Crane Richard F, Ruderman Joan V
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2006;322:435-43. doi: 10.1007/978-1-59745-000-3_31.
Xenopus oocytes are naturally arrested at G2/M in prophase I of meiosis. Stimulation with progesterone initiates a nontranscriptional signaling pathway that culminates in the activation of Cdc2/cyclin B and reentry into meiosis. This pathway presents a paradigm for nongenomic signaling by steroid hormones and for the G2/M cell cycle transition. It has been extensively studied using intact oocytes, which are amenable to microinjection and biochemical analyses described elsewhere in this book. However, there are several experimental advantages in using in vitro systems consisting of cytosolic fractions of prophase-arrested oocytes. Because of their homogeneous nature, extracts avoid the difficulties of signaling asynchrony between individual oocytes. They are also amenable to biochemical manipulations such as protein immunodepletions, and proteins and pharmacological agents can be added easily. Despite these features, oocyte extracts have yet to achieve the widespread utility of Xenopus egg extracts, which can proceed through rounds of deoxyribonucleic acid (DNA) replication and mitosis in vitro. Here, we review the historical development of oocyte extracts and discuss the factors most crucial to success in reproducing the signaling pathway and the G2/M transition in vitro.
非洲爪蟾卵母细胞在减数分裂前期I自然停滞于G2/M期。用孕酮刺激可启动一条非转录信号通路，最终导致Cdc2/细胞周期蛋白B激活并重新进入减数分裂。该通路为类固醇激素的非基因组信号传导以及G2/M细胞周期转变提供了一个范例。人们已经使用完整的卵母细胞对其进行了广泛研究，完整卵母细胞适用于本书其他地方所述的显微注射和生化分析。然而，使用由减数分裂前期停滞的卵母细胞的胞质部分组成的体外系统有几个实验优势。由于其性质均一，提取物避免了单个卵母细胞之间信号异步的困难。它们也适用于生化操作，如蛋白质免疫去除，并且可以轻松添加蛋白质和药物试剂。尽管有这些特点，卵母细胞提取物尚未达到非洲爪蟾卵提取物那样广泛的应用，后者可以在体外进行多轮脱氧核糖核酸（DNA）复制和有丝分裂。在这里，我们回顾了卵母细胞提取物的历史发展，并讨论了在体外重现信号通路和G2/M转变成功的最关键因素。
