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

School of Medicine, Cardiff University, Heath Park, Cardiff, United Kingdom.
Fertility and sterility (Impact Factor: 4.59). 01/2012; 97(3):742-7. DOI: 10.1016/j.fertnstert.2011.12.013
Source: PubMed


To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca(2+) oscillations during activation.
Test of a laboratory technique.
University medical school research laboratory.
Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles.
Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca(2+)-sensitive fluorescent dye.
Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca(2+) oscillations using a Ca(2+)-sensitive fluorescent dye.
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.
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.

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Available from: Michail Nomikos,
    • "This pattern of localization supports the role of PLCz as the SOAF, since after the acrosome reaction, the equatorial region of the sperm membrane is exposed, adheres to and fuses with the oolemma (Barroso et al., 2009). In strong support of the role of PLCz, is the fact that mammalian oocytes can be activated following microinjection of either recombinant PLCz or complementary RNA (cRNA) encoding PLCz (Yoon et al., 2008; Swann et al., 2012; Nomikos et al., 2013b). "
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    ABSTRACT: Background: Infertility affects between 10 and 16% of couples worldwide. Twenty to 30% of cases of infertility are due to a male factor, 20-35% to a female factor, and 25-40% are due to both male and female factors. In ∼10-25% of cases, the precise underlying cause remains unclear. IVF or ICSI followed by embryo transfer can be very appropriate treatment options in cases of female tubal damage, ovulatory failure or male-factor infertility. While the use of IVF has been reported to be suitable for many infertile couples, normal IVF cycles can fail in some cases. While ICSI can represent a powerful alternative in cases of IVF failure, complete fertilization failure can still occur in 1-5% of ICSI cycles. This can be due to a variety of factors and while commonly attributed to deficiency of sperm factors, it is very likely that abnormalities in crucial oocyte factors could also play a key role. Methods: A critical literature review using PubMed was performed between April 2014 and July 2015 targeting studies concerning sperm and oocyte factors that could account for oocyte activation deficiency, and including studies of in vitro oocyte maturation in human oocytes, and animal models. Results: Accumulating evidence indicates that phospholipase C zeta (PLCζ) is the sperm oocyte activation factor, although recent studies claim that another sperm protein known as post-acrosomal WWP-binding domain protein could also play a significant role in the activation of oocytes. The present review discusses our current understanding of these two proteins but emphasizes that defects in the molecular machinery within the oocyte that interacts with such sperm proteins may also represent an underlying cause of fertilization failure and infertility, especially in cases where there is no obvious indication for sperm deficiency. Abnormalities in such mechanisms are highly likely to exert influence over the pulsatile release of calcium within the ooplasm, the critical signal that controls oocyte activation events. These molecular targets within the oocyte are rarely, if ever, considered clinically. We therefore recommend that future diagnostic assays should be developed to consider the inositol triphosphate receptor, protein kinase C, proteins associated with stored operated calcium entry calcium/calmodulin-dependent protein kinase II and mitogen-activated protein kinase. Development of such assays would represent a significant step forward in the diagnosis of oocyte activation deficiency and may identify a series of potential therapeutic targets. Conclusions: The present review provides a general overview of how a combination of sperm and oocyte factors can underlie oocyte activation deficiency, but pays particular attention to the less appreciated role of the oocyte. Enhanced research within this realm is much warranted and may establish new approaches for the diagnosis and treatment of infertility.
    Human Reproduction Update 09/2015; DOI:10.1093/humupd/dmv040 · 10.17 Impact Factor
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    • "Another study showed that the microinjection of oocyte activation deficient spermatozoa along with exposure to calcium ionophore generated live offspring [133]. Furthermore, microinjection of oocytes, that failed to fertilize with ICSI, with PLC␨ cRNA resulted in the appearance of prolonged Ca 2+ oscillations where each transient Ca 2+ wave was accompanied by a small coordinated cytoplasmic movement [172]. These studies further solidified the idea that PLC␨ is a great candidate as a clinical therapeutic solution that could avoid potentially deleterious effects associated with AOA, which use the non-physiological stimuli. "
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    ABSTRACT: Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.
    Cell calcium 11/2013; 55(1). DOI:10.1016/j.ceca.2013.10.006 · 3.51 Impact Factor
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    • "Finally, recent reports have shown that particle image velocimetry (PIV) has significant potential to evaluate rhythmical cytoplasmic movements in the activating oocyte to predict the viability of zygote development and thus provide a potential means to select the best embryos for intra-uterine transfer (Adjuk et al., 2011). A follow-up study reported by Swann et al. (2012) revealed that the frequency of PLCz-induced cytoplasmic Ca 2þ oscillations and observed cytoplasmic movements in aged ICSI-failed human oocytes were simultaneous in nature, implying that the cytoplasmic movements observed in the activating oocyte were strongly associated with the specific pattern of Ca 2þ oscillations, and therefore the fundamental action of PLCz. It follows therefore, that PIV may represent an exciting and non-invasive technique for observing Ca 2þ oscillation patterns within human oocytes in a clinical setting, while simultaneously providing an effective indicator of zygote viability. "
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    ABSTRACT: Mounting scientific and clinical evidence supports the key role played by phospholipase C zeta (PLCζ), a sperm-specific protein, in the activation of oocytes following fertilisation. Lacking a pleckstrin homology domain, PLCζ remains the smallest known mammalian PLC and was first identified in 2002. Since then, PLCζ has been the target for a multitude of studies in both mammalian and non-mammalian species focused upon its fundamental biochemical activity and crucial role as the mediator of oocyte activation. The earliest event subsequent to gamete fusion is the onset of a series of intracellular calcium oscillations within the oocyte, which are known to modulate cortical granule exocytosis, release meiotic arrest, regulate gene expression, recruit maternal mRNA, and initiate embryogenesis. Collectively these processes are known as 'oocyte activation' and together, represent a fundamental mechanism for early embryonic development. Evidence suggests that these processes are initiated and controlled by calcium release from ooplasmic sources in response to PLCζ activity via the inositol-1,4,5-triphosphate (IP3) pathway. While the biochemical action of PLCζ has been extensively studied, especially in relation to the EF-hands, X-Y linker, and C2 domain, all of which play critical roles for in vivo activity, there are still key gaps in our knowledge, particularly in terms of regulation and interaction with other proteins within the oocyte. Moreover, increasing clinical evidence has revealed a strong correlation between certain types of male infertility and the aberrant expression, localisation, structure and function of PLCζ in human sperm, particularly in cases of recurrent intracytoplasmic sperm injection (ICSI) failure, globozoospermia, and oocyte activation deficiency (OAD). In addition, two heterozygous substitution mutations have been identified in the coding sequence of PLCζ in one particular patient causing disruption to the catalytic X and Y domains and resulting in infertility. Although, such cases can be treated via the use of artificial oocyte activators (AOAs) such as calcium ionophores, significant concern remains over the use of such chemical agents, largely due to the fact that calcium release manifests as a single transient, rather than a series of oscillations as observed during normal fertilisation. Current interest in PLCζ is thus to develop a series of prognostic, diagnostic and therapeutic approaches which could first identify male patients that are deficient in PLCζ and then rescue oocyte activation ability via assisted reproductive technology (ART) and a pure, functionally-active, recombinant human PLCζ protein. While significant progress has been made in such areas over recent years, there is a clear need to translate scientific findings to clinical settings in order to maximise successful outcome for patients.
    07/2013; 53(3). DOI:10.1016/j.jbior.2013.07.005
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