Confocal microscopy of the fertilization-induced calcium wave in sea urchins

Abstract

At fertilization, the egg undergoes a rapid rise in intracellular free calcium (=[Ca2+]i), which in turn triggers several important events, including cortical granule release and metabolic activation. In sea urchins, the fertilization induced increase in [Ca2+]i begins at the site of sperm fusion and sweeps through the egg in a wavelike fashion. Whether fertilization affects calcium levels only in the peripheral ooplasm or throughout the interior of the sea urchin egg is difficult to ascertain by conventional microscopic techniques, because out-offocus rays obscure subcellular organization in these relatively bulky cells. Recently, however, confocal microscopy has been introduced as a means of producing thin optical sections that reveal details not normally discernable by conventional light microscopy. In this paper, we use confocal laser scanning microscopy (CLSM) and time-lapse reconstructions to image calcium dynamics during fertilization in living sea urchin eggs labeled with the calcium-sensitive fluorescent dye fluo-3.