PLCζ Induced Ca²⁺ Oscillations in Mouse Eggs Involve a Positive Feedback Cycle of Ca²⁺ Induced InsP₃ Formation From Cytoplasmic PIP₂

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca²⁺ concentration, referred to as Ca²⁺ oscillations. It is widely accepted that these Ca²⁺ oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP₂ to produce the Ca²⁺ releasing messenger InsP₃. However, it is not clear whether PLCζ induced InsP₃ formation is periodic or monotonic, and whether the PIP₂ source for generating InsP₃ from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP₃ at different points of the Ca²⁺ oscillation cycle to show that PLCζ causes Ca²⁺ oscillations by a mechanism which requires Ca²⁺ induced InsP₃ formation. In contrast, incubation in Sr²⁺ media, which also induces Ca²⁺ oscillations in mouse eggs, sensitizes InsP₃-induced Ca²⁺ release. We also show that the cytosolic level Ca²⁺ is a key factor in setting the frequency of Ca²⁺ oscillations since low concentrations of the Ca²⁺ pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca²⁺ oscillations in eggs, even in Ca²⁺ free media. Given that Ca²⁺ induced InsP₃ formation causes a rapid wave during each Ca²⁺ rise, we use a mathematical model to show that InsP₃ generation, and hence PLCζ's substate PIP₂, has to be finely distributed throughout the egg cytoplasm. Evidence for PIP₂ distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP₂ in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr²⁺ induced, Ca²⁺ oscillations. These data suggest that the cytosolic Ca²⁺ level, rather than Ca²⁺ store content, is a key variable in setting the pace of PLCζ induced Ca²⁺ oscillations in eggs, and they imply that InsP₃ oscillates in synchrony with Ca²⁺ oscillations. Furthermore, they support the hypothesis that PLCζ and sperm induced Ca²⁺ oscillations in eggs requires the hydrolysis of PIP₂ from finely spaced cytoplasmic vesicles.