Prasad Shilpa, Koch Biplob, Chaube Shail K
Cell Physiology Laboratory, Biochemistry Unit, Department of Zoology, Banaras Hindu University, Varanasi 221005, UP, India.
Genotoxicology and Cancer Biology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, UP, India.
Biomed Pharmacother. 2016 Mar;78:216-225. doi: 10.1016/j.biopha.2016.01.013. Epub 2016 Feb 2.
Postovulatory aging-mediated spontaneous exit from metaphase-II (M-II) arrest deteriorates egg quality and limits assisted reproductive technologies outcome (ART) outcome. Present study was aimed to find out whether RO-3306, specific cyclin dependent kinase 1 (Cdk1) inhibitor could protect against postovulatory aging-mediated spontaneous exit from M-II arrest in rat eggs cultured in vitro.
Freshly ovulated M-II arrested eggs were exposed to various concentrations of RO-3306 for 3h in vitro. The morphological changes, percentage of spontaneous exit from M-II arrest, total and specific phosphorylation status of Cdk1, cyclin B1 level and Cdk1 activity were analyzed.
Data suggest that RO-3306 protected postovulatory aging-mediated spontaneous exit from M-II arrest in a concentration-dependent manner. Postovulatory aging increased Thr14/Tyr15 phosphorylated Cdk1 level, decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels and increased Cdk1 activity in aged eggs cultured in vitro. On the other hand, RO-3306 protected postovulatory aging-induced changes in specific phosphorylation of Cdk1, cyclin B1 level, inhibited the kinase activity and prevented spontaneous exit from M-II arrest.
Our results suggest that postovulatory aging destabilizes MPF by modulating specific phosphorylation of Cdk1 and cyclin B1 level. RO-3306 prevented these changes and maintained M-II arrest in rat eggs cultured in vitro. Hence, maintenance of M-II arrest in ovulated eggs using RO-3306 could be beneficial to increase the number of eggs available for various ART programs.
排卵后老化介导的从中期 II（M-II）阻滞中自发脱离会使卵子质量下降，并限制辅助生殖技术（ART）的结果。本研究旨在探究特异性细胞周期蛋白依赖性激酶 1（Cdk1）抑制剂 RO-3306 是否能防止体外培养的大鼠卵子因排卵后老化而从中期 II 阻滞中自发脱离。
将刚排出的处于 M-II 阻滞期的卵子在体外暴露于不同浓度的 RO-3306 中 3 小时。分析其形态变化、从中期 II 阻滞中自发脱离的百分比、Cdk1 的总磷酸化和特异性磷酸化状态、细胞周期蛋白 B1 水平以及 Cdk1 活性。
数据表明，RO-3306 以浓度依赖性方式防止排卵后老化介导的从中期 II 阻滞中自发脱离。在体外培养的老化卵子中，排卵后老化增加了苏氨酸 14/酪氨酸 15 磷酸化的 Cdk1 水平，降低了苏氨酸 161 磷酸化的 Cdk1 以及细胞周期蛋白 B1 水平，并增加了 Cdk1 活性。另一方面，RO-3306 保护了排卵后老化诱导的 Cdk1 特异性磷酸化、细胞周期蛋白 B1 水平的变化，抑制了激酶活性，并防止从中期 II 阻滞中自发脱离。
我们的结果表明，排卵后老化通过调节 Cdk1 的特异性磷酸化和细胞周期蛋白 B1 水平使成熟促进因子（MPF）不稳定。RO-3306 阻止了这些变化，并维持了体外培养的大鼠卵子的中期 II 阻滞。因此，使用 RO-3306 维持排卵卵子的中期 II 阻滞可能有利于增加可用于各种 ART 方案的卵子数量。
