Lin Tao, Diao Yun Fei, Kang Jung Won, Lee Jae Eun, Kim Dong Kyo, Jin Dong Il
Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
PLoS One. 2013 Dec 3;8(12):e82766. doi: 10.1371/journal.pone.0082766. eCollection 2013.
To determine whether chromosomes in the porcine first polar body (PB1) can complete the second meiotic division and subsequently undergo normal pre-implantation embryonic development, we examined the developmental competence of PB1 chromosomes injected into enucleated MII stage oocytes by nuclear transfer method (chromosome replacement group, CR group). After parthenogenetic activation (PA) or in vitro fertilization (IVF), the cleavage rate of reconstructed oocytes in the IVF group (CR-IVF group, 36.4 ± 3.2%) and PA group (CR-PA group, 50.8 ± 4.2%) were significantly lower than that of control groups in which normal MII oocytes were subjected to IVF (MII-IVF group, 75.8 ± 1.5%) and PA (MII-PA group, 86.9 ± 3.7%). Unfertilized rates was significantly higher in the CR-IVF group (48.6 ± 3.3%) than in the MII-IVF group (13.1 ± 3.4%). The blastocyst formation rate was 8.3 ± 1.9% in the CR-PA group, whereas no blastocyst formation was observed in the CR-IVF group. To produce tetraploid parthenogenetic embryos, intact MII stage oocytes injected with PB1 chromosomes were electrically stimulated, treated with 7.5 μg/mL cytochalasin B for 3 h (MII oocyte + PB1 + CB group), and then cultured without cytochalasin B. The average cleavage rate of reconstructed oocytes was 72.5% (48 of 66), and the blastocyst formation rate was 18.7% (9 of 48). Chromosome analysis showed similar proportions of haploid and diploid cells in the control (normal MII oocytes) and CR groups after PA; overall, 23.6% of blastocysts were tetraploid in the MII oocyte + PB1 + CB group. These results demonstrate that chromosomes in PB1 can participate in normal pre-implantation embryonic development when injected into enucleated MII stage oocytes, and that tetraploid PA blastocysts are produced (although at a low proportion) when PB1 chromosomes are injected into intact MII stage oocytes.
为了确定猪第一极体(PB1)中的染色体能否完成第二次减数分裂并随后经历正常的植入前胚胎发育,我们通过核移植方法检测了注入去核MII期卵母细胞中的PB1染色体的发育能力(染色体替代组,CR组)。孤雌激活(PA)或体外受精(IVF)后,IVF组(CR-IVF组,36.4±3.2%)和PA组(CR-PA组,50.8±4.2%)重构卵母细胞的分裂率显著低于正常MII期卵母细胞进行IVF的对照组(MII-IVF组,75.8±1.5%)和PA对照组(MII-PA组,86.9±3.7%)。CR-IVF组(48.6±3.3%)的未受精率显著高于MII-IVF组(13.1±3.4%)。CR-PA组的囊胚形成率为8.3±1.9%,而CR-IVF组未观察到囊胚形成。为了产生四倍体孤雌胚胎,对注入PB1染色体的完整MII期卵母细胞进行电刺激,用7.5μg/mL细胞松弛素B处理3小时(MII卵母细胞+PB1+CB组),然后在无细胞松弛素B的条件下培养。重构卵母细胞的平均分裂率为72.5%(66个中的48个),囊胚形成率为18.7%(48个中的9个)。染色体分析显示,PA后对照组(正常MII期卵母细胞)和CR组中,单倍体细胞和二倍体细胞的比例相似;总体而言,MII卵母细胞+PB1+CB组中23.6%的囊胚为四倍体。这些结果表明,将PB1中的染色体注入去核MII期卵母细胞时,其能够参与正常的植入前胚胎发育,并且当将PB1染色体注入完整的MII期卵母细胞时,能够产生四倍体PA囊胚(尽管比例较低)。