Nitric oxide is a critical messenger and effector of a wide range of physiological processes. Unlike many other messenger molecules it is highly membrane soluble and has a complicated concentration gradient due to its tertiary rate law in its reaction with oxygen. In order to measure and model these gradients, the investigators will develop new fluorescent probes for the direct detection of nitric oxide in vivo. In the first phase of this project new lipid soluble alkylated 2,3 diaminonaphthalene (DAN) and fluorescein derivatives will prepared and tested in the fish gamete model. In the second phase of this project new caged nitric oxide donor systems will be prepared for the time and location controlled release of nitric oxide. These systems will use fluorescent micro spheres derivatized with photolytically active poly(S- nitrosocysteines) which have been coated with gas permeable membranes. In the third phase of this project the investigators will prepare new fluorophores for the direct and oxygen independent detection of nitric oxide. These derivatives will mimic the known heme receptor in soluble nitric oxide synthase, which will be coupled to a fluorescent probe. Throughout the development of these new probes and donors they will be tested and calibrated against the fish gamete model developed in prior studies. In particular the investigators will use fluorescent and laser confocal microscopy to more precisely locate the egg NOS and its isoform. In addition to testing the new donors and probes, this research will allow the investigators to determine if the mammalian follicle produces nitric oxide, if so by which cell type, and is the nitric oxide a general stimulant of motility or could it act as a chemoattractant for mammal sperm. The significance of this research is, that for the first time, the question of egg derived factors which may regulate sperm motion (and thus be relevant to the success of fertilization) will be examined in both external and internal fertilizing vertebrates.
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