Bai Chunling, Liu Hui, Liu Ying, Wu Xia, Cheng Lei, Bou Shorgan, Li Guang-Peng
The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University , Hohhot 010021, People's Republic of China.
Cell Reprogram. 2011 Feb;13(1):37-45. doi: 10.1089/cell.2010.0038. Epub 2011 Jan 14.
Tetraploid embryos are a useful model for postimplantation development of polyploidy cells, and tetraploid cells are an advantage in studies for chimeras yielding offspring completely derived from embryo stem cells or induced pluripotent cells. This study was designed to investigate the effects of cytochalasin B (CB) on bovine oocyte meiosis, and to induce the formation of diploid oocytes and tetraploid embryos. The results showed that: (1) incubation of oocytes in CB at ≥2.0 μg/mL concentrations for 24 h significantly decreased oocyte maturation and the matured oocytes' haploid composition. Over 50% of the CB-treated oocytes did not expel PB1 (non-PB1), and most of the non-PB1 oocytes contained 2n (60) chromosomes. (2) Pretreatment of oocytes with CB at concentrations of 7.5 and 15 μg/mL for 10 h significantly decreased oocyte maturation. Posttreatment of oocytes with CB resulted in most of the oocytes containing 2n chromosomes. (3) The parthenogenetic blastocysts (25-28%) derived from the non-PB1 oocytes of posttreatment group was significantly higher than that from pretreatment, whole period treatment, and the control oocytes (12-16%). (4) Cytogenetic analysis of the embryos derived from CB-treated non-PB1 oocytes resulted in 74% of the one-cell stage embryos being 4n = 120 chromosomes, 82% of two-cell stage embryos contained 4n chromosomes in each blastomere, and 75% of the blastocysts were tetraploidy (4n = 120). (6) The stopped uncleaved one-cell embryos showed an amazing phenomenon of over 15% of them containing extra chromosomes, which suggested multiple DNA duplication occurred within 40 h after activation. In conclusion, CB inhibits PB1 extrusion, disfigures spindle structure, decreases oocyte maturation, and results in formation of diploid (2n or 4c) oocytes. The diploid oocytes resulted in a higher development of tetraploid embryos, which would be a unique approach for the production of tetraploid embryos in bovine.
四倍体胚胎是多倍体细胞植入后发育的有用模型，四倍体细胞在嵌合体研究中具有优势，可产生完全源自胚胎干细胞或诱导多能干细胞的后代。本研究旨在探讨细胞松弛素B（CB）对牛卵母细胞减数分裂的影响，并诱导二倍体卵母细胞和四倍体胚胎的形成。结果表明：（1）将卵母细胞在浓度≥2.0μg/mL的CB中孵育24小时，显著降低了卵母细胞成熟度和成熟卵母细胞的单倍体组成。超过50%经CB处理的卵母细胞未排出第一极体（非PB1），且大多数非PB1卵母细胞含有2n（60）条染色体。（2）用浓度为7.5和15μg/mL的CB预处理卵母细胞10小时，显著降低了卵母细胞成熟度。用CB对卵母细胞进行后处理，导致大多数卵母细胞含有2n条染色体。（3）后处理组非PB1卵母细胞来源的孤雌胚泡（25 - 28%）显著高于预处理组、全期处理组和对照卵母细胞（12 - 16%）。（4）对经CB处理的非PB1卵母细胞来源的胚胎进行细胞遗传学分析，结果显示74%的单细胞期胚胎为4n = 120条染色体，82%的二细胞期胚胎每个卵裂球含有4n条染色体，75%的胚泡为四倍体（4n = 120）。（6）停滞未分裂的单细胞胚胎出现了惊人的现象，超过15%的胚胎含有额外染色体，这表明在激活后40小时内发生了多次DNA复制。总之，CB抑制PB1排出，破坏纺锤体结构，降低卵母细胞成熟度，并导致二倍体（2n或4c）卵母细胞的形成。二倍体卵母细胞导致四倍体胚胎的发育率更高，这将是牛四倍体胚胎生产的独特方法。
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