oocyte activation deﬁciency: a role foran oocyte contribution?€¦ · oocyte activation...

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Oocyte activation deficiency: a rolefor an oocyte contribution?Marc Yeste*, Celine Jones, Siti Nornadhirah Amdani, Sheena Patel,and Kevin CowardNuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital,Headington, Oxford, UK

*Correspondence address. Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s Centre,John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. Tel: +44-1865-782829; Fax: +44-1865-769141; E-mail: [email protected]

Submitted on March 9, 2015; resubmitted on August 6, 2015; accepted on August 13, 2015

table of contents

† Introduction† Methods† ART: analysis of the current data† Activation of the oocyte is a step mediated by sperm proteins† PLCz: a critical SOAF

Phospholipases and PLCzPLCz is present in sperm from mammalian and non-mammalian species but may differ in its mechanism of actionMechanism of PLCz actionPLCz appears to be involved in functions other than oocyte activationOAD occurs in sperm exhibiting PLCz deficiencies

† Other sperm proteins as potential SOAF candidatesCitrate synthase and the truncated form of c-kit tyrosine kinase receptorPAWPControversies surrounding the relative importance of PLCz and PAWP

† Could oocyte factors explain some cases of OAD?† The SOAF triggers a cascade mediated by vital oocyte factors† PIP2 and DAG† The central role of InsP3R

Isoforms and regulationEvidence for the role of the InsP3R during oocyte activation and maturationInsP3Rs are present in oocytes from mammalian and non-mammalian species, but their intracellular distribution can

differ between species† PKC

The PKC familyPKC and the mammalian oocyteThe role of PKCa during oocyte activationPKCs, oocyte maturation and early embryo development

† Ca2+ oscillations and homeostasis in the oocyteMembrane channels and SOCE: STIM1 and ORAI1Sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases

† Other oocyte proteins that could underlie oocyte activation failureCaMKII and MAPKOther phospholipasesCDK1 and CHERP

& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]

Human Reproduction Update, Vol.22, No.1 pp. 23–47, 2016

Advanced Access publication on September 7, 2015 doi:10.1093/humupd/dmv040

† Oocyte mitochondria and Ca2+ oscillations† Lessons from the in vitro maturation of human oocytes† AOA

Mechanical, electrical and chemical activationSafety of AOA for child healthThe use of AOA as a routine practiceRescue of OAD with recombinant PLCz

† Conclusions

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 remainsunclear. IVF or ICSI followed by embryo transfer can be very appropriate treatment options in cases of female tubal damage, ovulatory failure ormale-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 bedue to a variety of factors and while commonly attributed to deficiency of sperm factors, it is very likely that abnormalities in crucial oocyte factorscould also play a key role.

methods: A critical literature review using PubMed was performed between April 2014 and July 2015 targeting studies concerning sperm andoocyte factors that could account for oocyte activation deficiency, and including studies of in vitro oocyte maturation in human oocytes, and animalmodels.

results: Accumulating evidence indicates that phospholipase C zeta (PLCz) is the sperm oocyte activation factor, although recent studiesclaim that another sperm protein known as post-acrosomal WWP-binding domain protein could also play a significant role in the activation ofoocytes. The present review discusses our current understanding of these two proteins but emphasizes that defects in the molecular machinerywithin the oocyte that interacts with such sperm proteins may also represent an underlying cause of fertilization failure and infertility, especially incases where there is no obvious indication for sperm deficiency. Abnormalities in such mechanisms are highly likely to exert influence over thepulsatile release of calcium within the ooplasm, the critical signal that controls oocyte activation events. These molecular targets within the oocyteare rarely, if ever, considered clinically. We therefore recommend that future diagnostic assays should be developed to consider the inositol tri-phosphate receptor, protein kinase C, proteins associated with stored operated calcium entry calcium/calmodulin-dependent protein kinase IIand mitogen-activated protein kinase. Development of such assays would represent a significant step forward in the diagnosis of oocyte activationdeficiency 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 oocyteactivation deficiency, but pays particular attention to the less appreciated role of the oocyte. Enhanced research within this realm is muchwarranted and may establish new approaches for the diagnosis and treatment of infertility.

Key words: infertility / oocyte activation deficiency / sperm-borne oocyte activation factor / phospholipase C zeta / post-acrosomalWWP-binding domain protein / Ca2+ homeostasis / protein kinase C

IntroductionInfertility is defined as the inability of a sexually active couple to achievepregnancyafter 1 yearof unprotected vaginal intercourse (Practice Com-mittee of the American Society for Reproductive Medicine, 2013) and isconsidered as a disease by the World Health Organization (Zegers-Hochschild et al., 2009). Infertility affects approximately one in sevencouples in the UK (National Collaborating Centre for Women’s andChildren’s Health, UK, 2013) and between 10 and 16% of couples world-wide (Fidlerand Bernstein, 1999; Boivin et al., 2007; Ombelet et al., 2008;Louis et al., 2013; Thoma et al., 2013; Chandra et al., 2014; ESHRE, ARTFact Sheet, June, 2014). Infertility figures differ between primary and sec-ondary causes, and amongst developed, developing and underdevel-oped countries. When considering the population worldwide, primaryinfertility affects between 1 and 8% of couples, while secondary infertility,defined as the inabilityof awoman to become pregnantor to carrya preg-nancy to term after the birth of one or more children, may occur in up to35% of cases in developing countries (Vayena et al., 2002).

The absence of pregnancy, or the experience of miscarriage, accountsfor many other consequences, including psychological distress (Bak et al.,2012), economic constraints (Wu et al., 2013) and social stigmatization(Slade et al., 2007). Furthermore, infertility and sperm quality have beensuggested as being markers of overall male health in addition to lifestyleand/or social factors, and men presenting a low semen volume, low per-centage of motile and morphologically normal sperm appear to havehigher mortality odds (Jensen et al., 2009).

Current data indicate that 20–30% of infertile cases are predominantlyrelated to a male factor, 20–35% of cases to a female factor, and 25–40%of cases to problems associated with both male and female factors.Moreover, the precise cause of infertility remains unexplained in around10–25% cases. Although figures can differ between studies, generalconsensus indicates that infertility is more likely to be related to a femalefactor, or male and female factors collectively, than a male only cause(Thonneau et al., 1991; De Kretser, 1997; Anderson et al., 2009; NationalCollaborating Centre for Women’s and Children’s Health, UK, 2013;ESHRE, ART Fact Sheet, 2014).

24 Yeste et al.

In the case of males, semen and sperm defects are a major cause ofinfertility including deficient spermatogenesis and/or abnormal epididy-mal maturation that may have a genetic basis (Matzuk and Lamb, 2008).These deficiencies are related not only to the basic cellular functionsrequired for mitosis, meiosis, sperm differentiation, and sperm chroma-tin integrity, but also to vital proteins that are of paramount importancefor the events that take place following sperm–oocyte fusion (Robinsonet al., 2012; Lopez et al., 2013; Pregl Breznik et al., 2013; Simon et al.,2013).

In women, abnormalities underlying infertility primarily involve ovarianand ovulatory disorders such as primary ovarian insufficiency and poly-cystic ovarian syndrome (25%), tubal damage (20%), and uterine or peri-toneal disorders (10%) (Data given here relate to the total percentage ofinfertile cases). Endometriosis, pelvic conditions, embryo defects, previ-ous infections from Chlamydia trachomatis and susceptibility to rubellamay also influence the ability to conceive (National CollaboratingCentre for Women’s and Children’s Health, (UK), 2013). Despite theidentification of genetic causes in some cases of infertility, further ad-vancement in their diagnosis and treatment is required. Indeed, disorderssuch as primary ovarian insufficiency premature and primary ovarianfailure (independent of hypothalamic and pituitary defects) have yet tobe determined (reviewed in Matzuk and Lamb, 2008).

Since factors that may underlie infertility are of a very broad nature,advice for treatment requirescorrect diagnosis of the specific cause. Cur-rently, three different types of treatment are available clinically: surgical,for example laparoscopy for ablation of endometriosis; medical, forexample the use of drugs to induce ovulation; assisted reproductive tech-nology (ART), which comprises any treatment in which conception isperformed by means other than vaginal coitus. IVF, ICSI and subsequentembryo transfer are the main ART procedures, and all involve handling ofgametes or embryos in a laboratory environment. Many cases of infertil-ity can now be rectified by ART, such as IVF (e.g. in cases of female tubaldamage, ovulatory failure; Stern, 2012) and ICSI (in male-factor infertilityfor conditions such as oligozoospermia; Kashir et al., 2010).

As this review focuses upon the factors that may lead to oocyte acti-vation deficiency (OAD) and subsequent fertilization failure, the system-atic analysis of published data arising from ART can shed significant lightupon the factors that may be involved in the failure of these techniquesand their relative association with OAD.

MethodsThis work is based upon information gathered from a critical review of theliterature arising from PubMed, performed between April 2014 and July2015, and targeting studies concerning sperm and oocyte factors thatcould account for OAD. This search included studies of in vitro oocyte mat-uration in human oocytes and work arising from animal models. In addition,we focused upon clinical data available for sperm- and oocyte-related OAD.We also include a section about artificial oocyte activation (AOA), whichsummarizes recent discussion about this technique, and the concerns asso-ciated with its clinical use.

ART: analysis of the current dataSince the birth of the first in vitro baby in 1978, ART has emerged as apowerful tool for those couples seeking treatment for infertility(Johnson, 2011). Globally, around 1.5 million ART cycles are performed

per year, with an estimated 350 000 babies born worldwide. Figuresreleased by the European Society for Human Reproduction and Embry-ology (ESHRE) show that pregnancy and delivery rates following IVF andICSI barely exceed 32 and 33%, respectively (De Mouzon et al., 2009;Kupka et al., 2014). According to Mascarenhas et al. (2012), pregnancyrates per embryo transfer are 23.4% after frozen embryo transfer and47.5% after oocyte donation.

To date, IVF and ICSI have been proven to be very successful, and 1.5%of all births in the UK now result from the use of ART (reviewed inRamadan et al., 2012; HFEA, 2014). However, while the use of IVF hasbeen reported to be a suitable treatment for many infertile couples,normal IVF cycles can still fail in some cases (Bhattacharya et al., 2013).In this scenario, ICSI, which consists of the direct injection of a singlespermatozoon into the oocyte (Palermo et al., 1992), is advised andhas proven to be very effective with a mean fertilization rate of �80%(Vanden Meerschaut et al., 2013, 2014a; Neri et al., 2014). Accordingto the latest figures released by the Human Fertilisation and EmbryologyAuthority (HFEA), �52% of ART cycles in the UK now involve ICSI(HFEA, 2014).

Figure 1 shows the main causes for total fertilization failure (TFF) fol-lowing IVF and ICSI treatments, using data obtained by the HFEA for2012 (HFEA, 2014). While male factors account for 10.0% of failedIVF cases and for 50.4% of failed ICSI cases, unexplained and uncate-gorised causes have been attributed to 50.3% (IVF) and 25.8% (ICSI)of TFF cases. The differences and similarities between IVF and ICSImay help us to understand what underlies the unexplained/uncate-gorised causes in both cases. In IVF, capacitated sperm have to interactwith the oocyte zona pellucida, the acrosome reaction needs to be trig-gered, and further fusion between sperm and oocyte membranes needsto occur (reviewed in Barroso et al., 2009). However, in the case of ICSI,there is no direct interaction between sperm and oocyte membranes,but rather the oocyte membrane is mechanically pierced and biologicalobstacles are surpassed (Palermo et al., 1992; Beck-Fruchter et al.,2014). Therefore, IVF failure can be associated with asthenozoospermia,inability of sperm to capacitate properly, failure to undergo acrosome re-action, or problems during gamete fusion, given the crucial interactionbetween Juno-Izumo1, and the role of lipid raft domains in the oocytemembrane (Van Blerkom and Caltrider, 2013; Bianchi et al., 2014). Incontrast, the main cause of ICSI failure is related to a male factor in�50% of cases (Pregl Breznik et al., 2013; Zhao et al., 2014a).

Although ICSI is a procedure that allowsany form of sperm to fertilizeanoocyte and thus rescue fertility in some cases, complete fertilization failurestill occurs in 1–5% of ICSI cycles (Mangoli et al., 2008; Yanagida et al.,2008; Nasr-Esfahani et al., 2010; Vanden Meerschaut et al., 2013,2014a), which in the case of the UK affects at least 1200 couples annually(Amdani et al., 2013). ICSI failure can be attributed to several factors, suchas technical factors (e.g. incorrect injection or sperm expulsion during in-jection), sperm defects and biochemical factors (e.g. poor sperm nuclearchromatin condensation, oocyte spindle defects) (Swain and Pool, 2008;Yanagida et al., 2008). However, OAD is generally regarded as the princi-pal cause of fertilization failure following ICSI, accounting for an estimated40% of failed cases (Flaherty et al., 1998; Rawe et al., 2000; Mahutte andArici, 2003; Vanden Meerschaut et al., 2013, 2014a). In this regard, it isworth mentioning that the causes of OAD are not necessarily restrictedto sperm defects but may also involve the oocyte, since oocyte qualityalso plays a pivotal role in fertilization success (Kashir et al., 2010;Ramadan et al., 2012; Neri et al., 2014).

Oocyte factors and activation deficiency 25

Therefore, the present work first considers recent oocyte activationstudies which have focused upon sperm factors, and in particular, therole of phospholipase c zeta (PLCz). After reviewing the crucial role ofthis protein for OAD and its clinical significance, we discuss whetheroocyte factors might also have a role to play. While this is a very relevantand pertinent issue, it has received far less attention in the literature, andmay open up several options for future research.

Activation of the oocyte is a stepmediated by sperm proteinsIt is well understood that mature oocytes remain arrested atmetaphase-II (M-II) when ovulated (Stricker, 1999; Kashir et al.,2013a) and are only released from this state when they are fertilizedby a spermatozoon in a process known as ‘oocyte activation’ (Jones,2007; Machaca, 2007; Horner and Wolfner, 2008; Dale et al., 2010).The presence of a sperm in the cytoplasm of a mammalian oocyteevokes a characteristic pattern of intracellular calcium (Ca2+) oscillations(Swann, 1990; Kline and Kline, 1992a) that orchestrate a series of furtherkey events, such as cortical granule exocytosis, prevention of poly-spermy, polar body extrusion, cytoskeletal rearrangements, resumptionof meiosis, formation of pronuclei, initiation of the first mitotic division inthe new zygote, recruitment of maternal mRNA, and regulation of geneexpression (Abassi and Foltz, 1994; Swann et al., 2004; Swann and Yu,2008; Kashir et al., 2014; Fig. 2). The release of these periodic Ca2+oscil-lations over a specific temporal window is critical not only for oocyte ac-tivation, but also for the initiation of embryogenesis (Ramadan et al.,2012; Amdani et al., 2013).

The precise mechanism by which fertilized oocytes are releasedfrom M-II arrest was the source of much debate that dates back

decades. Three theories were proposed to explain this event: The‘sperm factor theory’ which suggested that a catalytic substance inthe sperm head initiated Ca2+ release in the ooplasm followinggamete fusion (Dale et al., 1985; Fissore et al., 1999a); The ‘receptortheory’ in which a signal transduction pathway was proposed to be trig-gered after a receptor located on the oolemma interacted with a spermligand (Kline et al., 1988; reviewed in Parrington et al., 2007); The‘calcium bomb’ hypothesis in which Ca2+ would enter the oocyteupon fertilization either from stores in the sperm or through channelson the sperm plasma membrane (Jaffe, 1983). The ‘calcium bomb’theory is now largely rejected, as too is ‘the receptor theory’ (Williamset al., 1998). While the receptor theory gained some credence initially,a sperm ligand/oocyte receptor mechanism that can induce the releaseof Ca2+ in a mammalian oocyte has yet to be identified and ICSI cansuccessfully initiate activation bypassing any potential membrane mech-anism completely (Neri et al., 2014).

New technologies such as calcium imaging have allowed significantsteps forward in the last three decades (Roy and Hajnoczky, 2008;Swann et al., 2009; Bruton et al., 2012; Swann, 2013), with growing evi-dence for the ‘sperm factor theory’. Strikingly, sperm soluble extractsdirectly injected into the ooplasm were seen to induce Ca2+ oscillations(Jones et al., 1998; Parrington et al., 1998a), and the microinjection ofhamster and boar sperm cytoplasmic extracts into hamster oocyteswere also able to trigger Ca2+ oscillations similar to those observedduring IVF of hamster oocytes (Swann, 1990).

Further knowledge concerning the sperm activation mechanism innon-mammalian eggs was gained during the 1990s and the firstdecade of the 21st century (Swann and Whitaker, 1990). The firstprotein proposed as a sperm-borne oocyte activation factor (SOAF)in mammals was oscillin, this nomenclature being derived from theCa2+ oscillations believed to be evoked by this protein following

Figure 1 Principal causes of infertility following IVF and ICSI treatments. The data are from the latest figures released by the UK Human Fertilisation andEmbryology Authority (HFEA, 2014).

26 Yeste et al.

fertilization (Parrington et al., 1996; Montag et al., 1998). However, laterreports using a recombinant oscillin could not confirm this role, and add-itional evidence indicated that a homologue of oscillin (known as humanglucosamine-6-phosphate isomerise) was unable to induce Ca2+ oscilla-tions upon injection into mouse oocytes (Tesarik, 1998; Fissore et al.,1999a; Wolny et al., 1999). Accumulating biochemical and clinicalevidence now strongly indicates that PLCz (1-phosphatidylinositol4,5-bisphosphate phosphodiesterase zeta-1; accession number:Q86YW0) is the key SOAF that activates the oocyte upon gametefusion (Saunders et al., 2002; Kashir et al., 2010; Amdani et al., 2013).However, it is important to note that three other sperm proteins(citrate synthase, truncated c-Kit tyrosine kinase and post-acrosomalWWP-domain binding protein, PAWP) have either been put forwardas being SOAF candidates, or have been reported to be involved inthe oocyte activation mechanism (Sette et al., 2002; Harada et al.,2007; Wu et al., 2007a, b).

PLCz: a critical SOAF

Phospholipases and PLCz

PLC enzymes participate in one of the most common pathways in intra-cellular signal transduction (Kadamur and Ross, 2013; Rhee, 2013).In this general mechanism, PLCs hydrolyse phosphatidylinositol4,5-bisphosphate (PIP2) from plasma membrane sources into inositol1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) (Yang et al.,2013). Inositol 1,4,5-trisphosphate then interacts with its receptor(InsP3R) found on the membrane of the endoplasmic reticulum (ER),which in turn allows Ca2+ release from internal ER stores. DAG,which remains attached to the plasma membrane, together with theCa2+ released from ER, then activates protein kinase C (PKC) (Fukamiet al., 2010; Antal and Newton, 2014; Taylor et al., 2014).

Currently, evidence is rapidly accumulating to indicate that PLCz is thekey SOAF responsible for the activation of oocytes (Wu et al., 2001; Cox

Figure 2 Oocyte activation triggered by a sperm-specific factor, phospholipase C zeta (PLCz). Upon oocyte-sperm membrane fusion, PLCz is releasedinto the ooplasm and interacts with an as yet unknown oocyte-borne factor (or factors), thereby facilitating the hydrolysis of PIP2 into DAG and InsP3. InsP3

subsequently triggers Ca2+ release from intracellular stores and thus initiates a cascade of events that finally alleviates MII-arrest. Briefly, Ca2+ oscillationsmediate the release of cortical granules, deactivate MAPK which allows the formation of pronuclei, and activates CaMKII. Activation of CaMKII in turn inhi-bits CSF (Emi2) which then liberates APC. APC reduced the levels of Cyclin B1 in the maturation-promoting factor (MPF) complex comprising CDK1 andCyclin B1. Degradation of Cyclin B1 inactivates the MPF which, in turn, releases the oocyte from MII-arrest. APC: anaphase-promoting complex/cyclo-some; CaM/CaMKII: calcium/calmodulin-dependent protein kinase II; CSF: cytostatic factor; CNB1: cyclin B1; CDK1: cyclin-dependent kinase 1;DAG: diacylglycerol; InsP3: inositol 1,4,5-trisphosphate; InsP3R: InsP3 receptor; MAPK: mitogen-activated protein kinase; PIP2: phosphatidylinositol4,5-bisphosphate; PKC: protein kinase C.


et al., 2002; Saunders et al., 2002, 2007; Kouchiet al., 2004; Nomikoset al.,2013a; Escoffieret al., 2015). This spermprotein hydrolyses PIP2 into InsP3

and DAG, elicitsCa2+oscillations in oocytes, and coordinates movementsin the ooplasm shortly after gamete fusion (Swann et al., 2012). Both Ca2+

oscillations and cytoplasm movements terminate around the time of pro-nuclear formation (Kashir et al., 2010; Amdani et al., 2013; Fig. 2).

In fertile men, PLCz is found in the equatorial and post-acrosomalregions (Fujimoto et al., 2004; Yoon et al., 2008), and observationsusing transmission electron microscopy following immunogold staininghave suggested that this protein is located in the perinuclear theca andalong the inner acrosomal membrane (Escoffier et al., 2015). Uponcapacitation, and even following the acrosome reaction, PLCz remainslocalized at the post-acrosomal region (Grasa et al., 2008). Thispattern of localization supports the role of PLCz as the SOAF, sinceafter the acrosome reaction, the equatorial region of the sperm mem-brane is exposed, adheres to and fuses with the oolemma (Barrosoet al., 2009). In strong support of the role of PLCz, is the fact thatmammalian oocytes can be activated following microinjection of eitherrecombinant PLCz or complementary RNA (cRNA) encoding PLCz(Yoon et al., 2008; Swann et al., 2012; Nomikos et al., 2013b).

PLCz is present in sperm from mammalianand non-mammalian species but may differin its mechanism of actionPLCz has not only been identified in human sperm but also in othermammalian species, including hamster, pig, monkey and horse(Cox et al., 2002; Saunders et al., 2002; Yoneda et al., 2006; Parringtonet al., 2007; Bedford-Guaus et al., 2008; Young et al., 2009; Cooney et al.,2010). In addition, PLCz isoforms have also been identified in non-mammalian species including sperm from chicken and medaka fish, andthe ovary of pufferfish (Coward et al., 2005, 2011; Ito et al., 2008a;Kashir et al., 2013a). Therefore, not only are the role and activity ofPLCz highly conserved across mammalian species, but also PLCzappears to play a universal role across many taxonomic groups.

Human and rodent species differ in the temporal point at which theCa2+ oscillations elicited by PLCz terminate. Indeed, while Ca2+ oscilla-tions evoked by human PLCz continue far beyond the time of pronucleiformation, those triggered by mouse PLCz cease slightly before forma-tion of pronuclei. The nuclear translocation of PLCz has only beenreported for the mouse isoform and in mouse oocytes, so that onecould venture that this nuclear translocation could explain why oscilla-tions are terminated after pronuclei formation (Cooney et al., 2010).However, despite rat PLCz not entering the nucleus, Ca2+ oscillationsin rat oocytes also stop at the time of pronuclei formation, so itremains possible that PLCz sequestration into pronuclei may contributeto the termination of Ca2+ oscillations in mice but not in all mammalianspecies (Ito et al., 2008a, b). On the other hand, species-specific differ-ences exist in the frequency and pattern of Ca2+ oscillations followingPLCz cRNA injection into oocytes. Indeed, while the injection ofmurine PLCz cRNA into mouse oocytes induces Ca2+ oscillations at10-fold lower concentrations than bovine PLCz, the efficacy of bovinePLCz in inducing Ca2+ oscillations in bovine oocytes is higher than thatof murine PLCz, and bovine PLCz fails to accumulate in mouseoocytes (Ross et al., 2008; Cooney et al., 2010). Moreover, whenhuman PLCz is injected into mouse oocytes, it exhibits far greaterpotency than PLCz from either monkeys or mice (Cox et al., 2002).

Mechanism of PLCz actionTo date, the exact process by which PLCz activates the molecularmechanisms that promote oocyte activation still remains unclear, al-though the critical roles played by its discrete functional domains arehighly apparent (Nomikos et al., 2005). In this regard, one has to con-sider that PLCz is the smallest PLC isoform in mammals with a molecu-lar mass of 70.4 kDa in the case of humans (Nomikos et al., 2013a).PLCz comprises four EF hand domains, a C2 domain, and catalyticX and Y core domains. Kouchi et al. (2005) reported that EF1 andEF2 domains are important for protein activity, while EF3 is responsiblefor its high Ca2+ sensitivity (Yoda et al., 2004). Screening interactionsbetween the C2 domain and phosphoinositides revealed that theC2 domain exhibits substantial affinity for phosphatidylinositol3-phosphate and for phosphatidylinositol 5-phosphate but not forPIP2, and that phosphatidylinositol 3-phosphate and phosphatidylinosi-tol 5-phosphate reduce PLCz activity via a negative-regulatory mech-anism (Kouchi et al., 2005).

Most PLC isoforms (e.g. PLCd1), but not PLCz, reside in the plasmamembrane, are linked to G protein-coupled receptors and possess aPH domain (Williams et al., 1998; White et al., 2010). While the PHdomain typically binds to and hydrolyses PIP2 found in the plasma mem-brane (Suh et al., 2008; Bunney and Katan, 2011), PLCz is devoid of thisparticular domain and instead, targets its substrate via the X-Y linkerregion and PIP2-containing vesicles distributed across the ooplasm(Yu et al., 2012; Theodoridou et al., 2013). Therefore, upon thefusion of sperm and oocyte plasma membranes, PLCz has been pro-posed to diffuse across the ooplasm within the space of 10 min,target PIP2 located in oocyte vesicles and initiate the first calciumwave within 30 min of gamete fusion (Saunders et al., 2007; Swannand Lai, 2013).

Finally, and aside from the absence of a PH domain, there is anotherkey feature that makes PLCz different from the other somatic isoforms(-b, -g, -d and -1), since the XY-linker region of PLCz does notmediate catalytic auto-inhibition, but rather confers maximal enzymaticactivity (Nomikos et al., 2007, 2011b; Yu et al., 2012).

PLCz appears to be involved in functionsother than oocyte activationWhile it is clear that PLCz is involved in oocyte activation, its involvementin other vital reproductive events has also been suggested (Young et al.,2009). For example, there is emerging evidence for a potential role ofPLCzduring early embryonic division. In mouse, for example, Ca2+oscil-lations occur when PLCz is distributed throughout the ooplasm andcease when the nuclear localization signal drives PLCz into the develop-ing pronuclei (Larman et al., 2004). Once the first mitotic division isinitiated, Ca2+ oscillations are resumed, presumably because PLCz isreleased into the ooplasm following the breakdown of nuclear envelope(Larman et al., 2004; Yoda et al., 2004). Therefore, while the function ofthese mitotic Ca2+ spikes remains to be elucidated, it appears that theyexert an effect upon both pre- and post-implantation development in amanner that is independent of their ability to activate oocytes (Soneet al., 2005). In this context, it is worth mentioning that not only arethe oocytes able to decipher a specific pattern of Ca2+ oscillations, butalso gene expression profiles in the early embryo rely on this signallingpathway (Ozil et al., 2006).

28 Yeste et al.

OAD occurs in sperm exhibitingPLCz deficienciesDefective fertilizing sperm are known to cause arrest of development atmultiple levels during embryo preimplantation development (Colom-bero et al., 1999; Barroso et al., 2009). Thus, one of the most relevantaspects that merits particular attention is the relationship betweenPLCz and male infertility. Whilst fertile men present, overall, a significant-ly higher proportion of sperm exhibiting PLCz than infertile men (Kashiret al., 2013b; Yelumalai et al., 2015), deficiencies, abnormal localization,reduced activity/expression, or genetic mutations in PLCz have beenassociated with OAD and ICSI failure (Yoon et al., 2008; Heytenset al., 2009, 2010; Aghajanpour et al., 2011; Kashir et al., 2011, 2012a,c; Nomikos et al., 2011a; Lee et al., 2014; Yelumalai et al., 2015). Thepresence of morphologically abnormal spermatozoa has also beenrelated to deficient forms of PLCz, and the total absence of PLCz(Yoon et al., 2008; Heytens et al., 2009; Lee et al., 2014). One of themost severe and rare cases (incidence ,0.1%) is globozoospermia(see Dam et al., 2007) in which morphologically abnormal spermwithout acrosomes and significant DNA fragmentation can be totallydevoid of PLCz (Taylor et al., 2010; Kashir et al., 2012b; Yassine et al.,2015).

Furthermore, two point mutations in the PLCz gene have been iden-tified in a non-globozoospermic infertile patient.These mutations causedthe substitution of histidine to proline at residue 398 (H398P) withinthe Y domain, and the substitution of histidine to leucine (H233L)within the X domain, resulting in a significantly impaired ability to activatethe oocyte (Heytens et al., 2009; Kashir et al., 2012a, c).

OtherspermproteinsaspotentialSOAF candidatesTo date, four sperm-borne proteins have been put forward as predom-inant candidates for the SOAF. These four proteins are PLCz, a trun-cated form of c-kit tyrosine kinase receptor, citrate synthase, andPAWP. While there is extensive evidence for the role of PLCz(reviewed in previous section), the relative importance of the otherthree proteins is far less clear, and in the case of PAWP, rather contro-versial at present.

Citrate synthase and the truncated formof c-kit tyrosine kinase receptorThe truncated form of c-kit tyrosine kinase receptor (tr-kit) is present inthe equatorial and post-acrosomal sheath of sperm (Muciaccia et al.,2010), and has been reported to alleviate M-II arrest in mouse oocytesand trigger other events of early embryogenesis such as corticalgranule exocytosis and the formation of pronuclei (Sette et al., 1997,2002; Rossi et al., 2003). According to Sette et al. (2002), tr-kit mightinteract with Fyn, a Src-like tyrosine kinase residing in the oocytecortex, and Fyn (or perhaps another kinase) would then phosphorylatea tyrosine residue of tr-kit (Tyr161). This would allow tr-kit to interactwith the SH2 domain of Fyn and subsequently permit the phosphoryl-ation of a tyrosine residue (Tyr783) in the SH region (Carpenter and Ji,1999). The tr-kit/Fyn complex would thus finally activate the oocyteby interacting with the SH3 domain of PLCg1 (Sette et al., 1998,2002). However, while the role of PLCg has been investigated in non-

mammalian species (Sato et al., 2006), tr-kit has yet to be proven toelicit Ca2+ oscillations in oocytes. This makes it difficult to considertr-kit further as a SOAF in mammals.

On the other hand, the suggested role for citrate synthase arose fromthe work of Harada et al. (2007) in amphibians. These authors observedthat the protein responsible for egg activation in the newt Cynops pyrrho-gaster was 45 KDa and resided in the neck and intermediate piece of thesperm. This protein was partly homologous to citrate synthase inXenopus. However, injection of mouse PLCz was also able to activatenewt eggs. Although this may not explain what happens in mammals, itdoes highlight the variety of mechanisms present in the vertebratekingdom. In fact, general consensus of opinion amongst researchershas now discarded tr-kit and citrate synthase as SOAF candidates inmammals, with most evidence supporting the role of PLCz and emergingdata indicating a potential role for PAWP.

PAWPPAWP (also known as WBP2) is a sperm protein exclusively located inthe post-acrosomal sheath of the sperm perinuclear theca (Wu et al.,2007a, b), which surrounds the nucleus except in the caudal regionwhere the tail is implanted. This region consists of a condensedprotein layer that represents the major cytosolic fraction in the spermhead and is made up of two different regions: the sub-acrosomal layerand the post-acrosomal sheath (Oko and Sutovsky, 2009; Ferrer et al.,2012). Given the dynamics of sperm interaction with the oocyte, thepost-acrosomal sheath has been suggested to be involved in oocyteactivation (Sutovsky et al., 2003; Toshimori and Ito, 2003; Ito et al.,2009, 2010).

While some authors have found that injecting recombinant PAWP orprotein extracts from the perinuclear theca into porcine, bovine,macaque, human, and Xenopus oocytes alleviates M-II arrest (Wuet al., 2007a; Aarabi et al., 2010, 2014a; Kennedy et al., 2014), othersreport that PAWP does not trigger calcium oscillations in mouseoocytes (Nomikos et al., 2014, 2015a). Furthermore, total levels ofPAWP evaluated through immunohistochemistry have been associatedwith sperm quality and fertilizing ability in bulls (Kennedy et al., 2014),and with fertilization rates and embryo development following ICSI inhumans (Aarabi et al., 2014b).

Whether PAWP acts as a SOAF remains to be fully confirmed, but inthe context of the present review, evidence does appear to indicate apossible role in oocyte activation. If this was the case, failures in someof the oocyte proteins interacting with PAWP could also underliesome cases of OAD. The C-terminal PAWP region is rich in prolineand presents a functional PPXY consensus sequence that bindsWW-group I protein domains. These WW domains mediateprotein–protein interactions in a way that is similar to SH3 domains.When the PPXY region is blocked, PAWP does not activate theoocyte. For this reason, Aarabi et al. (2014a) suggested that the PPXYdomain present in PAWP interacts with the WW-group I proteindomain from PLCg following a non-canonical pathway. The activationof PLCg would then hydrolyse PIP2 and this would initiate Ca2+ oscilla-tions. This mechanism, however, requires further research in order tovalidate its authenticity and any relevance to oocyte activation. Such add-itional work is particularly important because other reviews have pointedout that PLCg does not mediate Ca2+ oscillations in mouse oocytes, andalso that the negative-control peptide used by Aarabi et al. (2014a) did


not affect the pattern of sperm-induced Ca2+ oscillations (Nomikoset al., 2015b).

Controversies surrounding the relativeimportance of PLCz and PAWPAlthough PLCz has been accepted as the SOAF for over a decade, theauthors that first proposed PAWP as the SOAF also raised concernsabout PLCz, largely due to the fact that PLCz is found in mouse andhuman sperm compartments other than the perinuclear theca whereSOAF should be expected to reside (Aarabi et al., 2012). In addition,Aarabi et al. (2012) challenged the role of PLCz as they failed to detectPLCz in spermatids at the end of spermiogenesis, and failed to demon-strate PLCz-immunostaining following the acrosome reaction, zona pel-lucida penetration, sperm–oocyte fusion, and sperm entry into theooplasm. Thus, in contrast to Escoffier et al. (2015), Aarabi et al.(2012) did not find PLCz in the perinuclear theca of mouse and humansperm.

Furthermore, Aarabi et al. (2012) also suggested that principal epididy-mal cells secrete PLCz. With regard to this, it is worth mentioning thatepididymal maturation involves the rearrangement of a variety of pro-teins, mainly on the sperm membrane surface (Dacheux and Dacheux,2013). However, PLCz has been identified in the perinuclear theca(Escoffier et al., 2015) and in the soluble-chromatin fraction of thehuman sperm head (Castillo et al., 2014). If PLCz is in the sperm chroma-tin soluble fraction, i.e. proteins that are weakly attached to the spermDNA and play a regulatory rather than structural role, it is quite unlikelythat it arises from epididymal maturation, but rather it should arise duringspermatogenesis.

Finally, while Aarabi et al. (2014b) demonstrated a link betweenPAWP and fertility outcomes from ICSI, reduced levels of PLCz(Kashir et al., 2013b) or genetic mutations in the PLCz coding region(Heytens et al., 2009; Kashir et al., 2012a) also affect fertility outcomesand can be rescued with PLCz cRNA and recombinant protein (Yoon

et al., 2008; Nomikos et al., 2013b; Yelumalai et al., 2015). Consequently,it is not yet clear whether there is one sole oocyte activation factor ormore, or whether different factors work together (Table I). In anycase, this issue remains the source of significant debate at present,because other research groups have failed to repeat the experimentsproviding evidence for PAWP (Nomikos et al., 2014, 2015b; Aarabiet al., 2015; Amdani et al., 2015; Vadnais and Gerton, 2015).

Could oocyte factors explainsome cases of OAD?In a recent study, total levels and proportions of sperm exhibiting PLCzwere found to be significantly correlated with ICSI fertilization outcomes;however, not all cases of TFF were explained by deficiencies in thisprotein (Yelumalai et al., 2015), at least using the tools and techniquesdeployed in this particular study, which relied heavily upon the use of apolyclonal antibody. While the specificity of the antibody used byYelumalai et al. (2015) has been confirmed in this and other studies(Grasa et al., 2008; Kashir et al., 2013b), and supported by appropriatepeptide blocking assays, it should, of course, be noted that the disparatespecificity of polyclonal antibodies could influence experimental out-comes (Ramadan et al., 2012; Amdani et al., 2013, 2015; Kashir et al.,2013b; Nomikos et al., 2013b). The entire body of work describedthus far in the literature for PLCz localization, by ourselves and otherresearch groups, relies solely upon the use of polyclonal antibodies. Itis therefore prudent to treat such data with a certain amount ofcaution until such work has been verified by a more powerful and reliablegeneration of monoclonal antibodies.

However, aside from this concern, and while it could be argued thatsperm factors other than PLCz could also account for OAD, it is entirelyreasonable to venture that oocyte factors may also contribute to suchfailure since the series of critical downstream signalling events triggeredby the sperm rely heavily upon oocyte-borne factors. This is supported

.............................................................................................................................................................................................

Table I PLCz, PAWP, and implications for oocyte activation deficiency (OAD).

Factor Description Possible contribution to OAD References

PLCz Significant evidence already exists for PLCz’s primary roleas the SOAF. PLCz hydrolyses PIP2 into InsP3 and DAG andelicits Ca2+ oscillations within the oocyte.In fertile men, PLCz is located along the inner acrosomalmembrane, and in the perinuclear theca of the equatorialand post-acrosomal regions.

Fertile men present a significantly higher proportion ofsperm exhibiting PLCz than infertile men.Deficiencies, abnormal localization, activity/expression, orgenetic mutations in PLCz have been linked to OAD andICSI failure.Two point mutations in the PLCz gene have been identifiedin a non-globozoospermic infertile patient resulting in asignificantly impaired ability to activate oocytes.

Cox et al. (2002)Escoffier et al. (2015)Fujimoto et al. (2004)Heytens et al. (2009)Heytens et al. (2010)Kashir et al. (2012a)Kashir et al. (2012c)Kouchi et al. (2004)Lee et al. (2014)Nomikos et al. (2011a)Saunders et al. (2002)

PAWP PAWP is another sperm-specific protein that has beenrecently proposed as a SOAF. This protein is exclusivelylocated in the post-acrosomal sheath of the spermperinuclear theca. Exact relevance has yet to be confirmedbut could play a role in oocyte activation, whether a SOAFrole is proven or not.

Total levels of PAWP evaluated via immunohistochemistryhave been associated with sperm quality and fertilizingability in bulls, and with fertilization rates and embryodevelopment following ICSI in humans.

Aarabi et al. (2010)Aarabi et al. (2014a)Aarabi et al. (2014b)Kennedy et al. (2014)Wu et al. (2007a)Wu et al. (2007b)

PLCz: phospholipase C zeta; PAWP: post-acrosomal WWP-domain binding protein; SOAF: sperm-borne oocyte activation factor; DAG: diacylglycerol; InsP3: inositol1,4,5-trisphosphate.

30 Yeste et al.

by outcomes from the mouse oocyte activation test (MOAT; Hein-dryckx et al., 2005) since in some couples that fail ICSI the malepartner presents a sufficient number of sperm exhibiting PLCz and theMOAT shows that such sperm are fully intact and functional. While itshould be noted that the results of Ca2+ imaging in mouse oocytescannot be directly extrapolated to human oocytes because humanPLCz presents higher potency than the mouse isoform, it is assumedthat MOAT-activation deficiency might be due to an oocyte-bornefactor (Heindryckx et al., 2005, 2008). In any case, reviewing the poten-tial contribution of the oocyte in OAD appears highly prudent at thistime.

Clinical data that specifically discuss the possibility of oocyte-relatedOAD remain scarce. In this regard, one needs to take into accountthat while sperm causes may be evaluated via the assessment of PLCzor by the MOAT, ethical issues and restrictions on the availability ofoocytes for research render it much more difficult to evaluate oocyteproblems. Therefore, one might suggest that an oocyte problem mightbe associated with cases of OAD if sperm factor problems have beeneliminated. In addition, it is difficult to define to what extent suchoocyte factors might specifically underlie OAD, or whether they areinvolved in events downstream of oocyte activation. Irrespective, bothscenarios will lead to fertilization problems, which is the main clinicaloutcome in this context.

There are a series of case reports that have suggested, but failed toprove, that TFF resulted from an oocyte-related OAD deficiency.Tesarik et al. (2002) and Eldar-Geva et al. (2003) were the first tosuggest that some OAD cases could be due to undisclosed oocyte-bornefactors. Two subsequent studies, using data derived from the MOAT,further suggested oocyte-related OAD in 8 out of 17 cases (47%), and7 out of 30 cases (23%), respectively (Heindryckx et al., 2005, 2008).In addition, Combelles et al. (2010) reported a case of oocyte-relatedOAD that could not be rescued by AOA, but which was explained bythe presence of multiple polar bodies and two disorganized spindle struc-tures. In contrast, Vanden Meerschaut et al. (2012) reported cases ofoocyte-related OAD that could be overcome by AOA. This led theseauthors to suggest that AOA could be only effective when an underlyingcytoplasmic defect, rather than a structural cytoskeletal-related ornuclear deficiency, was the cause of the oocyte-borne OAD. Finally, aretrospective study found that 75% of a cohort of patients with TFF fol-lowing ICSI was due to a male factor while 25% was attributed to unex-plained infertility (Shinar et al., 2014). It is worth noting that in theiranalysis, these authors did not include cases of poor ovarian reserverelated to advanced age, and included cycles in which five or moremature MII-oocytes were retrieved. Using these data, these authors spe-cifically highlighted the relevance of the oocyte in cases of TFF, since ICSIoutcome was positive when donor oocytes were used.

It should be acknowledged that the number of oocyte-related OADcases is still low and that there is some disparity in the data. In addition,care should be taken to interpret such cases since ‘cryptic’ sperm defects(apparently normal sperm that might underlie OAD and TFF), shouldalso be considered (Goudakou et al., 2012), especially in cases wherePLCz testing or the MOAT are not conducted. Consequently, the pos-sibility of an oocyte factor contribution to OAD is more complex than itmay appear. The identity of such oocyte factors, however, remainslargely unknown. For this reason, the following sections speculateupon which elements of the oocyte’s internal biological machinerymight be involved.

At first glance, a role for the oocyte during either oocyte activation, orindeed, downstream of this critical event, was first suggested by studiesemphasizing the relevance of oocyte quality. There are cases in whichTFF following ICSI occurs due to poor oocyte morphology, determinedby the presence of an abnormal spindle and interphase microtubules(Miyara et al., 2003; Xing et al., 2011; Kilani and Chapman, 2014).Other studies discuss factors that are produced by oocytes andcumulus cells which play a role in oocyte maturation and determine de-velopment potential and embryo quality (Ebner et al., 2006). Forexample, the expression of genes involved in meiosis, cell growth andapoptosis was altered in MII-oocytes from a patient presenting withTFF (Gasca et al., 2008). Li et al. (2014) further reported the relevanceof growth differentiation factor 9 (GDF9) and bone morphogeneticprotein 15 (BMP15) transcript levels in cumulus cells for successfuloocyte maturation. Finally, oocyte proteins involved in the resumptionof M-II are also of relevance, as are proteins involved in the cell cycle,such as cell division cycle proteins (CDC7, CDC20, CDC25), check-point kinase 2 (CHEK2) and cyclin B1 (CCNB1) (Grøndahl et al.,2013). Other oocyte proteins that could be involved in OAD are dis-cussed in the following sections.

The SOAF triggers a cascademediated by vital oocyte factorsIt is worth noting that although different sperm proteins have been pro-posed to be the SOAF, all candidates have been suggested to evoke Ca2+

oscillations and are known to be involved in a PLC/phosphoinositidetransduction pathway (Wu et al., 2001). So, either the SOAF itself is aPLC, as in the case of PLCz (Saunders et al., 2002; Kashir et al., 2010;Swann and Lai, 2013), or it involves the activation of oocyte PLCs, as sug-gested for PAWP (Aarabi et al., 2014a). In any case, both PLCz andPAWP trigger a downstream cascade that requires oocyte factors. Add-itionally, there are other signs indicating that the molecules that interactwith the SOAF are oocyte-specific. Indeed, while PLCz can be expressedin somatic cells, such as those from the Chinese hamster ovary, it isunable to elicit Ca2+ oscillations in these cells (Phillips et al., 2011).This suggests that either one or more unknown specific proteins thatare only present in the oocyte interact with PLCz resulting in Ca2+ oscil-lations, or that PLCz activity is inhibited by somatic cell factors.

On the other hand, specific peculiarities of PLCz render this phospho-lipase different from others, which in turn suggests a particular mechan-ism of action that has yet to be fully elucidated. As mentioned earlier,PLCz is the only PLC that does not possess a PH domain (Saunderset al., 2002). In other phospholipases, the PH domain targets PIP2

located on the inner leaflet of the plasma membrane (Garcia et al.,1995; Tuzi et al., 2003; Suh et al., 2008; Bunney and Katan, 2011).Instead, PLCz appears to hydrolyse PIP2 molecules that are present inoocyte vesicles distributed across the cytoplasm rather than thosepresent in the inner oolemma leaflet (Yu et al., 2012). This not onlymatches previous observations made by Yu et al. (2008), who reportedthat DAG generated by PLCz is not found in the oolemma, but also raisesfurther questions such as whether the targeting of PIP2 residing in oocytevesicles is mediated by one or more than one oocyte-specific protein/slocated in the membrane of those vesicles, as hypothesized recently(Swann and Lai, 2013). Although this theory has yet to be confirmed, itis entirely reasonable, and would explain the specificity of PLCz for


PIP2 from sources other than the plasma membrane, and why PLCz ac-tivity is oocyte-specific. While one could venture that the C2 domain isinvolved in targeting PLCz from these vesicles, as happens with thisdomain in other PLC isoforms (Swann and Lai, 2013), the existence ofthis oocyte protein would also explain why C2 domains from PLCz,but not those from other PLCs, are able to hydrolyse PIP2 fromoocyte vesicles (Fig. 2).

Finally, the existence of such an oocyte receptor could explain somecases of OAD and ICSI failure, in which the cause is not thought to bea deficiency in sperm PLCz. Therefore, more research is warranted toconfirm this hypothesis and to address whether this unidentifiedprotein could represent a prognostic marker with clinical and therapeuticimplications.

PIP2 and DAGApart from suggestions regarding a putative PLCz interactor in theoocyte, other oocyte factors that take part in the PLC pathway (suchas PIP2, DAG, InsP3R, or Ca2+ channel proteins) could also be involvedin OAD (Fig. 2 and Table II). Of these additional factors, one couldsuggest that PIP2 is a logical starting point as there is a considerablenumber of vesicles throughout the ooplasm containing PIP2 that are spe-cifically targeted by PLCz (Yu et al., 2012). As downstream events leadingto embryogenesis depend on the availability of InsP3 and DAG, and thesedepend upon the availability of sufficient amounts of PIP2, it could behypothesized that an abnormally low content of PIP2 in oocyte vesicles,or a reduced number of vesicles, could readily lead to OAD (Table II).This suggestion is supported by previous studies in which depletion ofPIP2 from cytoplasmic vesicles by inositol phosphatases led to the abol-ishment of Ca2+ oscillations following fertilization (Yu et al., 2008).However, comparative studies evaluating the number of vesicles in differ-ent oocytes have yet to be conducted.

Although there is agreement concerning PLCz and its ability to hydro-lyse PIP2, only a limited number of studies have focused on the role ofDAG. In this regard, it appears that not only PIP2 from the plasma

membrane is unaffected after injection of PLCz, but also DAG generationin the plasma membrane is relatively minimal (Yu et al., 2008). Significantproduction of DAG in the ooplasm occurs when PKCd1 (a PKC isoform)rather than PLCz, is over-expressed and this is concomitant with a seriesof unexpected secondary high-frequency Ca2+ oscillations. These sec-ondary Ca2+ oscillations can be mimicked in a variety of situations bythe stimulation of PKC and can be prevented by PKC inhibition, sothat overproduction of DAG in PKCd1-injected oocytes can lead toPKC-mediated Ca2+ influx and subsequent overloading of Ca2+ stores(Yu et al., 2008).

While PIP2 has been proven to exist in vesicles distributed across theoocyte cytoplasm, it remains to be confirmed whether PLCz-generatedDAG remains in the membrane of such vesicles. In addition, transversediffusion (or flip-flop) of DAG across the two leaflets of the lipid bilayerdepends on the fluidity of the membrane bilayer, which in turn relies uponthe composition and ratio of cholesterol:unsaturated glycerophospholi-pids in this membrane. This is important since the regulation of trans-bilayer movement of DAG may modify its binding to the C1 domain ofPKC and this could, in turn, affect the downstream PKC-activatedcascade (Ueda et al., 2014).

The central role of InsP3R

Isoforms and regulationInsP3 interacts with its receptor (InsP3R), a ligand-gated channel, via twophosphate groups that bind to opposite sides of the InsP3-binding site(Fig. 2). This leads to a conformational change that opens the channeland allows Ca2+-release from internal stores, such as the ER (Bhanum-athy et al., 2012). The final InsP3R structure arises from the assemblyof four identical (homotetramer) or different subunits (heterotetramer),and this is reliant upon the cell type (Alzayady et al., 2013). Up to threedifferent isoforms for this receptor (InsP3R1, InsP3R2, InsP3R3) havebeen described thus far (Ivanova et al., 2014).

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Table II Potential oocyte factors with implications for OAD (I): PIP2, InsP3 receptor and protein kinase C.


PIP2 PLCz specifically targets PIP2 found in the membraneof intracellular oocyte vesicles. PIP2 is thenhydrolysed into InsP3 and DAG.

Depletion of PIP2 in cytoplasmic vesicles by inositolphosphatases abolishes Ca2+ oscillations at fertilization.Defects/deficits in PIP2 and/or reduced amounts ofoocytes vesicles containing PIP2 could contribute to OAD.

Yu et al. (2008)Yu et al. (2012)

InsP3R InsP3 binds to InsP3R located on the endoplasmicreticulum membrane. InsP3R then opens an intrinsicchannel and allows Ca2+ release from the ER.

Inhibition of InsP3Rs abolishes Ca2+ oscillations. Geneticmutations leading to abnormal protein folding or abnormaltrafficking of InsP3Rs could result in OAD.

Cooney et al. (2010)Jellerette et al. (2000)Miyazaki et al. (1993)Ross et al. (2008)Wojcikiewicz et al. (1994)

PKC PKC is activated by DAG and phosphorylatesdifferent substrates, including MARCKS. PKC issuggested to phosphorylate and activate otherunidentified membrane channels.

Inhibition of PKC terminates sperm-induced Ca2+

oscillations and prevents Ca2+ influx that takes place afterCa2+ depletion in the ER. Genetic mutations leading toabnormal protein folding could contribute to OAD.

Gallicano et al. (1997)Gonzalez-Garcia et al. (2013)Halet et al. (2004)Luria et al. (2000)Tatone et al. (2003)Yu et al. (2008)

InsP3R: InsP3 receptor; PIP2: phosphatidylinositol 4,5-bisphosphate; PKC: protein kinase C; MARCKS: myristoylated alanine-rich C-kinase substrate; ER: endoplasmic reticulum.

32 Yeste et al.

Complexity across the InsP3R-signalling pathway implies that othermolecules are involved in the regulation of its activity, including kinasesand phosphatases, such as kinase PKA and phosphatases PP1 andPP2A (DeSouza et al., 2002). In addition, cyclic adenosine monopho-sphate (cAMP) also increases the sensitivity of InsP3Rs (Taylor et al.,2014), and calmodulin (CaM), a ubiquitous Ca2+ sensor protein, isalso involved in the regulation of InsP3R via binding a specific sitepresent in this receptor (Kasri et al., 2006). However, whether this oper-ates during oocyte activation has yet to be demonstrated.

Evidence for the role of the InsP3R duringoocyte activation and maturationInsP3R levels are correlated with variations in Ca2+ levels (retention ofCa2+ in the cytosol sensitizes the InsP3R) and, after fertilization, highlevels of InsP3 inevitably lead to the down-regulation of InsP3Rs (Wojci-kiewicz et al., 1994; Parrington et al., 1998b; Jellerette et al., 2000; Rosset al., 2008; Cooney et al., 2010). Indeed, the degradation of InsP3Rdepends upon uniform and sustained elevated levels of InsP3, whichresults in a diminished periodicity of calcium oscillations (Miyazakiet al., 1993; Lee et al., 2010). Further evidence of the role of InsP3 inInsP3R down-regulation is that adenophostin, a potent agonist forInsP3R (Sato et al., 1998), down-regulates InsP3R but does not triggerthe activation of oocytes (Brind et al., 2000). In addition, down-regulationof InsP3R may not completely prevent Ca2+ oscillations (Malcuit et al.,2005). Therefore, while Ca2+ oscillations are almost entirely mediatedby InsP3R, it remains to be determined whether all Ca2+ is released fol-lowing InsP3 interaction with InsP3R, or whether other calcium stores areinvolved (Table II).

On the other hand, InsP3R1 not only plays a relevant role for the ac-tivation of mammalian oocytes but also for their maturation and earlyembryo development. Indeed, the resumption of meiotic division in im-mature oocytes (i.e. at germinal vesicle stage) is also mediated by a phos-phoinositide pathway involving InsP3 present both in the ooplasm andnucleoplasm. In this context, it is worth mentioning that the nuclearmembrane also presents InsP3R1 and blocking these receptors leadsto the inhibition of germinal vesicle breakdown (Pesty et al., 1998).

In addition, during oocyte maturation there is an increase in the sensi-tivity of InsP3R1-mediated Ca2+-release, mediated by the phosphoryl-ation of the Thr2656 residue in InsP3R1 by polo-like kinase 1 (PLK1),that may ultimately affect the oocyte’s ability to be activated (Xu et al.,2003). The relevance of PLK1 is apparent from studies showing thatPLK1-inactivation in mouse oocytes at the early stages of maturation sig-nificantly reduced InsP3R1 phosphorylation (Vanderheyden et al., 2009).However, regulation of InsP3R1 is a far more complex process since itdoes not only involve phosphorylation by PLK1 but also by mitogen-activated protein kinase/extracellular regulated kinase 2 (MAPK/ERK2) (Lee et al., 2006).

Finally, InsP3R1 can be involved in the apoptosis pathway, sinceCaspase 3 is able to cleave InsP3R1, thereby generating a truncatedform of this receptor. This truncated InsP3R1 consists of a C-terminalfragment of 95 kDa that comprises the complete channel domain. Inter-estingly, aged mouse oocytes present this truncated form, which pre-vents Ca2+ oscillations to be evoked and induces an apoptoticphenotype when it is injected into mouse oocytes. This explains OADin aged oocytes, since they are diverted into an apoptosis pathway(Verbert et al., 2008).

InsP3Rs are present in oocytes frommammalian and non-mammalian species,but their intracellular distribution can differbetween speciesThe presence and relevance of InsP3Rs for oocyte activation was firstreported in Xenopus oocytes, where they were seen to organize intomicroscopic and macroscopic clusters (Mak and Foskett, 1997; Zhanget al., 2007). InsP3R1 is present in the ER and perinuclear region ofthese immature oocytes, and relocalises during oocyte maturation andfollowing fertilization (Kume et al., 1993, 1997; Toranzo et al., 2014).In addition, InsP3R3 is also present in the plasma membrane ofXenopus oocytes and for this reason is thought to be involved in Ca2+

entry from the extracellular space (Putney, 1997). However, this doesnot seem to be the case for mammalian oocytes, as discussed in the fol-lowing section.

In pre-ovulatoryand M-II mouse oocytes, InsP3R1 is distributed acrossthe cytosol (i.e. from cortex to perinucleus) and its expression significant-ly increases during oocyte maturation. This indicates that there is apropagating Ca2+ release from the cortex to the interior of the oocytethat is involved in oocyte maturation. In contrast, InsP3R2 (aggregatedin isolated islands in the cortex) and InsP3R3 are also present but inlower amounts than InsP3R1 (Fissore et al., 1999b).

In bovine species, the three isoforms are also expressed in M-IIoocytes, but again InsP3R1 is present in a larger quantity than theothers. InsP3R1-downregulation occurs following fertilization and pro-nuclei formation indicating that this isoform participates in oocyte activa-tion (He et al., 1999). In pigs, localization and expression changes ofInsP3R1 during oocyte maturation are thought to be mediated byPLK1, and no differences in the distribution of InsP3R1 were observedbetween in vivo and in vitro maturation. Furthermore, it is worth notingthat while InsP3R1 forms large clusters in the cortex of mouse oocytes,these structures are not observed in their pig counterparts (Sathana-wongs et al., 2015). In addition, it is worth noting that total expressionlevels and distribution of InsP3R1 in the oocyte cytoplasm have beenreported to be affected by vitrification-warming procedures in bothmouse and pig oocytes (Kim et al., 2011; Hirose et al., 2013).

In humans, InsP3R1 changes from a diffuse, irregular and granular dis-tribution in immature oocytes (i.e. at the germinal vesicle stage) to a re-ticular and peripheral localization at the M-II stage. After fertilization,InsP3R1 distribution changes again from a localization in the peripheryand centre in zygotes and 2–4-cell embryos to a predominant patternacross the cytosol in 6–8-cell embryos. Apart from changes in the distri-bution of InsP3R1, there is also an up-regulation of this receptor fromimmature to mature oocytes and from zygotes to 6–8-cell embryos(Goud et al., 1999).

PKC

The PKC familyFollowing the downstream cascade, another protein that plays a key roleduring oocyte activation and at the beginning of embryogenesis is PKC(Fig. 2). Protein kinases are involved in transduction pathways and phos-phorylate serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. Spe-cifically, PKC is a ubiquitous Ser/Thr-protein kinase that is involved in the


phosphoinositide pathway and is activated by Ca2+ and DAG (Inoueet al., 1977; Nishizuka, 1992).

Three different sub-families have been described according to theirstructural and activation features: conventional PKCs (a, bI, bII and g);non-classic, also known as novel, PKCs (d, 1, h and u); atypical PKCs(z and i/l). While conventional PKCs are activated by DAG andrequire phosphatidylserine and Ca2+ as cofactors, non-classic PKCsare activated by DAG and phosphatidylserine but not by Ca2+, and atyp-ical PKCs only depend upon phosphatidylserine (i.e. are independentfrom DAG or Ca2+) (reviewed by Kang et al., 2012). Some of these iso-forms are present in all tissues, while others are tissue-specific.

All members of the conventional family (a, bI, bII and g) present fourconserved domains (C1–C4). The first domain (C1) presentscysteine-rich zinc finger-like motifs and the recognition sites for DAG,phosphatidylserine, and phorbol ester. The second conserved domain,known as C2, contains the binding site for Ca2+ and is rich in acidic resi-dues. Finally, C3 and C4 domains are those that contain binding sites forATP and substrates (Newton, 1995; Kanashiro and Khalil, 1998). Al-though it is well understood that conventional PKCs are activated byDAG and by Ca2+ released from the ER after InsP3-stimulation, thefull list of substrates that PKC is able to phosphorylate has yet to bereported. In this regard, one could venture that substrates for PKCinclude unidentified membrane channels, since PKC is also involved instore-operated calcium influx (See below in Section about Ca2+ oscilla-tions and homeostasis in the oocyte; Halet et al., 2004).

PKC and the mammalian oocyteDifferent PKCs are known to be present in mammalian germinal vesicleand M-II arrested oocytes. Indeed, mouse and rat oocytes are known toexhibit conventional (PKCa, PKCb and PKCg), non-classic (PKCd andPKCu) and atypical isoforms (PKCz and PKCi/l) (Jones, 1998;Pauken and Capco, 2000; Baluch et al., 2004).

One interesting issue relates to the distribution of conventional PKCisoforms. Prior to oocyte activation, PKCa is uniformly distributed inthe cytoplasm and PKCbI and PKCbII are both present in the cytoplasmand in the plasma membrane. Following sperm–oocyte fusion, PKCatranslocates to the oocyte cortex. This translocation is considered akey feature of oocyte activation with kinetics similar to the Ca2+ oscilla-tions induced by sperm. This dependency in Ca2+ oscillations ismediated by the C2 domain of PKC (Luria et al., 2000; Halet et al.,2004). Furthermore, while levels of PKCbI in the ooplasm decreaseafter oocyte activation, the distribution of PKCbII remains unaffected.

On the other hand, Chen et al. (2014) have suggested that PKCd andPKCu are also involved in oocyte maturation. Indeed, these two proteinsappear to mediate the response to FSH in cumulus oocyte complexes,which ultimately results in the meiotic resumption of germinal vesicleoocytes.

The role of PKCa during oocyte activationThe role of PKCa in oocyte activation has been confirmed by its involve-ment in the generation of the second polar body and the exocytosis of cor-tical granules (Gallicano et al., 1997; Eliyahu and Shalgi, 2002; Fig. 2). In fact,not only does PKCa activity increase after fertilization and return to base-line levels following Ca2+ oscillations (Gallicano et al., 1997; Tatone et al.,2003), but also inhibiting PKCa with Bisindolylmaleimide-I leads toOAD (Halet, 2004). Increased PKCa activity has been observed in the

plasma membrane but also in the cytoplasm, the increase in the latter sup-porting the existence of vesicles containing PIP2 and hydrolysed by PLCz atfertilization (Gonzalez-Garcia et al., 2013) (Table II).

As mentioned previously, the extrusion of cortical granules is alsomediated by PKCa (Talmor-Cohen et al., 2004). Upon fertilization,PKCa translocation to the plasma membrane allows phosphorylationof myristoylated alanine-rich C-kinase substrate (MARCKS) proteins,which are involved in the regulation of actin network and thus in the pre-vention of polyspermy (Eliyahu et al., 2005, 2006). Indeed, phosphoryl-ation of MARCKS leads then to disassembly of the actin network at theoocyte cortex and allows cortical granules to reach the oolemma and toexocytose (Tsaadon et al., 2008). This explains why it is relevant for PKCto be translocated to the membrane following sperm-membrane fusionas an event of oocyte activation.

It is worth noting that conventional PKCs have been suggested aschronometers, because they first translocate to the oolemma duringoocyte activation where they mediate ‘store-operated calcium entry’(SOCE) and phosphorylate MARCKS. Subsequently, PKCs are con-verted into their catalytic subunits (PKC-M) that travel to the ooplasmand phosphorylate cytoskeletal components and other substrates(Capco, 2001; Kalive et al., 2010).

It has yet to be reported whether any calcium pump involved in pro-ducing Ca2+ oscillations in mammalian oocytes is phosphorylated byPKCs. This could also explain the failure of oocytes to activate. This sug-gestion comes from studies conducted with Xenopus oocytes, in whichvoltage-gated calcium (Cav2.2) channels were found to be a phosphor-ylation substrate for PKCs. It is known that Thr422, Ser1757 and Ser2132

residues within Cav2.2 channels are target sites for PKCa (Rajagopalet al., 2014).

Finally, some reports have questioned the role of PKCa during oocyteactivation, since CaMKII is able to trigger meiotic resumption and earlydevelopment (Madgwick et al., 2005; Knott et al., 2006), while othershave indicated that PKC activity in the plasma membrane upon fertiliza-tion is marginal (Yu et al., 2008). Therefore, more research is required toaddress whether abnormalities in PKC may underlie OAD.

PKCs, oocyte maturation and early embryodevelopmentThe role of PKC is also important in meiotic resumption of the oocyte in aprocess that involves zinc, as free zinc levels in the cytoplasm significantlyincrease during oocyte maturation and the depletion of zinc using ablocking agent reduces levels of both phosphorylated MAPK and PKC.In contrast, phosphorylation of PKC substrates and MAPK increasewhen oocytes are treated with a zinc agonist (Zhao et al., 2014b). More-over, in a study that evaluated the effect of age, germinal vesicle transferand modified nucleoplasmic ratio on PKCa distribution, it was observedthat while PKCa presented similar localization patterns within the cyto-plasm of M-II oocytes and early embryos, it mainly resided in the nucleusafter pronuclei formation and in 2-cell embryos (Cui et al., 2012).

As previously mentioned, non-classic and atypical PKCs have alsobeen reported to play a role during oocyte maturation, fertilization andcell differentiation during preimplantation development (Carracedoet al., 2013; Saiz et al., 2013). Indeed, non-classic PKCd and atypicalPKCz reside in the meiotic spindle and are suggested to play a roleduring meiosis (Tatone et al., 2003; Viveiros et al., 2003; Baluch et al.,2004). In humans, PKCd has been demonstrated to modulate the

34 Yeste et al.

epithelial mesenchymal transition from embryonic stem cells to cellsexpressing markers of parietal endoderm, an extraembryonic lineageformed before the formation of primitive ectoderm (Feng et al., 2012).Finally, Ezrin, an important protein involved in the compactation of8-cell embryos, possesses a Thr567 residue that is phosphorylated byan atypical PKC, which then leads to embryo compaction (Liu et al.,2013a).

Ca21 oscillations and homeostasisin the oocyteCalcium is a ubiquitous secondary messenger that plays a fundamentalrole in many reproductive processes, such as sperm capacitation andacrosome reaction (Costello et al., 2009; Alasmari et al., 2013; Yesteet al., 2015). We have already mentioned how important Ca2+ oscilla-tions are for the events that occur immediately after fertilization (i.e.oocyte activation and early embryo development), and the key role ofSOAF in evoking such oscillations. Indeed, these oscillations have beendescribed to present complex temporal and spatial properties, beginningsoon after gamete fusion and persisting beyond the completion ofmeiosis (Parrington et al., 1998b; Saunders et al., 2002; Swann et al.,2004, 2006; Kouchi et al., 2005; Sone et al., 2005; Ito et al., 2011). Fur-thermore, the Ca2+balance within and outside the oocyte is critically im-portant, since extracellular Ca2+ is needed for meiosis progression andblocking Ca2+ channels impacts oocyte maturation (Boni et al., 2007).

The pattern of Ca2+ release in artificially activated oocytes differs fromthat evoked by PLCz after normal fertilization (Yanagida et al., 2008). Inthe case of activation with strontium chloride, for example, this isbecause strontium from the external milieu, rather than activity of theER, is what underlies Ca2+ oscillations (Zhang et al., 2005; Carvachoet al., 2013). In addition, while there is a single Ca2+ spike after fertiliza-tion in non-mammalian species, this is not the case in mammalianoocytes, where periodic changes in Ca2+ levels in the ooplasm take

the form of oscillations (Kashir et al., 2013a). This implies that there isa well-established mechanism for emptying and refilling the internalstores. In this context, it is clear that the mission of Ca2+ influx fromthe extracellular medium is replenishing the Ca2+ content in the ER,since there is a premature termination of Ca2+-oscillations (triggeredby the SOAF) when the extracellular milieu is devoid of Ca2+ (Wakaiet al., 2013). For this reason, this section focuses upon the relevanceof Ca2+ homeostasis for mammalian oocytes (i.e. Ca2+ release fromthe ER and influx from outside the oocyte), since this could affect theirability to be activated and thus underlie OAD.

Membrane channels and SOCE:STIM1 and ORAI1It is clear that oocyte proteins that play a role in Ca2+ homeostasis canalso be involved in OAD. In this regard, there is a protein systemknown as SOCE that manages Ca2+ entry into intracellular stores(Wang and Machaty, 2013). In this system, which represents the majorCa2+ entry pathway in electrochemically non-excitable cells, there area series of gated-channels that are activated when Ca2+ in the ER isdepleted, thus providing a sustained cytoplasmic Ca2+ influx from theextracellular space (Putney, 1986, 2011; Smyth et al., 2010). Themajor components of this system are stromal interaction molecule-1(STIM1), Ca2+ release-activated Ca2+ channel protein 1 (ORAI1),sarco/endoplasmic Reticulum Ca2+ATPase (SERCA) and other uniden-tified membrane channels (Table III and Fig. 3). As InsP3 is known to inter-vene in the release of Ca2+ from ER, it is reasonable to suggest thatabnormalities in these proteins could affect Ca2+ release and thus theoocyte’s ability to be activated.

The relevance of SOCE, known to play vital functions in other cell types,has also been demonstrated in mammalian oocytes (Martın-Romero et al.,2012). Two components of this system, STIM1 and ORAI1, are importantduring oocyte maturation, when there is a progressive Ca2+-influx to theoocyte and a subsequent Ca2+-increase in the oocyte internal stores,

.............................................................................................................................................................................................

Table III Potential oocyte factors with implications for OAD (II): elements of the store-operated calcium-entry system.


STIM1 STIM1 senses Ca2+ levels in the ER using EF handspresent in the ER lumen. When Ca2+ levels in the ERlumen decrease, STIM1 translocates to the plasmamembrane and clusters with ORAI1 channels.

Genetic mutations in these proteins orabnormal trafficking resulting in loss offunction may lead to depletion of Ca2+

in the ER, thus preventing Ca2+

oscillations and contributing to OAD.

Cheon et al. (2013)Lee et al. (2012)Martın-Romero et al. (2012)Wang and Machaty (2013)

ORAI1 ORAI1 is a Ca2+ channel located in the plasmamembrane. Upon stimulation by STIM1, ORAI1channels open and allow Ca2+ influx.

Barr et al. (2009)Feske et al. (2006)Gudlur et al. (2014)Wang and Machaty (2013)Wang et al. (2012)

SERCA SERCA is a pump located in the ER membrane thatpumps Ca2+ into the ER lumen. Therefore, the ER canbe refilled with cytosolic Ca2+.

Kline and Kline (1992b)Wang and Machaty (2013)

Unidentifiedmembrane channels

Since inhibition of SOCE proteins does not completelyabrogate Ca2+ oscillations, the existence of other,unidentified Ca2+ membrane channels is suggested.However, more research is still required to address thispoint.

Miyazaki (1988)

ORAI: Ca2+ release-activated Ca2+ channel protein 1; SERCA: sarco/endoplasmic Reticulum Ca2+ATPase; STIM1: stromal interaction molecule-1; SOCE: store-operated calcium-entrysystem.


mainly the ER (Cheon et al., 2013). Such an increase has been related to thekey role of this second messenger during oocyte activation. STIM1 is asingle-pass, type-I transmembrane protein that activates Ca2+ entriesinto stores when luminal levels of this second messenger are low (Rooset al., 2005; Zhang et al., 2005). STIM1 is present in the ER membranebut can also be found in the plasma membrane (Soboloff et al., 2012).The primary sequence of this protein contains 685 amino acids and thecytosolic region presents two coiled-coil domains that are rich inproline/serine and lysine, respectively. The luminal region contains asignal peptide for ER, an EF-hand domain, and a sterile alpha motifdomain (Williams et al., 2002; Stathopulos et al., 2008). The EF-hand andsterile alpha motif domains act as a sensor of Ca2+ levels, since whenCa2+ levels in the ER decrease, the sterile alpha motif, which is adjacentto the EF-hand domain, mediates the oligomerization of STIM1 (Liouet al., 2007). Therefore, these two regions are crucial for a proper function-ing of the SOCE mechanism, and mutations in the EF-hands, or sterile alphamotif domains, may lead to active Ca2+ influx regardless of Ca2+ levelswithin the store. Considering that Ca2+ plays a crucial role in downstreamevents triggered by the SOAF and release from the ER, it could be the casethat alternations/deficiencies in this protein ultimatelyaffects the regulationof normal Ca2+ oscillations, as the folded/unfolded state of the Ca2+-sensor in STIM1 is vital for SOCE regulation (Stathopulos et al., 2008).

However, more research iswarranted in this respect sincesuchdeficienciescould impact upon other cell functions across the entire body.

STIM1 function is related to ORAI1. In a suggested general picture ofthe process (Fig. 3), the SOAF leads to the hydrolysis of PIP2 into InsP3

and DAG, and InsP3 interacts with its receptor located on the ER mem-brane. Ca2+ is then released from the ER, which concomitantly reducesCa2+ levels in the ER lumen. This reduction in Ca2+ is detected by theEF-hand and sterile alpha motif domains of STIM1, and this leads thisprotein to cluster and relocate to the plasma membrane. STIM1 theninteracts with the cytoplasmic region of the ORAI1 channel, a structuralcomponent of Ca2+ release-activated ion channels, and this leads to aconformation change near the external entrance to the pore thatopens the channel and allows Ca2+ influx (Feske et al., 2006; Barret al., 2009; Putney, 2009; Gudlur et al., 2014). Interestingly, STIM1 pre-sents a Ca2+ release-activated Ca2+ activation domain that consists of ahighly conserved region of 107 amino acids that binds directly to the Nand C termini of ORAI1. Therefore, the direct protein–protein inter-action between STIM1 and ORAI1 leads to set junctions between theER and plasma membrane (Park et al., 2009). Therefore, deficiencies inORAI1 could lead to the oocyte being unable to flux Ca2+ properly,and result in the inability of oocytes to elicit proper Ca2+ oscillations(Lee et al., 2012; Wang et al., 2012).

Figure 3 Oocyte mechanisms involved in the regulation of Ca2+. It is important to note the relevance of store-operated calcium entry for Ca2+ homeo-stasis, as well as the role of mitochondria. ORAI: Ca2+ release-activated Ca2+ channel protein 1; SERCA: sarco/endoplasmic Reticulum Ca2+ATPase;STIM1: stromal interaction molecule-1.

36 Yeste et al.

Sarco/endoplasmic reticulum and plasmamembrane Ca21-ATPasesSarco/endoplasmic reticulum Ca2+-ATPases (SERCA) are P-type ionpumps located in the ER membrane that pump Ca2+ from the cytosolto the lumen of the ER (Ullah et al., 2007; Fig. 3). The genes encodingthese proteins (SERCA1, SERCA2 and SERCA3) are located on three dif-ferent chromosomes and up to 14 SERCA-transcripts, with alphanume-rical identification, are generated by alternative splicing. This dependsupon cell type and developmental stage (Periasamy and Kalyanasun-daram, 2007). In addition, some of these isoforms have been reportedto be species-specific, such as SERCA2c, SERCA3d, SERCA3e andSERCA3f, which are only present in humans (Dally et al., 2010).During Ca2+ oscillations, the ER needs to be refilled with calcium afterthe luminal levels are depleted. In this context, SERCA pumps Ca2+ tothe ER in order to generate another Ca2+ oscillation that will be releasedupon interaction of InsP3 with its receptor (Wang and Machaty, 2013).

SERCA2b is an isoform present in mammalian oocytes that, wheninhibited with thapsigargin during oocyte maturation, prevents the in-crease of Ca2+ within the ER (Kline and Kline, 1992b). A two-stepmodel has been proposed to explain the replenishment of Ca2+ in theER, which consists of a rapid Ca2+ uptake from the cytosol by SERCAproteins and a gradual phase that relies upon Ca2+ influx from the exter-nal milieu (Wakai et al., 2013).

SERCA2b is redistributed in sub-cortical areas located close to theInsP3Rs of M-II oocytes and is organized into clusters when the oocytereaches the M-II stage (Wakai et al., 2013). While SERCA2b clustersare present within 3 h post-fertilization, they disappear when the pro-nuclear stage is reached. Since this matches with the actual duration ofCa2+ oscillations in mice (Jones et al., 1995), it confirms the relevanceof this protein in the events that take place after sperm–oocyte fusion(Wakai et al., 2013) (Table III).

Other vital proteins for Ca2+ oscillations include plasma membraneCa2+ ATPases (PMCAs), which pump Ca2+ from the cytosol to theextracellular milieu. Therefore, inappropriate functioning of SERCAand PMCA pumps has been suggested to disrupt Ca2+ oscillations andlead to an increase in cytosolic Ca2+ levels (Wakai et al., 2013).However, while deficiencies in PMCAs could feasibly underlie OAD,

more research is still required to investigate their presence and functionin mammalian oocytes.

Other oocyte proteins that couldunderlie oocyte activation failure

CaMKII and MAPKCa2+ oscillations allow the resumption of meiosis as they switch oncalcium/calmodulin-dependent protein kinase II (CaMKII) (Dupont,1998; Von Stetina and Orr-Weaver, 2011; Fig. 2). Indeed, upon activa-tion, CaMKII phosphorylates the oocyte-specific protein Emi2(Madgwick et al., 2005; Ducibella et al., 2006; Knott et al., 2006). Emi2allows the oocyte to remain arrested at M-II as it prevents the lossof M phase/maturation-promoting factor (MPF), a heterodimer ofcyclin-dependent kinase 1 (CDK1) and CCNB1, by blocking CCNB1-degradation (Jones, 2007). When Emi2 is phosphorylated by CaMKIIand thus degraded, MPF is also degraded and the oocyte is able to alle-viate its M-II arrest (Fig. 2). On the other hand, high cytoplasmic levelsof Ca2+, via CaM and CaMKII, inhibit InsP3Rs. Since this is followed bya reduction in the release of Ca2+ from internal stores and thus areduction in the concentration of Ca2+ in the ooplasm, InsP3Rinhibition terminates and Ca2+ release can be renewed from thestores (Matifat et al., 2001). Therefore, as CaMKII is a Ca2+-dependentprotein kinase, lack of appropriate Ca2+ homeostasis can prevent theprotein from functioning, and thus impact upon oocyte activation(Table IV).

On the other hand, following resumption and termination ofmeiosis-II, the pronuclei are formed in a process that is also dependenton Ca2+ levels and involves the MAPK pathway (Ducibella and Fissore,2008; Fig. 2). Thus, deficiencies in this transduction pathway couldprevent the oocyte from undergoing embryogenesis.

Other phospholipasesIn the debate to elucidate which sperm protein is the actual SOAF, it isfundamentally clear that any protein candidate needs to be intimatelyinvolved in Ca2+ metabolism and homeostasis. Different PLCs (b, g,

.............................................................................................................................................................................................

Table IV Potential oocyte factors with implications for OAD (III): other oocyte proteins.


CaMKII CaMKII is one of the proteins activated byCa2+. This protein plays a key role in theactivation of oocytes, since it initiates a cascadethat leads to alleviation of meiotic arrest.

Genetic mutations in these proteins resulting inloss of function may prevent events of oocyteactivation taking place, even when Ca2+

oscillations are generated successfully.

Ducibella et al. (2006)Dupont (1998)Knott et al. (2006)Madgwick et al. (2005)Von Stetina and Orr-Weaver (2011)

MAPK Inactivation of MAPK by Ca2+ oscillationsallows pronuclei formation.

Ducibella and Fissore (2008)Von Stetina and Orr-Weaver (2011)

Oocytephospholipases

PLCb, g and d are found in the ooplasm andmay have a role in generating Ca2+ oscillations.In addition, PAWP has been suggested tointeract with PLCg.

Reduced PLCb1 in mouse eggs is linked toCa2+ oscillations of lower amplitude, so loss/malfunction of oocyte PLCb1 could lead toOAD. If PAWP is confirmed to interact withPLCg, and PAWP plays a key role for oocytefertilization, a malfunction of oocyte PLCgcould lead to OAD and/or fertilization failure.

Aarabi et al. (2014a)Igarashi et al. (2007)Saunders et al. (2002)

CaMKII: calcium/calmodulin-dependent protein kinase II; MAPK: mitogen-activated protein kinase.


d), but not z, are found in the ooplasm of mammalian oocytes (Saunderset al., 2002). These PLC isoforms appear to play a role in Ca2+ oscilla-tions, since Igarashi et al. (2007) found that when levels of PLCb1 inmouse oocytes were reduced, Ca2+oscillations were of loweramplitude(Table IV). In amphibians, it was reported that Ca2+ can activate a cyto-solic phospholipase A2 (cPLA2), which releases arachidonic acid fromphospholipids of the ER as well as from the nuclear envelope, in a stepthat would make this protein able to participate in oocyte activation(Ajmat et al., 2013). For this reason, we could also suggest that altera-tions/deficiencies in other phospholipases within the oocyte couldalso lead to OAD.

CDK1 and CHERPIn ascidians, the sequestration of PLCz does not stop Ca2+ spikes.Instead, it seems that it is the blockage of CDK1 that causes terminationof Ca2+-induced spikes. Following these results, it has been suggestedthat CDK1, an oocyte protein, promotes InsP3 generation in the pres-ence of PLCz, thereby indicating that CDK1 is linked to phosphoinositidepathway (Levasseur et al., 2007; Fig. 2).

Anotherubiquitousprotein that isknowntobe involved inCa2+ traffick-ing in all cell types is Ca2+ homeostasis endoplasmic reticulum protein(CHERP). This protein has been suggested to regulate Ca2+ channels inthe ER membrane (i.e. InsP3Rs and ryanodine receptors), via regulatingthe function of the U2 small nuclear RNA spliceosomal complex in thenucleus rather than acting as a cytoplasmic regulator (Lin-Moshier et al.,2013). In Xenopus oocytes, CHERP is present in the nucleus rather thanin the ER, so its functional role in the cytoplasm needs to be demonstrated(Lin-Moshier et al., 2013). Studies investigating the presence and putativerole of CHERP have yet to be conducted in mammalian oocytes.

Oocyte mitochondria andCa21 oscillationsWhile fundamentally involved in oxidative phosphorylation, mitochon-dria also represent important internal stores of Ca2+ (Dumollardet al., 2004; Fig. 3). In this regard, it has been suggested that oocyteswith fewer mitochondria and/or inappropriate mitochondrial function,and thus reduced levels of ATP production, could fail to exhibit normalCa2+ oscillatory patterns and therefore influence activation ability. Inaddition, not only the absence of an appropriate balance betweenCa2+ release and sequestration has coincident effects upon levels ofmitochondrial ATP (Van Blerkom, 2011), but also excessive Ca2+ oscil-latory activity may result in the oocyte entering an apoptoticmitochondria-caspase mediated pathway, as demonstrated earlier inrat oocytes cultured in vitro (Tripathi and Chaube, 2012). These datahave led to the belief that mitochondrial abnormalities in the oocytemay underlie some cases of oocyte-related OAD (Van Blerkom, 2011).

Lessons from the in vitromaturation of human oocytesIn vitro maturation (IVM) of human oocytes is a promising technique forfertility preservation. Increasing success rates for this technique maybenefit, amongst others, young women suffering from cancer thatcannot delay chemo- or radiotherapy and/or cannot be offeredovarian hyperstimulation (Chian et al., 2013). Although success rates

for IVM in human oocytes have been increasing over recent times,there are still some issues that need to be addressed. While it is under-stood that oocyte maturation consists of complex changes that involveboth the nucleus and cytoplasm, it is also considered that alterations inorganelles and the cytoskeleton that occur during IVM are all possiblecauses for OAD, fertilization failure and reduced developmentalembryo development (Mao et al., 2014; Neri et al., 2014). Supportingthis, Ca2+ oscillations observed in IVM-fertilized human oocytes are oflower frequency and shorter duration than those observed in oocytesmatured in vivo (Nikiforaki et al., 2014). In addition, Liu et al. (2013b)showed that vitrified-warmed and subsequently in vitro maturedhuman oocytes presented higher ICSI-fertilization success when AOAwas used. These findings match the idea that the cytoplasmic changesthat take place during oocyte maturation affect the machinery that regu-late Ca2+ homeostasis and are vital for the sperm-triggered events fol-lowing sperm–oocyte fusion.

The asynchrony between nuclear and cytoplasmic maturation duringoocyte maturation, either in vivo or in vitro, can also account for the inabil-ity of the ooplasm to decondense the sperm chromatin (Eppig et al.,1994). In this context, the dialogue between the oocyte and thecumulus oophorus plays a key role during oocyte maturation, andseems to be responsible for allowing the oocyte cytoplasm to acquirethe developmental competence required for fertilization success (Neriet al., 2014). While this hypothesis matches other studies involvinghuman oocytes undergoing IVM (Hyun et al., 2007), more researchinto the IVM of human oocytes could also help to identify cases ofOAD that are not due to a sperm factor deficiency.

In general, however, fertilization, implantation and childbirth rateswith IVM are still lower than those achieved with conventional IVF(Son et al., 2008), and multinucleation rates following ICSI are higher inIVM than in in vivo-matured MII-oocytes (De Vincentiis et al., 2013). Anumber of factors might lead to lower implantation and childbirth rateswith IVM, including non-synchronization of oocyte nuclear and cytoplas-mic maturation, culture conditions, and timing of insemination. In par-ticular, non-synchronization of oocyte nuclear and cytoplasmicmaturation was considered to be a major reason for poor developmentalcompetence of IVM oocytes (Combelles et al., 2002 and Moor et al.,1998). Chian et al. (2000) reported that HCG administration in vivocan hasten the nuclear maturation process, whereas its effect on cyto-plasmic maturation is unclear. In addition, one of the important factorsregulating the number and quality of oocytes maturing in vitro are theculture conditions and timing of insemination used for IVM.

AOA

Mechanical, electrical and chemicalactivationAOA is a suitable technique for those couples that present a compro-mised fertilization rate below 30% following conventional ICSI (Hein-dryckx et al., 2005, 2008; Montag et al., 2012). This method involvesthe use of various mechanical, electric or chemical stimuli to help alleviateoocytes from MII-arrest (Alberio et al., 2001; Nasr-Esfahani et al., 2010;Vanden Meerschaut et al., 2014a). Not only has AOA been suggested tobe effective in cases of sperm-related fertilization deficiency (Heindryckxet al., 2005), but also in some cases in which an oocyte cytosolic factor issuspected to underlie OAD (Vanden Meerschaut et al., 2012).

38 Yeste et al.

Mechanical activation is a modified ICSI procedure which involves vig-orous cytoplasmic aspiration prior to injecting the sperm. This practiceincreases Ca2+ levels at the time of injection and helps the oocyte to ac-tivate (Ebner et al., 2004). Electrical activation works by opening pores inthe plasma membrane, thereby allowing Ca2+ influx from the surround-ing media, ultimately resulting in an elevation of Ca2+ levels in theooplasm. While these two approaches have been reported to be suc-cessful (Ebner et al., 2004; Yanagida et al., 2008; Egashira et al., 2009),their success rates are lower than those obtained with chemical activa-tion methods (Baltaci et al., 2010).

Chemical activation is the most commonly used procedure and con-sists of exposing oocytes to chemical agents that lead to an increase inintracellular Ca2+ levels in the oocyte. However, while some agentscause a single, prolonged Ca2+ transient in the oocyte, others causemultiple oscillations. Activating agents that cause a single Ca2+ transientinclude calcium ionophores, such as A23187 (also known as calcimycin)and ionomycin, and protein synthesis and kinase inhibitors, such aspuromycin and 6-dimethylaminopurine, respectively (Tesarik et al.,2000; Yamano et al., 2000; Nakagawa et al., 2001; Murase et al.,2004; Lu et al., 2006; Heindryckx et al., 2008, 2011; Nasr-Esfahaniet al., 2008; Terada et al., 2009). Within this category, calcimycin andionomycin are the most used clinically. Their mechanism of action con-sists of increasing the permeability of the plasma membrane to Ca2+,thereby allowing extracellular Ca2+ to flow into the oocyte. The firstcase of AOA using calcimycin was reported in 1997 (Rybouchkinet al., 1997), and recent data show fertilization rates from 25 to48%, with live birth rates of around 28% (Ebner et al., 2015). In add-ition, calcimycin was recently reported as being efficient for the artificialactivation of in vitro matured oocytes (Kim et al., 2015).

Chemical activators that cause multiple transients include strontiumchloride (SrCl2) (Kline and Kline, 1992a; Kishikawa et al., 1999; Kimet al., 2014), phorbol esters (Cuthbertson and Cobbold, 1985), thimer-osal (Fissore et al., 1995) and anhydrous alcohol (Liu et al., 2013b). WhileSrCl2 is the most used, and has been reported to be effective in mice, itsuse in humans is still under debate (Yanagida et al., 2006; Kyono et al.,2008; Chen et al., 2010; Vanden Meerschaut et al., 2014a). Recently,Kim et al. (2014) showed that AOA with SrCl2 may be a suitable strategyfor those couples in which calcimycin is not effective enough. The mech-anism by which SrCl2 induces Ca2+ oscillations is not fully understood,but appears to lead to Ca2+ release from the ER.

Safety of AOA for child healthAlthough AOA has been proven as an efficient strategy with which toovercome some cases of TFF, OAD may result from multi-factorialcauses, as discussed above. This should be taken into account when arti-ficial activators are used, since using artificial activators that increaseintracellular Ca2+ levels circumvents the normal oocyte machinery andCa2+ transients are not regulated by the appropriate endogenousmechanisms. In addition, the use of AOA could also underlie someeffects on gene expression and epigenetic regulation (Ozil et al., 2006;Ciapa and Arnoult, 2011). At present, a major concern over the use ofAOA is that we know very little about downstream events in theoocyte and early embryo. For this reason, the safety of artificial activatorshas been the source of much on-going debate (Nasr-Esfahani et al., 2010;Vanden Meerschaut et al., 2014a).

Although the number of live births registered after AOA still remainslow, the use of calcium ionophores has been demonstrated to be safeand has not been linked with any deleterious effects in terms of childhealth (Heindryckx et al., 2005; Ebner et al., 2012; Montag et al., 2012;Vanden Meerschaut et al., 2014b). While one study reported a babyborn after calcimycin AOA with a major malformation (anal atresia)(Ebner et al., 2015), it is entirely possible that this resulted from theuse of ART, since the risk of congenital malformations is known to beincreased following conventional IVF and ICSI (Kallen et al., 2010; Kana-sugi et al., 2013).

The first study investigating the subsequent health status of childrenconceived after combining AOA with ionomycin and ICSI has been pub-lished only very recently (Deemeh et al., 2014). Using a significant samplesize (79 children born following AOA-ICSI and 89 born by ICSI), thisstudy demonstrated the safety of AOA with ionomycin, since no signifi-cant differences between AOA-ICSI and conventional ICSI were found interms of intrauterine fetal death, preterm delivery, birthweight, growthrate, hospitalization in neonatal intensive care units, abnormal behaviouraccording to age, and the physical and mental health of children born. Inaddition, Deemeh et al. (2014) also showed that AOA did not causechromosomal abnormalities. This is highly relevant for safety becauseAOA takes place during meiotic spindle orientation and meiosis comple-tion (Alberio et al., 2001). It is worth noting that the study by Deemehet al. (2014) was conducted in couples that underwent ICSI withsevere teratozoospermia or sperm collected by testicular extraction.This population is different from couples with previous failed fertilizationcycles and, since the study by Deemeh et al. (2014) mainly focuses uponsperm, it does not address whether a problem in the relevant oocyte ma-chinery has any impact on the health status of children born.

In the case of SrCl2, data are less consistent. Indeed, while data in miceshow that this treatment reduces the birthweight of female pups (VandenMeerschaut et al., 2013), studies in humans have reported that it has noimpact on the physical and mental development of children from birth to60 months (Yanagida et al., 2006; Kyono et al., 2008; Kim et al., 2014).

The use of AOA as a routine practiceAlthough the data reported so far are encouraging, AOA is yet to be con-sidered an established treatment (Vanden Meerschaut et al., 2014a;Sfontouris et al., 2015). Indeed, while studies have reported noadverse effects, it must be noted that sample sizes are still too low andthat data on the follow-up of children born following AOA remainscarce. Since the oocyte machinery governing Ca2+ homeostasis isbypassed by AOA, one should be prudent when assessing potentialtreatments given in this scenario. Indeed, there is still a lack of knowledgeabout the exact mechanisms of action triggered by AOA agents. Whilethe impact of amplitude and frequency of artificially-induced Ca2+ oscil-lations on embryo development and gene expression has been reported(Ozil et al., 2006), the effects of calcium ionophores upon gene expres-sion have yet to be studied. In addition, although the risk of abortion afterconventional ICSI is higher than in natural conception (Deemeh et al.,2014), and there is an increased risk for some syndromes that hasbeen associated with imprinting errors (Kallen et al., 2010), future re-search should address whether the use of AOA increases these risks.Nevertheless, until a pure and active form of recombinant humanPLCz, or other SOAF protein(s), progresses through clinical trials andbecomes commercially available, AOA remains the only treatmentoption for patients diagnosed with OAD.


Rescue of OAD with recombinant PLCz

As mentioned above, Ca2+ oscillations generated by IVF and ICSI areknown to differ from those generated when artificial oocyte activatorsare used and there are thus concerns about their potential side-effects(Swann and Yu, 2008; Yanagida et al., 2008). Related to this, it isworth noting that differences between mammalian and non-mammalianspecies exist in the specific patterns of Ca2+ oscillations elicited by PLCz(Kashir et al., 2013a). Accordingly, injection of a non-mammalian PLCzinto a mouse oocyte evokes a single transient increase in Ca2+ levelsand generates excessive DAG, which in turn leads to elevated Ca2+

entry (Yu et al., 2008). This impairs oocyte development, and at thesame time suggests that an abnormal pattern of Ca2+ oscillations, suchas that caused by chemical activators or PLCz from a non-mammaliansource, can be detrimental for embryogenesis. In this context, the pro-duction of recombinant human PLCz protein, which should releaseCa2+ within the oocyte in a more efficient and controlled manner thanartificial activators, clearly represents a powerful therapeutic agent forOAD in those cases in which sperm exhibit PLCz deficiency (Kashiret al., 2010; Nomikos et al., 2012). Strikingly, Yoon et al. (2008) werethe first to demonstrate that cRNA encoding PLCz could rescue fertilityin an infertile patient whose sperm were both devoid of PLCz and unableto induce Ca2+ oscillations following ICSI. Kashir et al. (2011) later pub-lished the synthesis of the first recombinant human PLCz protein. Wheninjected into mouse and human oocytes, this recombinant protein notonly evoked Ca2+ oscillations that closely resembled those initiated bythe sperm after fertilization but also led to cleavage (Kashir et al.,2011; Yoon et al., 2012). Moreover, the ability of recombinant humanPLCz to initiate development in mice embryos was lower than inhuman counterparts, thereby demonstrating the specificity of this re-combinant form (Nomikos et al., 2013b).

ConclusionsWhile it is clear that sperm deficiencies can underlie a significant propor-tion of OAD cases, an intense debate has arisen concerning the trueidentity of the SOAF, with PLCz and PAWP being the two key candidates(Aarabi et al., 2015; Amdani et al., 2015; Nomikos et al., 2015b; Vadnaisand Gerton, 2015; Table I). However, far less attention has been paid tothe potential role of oocyte deficiencies in cases of OAD. The true rele-vance of oocyte-borne factors in OAD is clearly evident since total levelsand proportions of sperm exhibiting PLCz do not appear to explain allcases of TFF following ICSI (Yelumalai et al., 2015). Whether a specificoocyte receptor for PLCz or PAWP exists within mammalian oocytesis something that still needs to be proven. Nevertheless, it is clear thatapart from this putative receptor (or receptors), there are severalother key oocyte proteins that are involved in the cascade triggered bythe SOAF following sperm–oocyte fusion. In this regard, it would bevery beneficial to identify which oocyte protein/s may also contributeto OAD, as this may generate new targets for clinical diagnostic assaysor therapeutic intervention. In addition, Ca2+ trafficking is likely toinvolve many other, as yet unidentified, proteins. It follows then that de-ficiencies of oocyte activation could also be related to membrane chan-nels that have yet to be identified. Genetic deficiencies and/or anaberrant expression of such proteins could prevent activation of theoocyte upon fusion with the sperm membrane.

Given the clear imbalance in ongoing studies pertaining to the relativerole of sperm and oocyte proteins in OAD, it is clear that future researchprogrammes should turn their attention to the oocyte. However, study-ing human oocytes presents particular difficulties, both from an ethicaland practical point of view. For example, it is far more difficult to studya sufficient number of human oocytes in order to achieve statisticalpower than it is to study a sufficient number of sperm. In addition,while most existing studies utilize oocytes from animal models, it isalready clear that the mechanism of PLCz action may differ betweenspecies, and that this problem may also exist for other SOAF candidates.

AcknowledgementsThe authors would like to thank Servier Medical Art for their image bankused to assist the creation of Figures 2 and 3.

Authors’ rolesM.Y. wrote the manuscript. C.J., S.N.A., S.P. and K.C. revised themanuscript and approved the final version.

FundingM.Y. is funded by the European Commission, FP7-People Programme,Marie Curie-IEF (grant number: 626061). S.N.A. is funded by theChancellor’s Scholarship, Universiti of Brunei Darussalam.

Conflict of interestThe authors declare that there is no conflict of interest in regards to thismanuscript.

ReferencesAarabi M, Qin Z, Xu W, Mewburn J, Oko R. Sperm-borne protein, PAWP, initiates

zygotic development in Xenopus laevis by eliciting intracellular calcium release.Mol Reprod Dev 2010;77:249–256.

Aarabi M, Yu Y, Xu W, Tse MY, Pang SC, Yi YJ, Sutovsky P, Oko R. The testicular andepididymal expression profile of PLCz in mouse and human does not support its roleas a sperm-borne oocyte activating factor. PLoS One 2012;7:e33496.

Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, Oko R.Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillationsand oocyte activation in humans and mice. FASEB J 2014a;28:4434–4440.

Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, Oko R. Spermcontent of postacrosomal WW binding protein is related to fertilization outcomesin patients undergoing assisted reproductive technology. Fertil Steril 2014b;102:440–447.

Aarabi M, Sutovsky P, Oko R. Re: Is PAWP the ‘real’ sperm factor? Asian J Androl 2015;17:446–449.

Abassi YA, Foltz KR. Tyrosine phosphorylation of the egg receptor for sperm atfertilization. Dev Biol 1994;164:430–443.

Aghajanpour S, Ghaedi K, Salamian A, Deemeh MR, Tavalaee M, Moshtaghian J,Parrington J, Nasr-Esfahani MH. Quantitative expression of phospholipase C zeta,as an index to assess fertilization potential of a semen sample. Hum Reprod 2011;26:2950–2956.

Ajmat MT, Bonilla F, Hermosilla PC, Zelarayan L, Buhler MI. Role of phospholipase A2pathway in regulating activation of Bufo arenarumoocytes. Zygote 2013;21:214–220.

Alasmari W, Costello S, Correia J, Oxenham SK, Morris J, Fernandes L,Ramalho-Santos J, Kirkman-Brown J, Michelangeli F, Publicover S et al. Ca2+

signals generated by CatSper and Ca2+ stores regulate different behaviors inhuman sperm. J Biol Chem 2013;288:6248–6258.

40 Yeste et al.

Alberio R, Zakhartchenko V, Motlik J, Wolf E. Mammalian oocyte activation: lessonsfrom the sperm and implications for nuclear transfer. Int J Dev Biol 2001;45:797–809.

Alzayady KJ, Wagner LE II, Chandrasekhar R, Monteagudo A, Godiska R, Tall GG,Joseph SK, Yule DI. Functional inositol 1,4,5-trisphosphate receptors assembledfrom concatenated homo- and heteromeric subunits. J Biol Chem 2013;288:29772–29784.

Amdani SN, Jones C, Coward K. Phospholipase C zeta (PLCz): oocyte activation andclinical links to male factor infertility. Adv Biol Regul 2013;53:292–308.

Amdani SN, Yeste M, Jones C, Coward K. Sperm factors and oocyte activation:current controversies and considerations. Biol Reprod 2015; doi: 10.1095/biolreprod.115.130609.

Anderson JE, Farr SL, Jamieson DJ, Warner L, Macaluso M. Infertility servicesreported by men in the United States: national survey data. Fertil Steril 2009;91:2466–2470.

Antal CE, Newton AC. Tuning the signalling output of protein kinase C. Biochem SocTrans 2014;42:1477–1483.

Bak CW, Seok HH, Song SH, Kim ES, Her YS, Yoon TK. Hormonal imbalances andpsychological scars left behind in infertile men. J Androl 2012;33:181–189.

Baltaci V, Ayvaz OU, Unsal E, Aktas Y, Baltaci A, Turhan F, Ozcan S, Sonmezer M. Theeffectiveness of intracytoplasmic sperm injection combined with piezoelectricstimulation in infertile couples with total fertilization failure. Fertil Steril 2010;94:900–904.

Baluch DP, Koeneman BA, Hatch KR, McGaughey RW, Capco DG. PKC isotypes inpost-activated and fertilized mouse eggs: association with the meiotic spindle. DevBiol 2004;274:45–55.

Barr VA, Bernot KM, Shaffer MH, Burkhardt JK, Samelson LE. Formation of STIM andOrai complexes: puncta and distal caps. Immunol Rev 2009;231:148–159.

Barroso G, Valdespin C, Vega E, Kershenovich R, Avila R, Avendano C, Oehninger S.Developmental sperm contributions: fertilization and beyond. Fertil Steril 2009;92:835–848.

Beck-Fruchter R, Lavee M, Weiss A, Geslevich Y, Shalev E. Rescue intracytoplasmicsperm injection: a systematic review. Fertil Steril 2014;101:690–698.

Bedford-Guaus SJ, Yoon SY, Fissore RA, Choi YH, Hinrichs K. Microinjection of mousephospholipase C zeta complementary RNA into mare oocytes induces long-lastingintracellular calcium oscillations and embryonic development. Reprod Fertil Dev 2008;20:875–883.

Bhanumathy C, da Fonseca PC, Morris EP, Joseph SK. Identification of functionallycritical residues in the channel domain of inositol trisphosphate receptors. J BiolChem 2012;287:43674–43684.

Bhattacharya S, Maheshwari A, Mollison J. Factors associated with failed treatment: ananalysis of 121,744 women embarking on their first IVF cycles. PLoS One 2013;8:e82249. doi: 10.1371/journal.pone.0082249.

Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essentialfor mammalian fertilization. Nature 2014;508:483–487.

Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertilityprevalence and treatment-seeking: potential need and demand for infertilitymedical care. Hum Reprod 2007;22:1506–1512.

Boni R, Gualtieri R, Talevi R, Tosti E. Calcium and other ion dynamics during gametematuration and fertilization. Theriogenology 2007;68 Suppl 1:S156–S164.

Brind S, Swann K, Carroll J. Inositol 1,4,5-trisphosphate receptors are downregulated inmouse oocytes in response to sperm or adenophostin A but not to increases inintracellular Ca2+ or egg activation. Dev Biol 2000;223:251–265.

Bruton JD, Cheng AJ, Westerblad H. Methods to detect Ca(2+) in living cells. Adv ExpMed Biol 2012;740:27–43.

Bunney TD, Katan M. PLC regulation: emerging pictures for molecular mechanisms.Trends Biochem Sci 2011;36:88–96.

Capco DG. Molecular and biochemical regulation of early mammalian development. IntRev Cytol 2001;207:195–235.

Carpenter G, Ji QS. Phospholipase C-gamma as a signal-transducing element. Exp CellRes 1999;253:15–24.

Carracedo S, Braun U, Leitges M. Expression pattern of protein kinase C 1 duringmouse embryogenesis. BMC Dev Biol 2013;13:16. doi: 10.1186/1471-213X-13-16.

Carvacho I, Lee HC, Fissore RA, Clapham DE. TRPV3 channels mediate strontium-induced mouse-egg activation. Cell Rep 2013;5:1375–1386.

Castillo J, Amaral A, Azpiazu R, Vavouri T, Estanyol JM, Ballesca JL, Oliva R. Genomicand proteomic dissection and characterization of the human sperm chromatin.Mol Hum Reprod 2014;20:1041–1053.

Chandra A, Copen CE, Stephen EH. Infertility service use in the United States: datafrom the National Survey of Family Growth, 1982–2010. Natl Health Stat Report2014;73:1–21.

Chen J, Qian Y, Tan Y, Mima H. Successful pregnancy following oocyte activation bystrontium in normozoospermic patients of unexplained infertility with fertilisationfailures during previous intracytoplasmic sperm injection treatment. Reprod FertilDev 2010;22:852–855.

Chen Q, Zhang W, Ran H, Feng L, Yan H, Mu X, Han Y, Liu W, Xia G, Wang C. PKCd andupossibly mediate FSH-induced mouse oocyte maturation via NOX-ROS-TACE cascadesignaling pathway. PLoS One 2014;9:e111423. doi: 10.1371/journal.pone.0111423.

Cheon B, Lee HC, Wakai T, Fissore RA. Ca2+ influx and the store-operated Ca2+ entrypathway undergo regulation during mouse oocyte maturation. Mol Biol Cell 2013;24:1396–1410.

Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of humanchorionic gonadotrophin priming before immature oocyte retrieval fromunstimulated women with polycystic ovarian syndrome. Hum Reprod 2000;15:165–170.

Chian RC, Uzelac PS, Nargund G. In vitro maturation of human immature oocytes forfertility preservation. Fertil Steril 2013;99:1173–1181.

Ciapa B, Arnoult C. Could modifications of signalling pathways activated after ICSIinduce a potential risk of epigenetic defects? Int J Dev Biol 2011;55:143–152.

Colombero LT, Moomjy M, Sills ES, Rosenwaks Z, Palermo GD. The role of structuralintegrity of the fertilising spermatozoon in early human embryogenesis. Zygote 1999;7:157–163.

Combelles CM, Cekleniak NA, Racowsky C, Albertini DF. Assessment of nuclear andcytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod 2002;17:1006–1016.

Combelles CM, Morozumi K, Yanagimachi R, Zhu L, Fox JH, Racowsky C. Diagnosingcellular defects in an unexplained case of total fertilization failure. Hum Reprod 2010;25:1666–1671.

Cooney MA, Malcuit C, Cheon B, Holland MK, Fissore RA, D’Cruz NT. Species-specificdifferences in the activity and nuclear localization of murine and bovinephospholipase C zeta 1. Biol Reprod 2010;83:92–101.

Costello S, Michelangeli F, Nash K, Lefievre L, Morris J, Machado-Oliveira G, Barratt C,Kirkman-Brown J, Publicover S. Ca2+-stores in sperm: their identities and functions.Reproduction 2009;138:425–437.

Coward K, Ponting CP, Chang HY, Hibbitt O, Savolainen P, Jones KT, Parrington J.Phospholipase Czeta, the trigger of egg activation in mammals, is present in anon-mammalian species. Reproduction 2005;130:157–163.

Coward K, Ponting CP, Zhang N, Young C, Huang CJ, Chou CM, Kashir J, Fissore RA,Parrington J. Identification and functional analysis of an ovarian form of the eggactivation factor phospholipase C zeta (PLCz) in pufferfish. Mol Reprod Dev 2011;78:48–56.

Cox LJ, Larman MG, Saunders CM, Hashimoto K, Swann K, Lai FA. Sperm phospholipaseCzeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activationand development of mouse oocytes. Reproduction 2002;124:611–623.

Cui LB, Zhao ZJ, Zhou XY, Li Q, Huang XY, Sun FZ. Effect of age, GV transfer andmodified nucleocytoplasmic ratio on PKCa in mouse oocytes and early embryos.Zygote 2012;20:87–95.

Cuthbertson KS, Cobbold PH. Phorbol ester and sperm activate mouse oocytes byinducing sustained oscillations in cell Ca2+. Nature 1985;316:541–542.

Dacheux JL, Dacheux F. New insights into epididymal function in relation to spermmaturation. Reproduction 2013;147:R27–R42.

Dale B, DeFelice LJ, Ehrenstein G. Injection of a soluble sperm extract into sea urchineggs triggers the cortical reaction. Experientia 1985;41:1068–1070.

Dale B, Wilding M, Coppola G, Tosti E. How do spermatozoa activate oocytes? ReprodBiomed Online 2010;21:1–3.

Dally S, Corvazier E, Bredoux R, Bobe R, Enouf J. Multiple and diverse coexpression,location, and regulation of additional SERCA2 and SERCA3 isoforms in nonfailingand failing human heart. J Mol Cell Cardiol 2010;48:633–644.

Dam AH, Feenstra I, Westphal JR, Ramos L, van Golde RJ, Kremer JA.Globozoospermia revisited. Hum Reprod Update 2007;13:63–75.

De Kretser DM. Male infertility. Lancet 1997;349:787–790.De Mouzon J, Lancaster P, Nygren KG, Sullivan E, Zegers-Hochschild F, Mansour R,

Ishihara O, Adamson D, International Committee for Monitoring AssistedReproductive Technology. World collaborative report on Assisted ReproductiveTechnology, 2002. Hum Reprod 2009;24:2310–2320.


De Vincentiis S, De Martino E, Buffone MG, Brugo-Olmedo S. Use of metaphase Ioocytes matured in vitro is associated with embryo multinucleation. Fertil Steril2013;99:414–421.

Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificialoocyte activation: a pilot study. Reprod Sci 2014;22:322–328.

DeSouza N, Reiken S, Ondrias K, Yang YM, Matkovich S, Marks AR. Protein kinase Aand two phosphatases are components of the inositol 1,4,5-trisphosphate receptormacromolecular signaling complex. J Biol Chem 2002;277:39397–39400.

Ducibella T, Fissore R. The roles of Ca2+, downstream protein kinases, and oscillatorysignaling in regulating fertilization and the activation of development. Dev Biol 2008;315:257–279.

Ducibella T, Schultz RM, Ozil JP. Role of calcium signals in early development. Semin CellDev Biol 2006;17:324–332.

Dumollard R, Marangos P, Fitzharris G, Swann K, Duchen M, Carroll J. Sperm-triggered[Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absoluterequirement for mitochondrial ATP production. Development 2004;131:3057–3067.

Dupont G. Link between fertilization-induced Ca2+ oscillations and relief frommetaphase II arrest in mammalian eggs: a model based on calmodulin-dependentkinase II activation. Biophys Chem 1998;72:153–167.

Ebner T, Moser M, Sommergruber M, Jesacher K, Tews G. Complete oocyte activationfailure after ICSI can be overcome by a modified injection technique. Hum Reprod2004;19:1837–1841.

Ebner T, Moser M, Tews G. Is oocyte morphology prognostic of embryodevelopmental potential after ICSI? Reprod Biomed Online 2006;12:507–512.

Ebner T, Koster M, Shebl O, Moser M, Van der Ven H, Tews G, Montag M. Applicationof a ready-to-use calcium ionophore increases rates of fertilization and pregnancy insevere male factor infertility. Fertil Steril 2012;98:1432–1437.

Ebner T, Montag M, Montag M, Van der Ven K, Van der Ven H, Ebner T, Shebl O,Oppelt P, Hirchenhain J, Krussel J et al. Live birth after artificial oocyte activationusing a ready-to-use ionophore: a prospective multicentre study. Artificial oocyteactivation has been proposed as a suitable means to overcome the problem offailed or impaired fertilization after intracytoplasmic sperm injection (ICSI). ReprodBiomed Online 2015;30:359–365.

Egashira A, Murakami M, Haigo K, Horiuchi T, Kuramoto T. A successful pregnancy andlive birth after intracytoplasmic sperm injection with globozoospermic sperm andelectrical oocyte activation. Fertil Steril 2009;92:2037.e5–2037.e9.

Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M, Silber SJ. Successfulpregnancy and delivery after calcium ionophore oocyte activation in anormozoospermic patient with previous repeated failed fertilization afterintracytoplasmic sperm injection. Fertil Steril 2003;79 Suppl 3:1656–1658.

Eliyahu E, Shalgi R. A role for protein kinase C during rat egg activation. Biol Reprod 2002;67:189–195.

Eliyahu E, Tsaadon A, Shtraizent N, Shalgi R. The involvement of protein kinase C andactin filaments in cortical granule exocytosis in the rat. Reproduction 2005;129:161–170.

Eliyahu E, Shtraizent N, Tsaadon A, Shalgi R. Association between myristoylatedalanin-rich C kinase substrate (MARCKS) translocation and cortical granuleexocytosis in rat eggs. Reproduction 2006;131:221–231.

Eppig JJ, Schultz RM, O’Brien M, Chesnel F. Relationship between the developmentalprograms controlling nuclear and cytoplasmic maturation of mouse oocytes. DevBiol 1994;164:1–9.

Escoffier J, Yassine S, Lee HC, Martinez G, Delaroche J, Coutton C, Karaouzene T,Zouari R, Metzler-Guillemain C, Pernet-Gallay K et al. Subcellular localization ofphospholipase Cz in human sperm and its absence in DPY19L2-deficient spermare consistent with its role in oocyte activation. Mol Hum Reprod 2015;21:157–168.

ESHRE, ART fact sheet, June 2014. http://www.eshre.eu/Guidelines-and-Legal/ART-fact-sheet.aspx (5 November 2014, date last accessed).

Feng X, Zhang J, Smuga-Otto K, Tian S, Yu J, Stewart R, Thomson JA. Protein kinase Cmediated extraembryonic endoderm differentiation of human embryonic stem cells.Stem Cells 2012;30:461–470.

Ferrer M, Xu W, Oko R. The composition, protein genesis and significance of the inneracrosomal membrane of eutherian sperm. Cell Tissue Res 2012;349:733–748.

Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS,Daly M, Rao A. A mutation in Orai1 causes immune deficiency by abrogatingCRAC channel function. Nature 2006;441:179–185.

Fidler AT, Bernstein J. Infertility: from a personal to a public health problem. PublicHealth Rep 1999;114:494–511.

Fissore RA, Pinto-Correia C, Robl JM. Inositol trisphosphate-induced calcium release inthe generation of calcium oscillations in bovine eggs. Biol Reprod 1995;53:766–774.

Fissore RA, Reis MM, Palermo GD. Isolation of the Ca2+ releasing component(s) ofmammalian sperm extracts: the searchcontinues.Mol HumReprod1999a;5:189–192.

Fissore RA, Longo FJ, Anderson E, Parys JB, Ducibella T. Differential distribution ofinositol trisphosphate receptor isoforms in mouse oocytes. Biol Reprod 1999b;60:49–57.

Flaherty SP, Payne D, Matthews CD. Fertilization failuresand abnormal fertilization afterintracytoplasmic sperm injection. Hum Reprod 1998;13 Suppl 1:155–164.

Fujimoto S, Yoshida N, Fukui T, Amanai M, Isobe T, Itagaki C, Izumi T, Perry AC.Mammalian phospholipase Czeta induces oocyte activation from the spermperinuclear matrix. Dev Biol 2004;274:370–383.

Fukami K, Inanobe S, Kanemaru K, Nakamura Y. Phospholipase C is a key enzymeregulating intracellular calcium and modulating the phosphoinositide balance. ProgLipid Res 2010;49:429–437.

Gallicano GI, McGaughey RW, Capco DG. Activation of protein kinase C afterfertilization is required for remodeling the mouse egg into the zygote. Mol ReprodDev 1997;46:587–601.

Garcia P, Gupta R, Shah S, Morris AJ, Rudge SA, Scarlata S, Petrova V, McLaughlin S,Rebecchi MJ. The pleckstrin homology domain of phospholipase C-delta 1 bindswith high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.Biochemistry 1995;34:16228–16234.

Gasca S, Reyftmann L, Pellestor F, Reme T, Assou S, Anahory T, Dechaud H, Klein B, DeVos J, Hamamah S. Total fertilization failure and molecular abnormalities inmetaphase II oocytes. Reprod Biomed Online 2008;17:772–781.

Gonzalez-Garcia JR, Machaty Z, Lai FA, Swann K. The dynamics of PKC-inducedphosphorylation triggered by Ca2+ oscillations in mouse eggs. J Cell Physiol 2013;228:110–119.

Goud PT, Goud AP, Van Oostveldt P, Dhont M. Presence and dynamic redistribution oftype I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos duringin-vitro maturation, fertilization and early cleavage divisions. Mol Hum Reprod 1999;5:441–451.

Goudakou M, Kalogeraki A, Matalliotakis I, Panagiotidis Y, Gullo G, Prapas Y. Crypticsperm defects may be the cause for total fertilization failure in oocyte donorcycles. Reprod Biomed Online 2012;24:148–152.

Grasa P, Coward K, Young C, Parrington J. The pattern of localization of theputative oocyte activation factor, phospholipase Czeta, in uncapacitated,capacitated, and ionophore-treated human spermatozoa. Hum Reprod 2008;23:2513–2522.

Grøndahl ML, Borup R, Vikesa J, Ernst E, Andersen CY, Lykke-Hartmann K. Thedormant and the fully competent oocyte: comparing the transcriptome of humanoocytes from primordial follicles and in metaphase II. Mol Hum Reprod 2013;19:600–617.

Gudlur A, Quintana A, Zhou Y, Hirve N, Mahapatra S, Hogan PG. STIM1 triggers agating rearrangement at the extracellular mouth of the ORAI1 channel. NatCommun 2014;5:5164.

Halet G. PKC signaling at fertilization in mammalian eggs. Biochim Biophys Acta 2004;1742:185–189.

Halet G, Tunwell R, Parkinson SJ, Carroll J. Conventional PKCs regulate the temporalpattern of Ca2+ oscillations at fertilization in mouse eggs. J Cell Biol 2004;164:1033–1044.

Harada Y, Matsumoto T, Hirahara S, Nakashima A, Ueno S, Oda S, Miyazaki S, Iwao Y.Characterization of a sperm factor for egg activation at fertilization of the newtCynops pyrrhogaster. Dev Biol 2007;306:797–808.

He CL, Damiani P, Ducibella T, Takahashi M, Tanzawa K, Parys JB, Fissore RA. Isoformsof the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes andovaries: the type-1 isoform is down-regulated by fertilization and by injection ofadenophostin A. Biol Reprod 1999;61:935–943.

Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- oroocyte-related fertilization failure: assisted oocyte activation following diagnosticheterologous ICSI. Hum Reprod 2005;20:2237–2241.

Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of assistedoocyte activation as a solution for failed intracytoplasmic sperm injection. ReprodBiomed Online 2008;17:662–668.

Heindryckx B, Lierman S, Combelles CM, Cuvelier CA, Gerris J, De Sutter P. Aberrantspindle structures responsible for recurrent human metaphase I oocyte arrest withattempts to induce meiosis artificially. Hum Reprod 2011;26:791–800.

42 Yeste et al.

http://www.eshre.eu/Guidelines-and-Legal/ART-fact-sheet.aspx











Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, Fissore RA,Hamer R, Deane CM, Ruas M et al. Reduced amounts and abnormal forms ofphospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod2009;24:2417–2428.

Heytens E, Schmitt-John T, Moser JM, Jensen NM, Soleimani R, Young C, Coward K,Parrington J, De Sutter P. Reduced fertilization after ICSI and abnormalphospholipase C zeta presence in spermatozoa from the wobbler mouse. ReprodBiomed Online 2010;21:742–749.

HFEA. UK Fertility treatment in 2012: trends and figures. HFEA, 2014 (11 December2014, date last accessed).

Hirose M, Kamoshita M, Fujiwara K, Kato T, Nakamura A, Wojcikiewicz RJ, Parys JB, Ito J,Kashiwazaki N. Vitrification procedure decreases inositol 1,4,5-trisphosphatereceptor expression, resulting in low fertility of pig oocytes. Anim Sci J 2013;84:693–701.

Horner VL, Wolfner MF. Transitioning from egg to embryo: triggers and mechanisms ofegg activation. Dev Dyn 2008;237:527–544.

Hyun CS, Cha JH, Son WY, Yoon SH, Kim KA, Lim JH. Optimal ICSI timing after the firstpolar body extrusion in in vitro matured human oocytes. Hum Reprod 2007;22:1991–1995.

Igarashi H, Knott JG, Schultz RM, Williams CJ. Alterations of PLCbeta1 in mouse eggschange calcium oscillatory behavior following fertilization. Dev Biol 2007;312:321–330.

Inoue M, Kishimoto A, Takai Y, Nishizuka Y. Studies on a cyclic nucleotide-independentprotein kinase and its proenzyme in mammalian tissues. II. Proenzyme and itsactivation by calcium-dependent protease from rat brain. J Biol Chem 1977;252:7610–7616.

Ito M, Shikano T, Oda S, Horiguchi T, Tanimoto S, Awaji T, Mitani H, Miyazaki S.Difference in Ca2+ oscillation-inducing activity and nuclear translocation ability ofPLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human,and medaka fish. Biol Reprod 2008a;78:1081–1090.

Ito M, Shikano T, Kuroda K, Miyazaki S. Relationship between nuclear sequestration ofPLCzeta and termination of PLCzeta-induced Ca2+ oscillations in mouse eggs. CellCalcium 2008b;44:400–410.

Ito C, Akutsu H, Yao R, Kyono K, Suzuki-Toyota F, Toyama Y, Maekawa M, Noda T,Toshimori K. Oocyte activation ability correlates with head flatness and presenceof perinuclear theca substance in human and mouse sperm. Hum Reprod 2009;24:2588–2595.

Ito C, Yamatoya K, Yoshida K, Kyono K, Yao R, Noda T, Toshimori K. Appearance of anoocyte activation-related substance during spermatogenesis in mice and humans.Hum Reprod 2010;25:2734–2744.

Ito J, Parrington J, Fissore RA. PLCz and its role as a trigger of development invertebrates. Mol Reprod Dev 2011;78:846–853.

Ivanova H, Vervliet T, Missiaen L, Parys JB, De Smedt H, Bultynck G. Inositol1,4,5-trisphosphate receptor-isoform diversity in cell death and survival. BiochimBiophys Acta 2014;1843:2164–2183.

Jaffe F. Sources of calcium in egg activation: a review and hypothesis. Dev Biol 1983;99:265–276.

Jellerette T, He CL, Wu H, Parys JB, Fissore RA. Down-regulation of the inositol1,4,5-trisphosphate receptor in mouse eggs following fertilization orparthenogenetic activation. Dev Biol 2000;223:238–250.

Jensen TK, Jacobsen R, Christensen K, Nielsen NC, Bostofte E. Good semen quality andlife expectancy: a cohort study of 43,277 men. Am J Epidemiol 2009;170:559–565.

Johnson MH. Robert Edwards: the path to IVF. Reprod Biomed Online 2011;23:245–262.

Jones KT. Protein kinase C action at fertilization: overstated or undervalued? Rev Reprod1998;3:7–12.

Jones KT. Intracellular calcium in the fertilization and development of mammalian eggs.Clin Exp Pharmacol Physiol 2007;34:1084–1089.

Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-inducedCa2+ transients in mouse oocytes are cell cycle dependent. Development 1995;121:3259–3266.

Jones KT, Soeller C, Cannell MB. The passage of Ca2+ and fluorescent markersbetween the sperm and egg after fusion in the mouse. Development 1998;125:4627–4635.

Kadamur G, Ross EM. Mammalian phospholipase C. Annu Rev Physiol 2013;75:127–154.

Kalive M, Faust JJ, Koeneman BA, Capco DG. Involvement of the PKC family inregulation of early development. Mol Reprod Dev 2010;77:95–104.

Kallen B, Finnstrom O, Lindam A, Nilsson E, Nygren KG, Otterblad PO. Congenitalmalformations in infants born after in vitro fertilization in Sweden. Birth Defects ResA Clin Mol Teratol 2010;88:137–143.

Kanashiro CA, Khalil RA. Signal transduction by protein kinase C in mammalian cells.Clin Exp Pharmacol Physiol 1998;25:974–985.

Kanasugi T, Kikuchi A, Matsumoto A, Terata M, Isurugi C, Oyama R, Fukushima A,Sugiyama T. Monochorionic twin fetus with VACTERL association afterintracytoplasmic sperm injection. Congenit Anom (Kyoto) 2013;53:95–97.

Kang JH, Toita R, Kim CW, Katayama Y. Protein kinase C (PKC) isozyme-specificsubstrates and their design. Biotechnol Adv 2012;30:1662–1672.

Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K. Oocyte activation,phospholipase C zeta and human infertility. Hum Reprod Update 2010;16:690–703.

Kashir J, Jones C, Lee HC, Rietdorf K, Nikiforaki D, Durrans C, Ruas M, Tee ST,Heindryckx B, Galione A et al. Loss of activity mutations in phospholipase C zeta(PLCz) abolishes calcium oscillatory ability of human recombinant protein inmouse oocytes. Hum Reprod 2011;26:3372–3387.

Kashir J, Konstantinidis M, Jones C, Heindryckx B, De Sutter P, Parrington J, Wells D,Coward K. Characterization of two heterozygous mutations of the oocyteactivation factor phospholipase C zeta (PLCz) from an infertile man by use ofminisequencing of individual sperm and expression in somatic cells. Fertil Steril 2012a;98:423–431.

Kashir J, Sermondade N, Sifer C, Oo SL, Jones C, Mounce G, Turner K, Child T,McVeigh E, Coward K. Motile sperm organelle morphology evaluation-selectedglobozoospermic human sperm with an acrosomal bud exhibits novel patternsand higher levels of phospholipase C zeta. Hum Reprod 2012b;27:3150–3160.

Kashir J, Konstantinidis M, Jones C, Lemmon B, Lee HC, Hamer R, Heindryckx B,Deane CM, De Sutter P, Fissore RA et al. A maternally inherited autosomal pointmutation in human phospholipase C zeta (PLCz) leads to male infertility. HumReprod 2012c;27:222–231.

Kashir J, Deguchi R, Jones C, Coward K, Stricker SA. Comparative biology of spermfactors and fertilization-induced calcium signals across the animal kingdom. MolReprod Dev 2013a;80:787–815.

Kashir J, Jones C, Mounce G, Ramadan WM, Lemmon B, Heindryckx B, de Sutter P,Parrington J, Turner K, Child T et al. Variance in total levels of phospholipase Czeta (PLC-z) in human sperm may limit the applicability of quantitativeimmunofluorescent analysis as a diagnostic indicator of oocyte activation capability.Fertil Steril 2013b;99:107–117.

Kashir J, Nomikos M, Lai FA, Swann K. Sperm-induced Ca2+ release during eggactivation in mammals. Biochem Biophys Res Commun 2014;450:1204–1211.

Kasri NN, Torok K, Galione A, Garnham C, Callewaert G, Missiaen L, Parys JB, DeSmedt H. Endogenously bound calmodulin is essential for the function of theinositol 1,4,5-trisphosphate receptor. J Biol Chem 2006;281:8332–8338.

Kennedy CE, Krieger KB, Sutovsky M, Xu W, Vargovic P, Didion BA, Ellersieck MR,Hennessy ME, Verstegen J, Oko R et al. Protein expression pattern of PAWP inbull spermatozoa is associated with sperm quality and fertility following artificialinsemination. Mol Reprod Dev 2014;81:436–449.

Kilani S, Chapman MG. Meiotic spindle normality predicts live birth in patients withrecurrent in vitro fertilization failure. Fertil Steril 2014;101:403–406.

Kim BY, Yoon SY, Cha SK, Kwak KH, Fissore RA, Parys JB, Yoon TK, Lee DR.Alterations in calcium oscillatory activity in vitrified mouse eggs impact onegg quality and subsequent embryonic development. Pflugers Arch 2011;461:515–526.

Kim JW, Kim SD, Yang SH, Yoon SH, Jung JH, Lim JH. Successful pregnancy after SrCl2oocyte activation in couples with repeated low fertilization rates following calciumionophore treatment. Syst Biol Reprod Med 2014;60:177–182.

Kim JW, Yang SH, Yoon SH, Kim SD, Jung JH, Lim JH. Successful pregnancy and deliveryafter ICSI with artificial oocyte activation by calcium ionophore in in-vitro maturedoocytes: a case report. Reprod Biomed Online 2015;30:373–377.

Kishikawa H, Wakayama T, Yanagimachi R. Comparison of oocyte-activating agents formouse cloning. Cloning 1999;1:153–159.

Kline D, Kline JT. Repetitive calcium transients and the role of calcium in exocytosis andcell cycle activation in the mouse egg. Dev Biol 1992a;149:80–89.

Kline D, Kline JT. Thapsigargin activates a calcium influx pathway in the unfertilizedmouse egg and suppresses repetitive calcium transients in the fertilized egg. J BiolChem 1992b;267:17624–17630.

Kline D, Simoncini L, Mandel G, Maue RA, Kado RT, Jaffe LA. Fertilization eventsinduced by neurotransmitters after injection of mRNA in Xenopus eggs. Science1988;241:464–467.


Knott JG, Gardner AJ, Madgwick S, Jones KT, Williams CJ, Schultz RM.Calmodulin-dependent protein kinase II triggers mouse egg activation and embryodevelopment in the absence of Ca2+ oscillations. Dev Biol 2006;296:388–395.

Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, Miyazaki S.Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+oscillations in mouse eggs. J Biol Chem 2004;279:10408–10412.

Kouchi Z, Shikano T, Nakamura Y, Shirakawa H, Fukami K, Miyazaki S. The role ofEF-hand domains and C2 domain in regulation of enzymatic activity ofphospholipase Czeta. J Biol Chem 2005;280:21015–21021.

Kume S, Muto A, Aruga J, Nakagawa T, Michikawa T, Furuichi T, Nakade S, Okano H,Mikoshiba K. The Xenopus IP3 receptor: structure, function, and localization inoocytes and eggs. Cell 1993;73:555–570.

Kume S, Yamamoto A, Inoue T, Muto A, Okano H, Mikoshiba K. Developmentalexpression of the inositol 1,4,5-trisphosphate receptor and structural changes inthe endoplasmic reticulum during oogenesis and meiotic maturation of Xenopuslaevis. Dev Biol 1997;182:228–239.

Kupka MS, Ferraretti AP, de Mouzon J, Erb K, D’Hooghe T, Castilla JA, Calhaz-Jorge C,De Geyter C, Goossens V; European IVF-monitoring (EIM) Consortium, for theEuropean Society of Human Reproduction and Embryology (ESHRE). Assistedreproductive technology in Europe, 2010: results generated from Europeanregisters by ESHRE. Hum Reprod 2014;29:2099–2113.

Kyono K, Kumagai S, Nishinaka C, Nakajo Y, Uto H, Toya M, Sugawara J, Araki Y. Birthand follow-up of babies born following ICSI using SrCl2 oocyte activation. ReprodBiomed Online 2008;17:53–58.

Larman MG, Saunders CM, Carroll J, Lai FA, Swann K. Cell cycle-dependent Ca2+

oscillations in mouse embryos are regulated by nuclear targeting of PLCzeta. J CellSci 2004;117:2513–2521.

Lee B, Vermassen E, Yoon SY, Vanderheyden V, Ito J, Alfandari D, De Smedt H, Parys JB,Fissore RA. Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses atfertilization: a role for the MAP kinase pathway. Development 2006;133:4355–4365.

Lee B, Yoon SY, Malcuit C, Parys JB, Fissore RA. Inositol 1,4,5-trisphosphate receptor 1degradation in mouse eggs and impact on [Ca2+]i oscillations. J Cell Physiol 2010;222:238–247.

Lee K, Wang C, Machaty Z. STIM1 is required for Ca2+ signaling during mammalianfertilization. Dev Biol 2012;367:154–162.

Lee HC, Arny M, Grow D, Dumesic D, Fissore RA, Jellerette-Nolan T. Proteinphospholipase C Zeta1 expression in patients with failed ICSI but with normalsperm parameters. J Assist Reprod Genet 2014;31:749–756.

Levasseur M, Carroll M, Jones KT, McDougall A. A novel mechanism controls theCa2+ oscillations triggered by activation of ascidian eggs and has an absoluterequirement for Cdk1 activity. J Cell Sci 2007;120:1763–1771.

Li Y, Li RQ, Ou SB, Zhang NF, Ren L, Wei LN, Zhang QX, Yang DZ. Increased GDF9and BMP15 mRNA levels in cumulus granulosa cells correlate with oocytematuration, fertilization, and embryo quality in humans. Reprod Biol Endocrinol2014;12:81. doi: 10.1186/1477-7827-12-81.

Lin-Moshier Y, Sebastian PJ, Higgins L, Sampson ND, Hewitt JE, Marchant JS.Re-evaluation of the role of calcium homeostasis endoplasmic reticulum protein(CHERP) in cellular calcium signaling. J Biol Chem 2013;288:355–367.

Liou J, Fivaz M, Inoue T, Meyer T. Live-cell imaging reveals sequential oligomerizationand local plasma membrane targeting of stromal interaction molecule 1 afterCa2+ store depletion. PNAS 2007;104:9301–9306.

Liu H, Wu Z, Shi X, Li W, Liu C, Wang D, Ye X, Liu L, Na J, Cheng H et al. Atypical PKC,regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cellembryo compaction. Dev Biol 2013a;375:13–22.

Liu Y, Cao YX, Zhang ZG, Xing Q. Artificial oocyte activation and humanfailed-matured oocyte vitrification followed by in vitro maturation. Zygote 2013b;21:71–76.

Lopez G, Lafuente R, Checa MA, Carreras R, Brassesco M. Diagnostic value of spermDNA fragmentation and sperm high-magnification for predicting outcome ofassisted reproduction treatment. Asian J Androl 2013;15:790–794.

Louis JF, Thoma ME, Sørensen DN, McLain AC, King RB, Sundaram R, Keiding N, BuckLouis GM. The prevalence of couple infertility in the United States from a maleperspective: evidence from a nationally representative sample. Andrology 2013;1:741–748.

Lu Q, Zhao Y, Gao X, Li Y, Ma S, Mullen S, Critser JK, Chen ZJ. Combination of calciumionophore A23187 with puromycin salvages human unfertilized oocytes after ICSI.Eur J Obstet Gynecol Reprod Biol 2006;126:72–76.

Luria A, Tennenbaum T, Sun QY, Rubinstein S, Breitbart H. Differential localization ofconventional protein kinase C isoforms during mouse oocyte development. BiolReprod 2000;62:1564–1570.

Machaca K. Ca2+ signaling differentiation during oocyte maturation. J Cell Physiol 2007;213:331–340.

Madgwick S, Levasseur M, Jones KT. Calmodulin-dependent protein kinase II, and notprotein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs.J Cell Sci 2005;118:3849–3859.

Mahutte NG, Arici A. Failed fertilization: is it predictable? Curr Opin Obstet Gynecol 2003;15:211–218.

Mak DO, Foskett JK. Single-channel kinetics, inactivation, and spatial distribution ofinositol trisphosphate (IP3) receptors in Xenopus oocyte nucleus. J Gen Physiol1997;109:571–587.

Malcuit C, Knott JG, He C, Wainwright T, Parys JB, Robl JM, Fissore RA. Fertilization andinositol 1,4,5-trisphosphate (IP3)-induced calcium release in type-1 inositol1,4,5-trisphosphate receptor down-regulated bovine eggs. Biol Reprod2005;73:2–13.

Mangoli V, Dandekar S, Desai S, Mangoli R. The outcome of ART in males with impairedspermatogenesis. J Hum Reprod Sci 2008;1:73–76.

Mao L, Lou H, Lou Y, Wang N, Jin F. Behaviour of cytoplasmic organellesand cytoskeleton during oocyte maturation. Reprod Biomed Online 2014;28:284–299.

Martın-Romero FJ, Lopez-Guerrero AM, Alvarez IS, Pozo-Guisado E. Role ofstore-operated calcium entry during meiotic progression and fertilization ofmammalian oocytes. Int Rev Cell Mol Biol 2012;295:291–328.

Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National,regional, and global trends in infertility prevalence since 1990: a systematic analysisof 277 health surveys. PLoS Med 2012;9:e1001356.

Matifat F, Hague F, Brule G, Collin T. Regulation of InsP3-mediated Ca2+ release byCaMKII in Xenopus oocytes. Pflugers Arch 2001;441:796–801.

Matzuk MM, Lamb DJ. The biology of infertility: research advances and clinicalchallenges. Nat Med 2008;14:1197–1213.

Miyara F, Aubriot FX, Glissant A, Nathan C, Douard S, Stanovici A, Herve F,Dumont-Hassan M, LeMeur A, Cohen-Bacrie P et al. Multiparameter analysis ofhuman oocytes at metaphase II stage after IVF failure in non-male infertility. HumReprod 2003;18:1494–1503.

Miyazaki S. Inositol 1,4,5-trisphosphate-induced calcium release and guaninenucleotide-binding protein-mediated periodic calcium rises in golden hamstereggs. J Cell Biol 1988;106:345–353.

Miyazaki S, Shirakawa H, Nakada K, Honda Y. Essential role of the inositol1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+

oscillations at fertilization of mammalian eggs. Dev Biol 1993;158:62–78.Montag M, Parrington J, Swann K, Lai FA, van der Ven H. Presence and localization of

oscillin in human spermatozoa in relation to the integrity of the sperm membrane.FEBS Lett 1998;423:357–361.

Montag M, Koster M, van der Ven K, Bohlen U, van der Ven H. The benefit of artificialoocyte activation is dependent on the fertilization rate in a previous treatment cycle.Reprod Biomed Online 2012;24:521–526.

Moor RM, Dai Y, Lee C, Fulka J Jr. Oocyte maturation and embryonic failure. HumReprod Update 1998;4:223–236.

Muciaccia B, Sette C, Paronetto MP, Barchi M, Pensini S, D’Agostino A, Gandini L,Geremia R, Stefanini M, Rossi P. Expression of a truncated form of KIT tyrosinekinase in human spermatozoa correlates with sperm DNA integrity. Hum Reprod2010;25:2188–2202.

Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M, Araki Y. Pregnancyfollowing chemical activation of oocytes in a couple with repeated failure offertilization using ICSI: case report. Hum Reprod 2004;19:1604–1607.

Nakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T. Effect ofactivation with Ca ionophore A23187 and puromycin on the development ofhuman oocytes that failed to fertilize after intracytoplasmic sperm injection. FertilSteril 2001;76:148–152.

Nasr-Esfahani MH, Razavi S, Javdan Z, Tavalaee M. Artificial oocyte activation in severeteratozoospermia undergoing intracytoplasmic sperm injection. Fertil Steril 2008;90:2231–2237.

Nasr-Esfahani MH, Deemeh MR, Tavalaee M. Artificial oocyte activation andintracytoplasmic sperm injection. Fertil Steril 2010;94:520–526.

National Collaborating Centre for Women’s and Children’s Health (UK). Fertility:Assessment and Treatment for People with Fertility Problems. London: Royal

44 Yeste et al.

College of Obstetricians & Gynaecologists (UK); 2013 Feb. National Institute forHealth and Clinical Excellence: Guidance.

Neri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD. Understanding fertilizationthrough intracytoplasmic sperm injection (ICSI). Cell Calcium 2014;55:24–37.

Newton AC. Protein kinase C: structure, function, and regulation. J Biol Chem 1995;270:28495–28498.

Nikiforaki D, Vanden Meerschaut F, Qian C, De Croo I, Lu Y, Deroo T, Van denAbbeel E, Heindryckx B, De Sutter P. Oocyte cryopreservation and in vitroculture affect calcium signalling during human fertilization. Hum Reprod 2014;29:29–40.

Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation ofprotein kinase C. Science 1992;258:607–614.

Nomikos M, Blayney LM, Larman MG, Campbell K, Rossbach A, Saunders CM,Swann K, Lai FA. Role of phospholipase C-zeta domains in Ca2+-dependentphosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+

oscillations. J Biol Chem 2005;280:31011–31018.Nomikos M, Mulgrew-Nesbitt A, Pallavi P, Mihalyne G, Zaitseva I, Swann K, Lai FA,

Murray D, McLaughlin S. Binding of phosphoinositide-specific phospholipaseC-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructuredcluster of basic residues. J Biol Chem 2007;282:16644–16653.

Nomikos M, Elgmati K, Theodoridou M, Calver BL, Cumbes B, Nounesis G, Swann K,Lai FA. Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducingand PIP2 hydrolysis activity of sperm PLCz. Biochem J 2011a;434:211–217.

Nomikos M, Elgmati K, Theodoridou M, Georgilis A, Gonzalez-Garcia JR, Nounesis G,Swann K, Lai FA. Novel regulation of PLCz activity via its XY-linker. Biochem J 2011b;438:427–432.

Nomikos M, Swann K, Lai FA. Starting a new life: sperm PLC-zeta mobilizes the Ca2+

signal that induces egg activation and embryo development: an essentialphospholipase C with implications for male infertility. Bioessays 2012;34:126–134.

Nomikos M, Kashir J, Swann K, Lai FA. Sperm PLCz: from structure to Ca2+ oscillations,egg activation and therapeutic potential. Sperm PLCz: from structure to Ca2+

oscillations, egg activation and therapeutic potential. FEBS Lett 2013a;587:3609–3616.Nomikos M, Yu Y, Elgmati K, Theodoridou M, Campbell K, Vassilakopoulou V, Zikos C,

Livaniou E, Amso N, Nounesis G et al. Phospholipase Cz rescues failed oocyteactivation in a prototype of male factor infertility. Fertil Steril 2013b;99:76–85.

Nomikos M, Sanders JR, Theodoridou M, Kashir J, Matthews E, Nounesis G, Lai FA,Swann K. Sperm-specific post-acrosomal WW-domain binding protein (PAWP)does not cause Ca2+ release in mouse oocytes. Mol Hum Reprod 2014;20:938–947.

Nomikos M, Sanders JR, Kashir J, Sanusi R, Buntwal L, Love D, Ashley P, Sanders D,Knaggs P, Bunkheila A et al. Functional disparity between human PAWP and PLCz

in the generation of Ca2+ oscillations for oocyte activation. Mol Hum Reprod2015a;21:702–710.

Nomikos M, Swann K, Lai FA. Is PAWP the ‘real’ sperm factor? Asian J Androl 2015b;17:444–446.

Oko R, Sutovsky P. Biogenesis of sperm perinuclear theca and its role in spermfunctional competence and fertilization. J Reprod Immunol 2009;83:2–7.

Ombelet W, Cooke I, Dyer S, Serour G, Devroey P. Infertility and the provision ofinfertility medical services in developing countries. Hum Reprod Update 2008;14:605–621.

Ozil JP, Banrezes B, Toth S, Pan H, Schultz RM. Ca2+ oscillatory pattern in fertilizedmouse eggs affects gene expression and development to term. Dev Biol 2006;300:534–544.

Palermo G, Joris H, DevroeyP, Van Steirteghem AC. Pregnancies after intracytoplasmicinjection of single spermatozoon into an oocyte. Lancet 1992;340:17–18.

Park CY, Hoover PJ, Mullins FM, Bachhawat P, Covington ED, Raunser S, Walz T,Garcia KC, Dolmetsch RE, Lewis RS. STIM1 clusters and activates CRAC channelsvia direct binding of a cytosolic domain to Orai1. Cell 2009;136:876–890.

Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA. Calcium oscillationsin mammalian eggs triggered by a soluble sperm protein. Nature 1996;379:364–368.

Parrington J, Lai FA, Swann K. A novel protein for Ca2+ signaling at fertilization. Curr TopDev Biol 1998a;39:215–243.

Parrington J, Brind S, De Smedt H, Gangeswaran R, Lai FA, Wojcikiewicz R, Carroll J.Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and earlyembryos: the type I isoform is upregulated in oocytes and downregulated afterfertilization. Dev Biol 1998b;203:451–461.

Parrington J, Davis LC, Galione A, Wessel G. Flipping the switch: how a sperm activatesthe egg at fertilization. Dev Dyn 2007;236:2027–2038.

Pauken CM, Capco DG. The expression and stage-specific localization of protein kinaseC isotypes during mouse preimplantation development. Dev Biol 2000;223:411–421.

Periasamy M, Kalyanasundaram A. SERCA pump isoforms: their role in calciumtransport and disease. Muscle Nerve 2007;35:430–442.

Pesty A, Avazeri N, Lefevre B. Nuclear calcium release by InsP3-receptor channelsplays a role in meiosis reinitiation in the mouse oocyte. Cell Calcium 1998;24:239–251.

Phillips SV, Yu Y, Rossbach A, Nomikos M, Vassilakopoulou V, Livaniou E, Cumbes B,Lai FA, George CH, Swann K. Divergent effect of mammalian PLCz in generatingCa2+ oscillations in somatic cells compared with eggs. Biochem J 2011;438:545–553.

Practice Committee of the American Society for Reproductive Medicine. Definitions ofinfertility and recurrent pregnancy loss: a committee opinion. Fertil Steril 2013;99:63.

Pregl Breznik B, Kovacic B, Vlaisavljevic V. Are sperm DNA fragmentation,hyperactivation, and hyaluronan-binding ability predictive for fertilization andembryo development in in vitro fertilization and intracytoplasmic sperm injection?Fertil Steril 2013;99:1233–1241.

Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 1986;7:1–12.Putney JW Jr. Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium

entry. Cell Calcium 1997;21:257–261.Putney JW. Capacitative calcium entry: from concept to molecules. Immunol Rev 2009;

231:10–22.Putney JW. Origins of the concept of store-operated calcium entry. Front Biosci 2011;

S3:980–984.Rajagopal S, Fields BL, Burton BK, On C, Reeder AA, Kamatchi GL. Inhibition of protein

kinase C (PKC) response of voltage-gated calcium (Cav)2.2 channels expressed inXenopus oocytes by Cavb subunits. Neuroscience 2014;280:1–9.

Ramadan WM, Kashir J, Jones C, Coward K. Oocyte activation and phospholipase Czeta (PLCz): diagnostic and therapeutic implications for assisted reproductivetechnology. Cell Commun Signal 2012;10:12. doi: 10.1186/1478-811X-10-12.

Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, Vitullo AD. Cytoskeletalorganization defects and abortive activation in human oocytes after IVF and ICSIfailure. Mol Hum Reprod 2000;6:510–516.

Rhee SG. Reflections on the days of phospholipase C. Adv Biol Regul 2013;53:223–231.Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S, Kirkman-Brown J,

Coomarasamy A. The effect of sperm DNA fragmentation on miscarriage rates: asystematic review and meta-analysis. Hum Reprod 2012;27:2908–2917.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O,Kozak JA, Wagner SL, Cahalan MD et al. STIM1, an essential and conservedcomponent of store-operated Ca2+ channel function. J Cell Biol 2005;169:435–445.

Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA, Cibelli JB.Parthenogenetic activation of bovine oocytes using bovine and murinephospholipase C zeta. BMC Dev Biol 2008;8:16. doi: 10.1186/1471-213X-8-16.

Rossi P, Dolci S, Sette C, Geremia R. Molecular mechanisms utilized by alternative c-kitgene products in the control of spermatogonial proliferation and sperm-mediatedegg activation. Andrologia 2003;35:71–78.

Roy SS, Hajnoczky G. Calcium, mitochondria and apoptosis studied by fluorescencemeasurements. Methods 2008;46:213–223.

Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization andpregnancy after assisted oocyte activation and intracytoplasmic sperm injection in acase of round-headed sperm associated with deficient oocyte activation capacity.Fertil Steril 1997;68:1144–1147.

Saiz N, Grabarek JB, Sabherwal N, Papalopulu N, Plusa B. Atypical protein kinase Ccouples cell sorting with primitive endoderm maturation in the mouse blastocyst.Development 2013;140:4311–4322.

Sathanawongs A, Fujiwara K, Kato T, Hirose M, Kamoshita M, Wojcikiewicz RJ, Parys JB,Ito J, Kashiwazaki N. The effect of M-phase stage-dependent kinase inhibitors oninositol 1,4,5-trisphosphate receptor 1 (IP3 R1) expression and localization in pigoocytes. Anim Sci J 2015;86:138–147.

Sato Y, Miyazaki S, Shikano T, Mitsuhashi N, Takeuchi H, Mikoshiba K, Kuwabara Y.Adenophostin, a potent agonist of the inositol 1,4,5-trisphosphate receptor, isuseful for fertilization of mouse oocytes injected with round spermatids leading tonormal offspring. Biol Reprod 1998;58:867–873.

SatoK,FukamiY,StithBJ.Signal transductionpathways leadingtoCa2+ release inavertebratemodel system: lessons from Xenopus eggs. Semin Cell Dev Biol 2006;17:285–292.

Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K, Lai FA.PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryodevelopment. Development 2002;129:3533–3544.


Saunders CM, Swann K, Lai FA. PLCzeta, a sperm-specific PLC and its potential role infertilization. Biochem Soc Symp 2007;74:23–36.

Sette C, Bevilacqua A, Bianchini A, Mangia F, Geremia R, Rossi P. Parthenogeneticactivation of mouse eggs by microinjection of a truncated c-kit tyrosine kinasepresent in spermatozoa. Development 1997;124:2267–2274.

Sette C, Bevilacqua A, Geremia R, Rossi P. Involvement of phospholipase Cg1 in mouseegg activation induced by a truncated form of the c-kit tyrosine kinase present inspermatozoa. J Cell Biol 1998;142:1063–1074.

Sette C, Paronetto MP, Barchi M, Bevilacqua A, Geremia R, Rossi P. Tr-kit-inducedresumption of the cell cycle in mouse eggs requires activation of a Src-like kinase.EMBO J 2002;21:5386–5395.

Sfontouris IA, Nastri CO, Lima ML, Tahmasbpourmarzouni E, Raine-Fenning N,Martins WP. Artificial oocyte activation to improve reproductive outcomes inwomen with previous fertilization failure: a systematic review and meta-analysis ofRCTs. Hum Reprod 2015;30:1831–1841.

Shinar S, Almog B, Levin I, Shwartz T, Amit A, Hasson J. Total fertilization failure inintra-cytoplasmic sperm injection cycles—classification and management. GynecolEndocrinol 2014;30:593–596.

Simon L, Proutski I, Stevenson M, Jennings D, McManus J, Lutton D, Lewis SE. SpermDNA damage has a negative association with live-birth rates after IVF. ReprodBiomed Online 2013;26:68–78.

Slade P, O’Neill C, Simpson AJ, Lashen H. The relationship between perceived stigma,disclosure patterns, support and distress in new attendees at an infertility clinic. HumReprod 2007;22:2309–2317.

Smyth JT, Hwang SY, Tomita T, DeHaven WI, Mercer JC, Putney JW. Activation andregulation of store-operated calcium entry. J Cell Mol Med 2010;14:2337–2349.

Soboloff J, Rothberg BS, Madesh M, Gill DL. STIM proteins: dynamic calcium signaltransducers. Nat Rev Mol Cell Biol 2012;13:549–565.

Son WY, Chung JT, Demirtas E, Holzer H, Sylvestre C, Buckett W, Chian RC, Tan SL.Comparison of in-vitro maturation cycles with and without in-vivo matured oocytesretrieved. Reprod Biomed Online 2008;17:59–67.

Sone Y, Ito M, Shirakawa H, Shikano T, Takeuchi H, Kinoshita K, Miyazaki S. Nucleartranslocation of phospholipase C-zeta, an egg-activating factor, during earlyembryonic development. Biochem Biophys Res Commun 2005;330:690–694.

StathopulosPB,ZhengL,LiGY,PlevinMJ, Ikura M.Structural andmechanistic insights intoSTIM1-mediated initiation of store-operated calcium entry. Cell 2008;135:110–122.

Stern K. Assisted reproductive technology—what’s new and what’s important? AustFam Physician 2012;41:762–768.

Stricker SA. Comparative biology of calcium signaling during fertilization and eggactivation in animals. Dev Biol 1999;211:157–176.

Suh PG, Park JI, Manzoli L, Cocco L, Peak JC, Katan M, Fukami K, Kataoka T, Yun S,Ryu SH. Multiple roles of phosphoinositide-specific phospholipase C isozymes.BMB Rep 2008;41:415–434.

Sutovsky P, Manandhar G, Wu A, Oko R. Interactions of sperm perinuclear theca withthe oocyte: implications for oocyte activation, anti-polyspermy defense, and assistedreproduction. Microsc Res Tech 2003;61:362–378.

Swain JE, Pool TB. ART failure: oocyte contributions to unsuccessful fertilization. HumReprod Update 2008;14:431–446.

Swann K. A cytosolic sperm factor stimulates repetitive calcium increases and mimicsfertilization in hamster eggs. Development 1990;110:1295–1302. 259:353–369.

Swann K. Measuring Ca2+ oscillations in mammalian eggs. Methods Mol Biol 2013;957:231–248.

Swann K, Lai FA. PLCz and the initiation of Ca2+ oscillations in fertilizing mammalianeggs. Cell Calcium 2013;53:55–62.

Swann K, Whitaker MJ. Second messengers at fertilization in sea-urchin eggs. J ReprodFertil Suppl 1990;42:141–153.

Swann K, Yu Y. The dynamics of calcium oscillations that activate mammalian eggs. Int JDev Biol 2008;52:585–594.

Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic sperm factor that triggersCa2+ oscillations and egg activation in mammals is a novel phospholipase C:PLCzeta. Reproduction 2004;127:431–439.

Swann K, Saunders CM, Rogers NT, Lai FA. PLCz(zeta): a sperm protein that triggersCa2+ oscillations and egg activation in mammals. Semin Cell Dev Biol 2006;17:264–273.

Swann K, Campbell K, Yu Y, Saunders C, Lai FA. Use of luciferase chimaera to monitorPLCzeta expression in mouse eggs. Methods Mol Biol 2009;518:17–29.

Swann K, Windsor S, Campbell K, Elgmati K, Nomikos M, Zernicka-Goetz M, Amso N,Lai FA, Thomas A, Graham C. Phospholipase C-z-induced Ca2+ oscillations cause

coincident cytoplasmic movements in human oocytes that failed to fertilize afterintracytoplasmic sperm injection. Fertil Steril 2012;97:742–747.

Talmor-Cohen A, Tomashov-Matar R, Eliyahu E, Shapiro R, Shalgi R. Are Src familykinases involved in cell cycle resumption in rat eggs? Reproduction 2004;127:455–463.

Tatone C, Delle Monache S, Francione A, Gioia L, Barboni B, Colonna R.Ca2+-independent protein kinase C signalling in mouse eggs during the earlyphases of fertilization. Int J Dev Biol 2003;47:327–333.

Taylor SL, Yoon SY, Morshedi MS, Lacey DR, Jellerette T, Fissore RA, Oehninger S.Complete globozoospermia associated with PLCz deficiency treated with calciumionophore and ICSI results in pregnancy. Reprod Biomed Online 2010;20:559–564.

Taylor CW, Tovey SC, Rossi AM, Lopez Sanjurjo CI, Prole DL, Rahman T. Structuralorganization of signalling to and from IP3 receptors. Biochem Soc Trans 2014;42:63–70.

Terada Y, Hasegawa H, Takahashi A, Ugajin T, Yaegashi N, Okamura K. Successfulpregnancy after oocyte activation by a calcium ionophore for a patient with recurrentintracytoplasmic sperm injection failure, with an assessment of oocyte activation andsperm centrosomal function using bovine eggs. Fertil Steril 2009;91:935.e11–935.e14.

Tesarik J. Sperm-induced calcium oscillations. Oscillin-reopening the hunting season.Mol Hum Reprod 1998;4:1007–1012.

Tesarik J, Nagy ZP, Mendoza C, Greco E. Chemically and mechanically inducedmembrane fusion: non-activating methods for nuclear transfer in mature humanoocytes. Hum Reprod 2000;15:1149–1154.

Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified intracytoplasmicsperm injection technique to overcome sperm-borne and oocyte-borne oocyteactivation failures. Fertil Steril 2002;78:619–624.

Theodoridou M, Nomikos M, Parthimos D, Gonzalez-Garcia JR, Elgmati K, Calver BL,Sideratou Z, Nounesis G, Swann K, Lai FA. Chimeras of sperm PLCz reveal disparateprotein domain functions in the generation of intracellular Ca2+ oscillations inmammalian eggs at fertilization. Mol Hum Reprod 2013;19:852–864.

Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R, Buck Louis GM.Prevalence of infertility in the United States as estimated by the current durationapproach and a traditional constructed approach. Fertil Steril 2013;99:1324–1331.

Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, Lopes P, Tabaste JM,Spira A. Incidence and main causes of infertility in a resident population (1,850,000)of three French regions (1988–1989). Hum Reprod 1991;6:811–816.

Toranzo GS, Buhler MC, Buhler MI. Participation of IP3R, RyR and L-type Ca2+ channelin the nuclear maturation of Rhinella arenarum oocytes. Zygote 2014;22:110–123.

Toshimori K, Ito C. Formation and organization of the mammalian sperm head. ArchHistol Cytol 2003;66:383–396.

Tripathi A, Chaube SK. High cytosolic free calcium level signals apoptosis throughmitochondria-caspase mediated pathway in rat eggs cultured in vitro. Apoptosis2012;17:439–448.

Tsaadon L, Kaplan-Kraicer R, Shalgi R. Myristoylated alanine-rich C kinase substrate,but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in corticalgranules exocytosis. Reproduction 2008;135:613–624.

Tuzi S, Uekama N, Okada M, Yamaguchi S, Saito H, Yagisawa H. Structure anddynamics of the phospholipase C-delta1 pleckstrin homology domain located atthe lipid bilayer surface. J Biol Chem 2003;278:28019–28025.

Ueda Y, Ishitsuka R, Hullin-Matsuda F, Kobayashi T. Regulation of the transbilayermovement of diacylglycerol in the plasma membrane. Biochimie 2014;107 PartA:43–50.

Ullah G, Jung P, Machaca K. Modeling Ca2+ signaling differentiation during oocytematuration. Cell Calcium 2007;42:556–564.

Vadnais ML, Gerton LG. From PAWP to ‘Pop’: opening up new pathways tofatherhood. Asian J Androl 2015;17:443–444.

VanBlerkom J. Mitochondrial function in the human oocyte and embryo and their role indevelopmental competence. Mitochondrion 2011;11:797–813.

Van Blerkom J, Caltrider K. Sperm attachment and penetration competence in thehuman oocyte: a possible aetiology of fertilization failure involving the organizationof oolemmal lipid raft microdomains influenced by the DCm of subplasmalemmalmitochondria. Reprod Biomed Online 2013;27:690–701.

Vanden Meerschaut F, Nikiforaki D, De Gheselle S, Dullaerts V, Van den Abbeel E,Gerris J, Heindryckx B, De Sutter P. Assisted oocyte activation is not beneficial forall patients with a suspected oocyte-related activation deficiency. Hum Reprod2012;27:1977–1984.

Vanden Meerschaut F, Leybaert L, Nikiforaki D, Qian C, Heindryckx B, De Sutter P.Diagnostic and prognostic value of calcium oscillatory pattern analysis for patientswith ICSI fertilization failure. Hum Reprod 2013;28:87–98.

46 Yeste et al.

Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P. Assisted oocyteactivation following ICSI fertilization failure. Reprod Biomed Online 2014a;28:560–571.

Vanden Meerschaut F, D’Haeseleer E, Gysels H, Thienpont Y, Dewitte G,Heindryckx B, Oostra A, Roeyers H, Van Lierde K, De Sutter P. Neonatal andneurodevelopmental outcome of children aged 3–10 years born followingassisted oocyte activation. Reprod Biomed Online 2014b;28:54–63.

Vanderheyden V, Wakai T, Bultynck G, De Smedt H, Parys JB, Fissore RA. Regulation ofinositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation byMPM-2 phosphorylation. Cell Calcium 2009;46:56–64.

Vayena E, Rowe PJ, Peterson HB. Assisted reproductive technology in developingcountries: why should we care? Fertil Steril 2002;78:13–15.

Verbert L, Lee B, Kocks SL, Assefa Z, Parys JB, Missiaen L, Callewaert G, Fissore RA, DeSmedt H, Bultynck G. Caspase-3-truncated type 1 inositol 1,4,5-trisphosphatereceptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling. Biol Cell2008;100:39–49.

Viveiros MM, O’Brien M, Wigglesworth K, Eppig JJ. Characterization of protein kinaseC-delta in mouse oocytes throughout meiotic maturation and following eggactivation. Biol Reprod 2003;69:1494–1499.

Von Stetina JR, Orr-Weaver TL. Developmental control of oocyte maturation and eggactivation in metazoan models. Cold Spring Harb Perspect Biol 2011;3:a005553.

Wakai T, Zhang N, Vangheluwe P, Fissore RA. Regulation of endoplasmic reticulumCa(2+) oscillations in mammalian eggs. J Cell Sci 2013;126:5714–5724.

Wang C, Machaty Z. Calcium influx in mammalian eggs. Reproduction 2013;145:R97–R105.Wang C, Lee K, Gajdocsi E, Papp AB, Machaty Z. Orai1 mediates store-operated Ca2+

entry during fertilization in mammalian oocytes. Dev Biol 2012;365:414–423.White KL, Pate BJ, Sessions BR. Oolemma receptors and oocyte activation. Syst Biol

Reprod Med 2010;56:365–375.Williams CJ, Mehlmann LM, Jaffe LA, Kopf GS, Schultz RM. Evidence that Gq family G

proteins do not function in mouse egg activation at fertilization. Dev Biol 1998;198:116–127.

Williams RT, Senior PV, Van Stekelenburg L, Layton JE, Smith PJ, Dziadek MA. Stromalinteraction molecule 1 (STIM1), a transmembrane protein with growth suppressoractivity, contains an extracellular SAM domain modified by N-linked glycosylation.Biochim Biophys Acta 2002;1596:131–137.

Wojcikiewicz RJ, Furuichi T, Nakade S, Mikoshiba K, Nahorski SR. Muscarinic receptoractivation down-regulates the type I inositol 1,4,5-trisphosphate receptor byaccelerating its degradation. J Biol Chem 1994;269:7963–7969.

Wolny YM, Fissore RA, Wu H, Reis MM, Colombero LT, Ergun B, Rosenwaks Z,Palermo GD. Human glucosamine-6-phosphate isomerase, a homologue ofhamster oscillin, does not appear to be involved in Ca2+ release in mammalianoocytes. Mol Reprod Dev 1999;52:277–287.

Wu H, Smyth J, Luzzi V, Fukami K, Takenawa T, Black SL, Allbritton NL, Fissore RA.Sperm factor induces intracellular free calcium oscillations by stimulating thephosphoinositide pathway. Biol Reprod 2001;64:1338–1349.

Wu AT, Sutovsky P, Manandhar G, Xu W, Katayama M, Day BN, Park KW, Yi YJ,Xi YW, Prather RS et al. PAWP, a sperm-specific WW domain-binding protein,promotes meiotic resumption and pronuclear development during fertilization.J Biol Chem 2007a;282:12164–12175.

Wu AT, Sutovsky P, Xu W, van der Spoel AC, Platt FM, Oko R. The postacrosomalassembly of sperm head protein, PAWP, is independent of acrosome formation anddependent on microtubular manchette transport. Dev Biol 2007b;312:471–483.

Wu AK, Elliott P, Katz PP, Smith JF. Time costs of fertility care: the hidden hardship ofbuilding a family. Fertil Steril 2013;99:2025–2030.

Xing X, Zhao H, Li M, Sun M, Li Y, Chen ZJ. Morphologically abnormal oocytes notcapable of fertilization despite repeated strategies. Fertil Steril 2011;95:2435.e5–7.

Xu Z, Williams CJ, Kopf GS, Schultz RM. Maturation-associated increase in IP3 receptortype 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior inmouse eggs. Dev Biol 2003;254:163–171.

Yamano S, Nakagawa K, Nakasaka H, Aono T. Fertilization failure and oocyteactivation. J Med Invest 2000;47:1–8.

Yanagida K, Morozumi K, Katayose H, Hayashi S, Sato A. Successful pregnancy afterICSI with strontium oocyte activation in low rates of fertilization. Reprod BiomedOnline 2006;13:801–806.

Yanagida K, Fujikura Y, Katayose H. The present status of artificial oocyte activation inassisted reproductive technology. Reprod Med Biol 2008;7:133–142.

Yang YR, Follo MY, Cocco L, Suh PG. The physiological roles of primary phospholipaseC. Adv Biol Regul 2013;53:232–241.

Yassine S, Escoffier J, Martinez G, Coutton C, Karaouzene T, Zouari R, Ravanat JL,Metzler-Guillemain C, Lee HC, Fissore R et al. Dpy19l2-deficientglobozoospermic sperm display altered genome packaging and DNA damage thatcompromises the initiation of embryo development. Mol Hum Reprod 2015;21:169–185.

Yelumalai S, Yeste M, Jones C, Amdani SN, Kashir J, Mounce G, Fatum M, Barratt C,McVeigh E, Coward K. Total levels and proportions of sperm exhibitingphospholipase C Zeta (PLCz) are significantly correlated with fertilization ratesfollowing intracytoplasmic sperm injection. Fertil Steril 2015;104:561–568.e4.

Yeste M, Fernandez-Novell JM, Ramio-Lluch L, Estrada E, Rocha LG, Cebrian-Perez JA,Muino-Blanco T, Concha II, Ramırez A, Rodrıguez-Gil JE. Intracellular calciummovements of boar spermatozoa during ‘in vitro’ capacitation and subsequentacrosome exocytosis follow a multiple-storage place, extracellularcalcium-dependent model. Andrology 2015;3:729–747.

Yoda A, Oda S, Shikano T, Kouchi Z, Awaji T, Shirakawa H, Kinoshita K, Miyazaki S.Ca2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs isaccumulated to the pronucleus during egg activation. Dev Biol 2004;268:245–257.

Yoneda A, Kashima M, Yoshida S, Terada K, Nakagawa S, Sakamoto A, Hayakawa K,Suzuki K, Ueda J, Watanabe T. Molecular cloning, testicular postnatal expression,and oocyte-activating potential of porcine phospholipase Czeta. Reproduction2006;132:393–401.

Yoon SY, Jellerette T, Salicioni AM, Lee HC, Yoo MS, Coward K, Parrington J, Grow D,Cibelli JB, Visconti PE et al. Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+)release and are unable to initiate the first step of embryo development. J Clin Invest2008;118:3671–3681.

Yoon SY, Eum JH, Lee JE, Lee HC, Kim YS, Han JE, Won HJ, Park SH, Shim SH, Lee WSet al. Recombinant human phospholipase C zeta 1 induces intracellular calciumoscillations and oocyte activation in mouse and human oocytes. Hum Reprod2012;27:1768–1780.

Young C, Grasa P, Coward K, Davis LC, Parrington J. Phospholipase C zeta undergoesdynamic changes in its pattern of localization in sperm during capacitation and theacrosome reaction. Fertil Steril 2009;91:2230–2242.

Yu Y, Halet G, Lai FA, Swann K. Regulation of diacylglycerol production and proteinkinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation.Biol Cell 2008;100:633–643.

Yu Y, Nomikos M, Theodoridou M, Nounesis G, Lai FA, Swann K. PLCz causes Ca(2+)oscillations in mouse eggs by targeting intracellular and not plasma membranePI(4,5)P(2). Mol Biol Cell 2012;23:371–380.

Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K,Sullivan E, Vanderpoel S; International Committee for Monitoring AssistedReproductive Technology; World Health Organization. International Committeefor Monitoring Assisted Reproductive Technology (ICMART) and the WorldHealth Organization (WHO) revised glossary of ART terminology, 2009. FertilSteril 2009;92:1520–1524.

Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, Stauderman KA,Cahalan MD. STIM1 is a Ca2+ sensor that activates CRAC channels andmigrates from the Ca2+ store to the plasma membrane. Nature 2005;437:902–905.

Zhang D, Boulware MJ, Pendleton MR, Nogi T, Marchant JS. The inositol1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes. Biochem J 2007;404:383–391.

Zhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentationhas an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril2014a;102:998–1005.

Zhao MH, Kwon JW, Liang S, Kim SH, Li YH, Oh JS, Kim NH, Cui XS. Zinc regulatesmeiotic resumption in porcine oocytes via a protein kinase C-related pathway.PLoS One 2014b;9:e102097. doi: 10.1371/journal.pone.0102097.


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