Alberio R, Kubelka M, Zakhartchenko V, Hajdúch M, Wolf E, Motlik J
Department of Molecular Animal Breeding and Genetics, University of Munich, Oberschleissheim, Germany.
Mol Reprod Dev. 2000 Apr;55(4):422-32. doi: 10.1002/(SICI)1098-2795(200004)55:4<422::AID-MRD10>3.0.CO;2-C.
Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose-dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca(2+) influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine-activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine-supplemented medium (6-7 hr post-activation; hpa). A high synchrony in the S-phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine-supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine-activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin-bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6-8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation.
通过紧密模拟正常受精并能获得单倍体卵母细胞的实验程序激活牛卵母细胞，对于胞浆内单精子注射（ICSI）和核移植均至关重要。因此，为了产生单倍体激活卵母细胞，本研究评估了波希明单独或与离子霉素联合使用时，是否能够激活年轻的成熟牛卵母细胞。此外，还研究了波希明对原核形成后DNA合成模式的影响，以及激活过程中组蛋白H1激酶和丝裂原活化蛋白激酶（MAP激酶）活性的变化。我们关于波希明的研究结果表明，特异性抑制中期II牛卵母细胞中的细胞周期蛋白依赖性激酶（CDK）可诱导孤雌激活，且呈剂量依赖性（分别为25、50和100微摩尔），单独使用时激活率分别为3%、30%和50%，与离子霉素联合使用时激活率分别为30%、70%和87.5%。当在波希明存在的情况下刺激Ca(2+)内流时，观察到单个原核和第二极体的排出（97%），不过单独使用波希明时观察到了无极体排出的原核形成。波希明激活的卵母细胞在从添加波希明的培养基中取出后的第一小时开始合成DNA（37%）（激活后6 - 7小时；hpa）。在激活后8小时，超过85%的孤雌胚积极合成DNA，在S期出现了高度同步。相比之下，在波希明存在的情况下培养4、6和8小时的卵母细胞中未检测到DNA合成，而在添加波希明的培养基中培养18小时的卵母细胞中观察到较低的合成率（30%）。这证实了该抑制剂能够使细胞周期在G1/S边界停滞至少8小时。在波希明激活的卵母细胞中观察到组蛋白H1激酶活性下降。髓鞘碱性蛋白激酶（MBP激酶）的活性下降晚于组蛋白H1激酶，甚至在离子霉素 - 波希明处理后4小时，仍可检测到至少一半的该活性。然后，MBP激酶活性下降，在激活后6 - 8小时可见最低水平。总之，我们的研究表明，体外成熟的牛卵母细胞可被CDK的合成抑制剂成功激活。与离子霉素联合使用可增强这种效果。以这种方式靶向CDK来激活牛卵母细胞可能是提高哺乳动物卵母细胞激活效率的一种方法。
