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Cell Calcium 53 (2013) 63– 67

Contents lists available at SciVerse ScienceDirect

Cell Calcium

j ourna l ho me page: www.elsev ier .com/ locate /ceca

a2+ homeostasis and regulation of ER Ca2+ in mammalian oocytes/eggs

akuya Wakaia, Rafael A. Fissoreb,∗

Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, JapanDepartment of Veterinary and Animal Sciences, University of Massachusetts Amherst, 661 North Pleasant Street, Amherst, MA 01003, United States

r t i c l e i n f o

rticle history:eceived 11 October 2012eceived in revised form 2 November 2012ccepted 3 November 2012vailable online 21 December 2012

eywords:ocytes

a b s t r a c t

The activation of the developmental program in mammalian eggs relies on the initiation at the time offertilization of repeated rises in the intracellular concentration of free calcium ([Ca2+]i), also known as[Ca2+]i oscillations. The ability to mount the full complement of oscillations is only achieved at the end ofoocyte maturation, at the metaphase stage of meiosis II (MII). Over the last decades research has focusedon addressing the mechanisms by which the sperm initiates the oscillations and identification of thechannels that mediate intracellular Ca2+ release. This review will describe the up-to-date knowledge ofother aspects of Ca2+ homeostasis in mouse oocytes, such as the mechanisms that transport Ca2+ out of

2+

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the cytosol into the endoplasmic reticulum (ER), the Ca store of the oocyte/egg, into other organellesand also those that extrude Ca2+. Evidence pointing to channels in the plasma membrane that mediateCa2+ entry from the extracellular milieu, which is required for the persistence of the oscillations, is alsodiscussed, along with the modifications that these mechanisms undergo during maturation. Lastly, wehighlight areas where additional research is needed to obtain a better understating of the molecules andmechanisms that regulate Ca2+ homeostasis in this unique Ca2+ signaling system.

. Introduction

Before fertilization, mammalian eggs are arrested at theetaphase stage of the second meiosis (MII). Sperm entry triggers

series of increases in the intracellular free-Ca2+ concentration[Ca2+]i), termed [Ca2+]i oscillations, which enable exit from the

II arrest and induce egg activation [1]. Importantly, in mammalshe Ca2+ signal unfolds in a pattern of brief but periodical rises thatast for several hrs after sperm entry. The spatio-temporal informa-ion provided by the pattern of these [Ca2+]i responses is decodedy downstream effectors that underpin the distinct cellular eventsf egg activation, which include cortical granule (CG) exocytosis,xtrusion of the second polar body (2PB), pronuclear (PN) for-ation and entry into first mitosis [2]. Although the presence of

ong-lasting [Ca2+]i oscillations is a hallmark of mammalian fertil-zation, the underlying molecular changes that make them possibleemain elusive.

Inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release fromntracellular stores is primarily responsible for the generation ofhe [Ca2+]i wave and oscillations at fertilization [3]. Although the

eneral properties of Ca2+ release mechanism during oscillationsave been described in the context of IP3 production and regu-

ation of its cognate receptor (IP3R), the regulation of the [Ca2+]i

∗ Corresponding author. Tel.: +1 413 545 5548; fax: +1 413 545 6326.E-mail address: rfissore@vasci.umass.edu (R.A. Fissore).

143-4160/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.ceca.2012.11.010

© 2012 Elsevier Ltd. All rights reserved.

responses in these cells may be far more complex. For instance,to continue the long-term [Ca2+]i oscillations without attenuation,after each [Ca2+]i rise [Ca2+]i levels need to be rapidly returned tobaseline values, and the Ca2+ content of the endoplasmic reticu-lum (ER), the main Ca2+ reservoir in the cell [4], needs to be refilled([Ca2+]ER) in anticipation of the next [Ca2+]i rise. To bring [Ca2+]ito baseline levels, cells either reuptake the free cytosolic Ca2+ intothe ER refilling the store and/or remove it to other organelles or tothe extracellular space [5,6]. Nevertheless, to consistently replen-ish [Ca2+]ER and maintain oscillations, extracellular Ca2+ must enterinto the egg/oocyte across the plasma membrane by a varietyof channels and mechanisms, one of which is the store operatedCa2+ entry (SOCE) mechanism [7]. Despite the pivotal role of thesechannels/mechanisms on Ca2+ homeostasis and support of theoscillations, few studies have examined the underlying moleculesand their function in mammalian oocytes/eggs.

Prior to ovulation, during oocyte maturation, oocytes undergonuclear and cytoplasmic modifications in preparation for fertiliza-tion. Immature oocytes resume meiosis and transition from thegerminal vesicle (GV) stage to the MII stage. Importantly, the pre-cise spatio-temporal pattern of sperm-associated [Ca2+]i responsesin eggs is established during maturation. In fact, in vitro fertilizedGV oocytes show fewer [Ca2+]i oscillations and each [Ca2+]i rise

exhibits lesser duration and amplitude than those observed in fer-tilized MII eggs [8,9]. In spite of this knowledge, the mechanismsunderlying the enhanced Ca2+ releasing ability of matured eggs vs.GV oocytes are not well understood, although multiple parameters

6 ell Calcium 53 (2013) 63– 67

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Fig. 1. [Ca2+]ER and [Ca2+]i undergo simultaneous but opposite changes in con-centration during oscillations in mouse MII eggs. Ca2+ responses were induced byinjection of 0.05 �g/�l mouse PLC� cRNA into MII eggs. In vitro transcribed D1ERcRNA was injected into eggs 5 h before the initiation of [Ca2+]i measurements. Theemission ratio of D1ER (YFP/CFP) was used to estimate relative changes in [Ca2+]ER

(right axis, blue trace). [Ca2+]i (left axis, red trace) was recorded using Rhod-2. Rhod-2+

4 T. Wakai, R.A. Fissore / C

e.g. IP3R modifications, ER re-organization, increase in [Ca2+]ER) arehought to be involved in this process [10]. In addition, given thateveral of the parameters of Ca2+ homeostasis progressively changeuring maturation, for instance [Ca2+]ER steadily increases duringaturation, the molecules responsible for these adjustments in

a2+ homeostasis may experience dynamic modifications duringaturation such that some of the mechanisms/channels active at

he GV stage may not be so at the MII stage and vice versa.This review highlights the changes that [Ca2+]ER experiences

uring maturation and fertilization. The mechanisms that con-ribute to the regulation of [Ca2+]ER are also discussed, includinga2+ buffering systems and Ca2+ influx mechanisms as well as thehanges that these mechanisms undergo during oocyte maturation.

. Ca2+ homeostasis during fertilization

The initiation of [Ca2+]i oscillations during mammalian fertil-zation is thought to be triggered by the release from the spermf a male specific Phospholipase C (PLC) enzyme, PLC�, follow-ng fusion of the gametes [11,12]. More details about PLC� can beound in another chapter of this special issue. Therefore, our focusill be on other components of the Ca2+ toolkit that regulate Ca2+

omeostasis in oocytes and eggs.

.1. Ca2+ buffering mechanisms

To bring [Ca2+]i to baseline levels after a [Ca2+]i rise, cellsither reuptake the free cytosolic Ca2+ into the ER by the actionf the sarco-endoplasmic reticulum Ca2+ ATPases (SERCA) and/oremove it to the surrounding environment by the action of plasmaembrane Ca2+ ATPase (PMCA) and Na+/Ca2+ exchanger [5,6].

ew studies have addressed the function of these molecules inammalian oocytes/eggs. Three different SERCA genes (ATP2A1–3)

ncode three main isoforms (SERCA1–3), each of which under-oes tissue-specific splicing, further increasing the diversity ofhese pumps [13]. SERCA1a and SERCA1b variants are expressedn skeletal muscle. SERCA2a variant is expressed in cardiac mus-le, whereas SERCA2b variant is expressed nearly ubiquitously ands thus considered the housekeeping isoform. SERCA3 is insteadxpressed in a limited number of non-muscle cells [13]. In Xenopusocytes, expression of the SERCA2 protein was documented usingmmunofluorescence and it was shown to undergo reorganizationimilar to that described for IP3R during oocyte maturation, whichs consistent with the fact that both proteins are ER-resident pro-eins and with the reorganization that the ER undergoes during

aturation [14]. The molecular presence and cellular distributionf SERCA2b has not yet been characterized in mammalian oocytes,lthough transcripts and proteins have been reported [15,16]. Theunctional importance of SERCA can be surmised by the alterationf [Ca2+]i levels caused by exposure to thapsigargin, an specificnhibitor of SERCA [17]. For example, exposure of MII eggs to thap-igargin causes a slow and steady rise in [Ca2+]i followed by arotracted decline while in fertilized eggs, addition of thapsigarginr other SERCA inhibitors, prematurely terminates [Ca2+]i oscilla-ions [17,18]. Nonetheless, the impact that these inhibitors haven [Ca2+]ER has not been investigated, and therefore the regulatoryole of [Ca2+]ER on the pattern and/or persistence of oscillations isnknown in this species. Here, we made use of D1ER, a FRET-baseda2+ sensor [19], to directly examine [Ca2+]ER levels during oscilla-ions. To initiate [Ca2+]i oscillations, we injected mouse eggs with

ouse PLC� cRNA, a procedure that we and others have shown toause long-lasting and fertilization-like oscillations (Fig. 1) [11]. As

epicted in the figure, each [Ca2+]i rise is accompanied by a rapidecline in [Ca2+]ER that is followed by a slow recovery. Compared tohe quick return to baseline that cytosolic [Ca2+]i transients expe-ience, [Ca2+]ER levels return to near basal levels in a very gradual

2 is generally used to measure mitochondrial Ca , although given that it fails totarget into mitochondria in mouse eggs, it is possible to use it as a reported of [Ca2+]i

(Dumollard, R. et al.).

manner, suggesting that other Ca2+ buffering systems contributeto cytosolic [Ca2+]i clearance. Moreover, the upstroke of the next[Ca2+]i increase occurs before [Ca2+]ER levels are fully recovered,and for approximately the first 3 [Ca2+]i rises [Ca2+]ER levels pro-gressively decrease, although after that they seemingly reach aplateau, where each [Ca2+]i increase occurs from the same [Ca2+]ERlevel.

As mentioned, the lag time between the return of [Ca2+]i rises tobaseline and the recovery of [Ca2+]ER suggests the involvement ofother Ca2+ buffering mechanisms for clearing [Ca2+]i out of the cyto-sol. To this end, the functional activity of Na+/Ca2+ exchanger wasdemonstrated in mouse eggs [20,21]. It was shown that eliminationof Na+ from the external media caused [Ca2+]i responses, or acceler-ated existing ones, and these responses were ascribed to a reversemode of Na+–Ca2+ exchange. Importantly, in spite of the initialchanges in [Ca2+]i levels, even in the absence of external Na+, [Ca2+]ireturned to baseline levels, implying that the action of other mech-anisms, possibly plasma membrane Ca2+ ATPase (PMCA) may bemore physiologically relevant [21]. In spite of those initial studies,not until recently was the possible involvement PMCA in mam-malian oocytes tested. It was shown that inhibition of PMCA bymillimolar concentrations of gadolinium (Gd3+), generating a Ca2+

insulation system, as both the influx and efflux of Ca2+ are greatlyreduced, increased the amplitude and duration of the initial [Ca2+]irise and oscillations [22], suggesting the active presence of thismechanism in mouse eggs. Nonetheless, the molecular identifi-cation of PMCA was not documented in that study and thus themolecular presence of PMCAs in mammalian oocytes/eggs needsconfirmation. In contrast, in Xenopus oocytes, both the molecularpresence and function of PMCA1 were demonstrated [23].

Besides the aforementioned mechanisms, the mitochondriamay also contribute to shape [Ca2+]i rises during oscillations[24,25], as these organelles can uptake Ca2+ into their matrix,thereby alleviating the overall cytosolic Ca2+ load [26]. In support ofthis function, inhibition of mitochondrial function disrupted [Ca2+]i

oscillations and caused a sustained increase in [Ca2+]i in mouse eggs[27,28]. Nonetheless, this does not seem to be its main function interms of Ca2+ homeostasis, as inhibition of mitochondrial depolar-ization did not immediately terminate sperm-initiated oscillations

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28]. Instead, and possibly due to its vicinity to the IP3R1/ER, thea2+-driven ATP output may be the mitochondria’s most criticalontribution to Ca2+ homeostasis in MII eggs, as ATP produc-ion maintains SERCA activity, which is required for maintainingCa2+]ER and to sustain sperm-triggered Ca2+ oscillations [17].mportantly, direct assessment of mitochondrial Ca2+ uptake hasot been performed because the conventional inhibitors, such asuthenium red, are somehow ineffective in eggs. Recently, however,n RNAi screen showed that a protein named MICU1 is required foritochondrial Ca2+ uptake [29] and its discovery led to the molec-

lar identification of the mitochondrial Ca2+ unipoter [30,31]. Thus,sing knockdown approaches it will be worth investigating the rolef these molecules on [Ca2+]i oscillations in mammalian eggs.

.2. Ca2+ influx mechanisms

Given that only a fraction of Ca2+ from each [Ca2+]i rise iseposited back into the ER Ca2+ store by SERCA, extracellular Ca2+

ust be taken in to replenish [Ca2+]ER. Support for the notion thata2+ influx plays a pivotal role in fertilization is long-standing,s sperm-initiated [Ca2+]i oscillations cease prematurely in thebsence of external Ca2+ [22,32,33]. Nonetheless, the molecularechanisms underlining Ca2+ influx in mammalian eggs are poorly

nderstood. The cells input extracellular Ca2+ across the plasmaembrane through a variety of mechanisms, including receptor-

perated channels (ROCs), voltage-operated channels (VOCs) [34]nd store-operated Ca2+ channels (SOCs) [7,34,35]. The changesn membrane potential associated with [Ca2+]i responses in fertil-zed hamster eggs are of very low magnitude to suggest activationf VOCs during this process [36]. Further, several additional find-ngs suggest that the participation of these channels in mammalianertilization is likely minor, as [Ca2+]i rises precede changes in

embrane potential [33], Ca2+ influx continues between [Ca2+]iises [37] and, in the mouse, the changes in membrane potentialre nearly imperceptible [33]. These findings raise the prospect thata2+ influx in eggs may be attained, at least in part, by a differentechanism(s). Instead, SOCE, which is associated with [Ca2+]ER lev-

ls, may fulfill at least in part this role in mammalian eggs. Towardhis end, exposure of MII eggs to thapsigargin evoked a Ca2+ risefter addition of extracellular Ca2+, suggesting that SOCE is active inhese cells [17]. Subsequent studies implicated SOCE during [Ca2+]iscillations, as using the manganese-quenching technique it wasound that in mouse eggs the initiation of each [Ca2+]i rise coin-ided with divalent cation influx [37,38]. Nevertheless, additionalemonstration of the participation of this mechanism in mam-alian fertilization was prevented by lack of knowledge of theolecular components of this mechanism.Recently, the proteins responsible for SOCE have been identi-

ed; stromal interaction molecule 1 (STIM1) is located mostly inhe ER where it acts as a Ca2+ sensor, as its downregulation washown to decrease Ca2+ influx in response to thapsigargin [39,40].iven that STIM1 lacks an obvious channel, the search was on tond the required channel partner protein, one of which was quickly

dentified as Orai1 [41–43]. The expression and functional analy-is using siRNA knockdown of Stim1 and Orai1 were performedsing porcine eggs and the downregulation of them preventedersistent [Ca2+]i oscillations [44,45]. Although the involvementf Stim1 and Orai1 on [Ca2+]i oscillations has been demonstrated,heir regulation, for instance, the spatio-temporal patterns at eachCa2+]i response, and whether or not these molecules overlap dur-ng Ca2+ influx, remains obscure. In mouse oocytes, studies havelso reported the presence of these molecules and their possible

nvolvement in Ca2+ homeostasis both during maturation and fer-ilization. Nevertheless, the cellular organization, re-distributionnd expression of these proteins during maturation as well theirontribution to fertilization-associated oscillations needs to be

lcium 53 (2013) 63– 67 65

clarified [46]. Further, transient receptor potential (TRP) ion chan-nels [47], which are associated with both SOCs and ROCs andare expressed in mammalian eggs [15], may also participate inCa2+ influx during oscillations and their role needs to be furtherexplored.

Although the biological significance of [Ca2+]i oscillations inmammalian eggs seems to be ambiguous in terms of embryo devel-opment, it might be that episodic [Ca2+]i responses are importantfor the stepwise completion of the events of egg activation [10,48].Generally, different events of egg activation show differential sus-ceptibility to [Ca2+]i increases, and their initiation and completionalso have differential Ca2+ requirements. For instance, early eventssuch as CG exocytosis and 2PB extrusion require fewer [Ca2+]iresponses than later events such as PN formation and maternalrecruitment of mRNAs [49]. While these results associated the com-pletion of egg activation events to the number of [Ca2+]i rises, Miaoet al. recently demonstrated using the Ca2+ insulation system withmillimolar levels of extracellular Gd3+ that even if [Ca2+]i oscil-lations occur, eggs fails to extrude 2PB. Given that Ca2+ influx isobliterated under those conditions, the conclusion was drawn that,besides replenishing [Ca2+]ER, Ca2+ influx is required for completionof certain events of egg activation [22]. Up to now, the role of [Ca2+]irises on egg activation events has been considered in the contextof activation of the Ca2+/Calmodulin-dependent protein kinase II(CaMKII). It is well established that each sperm-induced [Ca2+]irise is accompanied by a parallel increase in Ca2+/Calmodulin-dependent protein kinase II (CaMKII) activity [50]. Further, the roleof CaMKII on egg activation in mammals was documented first bya series of studies where expression of constitutive active formsof CaMKII initiated all events of egg activation, except CG exocy-tosis, and promoted development to the blastocyst stage [51,52],and second by genetic studies whereby depletion of CaMKII� iso-form abrogated the ability of these eggs to exit MII in responseto [Ca2+]i stimulation [53,54] and caused infertility [54]. Thus, theauthors speculated that Ca2+ influx activates critical signaling path-ways upstream of CaMKII� that are required for 2PB emission [22].Since the detection of Ca2+ by target molecules can be highly selec-tive due to space, time and concentration constrains, as it should bewhen the signal involved regulates the completion of specific cel-lular events, the regulation of 2PB extrusion by Ca2+ influx couldbe viewed in the context of local vs. global Ca2+ sensing [55].Regardless, further studies are needed to address the signals link-ing [Ca2+]i increases and each egg activation event. Further, whilethe suggested role of Ca2+ influx on 2PB extrusion is an importantobservation, the effects of high Gd3+ on 2PB extrusion should alsobe examined after use of treatments that induce egg activation and2PB extrusion without increasing [Ca2+]i and presumably withoutinducing Ca2+ influx, such as the case after addition of the CDK1and MAPK inhibitors [56].

3. Ca2+ homeostasis during oocyte maturation

As described above, mouse eggs acquire fertilization-competence during oocyte maturation. Multiple factors areinvolved in the increased Ca2+ releasing ability of MII eggs, includ-ing IP3R1 phosphorylation and increased [Ca2+]ER content [18],although more in-depth studies are needed to determine thecontribution of each of these changes to the overall increase inCa2+ responsiveness. Following the theme of this review, herewe continue the discussion of factors that regulate the increasein [Ca2+]ER. The increase in [Ca2+]ER during maturation is a well-documented phenomenon [8,9,18] and here we show that addition

of ionomycin induces [Ca2+]i responses of increasing magnitudeas maturation progresses (Fig. 2). How the increase in [Ca2+]ERcontributes to [Ca2+]i oscillations in MII eggs was investigated byusing cyclopiazonic acid (CPA), a reversible SERCA inhibitor [18].

66 T. Wakai, R.A. Fissore / Cell Ca

Fig. 2. [Ca2+]ER content increases during mouse oocyte maturation. [Ca2+]ER wasestimated from the [Ca2+]i responses induced by the addition of 2 �M ionomycinu 2+

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nder Ca free conditions. The mean fluorescent peak (Fura-2) was compared at 0red), 4 (blue), 8 (green) and 12 (black) h of in vitro maturation, which correspondedith GV, GVBD, MI and MII stages of meiotic progression (n = 5).

PA-treated GV oocytes advanced to the MII stage without delay orross abnormalities, although without increasing their [Ca2+]ER. Inhis condition, CPA-matured eggs injected with PLC� cRNA showed

shortened first [Ca2+]i rise even though CPA was washed awayefore initiating the oscillations. These results suggest that the

ncrease in [Ca2+]ER directly impact [Ca2+]i oscillations, especiallyhe robust first [Ca2+]i rise.

Little is known about how oocytes accumulate Ca2+ in the storesuring maturation, although our preliminary results indicate thatxternal Ca2+ is the source of the increased [Ca2+]ER during matura-ion (unpublished observations). Further, how Ca2+ loading occursnder basal [Ca2+]i levels is also unclear as well as the mech-nisms/channels that might mediate this influx. Nevertheless, its noteworthy that GV oocytes display spontaneous [Ca2+]i oscil-ations [57]. Importantly, these oscillations cease around the GVreakdown (GVBD) stage, which is when the most drastic increase

n [Ca2+]ER is observed [8]. The temporal coincidence of these obser-ations implies the low levels of [Ca2+]ER at the GV stage, possiblyaused by a constitutive Ca2+ leak out of the ER, could promote Ca2+

nflux. While additional studies are needed to clarify how sponta-eous oscillations are terminated and the mechanism(s) that keeps

ow levels of [Ca2+]ER in GV mouse oocytes, given that Ca2+ influx isequired for the GV-stage [Ca2+]i oscillations and the known regu-ation of Ca2+ influx by Ca2+ store content [58], it stands to reasonhat Ca2+ influx might be regulated during oocyte mouse matu-ation. In this regard, in Xenopus oocytes Ca2+ homeostasis andolecules associated with it undergo profound modifications dur-

ng maturation and more precisely at the GVBD stage. For example,he function of the Ca2+ influx, SOCE, and efflux, PMCA, path-ays is significantly downregulated at the GVBD stage [14,59]. One

f the key mechanisms underlying these downregulations is theynamic reorganization of the plasma membrane during matura-ion, as channels and pumps are specifically internalized [14,60],epriving these oocytes from quick shuttling of Ca2+ to and fromhe external milieu; seemingly, the membrane trafficking mech-nism appear, at least in part, to be conserved between Xenopus

nd mammalian oocytes [61,62]. Nonetheless, the contribution oflasma membrane reorganization as a regulatory element of Ca2+

omeostasis in mouse oocytes needs additional investigation. Fur-her, the similarities between mouse and Xenopus oocytes must

[

lcium 53 (2013) 63– 67

end at some point during maturation, as mouse eggs at the MIIstage undergo oscillations after fertilization and, as noted before,these oscillations rely on Ca2+ influx. Therefore, important ques-tions remain unresolved regarding the regulation of Ca2+ influx inmouse oocytes and eggs. For example, is the same mechanism thatmediates Ca2+ influx at the GV stage, but that is inactivated at GVBD,capable of mediating influx in MII eggs following fertilization?Are there other Ca2+ channels that undergo differential regulationduring maturation and fertilization? Is it possible that some Ca2+

channels are synthesized/incorporated into the plasma membraneafter the GV stage? How conserved are the Ca2+ influx mechanismsduring maturation and fertilization among mammals, consideringthat other elements of the Ca2+ oscillation toolkit such as IP3R1 sen-sitivity and PlC� specific activity are widely different among thesespecies. Therefore, studies to answer these questions should occupyresearchers interested in this field for some time. Besides providinga deeper understanding of oocyte physiology, identification of thesemechanisms is likely to have clinical applications both to improvedevelopmental competence of in vitro matured oocytes as well asin the design of novel egg activation procedures.

Conflict of interest statement

All authors must disclose any financial and personal relation-ships with other people or organizations that could inappropriatelyinfluence (bias) their work, all within 3 years of beginning the worksubmitted. If there are no conflicts of interest, authors should statethat there are none.

There is no conflict of interests of Dr. R. Fissore or Dr. T. Wakai.

Acknowledgements

The authors want to thank Dr. Roger Tsien’s lab, UCD, for sharingthe D1ER construct. These studies were completed thanks to grantHD051872 from NIH to R.A.F. We thank Dr. Nan Zhang and BanyoonCheon for useful discussions. We apologize to those researcherswhose work was not cited due to space constraints.

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