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 (PLCζ) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca²⁺ oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) substrate in the plasma membrane. Previous studies failed to detect PLCζ in the plasma membrane, hence the means of PLCζ binding to PtdIns(4,5)P₂ is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P₂ or to liposomes containing near-physiological levels of PtdIns(4,5)P₂. The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca²⁺ oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P₂ hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca²⁺-oscillation-inducing activity and in vitro PtdIns(4,5)P₂ interaction of mouse PLCζ. Our data suggest that positively charged residues within the XY linker play an important role in the PLCζ interaction with PtdIns(4,5)P₂, a crucial step in generating the Ca²⁺ activation signal that is essential for fertilization in mammals.

A dramatic rise in intracellular calcium plays a vital role at the moment of fertilization, eliciting the resumption of meiosis and the initiation of embryo development. In mammals, the rise takes the form of oscillations in calcium concentration within the egg, driven by an elevation in inositol trisphosphate. The causative agent of these oscillations is proposed to be a recently described phosphoinositide-specific phospholipase C, PLCζ, a soluble sperm protein that is delivered into the egg following membrane fusion. In the present review, we examine some of the distinctive structural and functional characteristics of this crucial enzyme that sets it apart from the other known forms of mammalian PLC.

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.