College of Veterinary Medicine, Northwest A&F University, Shaanxi Stem Cell Engineering and Technology Center, Yangling, China.
Theriogenology. 2012 Oct 1;78(6):1171-81.e1. doi: 10.1016/j.theriogenology.2012.06.006. Epub 2012 Aug 15.
During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.
在哺乳动物卵母细胞成熟过程中，需要进行两次连续的减数分裂才能形成单倍体配子。对于每一次减数分裂，卵母细胞必须从中期转移到后期，但成熟促进因子（细胞周期蛋白依赖性激酶 1/细胞周期蛋白 B1）的活性会使卵母细胞保持在中期。因此，需要使成熟促进因子失活才能完成过渡并完成这两次分裂；这是通过细胞周期蛋白 B1 的后期促进复合物/细胞周期蛋白依赖性降解来提供的。本研究的目的是通过微注射其互补 RNA（cRNA）来检查牛卵母细胞中全长细胞周期蛋白 B1 和不可降解的 N 端 87 个氨基酸缺失的表达对减数分裂的影响，同时结合荧光素 Venus。全长细胞周期蛋白 B1-Venus 的过表达抑制了成熟卵母细胞中的同源体分离和第一极体形成（对照 70%对过表达 16%；P<0.05）。然而，在相同的表达水平下，它并没有阻止第二次减数分裂中期和孤雌激活后卵的分裂（对照：82%原核和 79%分裂；过表达：91%原核和 89%分裂）。全长细胞周期蛋白 B1 和不可降解的 N 端 87 个氨基酸缺失导致两次减数分裂都出现中期阻滞，而着丝粒蛋白的降解不受影响。Roscovitine 是一种有效的细胞周期蛋白依赖性激酶 1（CDK1）抑制剂，在 140μM 时可克服成熟卵母细胞中的这种中期阻滞，但需要更高的剂量（200μM）才能克服卵母细胞中的阻滞。总之，由于不可降解的细胞周期蛋白 B1 阻断的第一次减数分裂中期（MI）与第二次减数分裂中期（MII）在对触发 CDK1 失活的不同敏感性方面存在差异，我们得出结论，MI 阻滞的机制与 MII 阻滞不同。
