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Background: The mechanism underlying sperm PLCζ interaction with its target membrane is unresolved.Results: EF-hand mutations introduced into PLCζ reduce in vivo Ca2+ oscillation inducing activity and in vitro interaction with PIP2.Conclusion: EF-hand domain is essential for targeting PLCζ to PIP2-containing membranes.Significance: We propose a novel mechanism by which sperm PLCζ is anchored to its physiological membrane substrate.

Mature mammalian oocytes undergo a prolonged series of cytoplasmic calcium (Ca(2+)) oscillations at fertilization that are the cause of oocyte activation. The Ca(2+) oscillations in mammalian oocytes are driven via inositol 1,4,5-trisphosphate (IP3) generation. Microinjection of the sperm-derived phospholipase C-zeta (PLCζ), which generates IP3, causes the same pattern of Ca(2+) oscillations as observed at mammalian fertilization and it is thought to be the physiological agent that triggers oocyte activation. However, another sperm-specific protein, 'post-acrosomal WW-domain binding protein' (PAWP), has also been reported to elicit activation when injected into mammalian oocytes, and to produce a Ca(2+) increase in frog oocytes. Here we have investigated whether PAWP can induce fertilization-like Ca(2+) oscillations in mouse oocytes. Recombinant mouse PAWP protein was found to be unable to hydrolyse phosphatidylinositol 4,5-bisphosphate in vitro and did not cause any detectable Ca(2+) release when microinjected into mouse oocytes. Microinjection with cRNA encoding either the untagged PAWP, or yellow fluorescent protein (YFP)-PAWP, or luciferase-PAWP fusion proteins all failed to trigger Ca(2+) increases in mouse oocytes. The lack of response in mouse oocytes was despite PAWP being robustly expressed at similar or higher concentrations than PLCζ, which successfully initiated Ca(2+) oscillations in every parallel control experiment. These data suggest that sperm-derived PAWP is not involved in triggering Ca(2+) oscillations at fertilization in mammalian oocytes.

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Mammalian oocyte activation is mediated by cytosolic calcium (Ca(2+)) oscillations initiated upon delivery of a putative 'sperm factor' by the fertilizing sperm. Previous studies suggest the identity of this sperm factor as the testis-specific phospholipase C-zeta (PLCζ). Recently, a post-acrosomal sheath WW domain-binding protein (PAWP) has been proposed as an alternative sperm factor candidate, following a report that human PAWP protein and cRNA elicited Ca(2+) oscillations in mouse and human oocytes. Those Ca(2+) oscillations were inhibited by a PAWP-derived peptide corresponding to a functional PPGY binding motif. Herein, using a series of human PAWP expression constructs, we demonstrate that both human PAWP protein and cRNA are, in our experiments, unable to elicit Ca(2+) release following microinjection into mouse oocytes. Parallel experiments performed with human PLCζ elicited the characteristic Ca(2+) oscillations present at mammalian fertilization, which produced oocyte activation and embryo development. Furthermore, sperm-induced Ca(2+) oscillations were not inhibited by the PAWP-derived PPGY peptide following in vitro fertilization or intracytoplasmic sperm injection. Thus, the functional disparity with PLCζ leads us to conclude that human PAWP is neither sufficient nor necessary for the Ca(2+) oscillations that initiate mammalian oocyte activation at fertilization.

trigger exocytosis via a mechanism that does not involve CAMKii. we discuss some recent developments in our understanding of these triggers for egg activation within the framework of cytosolic Ca 2+ signaling.Reproduction (2016) 152 R41-R50

Initiated by luteinizing hormone and finalized by the fertilizing sperm, the mammalian oocyte completes its two meiotic divisions. The first division occurs in the mature Graafian follicle during the hours preceding ovulation and culminates in an extreme asymmetric cell division and the segregation of the two pairs of homologous chromosomes. The newly created mature egg rearrests at metaphase of the second meiotic division prior to ovulation and only completes meiosis following a Ca2+ signal initiated by the sperm at gamete fusion. Here, we review the cellular events that govern the passage of the oocyte through meiosis I with a focus on the role of the spindle assembly checkpoint in regulating its timing. In meiosis II, we examine how the egg achieves its arrest and how the fertilization Ca2+ signal allows the initiation of embryo development.

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca2+ concentration, referred to as Ca2+ oscillations. It is widely accepted that these Ca2+ oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP2 to produce the Ca2+ releasing messenger InsP3. However, it is not clear whether PLCζ induced InsP3 formation is periodic or monotonic, and whether the PIP2 source for generating InsP3 from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP3 at different points of the Ca2+ oscillation cycle to show that PLCζ causes Ca2+ oscillations by a mechanism which requires Ca2+ induced InsP3 formation. In contrast, incubation in Sr2+ media, which also induces Ca2+ oscillations in mouse eggs, sensitizes InsP3-induced Ca2+ release. We also show that the cytosolic level Ca2+ is a key factor in setting the frequency of Ca2+ oscillations since low concentrations of the Ca2+ pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca2+ oscillations in eggs, even in Ca2+ free media. Given that Ca2+ induced InsP3 formation causes a rapid wave during each Ca2+ rise, we use a mathematical model to show that InsP3 generation, and hence PLCζ's substate PIP2, has to be finely distributed throughout the egg cytoplasm. Evidence for PIP2 distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP2 in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr2+ induced, Ca2+ oscillations. These data suggest that the cytosolic Ca2+ level, rather than Ca2+ store content, is a key variable in setting the pace of PLCζ induced Ca2+ oscillations in eggs, and they imply that InsP3 oscillates in synchrony with Ca2+ oscillations. Furthermore, they support the hypothesis that PLCζ and sperm induced Ca2+ oscillations in eggs requires the hydrolysis of PIP2 from finely spaced cytoplasmic vesicles.