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

受精的标志之一是 新受精卵中游离钙的水平。 这 体外受精过程中已证明钙“峰值”， 一些有机体。 我们感兴趣的是隐杆线虫 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，线虫卵母细胞不能从 卵巢并保持活力，因此体外技术 施肥是不可能的。 我们计划规避这个问题 通过成像受精和子宫内的早期发育。 检测 钙瞬变我们将注入钙指示染料[钙绿 葡聚糖（IOK）和/或Ca-深红色葡聚糖（IOK），Molecular Probes， 股份有限公司、尤金，OR]直接转化为秀丽隐杆线虫obcytes。 蠕虫与 注射的卵母细胞随后在0. 1%三卡因 和0.01%的四咪唑以将它们溶解用于成像目的。 回肠麻醉剂不影响输卵管肌肉组织，因此允许 受精卵从卵巢进入子宫。 的 全固态多光子成像系统开发的MM将 用于对卵母细胞进行时移成像。 所述系统被配置 为了同时收集记录的两种荧光的图像， 信号和所传输的IR明场图像。 我们将观察 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，卵母细胞通过 受精囊和在早期发育（减数分裂和有丝分裂）的 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.