亮甲酚蓝染色法筛选卵母细胞对牛体细胞克隆胚体外发育的影响

Effect of BCB Selecting Oocytes Staining on the Developmental Competence of Bovine SCNT Embryos

为探索用亮甲酚蓝染色法筛选卵母细胞是否有利于牛体细胞克隆胚的体外发育,将采集的卵母细胞分成3组:对照组,采集后的卵母细胞直接放入成熟液培养,不用亮甲酚蓝处理;控制对照组,卵母细胞于PBS中放置90 min后放入成熟液中培养;试验组,将采集的卵母细胞在含有26μmol/L亮甲酚蓝的PBS中放置90 min。然后将处理过的卵母细胞根据着色情况分为两组,即BCB+(胞质呈蓝色,成熟卵母细胞)和BCB-(胞质未着色,生长期卵母细胞)。各组卵母细胞24 h体外成熟培养,统计各组卵母细胞成熟率。分别用成熟后的各组卵母细胞用于细胞核移植,统计体细胞核移植的卵裂数、囊胚数和囊胚细胞数。结果表明,BCB+组卵母细胞的成熟率明显高于BCB-组卵母细胞的成熟率(74.0%,53.4%),差异显著(P<0.05)。BCB+组的囊胚率(45.0%)与对照组、控制对照组和BCB-组的囊胚率(34.9%,36.4%和5.2%)相比差异显著(P<0.05)。另外BCB+组的囊胚细胞总数(114.5±7.5)较其他组差异显著(P<0.05)。表明通过亮甲酚蓝染色法筛选出成熟的高质量牛卵母细胞用于体细胞核移植,可以获得更高的克隆囊胚率。

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. Oocytes were divided into three groups： control （immediately in vitro maturation culture）; holding-control （kept in D-PBS for 90 min before culture） ; and experiment （incubated with 26 mmol/L BCB）. Treated oocytes were then divided into BCB-（colourless cytoplasm, increased G6PDH） and BCB＋ （coloured cytoplasm, low G6PDH）. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured to determine the cleavage rate and blastocyst rate. The percentages of maturation to metaphase II in BCB＋ group oocytes were higher than those of BCB- group oocytes （74. 0% versus 53.4%, P 〈0.05）. The BCB＋ oocytes yielded a significantly higher blastocyst rate （45.0%） than that in control （34.9%）, holding-control （36.4%） and BCB- oocytes （5.2%）. The BCB＋oocytes yielded a significantly higher number of nuclei in blastocysts （ 114. 5 ± 7. 5） than that in other groups. These results showed that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer.