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Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca2+ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1-10μmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45 Ca pre-loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization-induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.
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