Chatacteristics of PLC-zeta and induction of Ca²⁺ oscillations

Abstract

Intracellular Ca²⁺ forms spatial and temporal signals as a propagating Ca²⁺ wave and repetitive Ca²⁺ spikes, respectively. The mammalian egg is a good experimental system, since it has a large size and exhibits Ca²⁺ oscillations associated with a Ca²⁺ wave in each Ca²⁺ spike during fertilization as a pivotal signal for egg activation. Each Ca²⁺ spike is due to Ca²⁺ release from the ER mainly via the IP₃ receptor type 1. Ca²⁺ oscillations are induced by a cytosolic sperm factor that is introduced into the egg cytoplasm upon sperm-egg fusion. Recently, Saunders et al. have found a novel sperm-specific phospholipase C isoform, PLCζ which induces Ca²⁺ oscillations and early embryonic development when expressed in a mouse egg by injection of RNA encoding PLCζ. We expressed PLCζ fused with a fluorescent protein Venus and estimated PLCζ concentration for initiation of Ca²⁺ oscillations. Interestingly, expressed PLCζ-Venus translocated into the pronucleus formed after egg activation. We also synthesized recombinant PLCζ using baculovirus/Sf9-cell expression system. Ca²⁺ oscillations were produced by injection of PLCζ protein. PIP₂ hydrolysis assay in vitro showed that PLCζ has 70% maximal activity at 100 nM [Ca²⁺] that is usually the basal [Ca²⁺]_i level of cells. These characteristics not only qualify PLCζ for a strong candidate of the sperm factor but also provide a model of induction and regulation of Ca²⁺ oscillations. [Jpn J Physiol 54 Suppl:S38 (2004)]