Role of Phospholipase C- zeta Domains in Ca super(2+)-dependent Phosphatidylinositol 4,5-Bisphosphate Hydrolysis and Cytoplasmic Ca super(2+) Oscillations

The sperm-specific phospholipase C- zeta (PLC zeta ) elicits fertilization-like Ca super(2+) oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (CAMB:) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLC zeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLC zeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLC zeta structure-function, we assessed the ability of PLC zeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca super(2+) changes in intact mouse eggs. PLC zeta and the closely related PLC delta 1 had similar K sub(m) values for phosphatidylinositol 4,5-bisphosphate, but PLC zeta was around 100 times more sensitive to Ca super(2+) than was PLC delta 1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLC zeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca super(2+) sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca super(2+) oscillations. These data suggest that the exquisite Ca super(2+)-dependent features of PLC zeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca super(2+) oscillations in intact mouse eggs.