Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

Abstract

Results of numerous experiments indicate that the transient rise in intracellular Ca2+ following sperm-egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca2+ than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca2+, we manipulated buffered intracellular Ca2+ concentrations by microinjecting Ca2+-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca2+ concentrations between 0.5 and 1.0 microM, recruitment of maternal mRNAs is only partially stimulated at injected free Ca2+ concentrations of 2.5 microM, and no evidence for cell cycle resumption was observed (up to 2.5 microM Ca2+). Although the Ca2+- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca2+ that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.