The sperm-specific phospholipase C-ζ (PLCζ) elicits fertilization-like Ca²⁺ 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). PLCζ may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCζ 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 PLCζ structure-function, we assessed the ability of PLCζ and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca²⁺ changes in intact mouse eggs. PLCζ and the closely related PLCδ1 had similar K_m values for phosphatidylinositol 4,5-bisphosphate, but PLCζ was around 100 times more sensitive to Ca²⁺ than was PLCδ1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCζ constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca²⁺ 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 Ca²⁺ oscillations. These data suggest that the exquisite Ca²⁺-dependent features of PLCζ regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca²⁺ oscillations in intact mouse eggs.

To determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca(2+) oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCζ protein for rescuing failed oocyte activation and embryo development. Test of a novel therapeutic approach to male factor infertility. University medical school research laboratory. Donated unfertilized human oocytes from follicle reduction. Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca(2+)-sensitive fluorescent dye. Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca(2+) oscillations, activation, and early embryo development. In contrast to the wild-type protein, mutant forms of human sperm PLCζ display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCζ protein reliably triggers the characteristic pattern of cytoplasmic Ca(2+) oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage. Dysfunctional sperm PLCζ cannot trigger oocyte activation and results in male factor infertility, so a potential therapeutic approach is oocyte microinjection of active, wild-type PLCζ protein. We have demonstrated that recombinant human PLCζ can phenotypically rescue failed activation in oocytes that express dysfunctional PLCζ, and that this intervention culminates in efficient blastocyst formation.

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca²⁺oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca²⁺release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P₂targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P₂with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P₂from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca²⁺oscillations. Specific depletion of this PI(4,5)P₂pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca²⁺oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P₂, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P₂after PLCζ injection. Targeted depletion of vesicular PI(4,5)P₂by expression of ciPLCζ-fused Inp54p inhibited the Ca²⁺oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca²⁺mobilization by hydrolyzing internal PI(4,5)P₂stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.