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.
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