Mechanisms of PLCζ induced Ca²⁺ oscillations in mouse eggs at fertilisation

Abstract

All the events of egg activation in mammalian eggs are triggered physiologically by transient increases in cytosolic free Ca²⁺ referred to as Ca²⁺ioscillations. These oscillations are initiated by the sperm derived PLC isoform, PLCζ. PLCζ releases Ca²⁺ by hydrolysing its substrate PI(4,5)P₂ to produce IP₃, however, many of the mechanisms by which PLCζ elicits Ca²⁺release in eggs are poorly understood. The results of this thesis confirm that whilstPLCζ cRNA and recombinant protein is able to cause Ca²⁺ioscillations in mouse eggs the sperm derived protein PAWP does not cause any Ca²⁺ release in any circumstances. It is shown that EF hand domain and XY linker of PLCζ are important in determining its Ca²⁺i releasing ability by enabling PLCζ binding to its substrate PI(4,5)P₂ through electrostatic interactions. The C2 domain of PLCζ was also found to play a crucial role in the Ca²⁺ releasing ability of PLCζ, possibly by binding to lipids or proteins in the target membrane. The Ca²⁺releasing ability of eggs is acquired during oocyte maturation and a dramatic increase in PLCζ sensitivity of oocytes occurs after germinal vesicle breakdown.A variety of markers for PLCζ’s substrate PI(4,5)P₂ including fluorescent PI(4,5)P₂ and gelsolin based fluorescent probes suggests that this PI(4,5)P₂ is localised to intracellular vesicles that could derive from Golgi apparatus. Attempts are made to measure PI turnover in these intracellular compartments of eggs during PLCζ induced Ca²⁺i oscillations using several probes. The results of this thesis suggest that PLCζ releases Ca²⁺ by a novel IP₃ based signalling pathway that involves an intracellular source of PI(4,5)P₂