The objective is to define the in vitro effects of volatile anesthetics, particularly halothane, on gamete physiology and the processes of fertilization and early embryonic development using a mammalian model system. Experiments to define the effects of halothane on the physiology of mouse gametes prior to fertilization will focus on potential changes in sperm capacitation, the acrosome reaction and motility, as well as possible changes in the surface morphology and meiotic competence of mouse oocytes. In vitro fertilization techniques will be used to determine whether halothane can alter sperm incorporation (induce polyspermy) as found previously in sea urchin studies, or alter meiotic or mitotic processes that could lead to polyploidy. Embryo compaction and blastocyst formation will be studied to determine the effects of halothane on later stages of development and provide totally new information about the effects of volatile anesthetics on the fundamental events of embryonic cell growth, differentiation, and adhesion. Sea urchin gametes will be used in continuing studies to investigate the mechanism(s) by which halothane promotes polyspermy and inhibits microtubule-mediated events during early development. Planar cortex models of sea urchin egg membranes will be used to study the effect of halothane on the cortical reaction (slow block to polyspermy) and determine whether halothane affects membrane fusion events. Electrophysiological methods will be employed to determine whether halothane impairs the fast (electrically-mediated) block to polyspermy. Principle methods of study will include light microscopy (phase, interference contrast, darkfield, fluorescence), scanning and transmission electron microscopy, time-lapse and digitally-enhanced videomicroscopy, as well as immunolabelling techniques and electrophysiological recordings of single cells. 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, and those interested in the molecular biology of development.
