Thapsigargin induces cytoplasmic free Ca2+ oscillations in mouse oocytes

The mechanisms of calcium signalling in mammalian oocytes during maturation and fertilization are controversial. In this study we measured intracellular free Ca2+ concentrations ([Ca2+]i) with the photoprotein aequorin microinjected into immature mouse oocytes. Immature mouse oocytes typically produced [Ca2+]i responses to muscarinic acetylcholine (ACh) stimulation with two types of component. The first component consisted of a broad transient rise in [Ca2+]i lasting about 1 min. The second component consisted of pulsatile oscillations which could occur before, during or after the broad transient, but typically occurred on the rising phase of the broad transient, with a duration of about 5 s. Removal of external Ca2+ ([Ca2+]o) abolished the Ca2+ responses to ACh. Exposure of oocytes to the specific microsomal Ca(2+)-ATPase inhibitors thapsigargin (TG) and cyclopiazonic acid unexpectedly produced sustained oscillations in [Ca2+]i which were sensitive to the concentration of Ca2+ in the external milieu. The frequency of these oscillations was slow, and ceased, sometimes after several cycles, when Ca2+o was removed. Raised [Ca2+]o significantly increased the frequency in cells oscillating to TG and stimulated nonoscillating cells to begin oscillating. The majority of responsive oocytes which did not produce oscillations to ACh alone (70%), did so after TG treatment. Detailed data analysis indicated that these oscillations were identical to those generated by TG alone, with a similar sensitivity to changes in [Ca2+]o. Exposure of oocytes to ryanodine did not inhibit oscillatory behaviour. These results suggest that immature mouse oocytes possess a store which is insensitive to both TG and ryanodine and is capable of supporting [Ca2+]i oscillations.