ObjectiveTo determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca2+ 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.DesignTest of a novel therapeutic approach to male factor infertility.SettingUniversity medical school research laboratory.Patient(s)Donated unfertilized human oocytes from follicle reduction.Intervention(s)Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca2+-sensitive fluorescent dye.Main Outcome Measure(s)Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca2+ oscillations, activation, and early embryo development.Result(s)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 Ca2+ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage.Conclusion(s)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.

The XY-linker region of somatic cell PLC (phospholipase)-β, -γ, -δ and -ϵ isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLCζ XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLCζ XY-linker significantly diminishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+-oscillation-inducing activity, revealing evidence for a novel PLCζ enzymatic mechanism.

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

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.