FREE CALCIUM LEVELS DURING FERTILIZATION AND EARLY DVMT IN WILD TYPE C ELEGANS

受精的标志之一是体内的 新受精卵母细胞的游离钙水平。这 在中国的体外受精过程中发现了钙“尖峰” 一些生物体。我们感兴趣的是线虫感染是否 线虫卵母细胞在受精时也表现出类似的钙峰。 与其他生物不同，线虫的卵母细胞不能从 卵巢并保持存活，因此采用体外培养技术 不能受精。我们计划绕过这个问题 通过成像受精和子宫内早期发育。要检测 钙瞬变我们将注入钙指示剂染料[钙绿 葡聚糖(I OK)和/或钙深红色葡聚糖(I OK)，分子探针， Inc.，Eugene，OR]直接导入线虫Ob细胞。带着虫子 注射的卵母细胞随后将在0。1%三卡因 和0.01%的四咪唑来固定它们以供成像之用。 Ile麻醉剂不影响输卵管肌肉组织，因此允许 受精的卵母细胞从卵巢进入子宫。这个 MM Will研发的全固态多光子成像系统 用于对时间流逝的卵母细胞进行成像。系统已配置 以同时收集两种荧光的记录图像 信号和传输的红外亮场图像。我们将观察到 卵母细胞成熟过程中钙水平的变化 在受精之前，当卵母细胞驻留在卵巢中时， 在受精过程中，当卵母细胞通过 受精囊和早期发育(减数分裂和有丝分裂) 子宫内的胚胎。

One of the hallmarks of fertilization is a transient increase in the levels of free calcium in the newly fertilized oocyte. This calcium "spike" has been demonstrated during in vitro fertilization in a number of organisms. We are interested in whether Caenorhabditis elegans oocytes also exhibit a similar calcium spike at fertilization. Unlike other organisms, the C elegans oocyte can not be removed from the ovary and remain viable, therefore the techniques of in vitro fertilization are not available. We plan to circumvent this problem by imaging fertilization and early development in utero. To detect calcium transients we will inject calcium indicator dyes [Ca-green dextran (I OK) and/or Ca-crimson dextran (I OK), Molecular Probes, Inc., Eugene, OR] directly into C elegans obcytes. The worms with injected oocytes will be subsequently anesthetized in 0. 1% tricaine and 0.01 % tetramisole to immobilize them for the purpose of imaging. Ile anesthetic does not affect oviduct musculature, thus allowing fertilized oocytes to pass from the ovary to the uterus. The all-solid-state multi-photon imaging system developed at the MM will be used to image the oocytes in timelapse. The system is configured to collect simultaneously recorded images of both the fluorescence signal and the transmitted IR brightfield image. We will observe the changes in calcium levels in oocytes during the process of maturation prior to fertilization while the oocyte is resident in the ovary, during the process of fertilization as the oocyte passes through the spermatheca and during early development (meiosis and mitosis) of the embryo in the uterus.