The overall objective of this research plan is to define the effects of volatile anesthetics, particularly halothane, on the earliest aspects of embryonic development. Sea urchin gametes will be used as a model system to 1) examine the effects of volatile anesthetics on motile events associated with fertilization and syngamy, 2) determine the mechanism by which halothane reversibly inhibits the growth of the mitotic apparatus in dividing cells, and 3) study the effects of volatile anesthetics on the structure and function of membrane-associated actin. The effects of halothane on the acrosome reaction, sperm-egg adhesion, fertilization, and pronuclear movements will be studied by light and electron microscopy and differential interference contrast video microscopy. The mechanism by which volatile anesthetics reversibly in hibit the growth of the mitotic apparatus will be studied in two ways: 1) detergent-isolated MAs will be exposed to halothane and changes in birefringence montitored, and 2) mechanically-isolated MAs will be examined for 45Ca-sequestering activity and Ca++ stimulated ATPase in the presence and absence of halothane. The effect of halothane on membrane-associated actin will be investigated using DNAase inhibition assays, detergent-extracted models of sea urchin egg cortices, and the fluorescent conjugate NBD-phallacidin. These studies will define the effects of volatile anesthetics on microtubule- and actin-dependent processes which occur during early development and which may play a role in the purported teratogenicity of volatile anesethetics. Simultaneously, these studies will provide new information about the effects of anesthetic agents on the structure and function of microtubles and microfilaments in vitro. Such studies will be of interest to a variety of investigators, including those interested in the clinical practice of anesthesia, those researching molecular mechanisms of anesthetic action, as well as those interested in the molecular biology of cell division and intracellular contractile processes.
