The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs.

At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1,4,5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here, we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI. Mouse eggs incubated in Ca2+-free, Sr2+-containing medium immediately underwent Ca2+ oscillations but human eggs consistently failed to undergo Ca2+ oscillations in the same Sr2+ medium. We tested the InsP3-receptor (IP3R) sensitivity directly by photo-release of caged InsP3 and found that mouse eggs were about 10 times more sensitive to InsP3 than human eggs. There were no major differences in the Ca2+ store content between mouse and human eggs. However, we found that the ATP concentration was consistently higher in mouse compared to human eggs. When ATP levels were lowered in mouse eggs by incubation in pyruvate-free medium, Sr2+ failed to cause Ca2+ oscillations. When pyruvate was added back to these eggs, the ATP levels increased and Ca2+ oscillations were induced. This suggests that ATP modulates the ability of Sr2+ to stimulate IP3R-induced Ca2+ release in eggs. We suggest that human eggs may be unresponsive to Sr2+ medium because they have a lower level of cytosolic ATP.

Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca(2+) oscillations required for mammalian oocyte activation.

A sperm-specific phospholipase C-zeta (PLCζ) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLCζ has been shown to trigger a prolonged series of repetitive Ca(2+) transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca(2+) signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLCζ from disparate mammalian species to trigger Ca(2+) oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLCζ species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLCζ relative to mouse PLCζ in generating Ca(2+) oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLCζ chimeras to enable study of the potential role of discrete PLCζ domains in conferring the enhanced Ca(2+) signalling potency of human PLCζ. Functional analysis of these human/mouse PLCζ domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLCζ activity. Our empirical observations are compatible with a basic mathematical model for the Ca(2+) dependence of generating cytoplasmic Ca(2+) oscillations in mammalian oocytes by sperm PLCζ.

Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization.

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.

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 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. 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 Ca(2+)-sensitive fluorescent dye. MAIN OUTCOME MEASURE(S): Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca(2+) 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 Ca(2+) 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-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 Ca(2+) 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 Ca(2+)-sensitive fluorescent dye. MAIN OUTCOME MEASURE(S): Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca(2+) oscillations using a Ca(2+)-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 Ca(2+) oscillations. Each transient Ca(2+) 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 Ca(2+) 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 Ca(2+) oscillations in human zygotes.

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 Ca(2+) oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) 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)P(2) is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P(2) or to liposomes containing near-physiological levels of PtdIns(4,5)P(2). 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 Ca(2+) oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P(2) hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca(2+)-oscillation-inducing activity and in vitro PtdIns(4,5)P(2) 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)P(2), a crucial step in generating the Ca(2+) activation signal that is essential for fertilization in mammals.

Novel regulation of PLCζ activity via its XY-linker.

The XY-linker region of somatic cell PLC (phospholipase)-ß, -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.

Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP(2) hydrolysis activity of sperm PLCζ.

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP(2) (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.