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.

Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization.

Calcium has an important role in the events of egg activation and early preimplantation development. We investigated changes in intracellular calcium concentration in human oocytes at fertilization using the calcium-sensitive photoprotein aequorin. Oocytes were donated for research by patients undergoing in-vitro fertilization treatment in the Department of Obstetrics and Gynaecology. Cumulus cells, and in some cases zonae pellucidae, were removed by appropriate enzyme treatment. Single oocytes were micro-injected with aequorin and incubated in a chamber perfused with pre-equilibrated culture medium in a photomultiplier system. Eleven zona-intact and 15 zona-free oocytes were incubated with sperm, and oocytes from each group were incubated without sperm as controls. Dramatic transient increases in intracellular free calcium concentration were recorded in three zona-intact and seven zona-free oocytes, thought to be the first direct measurements of intracellular changes in human oocytes at fertilization. The amplitude (up to 2.5 microM), duration (120 s) and frequency (every 10-35 min) of these transients were similar in zona-intact and zona-free oocytes. They resemble those recorded in mouse oocytes, which may therefore be a suitable model for biochemical events at human fertilization.