Departmental Bulletin Paper 〈Original Papers〉ウサギ半数体ES細胞樹立に向けた雌性半数体胚の解析

堂本, 勇磨  ,  八尾, 竜馬  ,  山縣, 一夫  ,  細井, 美彦

半数体胚性幹(Embryonic stem:ES)細胞は、染色体を一組しか持たないという特徴を有している。そのため、遺伝子導入や破壊が容易であり、遺伝子型と表現型との関係を直接的に知ることができる。そこで本研究では、ウサギ半数体ES 細胞の樹立を最終的な目的として、まず、ウサギ雌性半数体胚の作製とその詳細な観察を行った。マニピュレーション操作によって2 前核(Pronuclear:PN)胚である受精卵から雄性前核の除核を行い、1 つの前核を有する胚(1PN 胚)を得た。この胚の発生過程をライブセルイメージング技術を用いて3 次元的に観察し、卵割時における1PN 胚の染色体及び紡錘体の動態の多次元画像情報を得た。これを元に、分裂期のM 期染色体の体積を比較すると、1PN 胚における核の体積が2PN 胚と比較して有意に減少していることが確認できた。次に、1PN 胚の卵割過程を解析したところ、8 細胞期以降で発生が停止することが明らかとなった。さらに、2 細胞期及び4 細胞期に要する時間を計測し、比較した結果、1PN 胚の4 細胞期に要する時間が有意に増加していることが確認された。また、4細胞期までの卵割における染色体分配異常(Abnormal chromosome segregation : ACS)の観察を行ったが、1PN 胚及び2PN 胚において、差は無かった。以上の結果から、作製された1PN 胚の各細胞期における発生遅延の蓄積が8 細胞期以降の発生に影響していると考えられる。また、ウサギ胚においてもマウス胚と同様、4 細胞期までの染色体分配の安定性が保たれていることが示唆された。Haploid embryonic stem(ES)cells have a unique feature that single set of chromosomes exist and function in them. Therefore, from the viewpoint of gene manipulation and developmental engineering, it is feasible to transfer and knockout the genes, and then, it can be liked between genotypes and phenotypes directly without making the homozygous cell lines and animals. Although the haploid ES cells were known to be derived from haploid blastocyst embryos, the procedures and developmental characters of haploid embryos have been unclear. In this study, we produced and analyzed female haploid embryos of rabbit, towards to establish the haploid rabbit ES cells in future. Male pronucleus was removed from fertilized rabbit oocytes by piezo-driven micromanipulator and embryo with one pronucleus(1PN)was obtained. By means of long-term three-dimensional live-cell imaging technology we previously developed, time-dependent changes in chromosome and spindle structure during cleavage were monitored in the rabbit 1PN embryos. When comparing volume of metaphase chromosomes during first mitosis, it was apparent that the volume in 1PN embryos was significantly smaller than that of fertilized eggs with two pronuclei(2PN), suggesting the validity of procedures to make the haploid embryo. Expectedly, the cleavage of the 1PN embryos was retarded and arrested at the 8-cell stage or later. Furthermore, the time durations at 2- and 4-cell stages of 1PN embryos were measured. As a result, the time required for 4-cell stage of 1PN embryos was significantly increased when compared with that of 2PN embryos. In addition, when the chromosomal integrity during the each cleavage was assessed, the occurrence of abnormal chromosomal segregation, such as lagging chromosome and misaligned chromosome, was comparable among these two groups of embryos and also that of mouse in vitro fertilized embryo, previously reported by us. These results demonstrated that the accumulation of slight delays in each stage may result in lower rate of development in 1PN rabbit embryos, and that the chromosome integrity during the segregation seem to be sustained also in rabbit embryo likely to mouse regardless of its karyotype.

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