Michail Nomikos

Binding of Phosphoinositide-specific Phospholipase C-ζ (PLC-ζ) to Phospholipid Membranes POTENTIAL ROLE OF AN UNSTRUCTURED CLUSTER OF BASIC RESIDUES

Phospholipase C-ζ (PLC-ζ) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-δ1, but lacks the PH domain that anchors PLC-δ1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-ζ interacts with membranes. The linker region between the X and Y catalytic domains of PLC-ζ, however, contains a cluster of basic residues not present in PLC-δ1. Application of electrostatic theory to a homology model of PLC-ζ suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-ζ 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 5 × 103 m-1, a biologically relevant value. Expressed fragments corresponding to the PLC-ζ 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-ζ-(374-385), binds to PC/PS vesicles with higher affinity than PLC-ζ, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-ζ 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.

Starting a new life: Sperm PLC‐zeta mobilizes the Ca2+ signal that induces egg activation and embryo development

We have discovered that a single sperm protein, phospholipase C‐zeta (PLCζ), can stimulate intracellular Ca2+ signalling in the unfertilized oocyte (‘egg’) culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca2+ concentration. The fertilized egg responds to the intracellular Ca2+ rise by completing meiosis. In mammalian eggs, the Ca2+ signal is delivered as a train of long‐lasting cytoplasmic Ca2+ oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca2+ release from egg intracellular stores by hydrolyzing the membrane lipid PIP2 and consequent stimulation of the inositol 1,4,5‐trisphosphate (InsP3) receptor Ca2+‐signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca2+ oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.

PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2

At fertilization the sperm causes InsP3 production and Ca2+ release by introducing phospholipase C ζ (PLCζ) into the egg. This sperm PLCζ does not cause hydrolysis of plasma membrane PIP2. Instead it hydrolyzes PIP2 found in intracellular membranes. Fertilization may involve a novel aspect of the InsP3 signaling pathway.

Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility.

Objective To 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. Design Test of a novel therapeutic approach to male factor infertility. Setting University 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 Cζ binding to PtdIns(4,5)P2 requires the XY-linker region.

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.

Sperm PLCζ: From structure to Ca2+ oscillations, egg activation and therapeutic potential

Significant evidence now supports the assertion that cytosolic calcium oscillations during fertilization in mammalian eggs are mediated by a testis‐specific phospholipase C (PLC), termed PLC‐zeta (PLCζ) that is released into the egg following gamete fusion. Herein, we describe the current paradigm of PLCζ in this fundamental biological process, summarizing recent important advances in our knowledge of the biochemical and physiological properties of this enzyme. We describe the data suggesting that PLCζ has distinct features amongst PLCs enabling the hydrolysis of its substrate, phosphatidylinositol 4,5‐bisphosphate (PIP2) at low Ca2+ levels. PLCζ appears to be unique in its ability to target PIP2 that is present on intracellular vesicles. We also discuss evidence that PLCζ may be a significant factor in human fertility with potential therapeutic capacity.

Novel regulation of PLCζ activity via its XY-linker

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.

Sperm-induced Ca2+ release during egg activation in mammals.

This review discusses the role that the sperm-specific phospholipase C zeta (PLCζ) is proposed to play during the fertilization of mammalian eggs. At fertilization, the sperm initiates development by causing a series of oscillations in cytosolic concentrations of calcium [Ca(2)] within the egg. PLCζ mimics the sperm at fertilization, causing the same pattern of Ca(2+) release as seen at fertilization. Introducing PLCζ into mouse eggs also mimics a number of other features of the way in which the fertilizing sperm triggers Ca(2+) oscillations. We discuss the localization of PLCζ within the egg and present a hypothesis about the localization of PLCζ within the sperm before the initiation of fertilization.

Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection

Objective To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca2+ oscillations during activation. Design Test of a laboratory technique. Setting University medical school research laboratory. Patient(s) Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles. Intervention(s) Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca2+-sensitive fluorescent dye. Main Outcome Measure(s) Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca2+ oscillations using a Ca2+-sensitive fluorescent dye. Result(s) Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca2+ oscillations. Each transient Ca2+ concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis. Conclusion(s) The occurrence and frequency of cytoplasmic Ca2+ oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca2+ oscillations in human zygotes.

Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca2+ ionophores to produce a single cytosolic Ca2+ increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca2+ oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes. We show that increasing periods of 56°C exposure of sperm produces a progressive loss of Ca2+ oscillations after ICSI. The decrease in Ca2+ oscillations produces a reduction in oocyte activation and embryo development to the blastocyst stage. We treated such oocytes that failed to activate after ICSI either with Ca2+ ionophore, or with Sr2+ media which causes Ca2+ oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr2+ and PLCζ gave the highest rates of development to blastocyst. When recombinant PLCζ was given to oocytes previously injected with control sperm, they developed normally to the blastocyst stage at rates similar to that after control ICSI. The data suggest that recombinant human PLCζ protein is an efficient means of rescuing oocyte activation after ICSI failure and that it can be effectively used even if the sperm already contains endogenous Ca2+ releasing activity.