Graduate School of Science, Kobe University, Nada, Kobe, Japan.
Methods. 2010 May;51(1):177-82. doi: 10.1016/j.ymeth.2010.01.008. Epub 2010 Jan 15.
Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.
卵激活/受精过程中的细胞内信号转导已经通过完整的卵进行了广泛的研究，可以通过微注射不同的 mRNA、蛋白质或化学药物来操纵这些卵。此外，还开发了保留高 CSF 活性（CSF 停滞提取物）的卵提取物，用于研究受精/激活信号转导，该系统作为模型系统具有显著优势。向 CSF 停滞提取物中添加钙会引发许多在卵激活过程中发生的信号事件。因此，钙动员下游的信号转导已在卵提取物中成功研究。此外，尽管在提取物制备过程中破坏了膜相关信号区室和有序的区室化，但 CSF 停滞提取物仍可成功用于研究卵激活/受精过程中钙释放之前发生的早期信号事件。与 CSF 停滞提取物结合使用，激活的卵筏可以复制卵激活的一些事件，包括 PLCγ 激活、IP3 产生、瞬时钙释放、MAPK 失活和减数分裂退出。这是由于筏中存在的精子诱导的卵激活信号机制与提取物中定位的信号转导系统组件互补所致。在此，我们描述了使用从中期阻滞的爪蟾卵中制备的无细胞提取物和膜筏来研究卵受精/激活的分子机制的方案。
