Ohsumi Keita, Yamamoto Tomomi M, Iwabuchi Mari
Laboratory of Cell and Developmental Biology, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Methods Mol Biol. 2006;322:445-58. doi: 10.1007/978-1-59745-000-3_32.
In meiotic cell cycles, meiosis I (MI) is followed by meiosis II (MII) without an intervening S phase, whereas in mitotic cell cycles, an S phase necessarily alternates with an M phase. For the study of mitotic cell cycles, extracts prepared from unfertilized and parthenogenetically activated Xenopus eggs have been very useful as they can perform the progression of mitotic cycles in vitro. To establish a cell-free system to study the regulatory mechanisms of meiotic transition from MI to MII, extracts have been prepared from maturing Xenopus oocytes isolated from ovaries, stimulated with progesterone to induce the resumption of meiosis, and arrested at meiotic metaphase I by cold treatment. In oocyte extracts, the activity of cyclin B-Cdc2 complexes, the M phase inducer, fluctuates in the same manner as it does in maturing oocytes during the MI to MII transition period. By the use of oocyte extracts, it has been found that incomplete inactivation of Cdc2 at the end of MI is required for meiotic M-M transition. The meiotic extract should provide a useful tool to elucidate molecular mechanisms of meiotic M to M transition, including a role of Mos/mitogen-activated protein kinase cascade in the suppression of S phase entry after MI exit. In this chapter, we describe methods for the preparation and the uses of meiotic extracts. As a comparison, we also include a protocol for the preparation of mitotic extracts.
在减数分裂细胞周期中，减数分裂I（MI）之后紧接着是减数分裂II（MII），中间没有插入的S期，而在有丝分裂细胞周期中，S期必然与M期交替出现。对于有丝分裂细胞周期的研究，从未受精和孤雌生殖激活的非洲爪蟾卵母细胞中制备的提取物非常有用，因为它们能够在体外进行有丝分裂周期的进程。为了建立一个无细胞系统来研究从MI到MII的减数分裂转变的调控机制，已经从从卵巢中分离出的成熟非洲爪蟾卵母细胞制备了提取物，用孕酮刺激以诱导减数分裂的恢复，并通过冷处理使其停滞在减数分裂中期I。在卵母细胞提取物中，M期诱导剂细胞周期蛋白B-Cdc2复合物的活性在MI到MII转变期间与成熟卵母细胞中的波动方式相同。通过使用卵母细胞提取物，已经发现MI末期Cdc2的不完全失活是减数分裂M-M转变所必需的。减数分裂提取物应该为阐明减数分裂M到M转变的分子机制提供一个有用的工具，包括Mos/丝裂原活化蛋白激酶级联在MI退出后抑制S期进入中的作用。在本章中，我们描述了减数分裂提取物的制备方法和用途。作为比较，我们还包括了有丝分裂提取物的制备方案。
