Meng Qinggang, Bai Chunling, Liu Ying, Wu Xia, Bunch Thomas D, Li Guang-Peng
The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, People's Republic of China.
Cell Reprogram. 2010 Aug;12(4):481-90. doi: 10.1089/cell.2009.0114.
This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MII spindle, 94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MII spindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9%) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs. 23.1%; 27.5 vs. 67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n, 2n/8n, and 2n/4n/8n. (6) Nuclear transfer preceding the oocyte enucleation experiment indicated that prolonged presence of oocyte nuclei induced abnormal DNA configuration and reduced in vitro development of transferred somatic nuclei, but short time presence of oocyte nuclei did not affect the in vitro development of cloned embryos. We conclude that oocyte MII spindles induce donor cell PCC, the developmental capacity of cloned embryos reconstituted with nonenucleated oocytes is inferior to those with enucleated oocytes, and that all such derived blastocysts are polyploidy.
本研究旨在探讨卵母细胞核的存在对供体细胞的核重塑（包括早熟染色体凝集（PCC）和DNA构型）以及后续胚胎发育的影响。结果表明：（1）卵母细胞MII纺锤体的存在更易诱导供体细胞出现PCC。（2）融合后的供体细胞与卵母细胞中期纺锤体的位置关系对卵母细胞第二极体（PB2）的排出有影响。当融合后的供体细胞与MII纺锤体相距较远时，94.4%的重构卵母细胞排出PB2。当供体细胞与MII纺锤体相邻融合时，几乎所有重构卵母细胞都不排出PB2；大多数（67.9%）形成一个非常大的M期纺锤体，其中卵母细胞和供体细胞的染色体融合在一起。（3）激活后，卵母细胞核和供体细胞核呈现出多种原核模式且发育不同步。（4）用未去核卵母细胞重构的胚胎与用去核卵母细胞重构的对照胚胎的卵裂率相似。未去核和去核克隆胚胎的囊胚发育率无差异；然而，与去核对照组相比，未去核组从早期囊胚到孵化囊胚的发育率显著降低（0%对23.1%；27.5%对67.8%），无论供体细胞是卵丘细胞还是成纤维细胞。（5）所有未去核卵母细胞来源的囊胚都含有多种组成的混合多倍体，包括2n/4n、2n/6n、2n/8n和2n/4n/8n。（6）卵母细胞去核实验之前的核移植表明，卵母细胞核的长时间存在会诱导异常的DNA构型并降低移植体细胞核的体外发育能力，但卵母细胞核的短时间存在并不影响克隆胚胎的体外发育。我们得出结论，卵母细胞MII纺锤体诱导供体细胞PCC，用未去核卵母细胞重构的克隆胚胎的发育能力低于用去核卵母细胞重构的胚胎，且所有此类来源的囊胚都是多倍体。
