Nanassy L, Lee K, Javor A, Machaty Z
Department of Animal Breeding Science, Center of Agricultural Sciences, University of Debrecen, Böszörményi Street 138, Debrecen, Hungary.
Theriogenology. 2007 Jul 15;68(2):146-52. doi: 10.1016/j.theriogenology.2007.04.044. Epub 2007 May 23.
The effect of different oocyte activation methods on the dynamics of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity in porcine oocytes were examined. Three activativation methods were tested: (1) electroporation (EP); (2) electroporation combined with butyrolactone I (BL), an inhibitor of cdc2 and cdk2 kinases; (3) electroporation followed by a treatment with cycloheximide (CHX), a protein synthesis blocker. The activity of cdc2 in MII oocytes was 0.067+/-0.011pmol/oocyte/min (mean+/-S.E.M.), which by 1h decreased in every treatment group (P<0.05) and stayed at low levels until 6h post-activation, approximately the time of pronuclear formation. The initial MAPK activity (0.123+/-0.017pmol/oocyte/min) also decreased 1h after each type of activation treatment (P<0.005). However, in the electroporation only group, activity reached its lowest level at 3h; thereafter, it started to recover and at later time points, MAPK activity did not differ from that in non-treated oocytes (P>0.1). In contrast, oocytes where electroporation was followed by protein kinase or protein synthesis inhibition had low MAPK activity by the time pronuclei were to be formed. Pronuclear formation in these groups (86.3+/-3.3% for EP+BL and 87.6+/-3.7% for EP+CHX) was higher compared to that found in the EP-only oocytes (69.4+/-3.3%; P<0.05). These findings demonstrated that electroporation alone efficiently triggered the inactivation of MPF but not that of MAPK. In order to achieve low MAPK activity to allow high frequency of pronuclear formation, electroporation should be followed by a treatment that inhibits protein synthesis or specific protein kinases. The combined activation methods provided stimuli that efficiently induced both MPF and MAPK inactivation and triggered pronuclear formation with high frequencies.
研究了不同的卵母细胞激活方法对猪卵母细胞中M期促进因子（MPF）和丝裂原活化蛋白激酶（MAPK）活性动态变化的影响。测试了三种激活方法：（1）电穿孔（EP）；（2）电穿孔联合丁内酯I（BL），一种cdc2和cdk2激酶的抑制剂；（3）电穿孔后用蛋白质合成阻滞剂环己酰亚胺（CHX）处理。MII期卵母细胞中cdc2的活性为0.067±0.011pmol/卵母细胞/分钟（平均值±标准误），在每个处理组中，1小时后均下降（P<0.05），并在激活后6小时一直保持在低水平，这大约是原核形成的时间。每种激活处理1小时后，初始MAPK活性（0.123±0.017pmol/卵母细胞/分钟）也下降（P<0.005）。然而，仅电穿孔组的活性在3小时达到最低水平；此后，它开始恢复，在随后的时间点，MAPK活性与未处理的卵母细胞无差异（P>0.1）。相反，在原核形成时，电穿孔后进行蛋白激酶或蛋白质合成抑制的卵母细胞具有较低的MAPK活性。与仅电穿孔的卵母细胞（69.4±3.3%）相比，这些组中的原核形成率更高（EP+BL组为86.3±3.3%，EP+CHX组为87.6±3.7%；P<0.05）。这些发现表明，单独的电穿孔能有效触发MPF的失活，但不能触发MAPK的失活。为了实现低MAPK活性以允许高频率的原核形成，电穿孔后应进行抑制蛋白质合成或特定蛋白激酶的处理。联合激活方法提供了能有效诱导MPF和MAPK失活并以高频率触发原核形成的刺激。
