PLCζ Induced Ca(2+) Oscillations in Mouse Eggs Involve a Positive Feedback Cycle of Ca(2+) Induced InsP(3) Formation From Cytoplasmic PIP(2)

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca(2+) concentration, referred to as Ca(2+) oscillations. It is widely accepted that these Ca(2+) oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP(2) to produce the Ca(2+) releasing messenger InsP(3). However, it is not clear whether PLCζ induced InsP(3) formation is periodic or monotonic, and whether the PIP(2) source for generating InsP(3) from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP(3) at different points of the Ca(2+) oscillation cycle to show that PLCζ causes Ca(2+) oscillations by a mechanism which requires Ca(2+) induced InsP(3) formation. In contrast, incubation in Sr(2+) media, which also induces Ca(2+) oscillations in mouse eggs, sensitizes InsP(3)-induced Ca(2+) release. We also show that the cytosolic level Ca(2+) is a key factor in setting the frequency of Ca(2+) oscillations since low concentrations of the Ca(2+) pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca(2+) oscillations in eggs, even in Ca(2+) free media. Given that Ca(2+) induced InsP(3) formation causes a rapid wave during each Ca(2+) rise, we use a mathematical model to show that InsP(3) generation, and hence PLCζ's substate PIP(2), has to be finely distributed throughout the egg cytoplasm. Evidence for PIP(2) distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP(2) in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr(2+) induced, Ca(2+) oscillations. These data suggest that the cytosolic Ca(2+) level, rather than Ca(2+) store content, is a key variable in setting the pace of PLCζ induced Ca(2+) oscillations in eggs, and they imply that InsP(3) oscillates in synchrony with Ca(2+) oscillations. Furthermore, they support the hypothesis that PLCζ and sperm induced Ca(2+) oscillations in eggs requires the hydrolysis of PIP(2) from finely spaced cytoplasmic vesicles.