し，クロマチンの緩さの指標とした。クロマチンが緩い（MFが高い）解析胚から順に1細胞期ではA-Fの6グループ，2細胞期ではa-dの4グループに分け，グループ間の胚盤胞率および産仔率を比較した。【結果】1細胞期と2細胞期共に，クロマチンが最も締まったF, dグループの胚盤胞率が最も低くなった（1細胞期：A: 82, B: 91, C: 94, D: 91, E: 89, F: 66%；2細胞期：a: 93, b: 100, c: 93, d: 77%）。また有意差は見られなかったが，1細胞期と2細胞期共に，クロマチンが最も緩いA, aグループの胚盤胞率がやや低い値を示した（上記）。さらに，1細胞期でFRAP解析を行った胚についてはC-Fの4グループで胚移植を行ったが，産仔への発育も胚盤胞率と同様の傾向を示した（C: 44, D: 41, E: 29, F: 14%）。【考察】以上の結果から，胚のクロマチンは緩すぎても締まりすぎても悪影響であるが，特に締まりすぎた際に発生を強く阻害し，適切な胚発生にはクロマチンの適度な緩みが重要であると考えられる。これにより，クロマチンの緩さから個体発生能を評価できる可能性が見出された。

DOI： 10.14882/jrds.114.0_p-47

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【背景】ライブセルイメージング技術は着床前胚を非侵襲的に評価することが可能であり，ヒトを含め多くの哺乳類で使用されている。しかし，ライブセルイメージング機器は高額であり，どの研究室でも購入して実施できる一般的な解析手段にはなっていない。本研究では，我々が以前開発したCO₂インキュベーターを用いない密閉系での培養システムを，観察かつ密閉が可能なガラスキャピラリーと組み合わせ，簡易なライブセルイメージング技術の開発を試みた。【方法】CO₂分圧を最適化した培地を用意し，胚とともにガラスキャピラリーに封入した。市販のガラスキャピラリー6種を使用し，培養に最適なものを決定した。決定したガラスキャピラリーを使用し，顕微鏡にセットしたサーモプレート上で，キャピラリーの保温条件および観察方法を検討した。最後に，顕微鏡に市販のタイマー式電源スイッチを取り付け，顕微鏡全体を暗幕で覆い，30分に1回のタイムラプス撮影による1細胞期から胚盤胞までのライブセルイメージングを試みた。【結果】サーモプレート上に直にガラスキャピラリーを置くと，キャピラリー内の胚をはっきりと観察できず，培養もできなかった。しかし，ディッシュにオイルを張り，その中でキャピラリーを保温したところ，キャピラリー内の胚を鮮明に観察することが可能になっただけでなく，胚盤胞へ発生させることにも成功した（65％）。また，タイムラプス撮影によるライブセルイメージングを行った結果，発生率は低下してしまった。【考察】本研究では，従来高額な機器を必要としたライブセルイメージングを低コストで実施することに成功した。この技術は胚培養が行える研究室には常備されている器具を組み合わせて用いるため，多くの施設ですぐに実施することが可能である。今後は発生率の低下の原因と考えられる温度管理や光の影響を減らすことで改善を試み，本培養法とライブセルイメージング機器であるCV1000（横河電機）での培養と比較し，発生速度および移植による産仔率の正常性を検証する。

DOI： 10.14882/jrds.114.0_p-53

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【背景・目的】哺乳類精子の保存及び輸送には液体窒素が従来用いられているが，タンクやドライシッパーなどの特殊容器が必要なうえ運搬には特別料金がかかり，作業には窒息や超低温による怪我の恐れを伴う。我々は第112・113回大会において，プラスチックシートを用いたマウス凍結乾燥精子の保存技術を発表した。本研究では，このシート保存技術がはがきや封筒を用いた簡便な精子の輸送技術として応用可能か検討した。【方法】精子はICR及びBCF1系統の雄マウスから採取し，10分間の凍結処理と6時間の真空乾燥処理を行った後，2枚のプラスチックシートを接着させながらその間に挟み保存した。実験1では，–30℃で最長3ヶ月，室温で最長1週間保存し，シート保存の限界を調べた。実験2では，実際に精子を挟んだシートをはがきに貼り付けるか，あるいは封筒に入れてポストに投函し，山梨県内，あるいは東京大（千葉県）から山梨大へ一般郵送した。対照区として郵送と同期間，研究室内の室温でシートを保存した。回収した精子はICRマウスの卵子へ顕微注入し，受精率及び産仔率の比較を行った。【結果】–30℃であれば3ヶ月間シートで保存しても産仔率は低下しなかったが，室温保存の場合産仔が得られる限界は3日間であった。山梨県内及び千葉-山梨県の郵送はいずれも2日以内で到着した。郵送した精子の産仔率（県内：産仔数2匹，4％，千葉-山梨県：産仔数4匹，7％）は，研究室で室温保存した対照区の精子の産仔率（産仔数2匹，3%）と差がなかった。【考察】マウス精子は本方法によりはがきで簡単に国内郵送できるようになった。本方法は，研究室間の簡便かつ低コストでの遺伝子改変マウスなどの分与を可能にし，研究の円滑な遂行に役立つと考えられる。しかし本方法が悪用された場合，例えば和牛の精子をはがきに貼り付けて海外へ不正輸出した場合，検疫で見つけることは困難であり，貴重な遺伝資源の違法流出さえも促進しかねない。本方法の実用化には，技術改善に加え国際的な法整備が不可欠である。

DOI： 10.14882/jrds.114.0_or-43

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2020-059

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/dev.190777.

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：The Company of Biologists  

The reason for the poor development of cloned embryos is not yet clear. Several reports have suggested that some nuclear remodeling/reprogramming factors (RRFs) are removed from oocytes at the time of enucleation, which might cause the low success rate of animal cloning. However, there is currently no method to manipulate the amount of RRFs in oocytes. Here, we describe techniques we have developed to gradually reduce RRFs in mouse oocytes by injecting somatic cell nuclei into oocytes. These injected nuclei were remodeled and reprogrammed using RRFs, and then RRFs were removed by subsequent deletion of somatic nuclei from oocytes. The size of the metaphase II spindle reduced immediately, but did recover when transferred into fresh oocytes. Though affected, the full-term developmental potential of these RRF-reduced oocytes with MII-spindle shrinkage was not lost after fertilization. When somatic cell nuclear transfer was performed, the successful generation of cloned mice was somewhat improved and abnormalities were reduced when oocytes with slightly reduced RRF levels were used. These results suggest that a change in RRFs in oocytes, as achieved by the method described in this paper or by enucleation, is important but not the main reason for the incomplete reprogramming of somatic cell nuclei.

DOI： 10.1242/dev.190777

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1530/REP-20-0054.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

DOI： 10.1530/REP-20-0054

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-019-13830-x

PubMed

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】第112回札幌大会のポスターセッションにて我々は，世界で初めて薬包紙の上で凍結乾燥保存したマウス精子からの産仔作出例を発表した。だが本技術の保存成績にはばらつきが見られ（産仔率0–47%，平均28%），作製方法を確立できたとはいえない。従来のガラスアンプルを用いた保存方法に代わる新たな精子の保存技術として本技術を確立するために本研究では，①ガラスアンプルで作製した凍結乾燥精子との比較による本技術を用いた場合の精子DNAの損傷率，②本技術の保存成績が安定しなかった原因をサンプル内への空気混入と仮定し産仔率に対する空気の影響，の2点を調べた。【方法】実験にはICR系統のマウス精子と卵子を用い，精子の凍結乾燥処理には前回大会と同様の方法を用いた。①採精後同日中にガラスアンプルと薬包紙を用いて凍結乾燥精子を作製し，コメットアッセイおよび雄性前核のγ-H2A.x染色により精子DNAの損傷率を比較した。②前回大会で発表した方法では，ラミネートの封をする際に試料内に空気が混入していた。そこで今回新たに，真空のシート状で凍結乾燥精子を保存する技術を開発した。本方法により冷凍保存した精子を顕微授精に使用し，得られた2細胞期胚を卵管へ移植し産仔率を調べた。【結果】①ガラスアンプルと薬包紙の上で作製したそれぞれの凍結乾燥精子のDNA損傷率に有意な差はなく，ガラスアンプルを用いた従来の凍結乾燥方法と薬包紙を用いた本技術は精子DNAに対し同程度のダメージを与えていた。②空気を混入させて保存した精子を用いた場合の産仔率は28%であるのに対し，真空保存した精子を用いた場合の産仔率は8%であり，空気の混入は本技術において凍結乾燥精子の産仔率を低下させる原因ではなかった。【考察】凍結乾燥処理が精子DNAに及ぼすダメージや空気の混入に起因する産仔率の低下は見られなかったことから，本技術の保存成績に影響を与える要因として，ラミネートの材質やラミネートに挟む際に精子にかかる圧力など本研究で調べた項目以外の要因が考えられる。

DOI： 10.14882/jrds.113.0_p-85

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/rmb2.12312

PubMed

記述言語：日本語  

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【背景】CO₂インキュベーターは初期胚の培養に必須だと考えられてきたが，近年それを用いない培養方法が報告されている。我々はCO₂分圧を最適化した培地（CO₂最適化培地）を密閉容器で使用すれば，インキュベーターを使用せず胚の培養や産仔作出が可能になり，経費削減だけでなく胚の輸送などにも応用可能と考えた。【方法】①CO₂最適化培地の作成：CO₂インキュベーターあるいは炭酸ガス発生剤「アネロパウチ」を用いてCZB培地のCO₂分圧が4–5％になる時間を決定した。②密閉環境での胚培養：密閉可能なチューブやフラスコを用いマウス体外受精胚をCO₂最適化培地内で4日間，37℃で培養し，胚移植を行った。③胚培養の実践：胚輸送を想定し，38.5℃のお湯を入れた水筒の中で胚を1–2日間培養した。回収した胚は従来法で培養し，発生率を確認した後に移植し産仔率を調べた。一部の胚は実際に輸送を行った。【結果】①CO₂インキュベーター内では24時間でCO₂分圧が約4%の気液平衡状態となった。アネロパウチを用いた場合，24 時間で分圧は12%まで上昇してしまったが，3時間の処理でCO₂最適化培地を作成できることが分かった。②チューブでの胚盤胞および産仔への発生率は正常であった（胚盤胞率100%，産仔率38%）。フラスコをサーモプレート上で加温した実験では，胚培養だけでなく観察も可能であった。③水筒での培養は，1細胞期胚の培養は1日が限界だったが，2細胞期胚の培養は2日間可能であり，産仔を得ることが出来た（胚盤胞率86%，産仔率35%）。また，輸送した区でも同様な結果となった。【考察】従来必須とされたCO₂インキュベーターを使用せずに，胚盤胞や産仔への発生率に影響を与えずに胚を培養することが可能となった。施設費，維持費，スペースなどを削減するだけでなく，国内の輸送も容易になる。また，フラスコを用いればライブセルイメージングも可能になるだろう。本研究で示した密閉系での培養方法は今後様々な実験で利用されるのではないだろうか。

DOI： 10.14882/jrds.113.0_p-54

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2019-043

PubMed

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】クローン胚を作製する際に人為的活性化は必要不可欠であるが受精による活性化と異なる。そこで精子頭部に存在する卵子活性化因子，PLCζを卵子に注入して活性化させる方法の検討が進められている。この方法は卵子を最も自然に近い方法で活性化させる一方，mRNAの注入のダメージという新たな問題が生じる。そこで本実験は，PLCζ-mRNA注入と核移植を同時に行うことで実験の効率改善を目的とした。【方法】①PLCζ-mRNAが2, 20, 100 ng/µlの濃度で添加されているPVP溶液を作製し，この中へ卵丘細胞を混ぜ，常法に従って核移植を行った。その後，再構築卵の活性化率および桑実胚/胚盤胞への発生率を観察することで最適濃度を調べた。②PLCζ-mRNAを体細胞と同時，あるいは分けて注入して活性化したクローン胚を，SrCl₂で活性化した従来法のクローン胚と比較した。1細胞期胚ではエピジェネティクス修飾（H3K4me3，H3K9me3）について，2細胞期胚では産仔率について調べた。【結果】①卵子の活性化率は，2, 20, 100 ng/μlでそれぞれ，82, 95, 96％となり，全区において大部分が活性化された。桑実胚期/胚盤胞期までの発生率22, 48, 21％であり，低濃度および高濃度のPLCζ-mRNAにより活性化した場合は発生が低下する傾向がみられた。②活性化後10時間でH3K4me3およびH3K9me3の修飾量を調べたところ，いずれの区でも有意な差はみられなかった。クローン産仔は，PLCζ-mRNAを分けて注入した方法で産子が得られ，同時注入法は現在移植中である。まだ最終結果は出ていないが，おそらくPLCζを用いた同時活性化法は，SrCl₂の代替になるだけでなく，手間とダメージを軽減できる簡便な方法になりえる可能性が示された。

DOI： 10.14882/jrds.112.0_p-96

CiNii Research

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】従来，凍結乾燥精子は真空状態が保たれたガラスアンプルでの保存が大前提であったが，アンプル瓶の作製ミスや破損などの懸念が伴う。本研究では，凍結乾燥精子をアンプル瓶よりも安全に長期間保存できる新たな保存媒体と最適処理方法の探索，及びその技術を用いた産仔の作出を試みた。【方法】実験にはICR系統のマウス精子と卵子を用いた。保存媒体には薬包紙，オブラート，プラスチックシート，ラップフィルム，和紙を用いた。精子の凍結乾燥は，キャパシテーションをした精子懸濁液を，液体窒素上に置いた媒体の上に滴下し凍結させ，直ちにLABCONCO FreezOne2.5®にて乾燥処理を行った。凍結処理は10分～2時間，乾燥処理は2～24時間行った。乾燥処理後，精子を載せた媒体をラミネート加工し，–30℃で数日から1ヵ月間保存した。各処理条件，各媒体で保存した精子について，加水後の精子回収率，ICSI後の発生率と産仔率，DNA損傷率を調べた。【結果・考察】薬包紙と和紙は媒体に精子が固くこびりつき回収が困難なほか，繊維などのゴミも多かった。オブラートとラップフィルムも同様の結果であったうえ，ラップフィルムはちぎれやすく回収をより困難にした。プラスチックシートを用いた場合，精子はシートに全く貼りつかず回収が容易であり，かつ繊維などの混入も見られなかった。回収した凍結乾燥精子を用いて顕微授精を行った結果，薬包紙で26日間−30℃で保存した精子から産仔を得ることに成功した（産仔率47%）。他の保存媒体による産仔率やDNA損傷への影響は現在実験中である。長期保存のためには空気の混入量やラミネートの接着剤による精子への毒性等も調査が必要だが，紙などシート状の媒体で保存した凍結乾燥精子からの産仔作出の成功は，“アルバム”のような形状でも精子が保存できることを示している。将来的には数万種にも及ぶような遺伝資源の一括管理やより安全な長期保存を実現することで，精子の凍結乾燥保存技術の利用価値はさらに高まると考えられる。

DOI： 10.14882/jrds.112.0_p-128

CiNii Research

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】希少動物や貴重な遺伝子組換え動物などの卵子を用いる研究において，採卵対象の個体を殺すことなく手術により採卵（手術採卵）することが可能となれば，限られた実験材料をより有効に活用可能となる。そこで，本研究ではモデル実験としてICR系統のマウスを用い，手術採卵後の再利用の可否とその最適条件の探索を行った。【方法】手術採卵 過排卵処理を施した雌マウスを用意し，アバチンあるいは三種混合で麻酔したのち背側から卵巣と卵管を露出させ，マイクロ剪刀を用いて卵管膨大部の下部に切り込みを入れ，ピンセットにより卵丘卵子複合体を摘んで取り出した。その後，体内に卵巣，卵管を戻し，腹膜，皮膚を縫合した後，覚醒させた。実験1 手術採卵した卵子と従来法で採取した卵子（安楽死後に採取：通常採卵）について，採卵数，IVF，ICSI，ROSI後の受精率と胚盤胞発生率，および産仔率について比較した。実験2 手術採卵を行った雌マウスに対し，術後10，20，30日目に再び過排卵処理を行い，採卵数および卵子の質について実験1と同様に比較した。【結果と考察】実験1 手術採卵と通常採卵とでは，排卵数，受精率，胚盤胞発生率および産仔率はすべての区において差は無く，また2種類の麻酔薬間でも差はなかったことから，採卵における手術の影響はないと判断できた。実験2 手術採卵により卵管が塞がるために，術後全期間において70％程度の卵管からは再採卵が不可能であった。しかし，再採卵に成功した卵子では術後全期間において胚盤胞発生率はIVFで80％以上，ICSIで70％程度，産仔率はIVFで60％程度，ICSIで50％程度となり，通常採卵と比べて悪影響は見られなかったため，問題なく実験に使用可能だと考えられた。今後は手術採卵後の雌マウスについて，卵巣から未成熟卵を採取後，体外成熟実験への利用や，子宮への胚移植の際の代理出産母としての利用が可能かを確認していく予定である。

DOI： 10.14882/jrds.112.0_p-115

CiNii Research

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】人類の宇宙開発が進む中，宇宙空間で哺乳類が正常に生殖を行えるかの可否は人類の宇宙進出において重要な知見となる。我々は実際に宇宙ステーションを利用してマウス初期胚の培養を行う実験を計画しているが，一般的な哺乳類初期胚の培養方法は宇宙で行うことができない。胚の扱いに不慣れな宇宙飛行士が無重力空間で実施するため，耐圧性がありガス透過がない密閉容器で胚を培養することになるだろう。そこで我々は，あらかじめ必要なガスを取り込んだ培地（以下平衡化培地）を用い，完全密閉容器内で胚を培養する方法の開発を試みた。【方法】本実験では密閉容器として蓋の周りをパラフィルムでシールしたアシストチューブを用いた。最初に，培地を入れ蓋を緩めたアシストチューブをCO₂インキュベーター内に入れ，培地のCO₂分圧の変化を経時的に測定した。次に，ICRあるいはB6D2F1マウスのIVFを行い，1細胞期胚を平衡化培地の入ったアシストチューブに入れ密封し，恒温槽内で72時間または96時間培養した。72 時間の培養で得られた胚は子宮へ移植し産仔率を，96 時間の培養で得られた胚は胚盤胞への発生率を算出し，得られた胚盤胞は免疫蛍光染色により，全細胞数およびICMとTEの細胞計数を行った。【結果と考察】インキュベーターに入れた培地のCO₂分圧は約24 時間で平衡化することがわかった。この培地を用いて密閉容器で培養した胚の胚盤胞までの発生率はどちらの系統でも90％以上であり対照区のディッシュでの培養と変わらなかった。また，胚盤胞のICMとTEの細胞数も対照区と差はなかった。胚移植は現在実施中だが，すでに密閉容器培養から産仔を得ることに成功している。これらの結果から，宇宙ステーションで胚を培養する実験は，予め平衡化した培地を使うことができれば実現可能であることが明らかとなった。また，この方法は温度のみの維持で培養できるため胚の輸送にも適しており，宇宙実験だけでなく地上での発生工学の研究や生殖補助医療にも応用できると思われる。

DOI： 10.14882/jrds.112.0_p-66

CiNii Research

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】哺乳類は高温や乾燥などの極限環境にさらされると，ほぼすべての個体は死んでしまう。しかし，フリーズドライ精子（FD精子）は完全に乾燥し，死んでいるにもかかわらず顕微授精により産仔を得ることが可能である。一方，乾眠することで究極の耐性を得るクマムシにも同じように乾燥耐性があるが，さらにそれらには耐熱耐性も備わっている。そこで我々はFD精子にも同じ性質があるかを検証するために様々な温度処理を行い，極限環境耐性能を調べた。【方法】①急激な温度変化に対する耐性を調べるためにFD精子を液体窒素へ浸してから室温に戻す処理を10回繰り返した。②FD精子を30分間，65℃，80℃，95℃に曝すことで，100℃以下の温耐性を調べた。③95℃加熱処理の限界時間を検討した。④100℃以上の高温での限界温度を検討した。ＦＤ精子はそれぞれ核のＤＮＡダメージを調べた後，顕微授精による産仔率を比較した。【結果】①FD精子を液体窒素へ10回浸しても顕微授精により多数の産仔が得られたことから急激な温度変化に対して耐性があることが示された。②新鮮精子の場合卵子へ活性化処理を行っても65℃加熱が産仔を得られる限界だったのに対して，FD精子の場合，95℃処理でも出産率に影響がなかった。③焦げの原因となる糖をトレハロースに変更したところ，最長で6時間，95℃に加熱し続けた精子からでも産仔を得ることに成功した。④100℃以上の耐性は，120℃であれば10分間，150℃であれば3分間加熱処理した精子からでも産仔が得られた。同様な耐性は他の系統でも観察された。【考察】哺乳類の細胞は急激な温度変化や高熱により死んでしまうため極限耐性はないと思われていた。しかし本実験によりFD化すれば，クマムシに匹敵するほどの強い熱耐性があることが分かった。このことから，フリーズドライ精子は火事や災害などの有事の際でも動物の遺伝子資源として有効な方法と言えるのではないだろうか。（Wakayama S et al., Sci. Rep. 2019）

DOI： 10.14882/jrds.112.0_p-120

CiNii Research

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2019-058

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-019-42062-8

PubMed

その他リンク： http://www.nature.com/articles/s41598-019-42062-8

記述言語：英語  

DOI： 10.1007/978-1-4939-8831-0_12

PubMed

記述言語：英語  

DOI： 10.1038/s41598-018-33304-2

PubMed

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】受精卵の割球は16細胞期にICMとTEへ分化する。その運命決定は，①先に分裂した割球はICM，遅れた割球はTEに分化する，②16細胞期に外側に配置された割球がTE，内側に配置された割球がICMに分化する，とされている。当時は2つの胚を区別する技術は無く，生まれた産仔のキメリズムで確認された。そこで本研究では2色のマウス胚を用いて集合キメラを作製し，蛍光ライブセルイメージングにより運命決定の再評価を試みた。【方法】全身でGFPあるいはRFPを発現するマウスから交尾後2日目の胚を回収し，それぞれの胚をPHPで集合させ，ライブセルイメージング（CV1000）で2日間観察しながら胚盤胞への細胞系譜を調べた。実験区では発育ステージが異なる2つの胚の間でのキメラ，同じ発育ステージだが1つの胚を2つの胚で挟んだサンドイッチキメラなどを行った。胚盤胞へ発生したキメラ胚の一部は移植し，残りは固定し免疫染色を行っている。【結果】キメラ胚をライブセルイメージングしたところ，キメラ胚の割球ははっきりと緑と赤に区別でき，観察したまま胚盤胞まで発生させることが出来た。対照区ではICM・TEへの寄与率は両胚で同程度であった。一方，発生ステージの異なる2つの胚を集合させたキメラ胚の場合は67％が，3つの胚を使ったサンドイッチキメラの場合は80％が，胚の発生ステージや配置とは無関係にICM・TEへ分化した。現在，免疫染色及び胚移植により詳細を確認中である。【考察】本研究によりキメラ胚の発生の詳細を観察することが出来たことから，我々の方法が初期胚での細胞系譜を視覚的に明瞭に示す有効な手段であることが証明された。その結果，一部の胚は従来説を裏付けていたが，半数以上の胚は従来説とは無関係に分化していることが明らかとなった。今後は発育ステージのより細かな組み合わせやOct-RFPとCdx-GFPマウスのキメラ胚を用いて，割球の運命決定をより正確に明らかにしていきたい。

DOI： 10.14882/jrds.111.0_p-67

CiNii Research

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語   出版者・発行元：公益社団法人 日本繁殖生物学会  

【目的】卵子の人為的活性化処理は円形精子細胞による受精やクローン技術において不可欠であるが，最適な活性化法は確立されていない。そこで精子頭部に存在する卵子活性化因子PLCζを用いることで，より通常の受精に近い卵子活性化方法の検討が進められている。しかしこの方法では，細胞の注入の他にPLCζタンパクあるいはPLCζ-mRNAの注入が必要であり，計2回の注入は手間がかかるだけでなく卵子へ大きなダメージを与えてしまう。そこで本実験では，細胞とPLCζ-mRNAを同時に注入することで，胚発生を向上させると同時に作業効率を改善することを目的とした。【方法】①PLCζ-mRNAをPVPに加え最終濃度が2 ng/µl，20 ng/µlおよび100 ng/µlになるよう調整し，マウス卵子へ注入した。その後，活性化率および2倍体化処理後の胚盤胞への発生率を観察することで最適濃度を調べた。②円形精子細胞を上記PLCζ-mRNA -PVP溶液と混ぜ卵子へ注入し，活性化率，胚発生率，および産仔作出率を調べることで新規活性化方法の有用性を検討した。【結果】①卵子の活性化率は，2 ng/μlでは33%，20 ng/μlでは87%，100 ng/μlでは95％となり，20 ng/μlおよび100 ng/μlで高い活性化率を示した。しかしながら，桑実胚期/胚盤胞期までの発生率において20 ng/µlでは49%，100 ng/μlでは42％であり発生が低下する傾向がみられた。②円形精子細胞と20 ng/μlのPLCζ-mRNAを混ぜ，同時に卵子へ注入したときの2前核率は44％，桑実胚期/胚盤胞期への発生率は42%であり，従来法で活性化した対照区（47％および54％）と有意な差はみられなかった。胚盤胞の品質および移植後の産仔作出率は現在実施中である。これらの結果から，細胞とPLCζ-mRNAの同時注入は従来法より作業効率が向上し，胚へのダメージを削減することに繋がることが示唆された。今後はクローン胚についても効果があるか検討する。

DOI： 10.14882/jrds.111.0_p-75

CiNii Research

J-GLOBAL

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-018-28896-8

PubMed

記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

J-GLOBAL

記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

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

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR-outbred mouse strain. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/ embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

DOI： 10.1530/REP-17-0372

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/nature23286.

記述言語：英語   出版者・発行元：NATL ACAD SCIENCES  

DOI： 10.1073/pnas.1711468114

Web of Science

PubMed

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

Inhibitors of Mek1/2 and Gsk3 beta, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents(1,2). Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

DOI： 10.1038/nature23286

Web of Science

PubMed

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

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 degrees C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

DOI： 10.1073/pnas.1701425114

Web of Science

PubMed

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

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells2017;35:1189-1196

DOI： 10.1002/stem.2601

Web of Science

PubMed

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

Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.

DOI： 10.1089/cell.2016.0026

Web of Science

PubMed

記述言語：日本語  

J-GLOBAL

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

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

DOI： 10.1095/biolreprod.116.138677

Web of Science

PubMed

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

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

DOI： 10.1038/srep23808

Web of Science

PubMed

記述言語：日本語  

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語  

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

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

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

DOI： 10.1371/journal.pone.0138854

Web of Science

PubMed

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】生殖系列におけるリプログラミング機構は，受精を介して全能性を獲得する卵子と精子を生じさせる。体細胞クローン技術は，終末まで分化した細胞に全能性を再獲得させるが，産子への発生率は低く，頻繁に胎仔・胎盤の形成異常を伴うことが知られている。体細胞クローンマウスではエピジェネティックな異常が見られることが報告されている。本研究では，クローンマウス精子の包括的DNAメチローム解析を実施し，DNAメチル化の正常性について検討した。 【方法】BDF1マウスの尾端線維芽細胞をドナーとして用い，同系統の除核卵に核移植し，7匹の正常な表現型を示すクローンマウスおよび5匹のBDF1野生型マウスから得られた精子をサンプルとした。DNAメチル化解析は，Post-bisulfite adaptor tagging法を用いたターゲットメチロームシークエンス法により実施し，メチル化に関係する領域についてのメチロームデータを作成した。23021個のCpGアイランドとそれらのアイランドショアについて，野生型から外れ値を除いて平均化したものとクローンの各個体との間でメチル化率が統計学的に有意な差異を示す領域を抽出した。 【結果】既知のインプリント制御領域（ICR）のメチル化率は，リードがマッピングされた2個の父方メチル化ICRにおいて，クローン全個体で95%以上，19個の母方メチル化ICRにおいて6%以下であり，これらの領域は正常にリプログラムされていること確認された。全CpGアイランド中で統計学的に有意にメチル化率が異なる領域について，クローンが野生型に対して高メチル化となる領域が5個体に計11領域，低メチル化となる領域が3個体に計16領域検出された。さらにCpGアイランドショアについても同様の解析により，高メチル化領域が3個体に計6個，低メチル化領域が1個体に計3個検出された。本研究の結果から，生殖細胞系列におけるリプログラミングの影響を受けにくい領域の存在が示唆された。

DOI： 10.14882/jrds.108.0_P-82

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：論文集（書籍）内論文   出版者・発行元：Springer New York  

The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

DOI： 10.1007/978-1-4939-1594-1_8

Scopus

PubMed

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】生殖系列におけるリプログラミング機構は，受精を介して全能性を獲得する卵子と精子を生じさせる。体細胞クロー技術は，終末まで分化した細胞に全能性を再獲得させるが，産子への発生率は低く，頻繁に胎仔・胎盤の形成異常を伴うことが知られている。しかし，体細胞クローン個体から誕生した産子には，親世代に生じた異常は認められない。果たしてクローン個体の生殖系列で，不完全なエピゲノムエラーが修正されるのであろうか？仮に，生殖細胞にエピゲノムエラーが潜在しているとすればトランスジェネレーショナルな伝達の可能性も否定できない。そこで，本研究では体細胞クローン個体が生産する精子のDNAメチロームを明らかにしようとした。【方法】 成熟雄マウス（BDF1）の尾から調整した体細胞をドナー細胞として排卵卵子（BDF1）に核移植して体細胞クローンマウスを作出した。作出された体細胞クローンは10–12週まで飼育したのち，精巣上体尾部より精子を採取し，DNAメチローム解析用試料とした。DNAメチローム解析は，SureSelect-Methyl-seq (Agillent社)を用いてライブラリーを作製後，次世代シークエンサー（HiSeq2500, Illumina社）を用いて解析した。【結果】受精卵由来雄マウス（n=2）精子と体細胞クローンマウス（n=9）の精子において計23,116のCpG領域を比較したところ，メチル化状態が異なる領域が検出された。たとえば，コントロール個体のメチル化状態が90%を越えるCpG領域で50%以下のメチル化を示すクローン個体が存在した。一方，コントロール個体のメチル化状態がいずれも30％以下のCpG領域で70%以上のメチル化を示すクローン個体も存在した。これらのCpG領域は体細胞クローンマウス精子に特有なメチル化変異である可能性が高い。また，メチル化異常を示した領域は，必ずしも共通する領域ではないが，遺伝子発現に何らかの影響を及ぼす可能性も否定できない。

DOI： 10.14882/jrds.107.0_OR1-25

J-GLOBAL

記述言語：日本語   出版者・発行元：エヌ・ティー・エス  

CiNii Books

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：論文集（書籍）内論文   出版者・発行元：Elsevier Inc.  

Since the first cloned mammal, Dolly the sheep, the success rates of somatic cell nuclear transfer (SCNT) have been extremely low. This inefficiency has been limiting practical applications of SCNT. In the past decade, many trials aimed to enhance SCNT have led to the development of new modified SCNT methods, resulting in an increased success rate. These trials can be mainly divided into four types: type 1, improvement by optimizing SCNT protocols in each condition such as the methods of oocyte activation
type 2, improvement by modified donor cell preparation, including optimization of the cell cycle
type 3, improvement by chemical treatment of reconstructed oocytes
and type 4, improvement by gene manipulation of donor cells.In 2006, treatment of SCNT embryos with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), was found to improve significantly the development of SCNT embryos in mice
this was the first example of success (of type 3) using chromatin remodeling agents. Since then, a variety of modified SCNT protocols with HDACs have been examined in many species. Thus, the success of significant improvement in SCNT with HDACi has not only contributed to development of new method of SCNT, but also provided a lot of insights into nuclear reprogramming. In this chapter, we review recent advances in modified SCNT methods using chemical agents for chromatin remodeling, focusing particularly on modified SCNT with HDACi in mice. © 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/B978-0-12-386541-0.00011-4

Scopus

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

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

DOI： 10.1371/journal.pone.0078380

Web of Science

PubMed

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】哺乳動物の生殖細胞保存技術は畜産，医療，基礎生物学分野さらに生物種の保全など多くの分野で利用されている重要な技術である。我々が開発したマウス精子の凍結乾燥保存方法は，精子を液体窒素や冷凍庫なしでも1か月以上，–80℃では長期保存可能であり，サンプルの輸送も簡便であるため，新規生殖細胞保存法としての利用が期待される。ところが凍結乾燥精子を顕微授精した場合，産仔率が新鮮精子と比べて非常に低いという問題がある。そこで本研究では凍結乾燥精子で受精した胚の初期発生過程をライブセルイメージング解析し，発生停止の原因を探った。 【方法】複数のBDF1マウスから精子を採取し，個体別に凍結乾燥精子を作成した。初めにこれらの精子を顕微授精して個体間の産仔率の違い調べた。次に高い産仔率だったもの(BDF1–1)と，産仔が1匹も得られなかったもの(BDF1–2)を選んで顕微授精し，前核期にH2B-mRFP1のmRNAを注入後，ライブセルイメージングにより観察を行った。一部の胚は発生促進を期待して人為的に活性化し，同様にライブセルイメージングにより観察した。また新鮮精子の顕微授精胚をコントロールとして用いた。 【結果】イメージング解析に用いたサンプルBDF1–1および2の産仔率はそれぞれ22％と0％であった。ライブセルイメージングの結果，産仔率によらず凍結乾燥精子で受精した胚は新鮮精子を用いた場合と比較して第一卵割までにかかる時間が長く，各胚間でのばらつきも多かった。凍結乾燥精子由来胚では第一卵割での染色体分配異常が新鮮精子 (7％)と比べて非常に多く，BDF1–1が78％, BDF1–2では87％で両者の間に有意な差はなかったものの産仔率が高いもので少ない傾向があった。人為的活性化刺激を加えた胚では染色体分配異常が増える傾向が見られた(BDF1–1 88%, BDF1–2 100%)。以上より，第一卵割における染色体分配異常が凍結乾燥精子の発生停止の原因であり，この頻度が産仔率に影響していることが示唆された。

DOI： 10.14882/jrds.106.0.P-97.0

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】哺乳動物のクローン作出は，優良家畜の大規模な生産や絶滅危惧種の保全を可能にする新しい技術として期待されてる。しかし現在の成功率では一度に大量のクローン動物を作ることは出来ないため，クローン動物の体細胞から再びクローン動物を作り出す連続核移植（再クローニング）技術が必要だと考えられていた。ところがこれまでの報告では，再クローニングを繰り返すごとに出産率は低下し，マウスで6世代，ウシやネコで2世代までが限界だった。この原因は，クローン技術特有の「初期化異常」が，核移植を行うたびに蓄積するためと考えられていたが，成功率が低すぎるため検証できていなかった。そこで今回我々は，最新の技術を用いて再クローニングに限界があるのか確かめてみた。 【方法】我々は2005年にトリコスタチン A（TSA）が初期化を促進し，クローンマウスの出産率を大きく改善できることを発見した。そこでTSAを用いて1匹のドナーマウス（BD129F1)からクローンマウスを作り（G1と呼ぶ），このクローンマウスが3カ月齢になった段階で再びクローンマウスを作製した（これをG2と呼ぶ）。以降これを繰り返した。生まれた再クローンマウスについて，テロメアや妊性，網羅的遺伝子発現などを調べ，自然マウスおよびG1クローンマウスと比較し，エピジェネティック異常が蓄積されるか調べた。 【結果】現時点で27世代，合計645匹のクローンを作ることに成功している。核移植の出産率は1世代目の7％から上昇傾向を示し，最高で15%を記録している。G20クローンの繁殖能力，寿命，テロメアの長さなどに異常は見られなかった。また網羅的遺伝子発現解析により核移植を繰り返しても初期化異常は蓄積しないことが明らかとなった。これらの結果は，再クローニングはほぼ無限に繰り返せることを示している。Wakayama et al., Cell Stem Cell 2013.

DOI： 10.14882/jrds.106.0.P-102.0

J-GLOBAL

記述言語：英語   掲載種別：（MISC）その他記事   出版者・発行元：WILEY-BLACKWELL  

DOI： 10.1002/stem.1346

Web of Science

記述言語：日本語  

CiNii Books

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

Previous studies of serial cloning in animals showed a decrease in efficiency over repeated iterations and a failure in all species after a few generations. This limitation led to the suggestion that repeated recloning might be inherently impossible because of the accumulation of lethal genetic or epigenetic abnormalities. However, we have now succeeded in carrying out repeated recloning in the mouse through a somatic cell nuclear transfer method that includes a histone deacetylase inhibitor. The cloning efficiency did not decrease over 25 generations, and, to date, we have obtained more than 500 viable offspring from a single original donor mouse. The reprogramming efficiency also did not increase over repeated rounds of nuclear transfer, and we did not see the accumulation of reprogramming errors or clone-specific abnormalities. Therefore, our results show that repeated iterative recloning is possible and suggest that, with adequately efficient techniques, it may be possible to reclone animals indefinitely.

DOI： 10.1016/j.stem.2013.01.005

Web of Science

PubMed

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】人類は宇宙で繁栄できるだろうか。これまでの研究から，両生類や鳥類の胚は宇宙でも発生できることが確認されている。しかし，肝心の哺乳動物を用いた繁殖実験はすべて失敗に終わっている。これは動物を宇宙で飼育することが困難なことや，宇宙飛行士にとって初期胚を使った実験は難しすぎることが原因だと思われる。我々が行った疑似微小重力発生装置を用いた実験でも，マウス胚は無重力環境下で発育できない可能性を示していた（Wakayama et al. Plos One 2009）。だがシミュレーションでは本当のことはわからない。宇宙での本当の実験が不可欠である。【方法】我々は2009年にJAXAが公募していた国際宇宙ステーション「きぼう」第二次後期利用実験計画に，フリーズドライにしたマウス精子を宇宙で長期間保存し，生殖細胞への宇宙放射線の影響を調べるというテーマで応募した。この方法ならロケットの打ち上げ時と回収時を常温で行え，また宇宙飛行士は容器を運ぶだけなので難しい技術の習得も時間も必要ない。これらの利点が評価され，我々のテーマは2010年に「打ち上げ候補」として採択された。次に我々は1．実際の打ち上げを想定した温度変化を与えても生まれることの確認，2．振動・衝撃が加わっても精子の保存が可能な容器の決定3．エックス線やガンマ線照射を行い，生まれる限界放射線量を決定，4．詳細な実験計画とその評価方法の技術的確立など膨大な量の実験を行い，2012年についに打ち上げ決定となった。実験ではBDF1やB6など4系統のマウスそれぞれ3−4個体からフリーズドライ精子を作成し，宇宙ステーションと地上（コントロール）でそれぞれ3か月間，1年間および2年間保存する。回収後，精子のDNAダメージ度の測定や顕微授精による産仔作出を試み，生殖細胞への宇宙放射線の影響を明らかにする。現在，打ち上げ用として最も出産率の高い試料を選定中である。【結果】2013年8月4日，種子島からH2Bロケット4号機で打ち上げ予定。

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】我々は既にマウスおよびブタ胚のガラス化保存において，中空糸法により高い生存性が得られることを報告した。本研究は，中空糸法の融解条件が胚生存性に及ぼす影響を検証することを目的とした。 【方法】過排卵処理したBDF1マウスから採取した2細胞期胚を実験に用いた。既報(Matsunariら，2012)に従い，7.5% DMSO，7.5% エチレングリコール(EG)を含む平衡液中で，セルローストリアセテート製中空糸内に10個の胚を収容した。5–7分の平衡後，中空糸を15%DMSO，15%EG，0.5M sucroseを含むガラス化液に1分間維持し，その後液体窒素(LN)中に投入した。3種の融解条件を比較した。通常法：LNから取り出した中空糸を37.5℃に温めた融解液(1M sucrose含)に素早く投入した。加温盤法：中空糸を37.5℃に温めた加温盤上に約3秒静置して融解し，その後室温の融解液に投入した。空気中法：中空糸を室温の空気中に5秒間保持して融解し，その後室温の融解液に投入した。融解液投入以降の凍害保護剤の段階的希釈および洗浄は常温で行った。胚の生存判定は培養および移植試験により行った。 【結果】通常法，加温盤法，空気中法および非ガラス化区の胚盤胞形成率には，有意差は見られなかった(105/110, 95.5% vs. 107/110, 97.3% vs. 94/100, 94.0% vs. 109/110, 99.1%)。各区のガラス化胚を発情同期化したレシピエント雌に移植した結果，妊娠率はいずれも100% (4/4；非ガラス化区のみ2/2)であり，移植胚の胎仔への発達率は，55/80(68.8%)，56/80(70.0%)，55/80(68.8%)，35/40(87.5%)であった(有意差なし)。 【考察】一般にガラス化法においては，超急速な融解条件が必須と考えられている。中空糸ガラス化法では，比較的緩除な条件で融解が行われた場合にも，胚の生存性が高く保たれることが明らかとなった。

DOI： 10.14882/jrds.106.0.OR2-26.0

その他リンク： http://search.jamas.or.jp/link/ui/2014046938

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

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.

DOI： 10.1095/biolreprod.111.098764

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PubMed

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

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

DOI： 10.1371/journal.pone.0031638

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PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

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

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P &lt; 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in MI6 or CZB media (P &lt; 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2011.06.028

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PubMed

記述言語：日本語  

CiNii Books

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（研究会，シンポジウム資料等）  

CiNii Books

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

The successful generation of cloned animals and the establishment of embryonic stem (ES) cell lines from somatic cells suggest that these techniques may be used in human regenerative medicine. However, the fact that oocytes must be donated by women undergoing infertility treatment remains a fundamental ethical objection, as they might be concerned about the potential exploitation of their genome. Here, we investigated the reprogramming potential of enucleated and cryopreserved oocytes for the development of full-term cloned mice. BDF1 strain mouse oocytes were cryopreserved at metaphase II, before and after enucleation. After thawing, cumulus cell nuclei were microinjected to generate clones. Although the rate of development of cloned embryos to the blastocyst stage using the treated oocytes was lower than that obtained using fresh oocytes, three live pups were delivered after embryo transfer into pseudopregnant females (0.4% of the oocytes used). Thus, although cryo-preservation reduces the potential of oocytes, these cells retain the ability to support the full-term development of cloned embryos. In addition, the removal of DNA from human oocytes may alleviate the ethical and psychological problems for women who are undergoing infertility treatment and are considering oocyte donation for research or therapeutic purposes.

DOI： 10.1089/cell.2010.0059

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PubMed

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

Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis. Reproduction (2011) 141 67-77

DOI： 10.1530/REP-10-0109

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PubMed

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

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results. in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

DOI： 10.1095/biolreprod.109.083337

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PubMed

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

Mammalian parthenogenetic embryos invariably die in mid-gestation from imprinted gene defects and placental hypoplasia. Based on chimera experiments, trophoblastic proliferation is supposed to be inhibited in the absence of a male genome. Here, we show that parthenogenetic mouse embryonic cell nuclei can be reprogrammed by serial rounds of nuclear transfer without using any genetic modification. The durations of survival in uteri of cloned foetuses derived from green fluorescent protein (GFP)-labelled parthenogenetic cell nuclei were extended with repeated nuclear transfers. After five repeats, live cloned foetuses were obtained up to day 14.5 of gestation; however, they did not survive longer even when we repeated nuclear transfer up to nine times. All foetuses showed intestinal herniation and possessed well-expanded large placentas. When embryonic stem (ES) cells derived from fertilised embryos were aggregated with the cloned embryos, full-term offspring with large placentas were obtained from the chimeric embryos. Those placentas were derived from parthenogenetic cell nuclei, judging from GFP expression. The patterns of imprinted gene expression and methylation status were similar to their parthenogenetic origin, except for Peg10, which showed the same level as in the normal placenta. These results suggest that there is a limitation for foetal development in the ability to reprogramme imprinted genes by repeated rounds of nuclear transfer. However, the placentas of parthenogenetic embryos can escape epigenetic regulation when developed using nuclear transfer techniques and can support foetal development to full gestation.

DOI： 10.1242/dev.051375

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PubMed

担当区分：筆頭著者   記述言語：日本語   出版者・発行元：エヌ・ティー・エス  

CiNii Books

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：UNIV BASQUE COUNTRY UPV-EHU PRESS  

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

DOI： 10.1387/ijdb.103205sw

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PubMed

記述言語：英語   掲載種別：論文集（書籍）内論文   出版者・発行元：ELSEVIER ACADEMIC PRESS INC  

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach'' and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold.

DOI： 10.1016/S0076-6879(10)76009-2

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PubMed

担当区分：筆頭著者   記述言語：日本語   出版者・発行元：オーム社  

CiNii Books

J-GLOBAL

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

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (mu G) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under mG. However, although we obtained 75 healthy offspring from mu G-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under mG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under mG environment in a mammal, but normal preimplantation embryo development might require 1G.

DOI： 10.1371/journal.pone.0006753

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PubMed

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

Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos. Reproduction (2009) 138 309-317

DOI： 10.1530/REP-08-0299

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PubMed

記述言語：日本語  

CiNii Books

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-59745-471-1_13

Scopus

PubMed

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

In mammalian cloning, evidence suggests that genomic reprogramming factors are located in the nucleus rather than the cytoplasm of oocytes or zygotes. However, little is known about the mechanisms of reprogramming, and new methods using nuclear factors have not succeeded in producing cloned mice from differentiated somatic cell nuclei. We aimed to determine whether there are functional reprogramming factors present in the cytoplasm of germinal vesicle stage (GV) oocytes. We found that the GV oocyte cytoplasm could remodel somatic cell nuclei, completely demethylate histone H3 at lysine 9 and partially deacetylate histone H3 at lysines 9 and 14. Moreover, cytoplasmic lysates of GV oocytes promoted somatic cell reprogramming and cloned embryo development, when assessed by measuring histone H3-K9 hypomethylation, Oct4 and Cdx2 expression in blastocysts, and the production of cloned offspring. Thus, genomic reprogramming factors are present in the cytoplasm of the GV oocyte and could facilitate cloning technology. This finding is also useful for research on the mechanisms involved in histone deacetylation and demethylation, even though histone methylation is thought to be epigenetically stable.

DOI： 10.1242/dev.023747

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PubMed

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

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the "resurrection" of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20 degrees C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to "resurrect" animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.

DOI： 10.1073/pnas.0806166105

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PubMed

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

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.

DOI： 10.1017/S0967199408004620

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PubMed

記述言語：英語   出版者・発行元：ELSEVIER INC  

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2008.01.027

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PubMed

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

Cloning methods in mice are now well described and are becoming routine. However, the frequency at which cloned mice are produced remains below 5%, irrespective of the nucleus donor species or cell type. Only a few laboratories have made clones from adult mouse somatic cells and most strains have never produced cloned mice. On the other hand, nuclear transfer can be used to generate human embryonic stem (ntES) cell lines from a patient&apos;s own somatic cells. It has been shown that such cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. This technique could be used in regenerative medicine and, in theory, in infertility clinics to treat completely infertile individuals. However, these results suggest that the reprogramming integrity of each cloned embryo differs: some cloned embryos can be converted to ntES cells, but these embryos cannot achieve full term development. This review outlines the nature of genomic reprogramming potential and its application, and suggests new approaches to avoid the ethical problems of creating embryos by nuclear transfer.

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PubMed

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

Cell transplantation with embryonic stem (ES) cell progeny requires immunological compatibility with host tissue. 'Therapeutic cloning' is a strategy to overcome this limitation by generating nuclear transfer (nt) ES cells that are genetically matched to an individual. Here we establish the feasibility of treating individual mice via therapeutic cloning. Derivation of 187 ntES cell lines from 24 parkinsonian mice, dopaminergic differentiation, and transplantation into individually matched host mice showed therapeutic efficacy and lack of immunological response.

DOI： 10.1038/nm1732

Web of Science

PubMed

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

Recent cloning technology has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem (ES) cells. Mice can be cloned from adult somatic cells or ES cells by NT, and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remain unclear. In this study, our goal was to clarify why the contribution rate of ES cells derived from parthenogenetic embryos (pES) cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was significantly increased in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status of U2af1-rs1 could be used as a genetic marker to investigate the effects of NT.

DOI： 10.1634/stemcells.2007-0907

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PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

記述言語：日本語   出版者・発行元：共立出版  

PubMed

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2008108304

記述言語：日本語  

DOI： 10.14882/jrds.100.0.20103.0

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語  

CiNii Books

担当区分：筆頭著者   記述言語：日本語   掲載種別：（MISC）総説・解説（学術雑誌）  

CiNii Books

記述言語：英語   出版者・発行元：CELL PRESS  

DOI： 10.1016/j.cub.2006.12.020

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PubMed

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

Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting. The objective of this study was to investigate changes in histone H3 modifications in relation to chromatin/chromosome morphology in pig oocytes during their growth, maturation, and activation. During the growth phase, histone H3 was acetylated at lysines 9, 14, and 18 (K9, K14, and K18), and became methylated at K9 when the follicles developed to the antral stage (oocyte diameter, 90 gm). When the fully grown oocytes (diameter, 120 gm) started their maturation, histone H3 became phosphorylated at serine 28 (S28) and then at S10, and deacetylated at K9, K14, and K18 as the chromosomes condensed. After the electroactivation of mature oocytes, histone H3 was reacetylated and dephosphorylated concomitant with the decondensation of the chromosomes. Histone H3 kinase activity increased over a similar time course to that of the phosphorylation of histone H3-S28 during oocyte maturation, and this activity decreased as histone H3-S10 and H3-S28 began to be dephosphorylated after the activation of the mature oocytes. These results suggest that the chromatin morphology of pig oocytes is regulated by the acetylation/deacetylation and the phosphorylation/dephosphorylation of histone H3, and the phosphorylation of histone H3 is the key event in meiotic chromosome condensation in oocytes. The inhibition of histone deacetylase with trichostatin A (TSA) inhibited the deacetylation and phosphorylation of histone H3, and chromosome condensation. Therefore, the deacetylation of histone H3 is thought to be required for its phosphorylation in meiosis. Although histone H3 acetylation and phosphorylation were reversible, the histone methylation that was established during the oocyte growth phase was stable throughout the course of oocyte maturation and activation.

DOI： 10.1530/REP-06-0099

Web of Science

PubMed

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

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

DOI： 10.1262/jrd.18098

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PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

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

Recently, ES cell lines were established from single blastomeres taken from eight-cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two-cell (establishment rate, 50%-69%), early four-cell (28%-40%), late four-cell (22%), and eight-cell (14%-16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%-40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.

DOI： 10.1634/stemcells.2006-0615

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PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：CSIRO PUBLISHING  

Web of Science

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

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.50%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.

DOI： 10.1530/rep.1.01010

Web of Science

PubMed

記述言語：英語  

PubMed

記述言語：日本語   出版者・発行元：共立出版  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2007144692

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

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT-ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT-ESCs, we established more than 150 NT-ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT-ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst-derived ESCs in terms of their expression of pluripotency markers in the presence of tissue-dependent differentially DNA methylated regions, in DNA microarray profiles, and in high-coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT-ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

DOI： 10.1634/stemcells.2005-0537

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PubMed

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

Trichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantantion period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embyros generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embyros and found that TSA treatment inhibited the preimplantation development of ICSI embyros but not ROSI or parthenogenetic embyros. And, although we often observed hypomorphism following TSA treatment in embyros grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their procedure in current Assisted Reproductive Technologies.(c) Versita Warsaw and Spring-Verlag Berlin Heidelberg. All rights reserved.

DOI： 10.2478/s11535-006-0023-5

Web of Science

記述言語：日本語  

DOI： 10.14882/jrds.99.0.150.0

J-GLOBAL

記述言語：日本語  

DOI： 10.14882/jrds.99.0.162.0

J-GLOBAL

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

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 mu m, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

DOI： 10.1095/biolreprod.105.044677

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PubMed

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

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and fullterm development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P &lt; 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P &lt; 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

DOI： 10.1095/biolreprod.105.047803

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PubMed

記述言語：日本語  

CiNii Books

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

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressmg blastocysts were as low as 50%, and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (&gt; 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.02.036

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PubMed

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

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of historic deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.11

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PubMed

記述言語：英語  

DOI： 10.1111/j.1749-0774.2005.00001.x

PubMed

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

Although round spermatid injection can be used to create progeny for males who do not produce mature sperm, the rate of successful embryogenesis after such procedures is significantly lower than that for similar procedures using mature spermatozoa. The mechanisms underlying this difference are unknown. In this study, we demonstrate that, unlike the normal paternal genome, the paternal zygotic genome derived from a round spermatid is highly remethylated before first mitosis after demethylation. Genomes from elongated spermatids exhibited an intermediate level of DNA methylation, between those of round spermatids and mature spermatozoa, suggesting that the male germ cell acquires the ability to maintain its undermethylated state in the paternal zygotic genome during this phase of spermiogenesis. In addition, treatment of zygotes with trichostatin A led to a significant reduction in DNA methylation, specifically in the spermatid-derived paternal genome, except for the pericentromeric regions enriched by trimethylation of Lys9 of histone H3. These data provide insight into epigenetic errors that may be associated with the poor development of embryos generated from immature spermatozoa. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2005.10.026

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PubMed

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

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains &lt; 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo- actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell&apos;s genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in &gt;= 3 months.

DOI： 10.1038/nprot.2006.21

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PubMed

記述言語：英語  

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

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

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.

DOI： 10.1262/jrd.17061

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PubMed

記述言語：日本語   出版者・発行元：Japanese Society of Animal Reproduction  

体細胞からのクローンマウスの成功率は低いが、ES細胞からは比較的容易に作出できる。そこで我々は体細胞核移植胚より得られた胚性幹細胞（nES細胞）を用いてクローンマウスを作成することにより、作成効率が体細胞を用いた場合より向上するか検討した。その結果、7個体のマウスの体細胞核から11匹のクローンマウスと68のnES細胞株、およびそれらのnES細胞核から41匹のクローンマウスを作出した。クローンマウスの成功率は、予想に反してnES細胞を用いた場合でも改善はみられなかったが、体細胞核移植ではクローンマウスの作出に失敗した2個体からでも、nES細胞の核移植によってクローンマウスの作出に成功した。最終的に7個体中6個体から体細胞あるいはnES細胞をドナー核として用いることでクローンマウスの作出に成功した。このように、nES細胞をドナーとしてもクローンマウスの成功率は改善されないが、重要なマウスの個体をクローン技術で維持するためには、nES細胞株も同時に樹立しておいた方が有効である。それらのnES細胞は核移植のドナーとして無限に増やすことができ、現在の核移植技術を補完するであろう。

CiNii Books

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

Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.

DOI： 10.1002/gene.20153

Web of Science

PubMed

担当区分：筆頭著者   記述言語：日本語  

DOI： 10.14882/jrds.98.0.43.0

J-GLOBAL

記述言語：日本語  

DOI： 10.14882/jrds.98.0.97.0

J-GLOBAL

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

Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F(1)mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.

DOI： 10.1095/biolreprod.104.035105

Web of Science

PubMed

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

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27degreesC); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4degreesC, RT, or 37degreesC (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4degreesC; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20degreesC. Preservation of mouse spermatozoa at VC in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4degreesC. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20degreesC for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.

DOI： 10.1095/biolreprod.104.034678

Web of Science

PubMed

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

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based. on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.

DOI： 10.1017/S0967199405003023

Web of Science

PubMed

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

Animals generated by systematic mutagenesis and routine breeding are often infertile because they lack germ cells, and maintenance of such lines of animals has been impossible. We found a hermaphrodite infertile mouse in our colony, a genetic male with an abnormal Y chromosome lacking developing germ cells. We tried to clone this mouse by conventional nuclear transfer but without success. ES cells produced from blastocysts, which had been cloned by using somatic cell nuclear transfer (ntES cells) from this mouse, were also unable to produce offspring when injected into enucleated oocytes. Although we were able to produce two chimeric offspring using these ntES cells by tetraploid complementation, they were infertile, because they also lacked developing germ cells. However, when such ntES cells were injected into normal diploid blastocysts, many chimeric offspring were produced. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome-bearing sperm. Thus, ntES cells were used to propagate offspring from infertile mice lacking germ cells.

DOI： 10.1073/pnas.0408548102

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PubMed

記述言語：英語   出版者・発行元：JAPANESE SOCIETY OF OVA RESEARCH  

The progress of nuclear transfer technology, we can create cloned animals. And recently from this same technology we can possible to establish nuclear transfer embryonic stem cells (ntES cell). From our experiments we can establish mouse ntES cells, which any kind of cell type and strain and sex. And ntES cell has very similar ability like ES cells that has capacities for in vitro differentiation and in vivo germline transmission. The ntES cell made from donor somatic cells, which are very useful for therapeutic cloning Because of no immune rejection. Especially for human, it have high expectation from this new opportunity for rejuvenation of the ageing or diseased body. However, even for cloned animals the efficiency is so low and that have many problems. So, it necessary to do more analysis of ntES cell can work normal and safe before clinic.<br>

DOI： 10.1274/jmor.22.152

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

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

To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromo some complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or lVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.

DOI： 10.1262/jrd.50.627

Web of Science

PubMed

記述言語：日本語   出版者・発行元：Japanese Society of Animal Reproduction  

マウス未受精卵はガラス化保存などで超低温保存されているが、液体窒素などを利用するため、簡単な輸送であっても極低温を維持しなければならない。そこで我々はマウス卵母細胞の4C以上の温度下で保存の可能性を検討した。未受精卵を数種類の培地で4C、27Cおよび37Cで24時間保存し、保存後の形態を光学および共焦点顕微鏡で観察した。4Cでは約70％の卵が細胞質中に水疱を形成し、単為発生刺激を与えると5％程度が胚盤胞まで発生した。27Cでは比較的正常だったが紡錘体のα-チューブリンはほとんどの卵で異常を示した。単為発生刺激後、30％程度が胚盤胞まで発生した。37Cでは形態は正常なものが多かったが細胞質の色が濃くなり、単為発生刺激を与えても胚盤胞へはまったく発生しなかった。培地による違いは見られなかったが、HYが比較的よかったため、HYで室温保存した卵にIVFあるいはICSIをしたところ、染色体の不等分離が数多く観察されたものの、わずかながら産仔を得ることができた。成績を改善するために、保存卵の紡錘体を新鮮卵の紡錘体と置換してみたが、紡錘体異常は改善されなかった。本研究により少なくともマウス未受精卵の一部を室温で24時間保存できることが明らかになった。また、発生率の低下は細胞質だけでなく核にも原因があることが示された。

CiNii Books

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

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.

DOI： 10.1017/S0967199404002965

Web of Science

PubMed

記述言語：日本語  

J-GLOBAL

担当区分：筆頭著者   記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

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

The injection of male haploid germ cells, such as spermatozoa and round spermatids, into preactivated mouse oocytes can result in the development of viable embryos and offspring. However, it is not clear how the timing of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) affects the production of offspring. We carried out ICSI and ROSI every 20 min for up to 4 h after the activation of mouse oocytes by Sr2+ and compared the late-stage development of ICSI- and ROSI-treated oocytes, including the formation of pronuclei, blastocyst formation, and offspring production. The rate of pronucleus formation (RPF) after carrying out ICSI started to decrease from &gt;95% at 100 min following oocyte activation and declined to &lt;20% by 180 min. In comparison, RPF by ROSI decreased gradually from &gt;70% between 0 and 4 h after activation. The RPFs were closely correlated with blastocyst formation. Offspring production for both ICSI and ROSI decreased significantly when injections were conducted after 100 min, a time at which activated oocytes were in the early G1 stage of the cell cycle. These results suggest that spermatozoa and round spermatids have different potentials for inducing the formation of a male pronucleus in activated oocytes, but ICSI and ROSI are both subject to the same time constraint for the efficient production of offspring, which is determined by the cell cycle of the activated oocyte.

DOI： 10.1095/biolreprod.103.025171

Web of Science

PubMed

記述言語：日本語  

CiNii Books

J-GLOBAL

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science

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

Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr2+-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.

DOI： 10.1089/153623003769645848

Web of Science

PubMed

記述言語：英語   掲載種別：（MISC）研究発表要旨（国際会議）   出版者・発行元：SOC STUDY REPRODUCTION  

Web of Science