Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes
Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes
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DOI：
10.1530/rep.1.00431
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发表时间：
2005-02-01
期刊：
影响因子：
3.8
通讯作者：
Hochi, S
中科院分区：
文献类型：
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作者：
Ito, J;Hirabayashi, M;Hochi, S
本研究旨在阐明体外衰老或去核大鼠卵母细胞p34（cdc2）激酶活性与微注射卵丘细胞核过早染色体凝聚（PCC）的关系。Wistar大鼠卵母细胞在动物死亡后放置体外120分钟。卵母细胞p34（cdc2）激酶活性呈时间依赖性下降。完整卵母细胞核注射完成时间为15 ~ 45 min， PCC的发生率高于46 ~ 120 min。当大鼠卵母细胞去核后进行核注射时，p34（cdc2）激酶活性在去核后不久短暂升高，但在30分钟后急剧下降。即使在60分钟后，去除细胞质而不是中期板也不影响p34（cdc2）激酶的活性。PCC发生在完整和去细胞质的卵母细胞中，而不在去核卵母细胞中。相比之下，BDF1小鼠卵母细胞的p34（cdc2）激酶水平是大鼠卵母细胞的两倍，即使去核也能支持PCC。完整大鼠卵母细胞的p34（cdc2）激酶水平通过45分钟的罗斯科维汀（p34（cdc2）激酶抑制剂）处理降低到与衰老（120分钟）或去核（+ 60分钟）卵母细胞相当的水平。罗斯科维汀处理的卵母细胞没有支持PCC，而一半的对照卵母细胞支持PCC。当用蛋白酶体抑制剂MG132处理大鼠卵母细胞时，在MG132处理的卵母细胞中观察到p34（cdc2）激酶的延迟失活。与对照卵母细胞相比，mg132处理的卵母细胞支持PCC的比例明显更高。此外，mg132处理和去核的卵母细胞在丘细胞注射后携带两个伪原核的比例高于去核的卵母细胞在末期ii期发育到两细胞期。这些结果表明，衰老或去核大鼠卵母细胞中p34（cdc2）激酶活性水平的降低是它们无法支持微注射供体细胞核的PCC的原因，而化学物质如MG132抑制p34（cdc2）激酶失活在一定程度上对大鼠卵母细胞促进PCC和进一步发育有效。
The present study was undertaken to clarify the relationship between the p34(cdc2) kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34(cdc2) kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15-45 min rather than 46-120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34(cdc2) kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the metaphase-plate did not affect the p34(cdc2) kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34(cdc2) kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34(cdc2) kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+ 60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34(cdc2) kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34(cdc2) kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34(cdc2) kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34(cdc2) kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development.