RESEARCH ARTICLE

Constitutive IP3R1-mediated Ca2+ release reduces Ca2+ storecontent and stimulates mitochondrial metabolism in mouseGV oocytesTakuya Wakai* and Rafael A. Fissore‡

ABSTRACTIn mammals, fertilization initiates Ca2+ oscillations in metaphase IIoocytes, which are required for the activation of embryo development.Germinal vesicle (GV) oocytes also display Ca2+ oscillations,although these unfold spontaneously in the absence of any knownagonist(s) and their function remains unclear. We found that the mainintracellular store of Ca2+ in GV oocytes, the endoplasmic reticulum([Ca2+]ER), constitutively ‘leaks’Ca2+ through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca2+]ER leak ceases around theresumption of meiosis, the GV breakdown (GVBD) stage, whichcoincides with the first noticeable accumulation of Ca2+ in the stores.It also concurs with downregulation of the Ca2+ influx and terminationof the oscillations, which seemed underpinned by the inactivation ofthe putative plasma membrane Ca2+ channels. Lastly, wedemonstrate that mitochondria take up Ca2+ during the Ca2+

oscillations, mounting their own oscillations that stimulate themitochondrial redox state and increase the ATP levels of GVoocytes. These distinct features of Ca2+ homeostasis in GVoocytes are likely to underpin the acquisition of both maturation anddevelopmental competence, as well as fulfill stage-specific cellularfunctions during oocyte maturation.

KEY WORDS: Calcium oscillations, Inositol 1, 4, 5-trisphosphatereceptor, Mammals, Mitochondria, Oocyte maturation

INTRODUCTIONPrior to ovulation and following a systemic luteinizing hormone(LH) surge, prophase-arrested germinal vesicle (GV) oocytesresume meiosis and progress to the metaphase stage of the secondmeiosis (MII), completing a process that is commonly referred to asoocyte maturation. The arrest that ensues in MII oocytes is relievedby fertilization, as following gamete fusion the sperm promotesperiodical increases in the intracellular concentration of free Ca2+,which are termed Ca2+ oscillations (Stricker, 1999). The ability ofMII oocytes to mount precise spatiotemporal patterns of Ca2+

oscillations in response to fertilization is progressively acquiredduring oocyte maturation (Wakai et al., 2011). Other parameters ofCa2+ homeostasis also undergo marked changes during maturation,which are likely to impact how oscillations unfold (Wakai and

Fissore, 2013). For example, the content of the main intracellularCa2+ storewithin the endoplasmic reticulum ([Ca2+]ER) (Jones et al.,1995; Mehlmann and Kline, 1994; Wakai et al., 2012), markedlyincreases during maturation and there is a pronounceddownregulation of Ca2+ influx as maturation progresses (Cheonet al., 2013; Lee et al., 2013). Remarkably, the molecules andregulatory mechanisms that underlie this optimization of Ca2+

oscillations and changes in Ca2+ homeostasis during oocytematuration are largely unknown and may experience dynamicmodifications such that mechanisms active at the GV stage may notbe so at the MII stage, and vice versa.

GV oocytes also exhibit oscillatory Ca2+ responses (Carroll andSwann, 1992; Carroll et al., 1994). In contrast to sperm-induced Ca2+

oscillations, the spontaneous, repetitive Ca2+ rises in GV oocytesare of small amplitude and occur approximately every 1 to 3 min.These Ca2+ oscillations appear to be agonist-independent and areconstitutive, although the mechanisms that underpin them and theirfunctions remain poorly investigated. It is noteworthy that thesespontaneous Ca2+ oscillations persist for a few hours and ceasearound the time of GV breakdown (GVBD), which is when oocytessimultaneously undergo the most drastic increase in [Ca2+]ER storecontent and experience a sharp downregulation in Ca2+ entry (Cheonet al., 2013; Wakai and Fissore, 2013). The temporal coincidence ofthese phenomena implicates [Ca2+]ER-associated mechanisms in theoccurrence of spontaneous Ca2+ oscillations.

Inositol 1,4,5-trisphosphate receptor family (IP3R)-mediated Ca2+

release from the [Ca2+]ER is primarily responsible for the Ca2+

oscillations during fertilization (Miyazaki et al., 1992). Thespontaneous Ca2+ oscillations at the GV stage are also mediatedby IP3R1 (also known as ITPR1), as inhibition of IP3R1 function byheparin blocks the oscillations (Carroll and Swann, 1992).Nevertheless, the regulation of these Ca2+ oscillations is likely tobe more complex, as given that they unfold in the absence of aspecific agonist, they must be regulated by other factors of cellularCa2+ homeostasis such as Ca2+ influx and Ca2+ clearingmechanisms. To this end, continued Ca2+ oscillations requirereplenishment of the [Ca2+]ER, a function that is mostly carried outby the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA)pump protein family (Wakai et al., 2013). In this regard, the contentof the [Ca2+]ER is vital to maintain the oscillations because it is themain source of Ca2+. The mitochondria also take up cytosolic Ca2+,thereby contributing to shaping of the spatiotemporal patterns ofCa2+ responses, while simultaneously promoting a number of eventsthat sustain ATP levels in cells (Hajnóczky et al., 1995). In fact, theCa2+-driven ATP output is likely to be the mitochondria’s mostcritical contribution towards Ca2+ homeostasis in MII oocytes, asATP production maintains SERCA activity, which is required formaintaining [Ca2+]ER levels and sustaining sperm-triggered Ca

2+

oscillations (Dumollard et al., 2004; Wakai et al., 2013). It isReceived 16 September 2018; Accepted 2 January 2019

Department of Veterinary and Animal Sciences, University of MassachusettsAmherst, 661 North Pleasant Street, Amherst, MA 01003, USA.*Present address: Department of Animal Science, Graduate School of Environmentand Life Science, Okayama University, Okayama 700-8530, Japan.

‡Author for correspondence (rfissore@vasci.umass.edu)

T.W., 0000-0003-4705-8974; R.A.F., 0000-0001-5655-0915

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mailto:rfissore@vasci.umass.eduhttp://orcid.org/0000-0003-4705-8974http://orcid.org/0000-0001-5655-0915

nevertheless unknown whether the spontaneous oscillationsobserved in GV oocytes play a similar role in mitochondrialfunction. In addition, the mechanisms and underlying moleculesinvolved in the regulation of Ca2+ homeostasis in GVoocytes remainpoorly investigated.In the present study, we uncover a unique regulatory mechanism

of [Ca2+]ER content in GV oocytes, which is reversed in MIIoocytes; namely, in GV oocytes [Ca2+]ER levels are maintainedpersistently low by a constitutive Ca2+ ‘leak’ through IP3R1 channelsthat is manifested in the form of spontaneous Ca2+ oscillations. TheCa2+ oscillations are actuated by nearly constitutive Ca2+ influx, anddownregulation of this influx, which terminates the oscillations,appears linked to the progressive increase in the content of the[Ca2+]ER that occurs during oocyte maturation. Further, wedemonstrate a novel role for the spontaneous Ca2+ oscillations, aspart of the cytosolic Ca2+ is transferred into the mitochondria whereit stimulates mitochondrial metabolism, thereby increasing the levelsof ATP in GV oocytes. These distinct regulatory mechanisms oforganellar Ca2+ homeostasis in GVoocytes are likely to underpin theacquisition of both maturation and developmental competence,as well as fulfill stage-specific cellular function during oocytematuration.

RESULTSGV oocytes constitutively leak Ca2+ from [Ca2+]ER storesIt is a well-known phenomenon that GV-arrested mouse oocytes showreduced intracellular Ca2+ stores content (Jones et al., 1995;Mehlmann and Kline, 1994; Wakai et al., 2012). This wasdemonstrated using ionomycin to empty the ER stores, and measureCa2+ responses in the presence of Ca2+-free conditions containingEGTA. Further, those studies showed that the Ca2+ store content, asassessed by the same procedure, sharply increased between the GVand GVBD stages and less so thereafter until the MII stage (Fig. S1).The underlying reason(s) by which Ca2+ stores have less Ca2+ at

the GV stage is not known, although the occurrence of spontaneousCa2+ oscillations suggests a possible molecular mechanism. Hence,we propose that a Ca2+ leak out of the ER, which lowers its Ca2+

content, induces transient Ca2+ rises by stimulating Ca2+-induced

Ca2+ release (CICR) through IP3R1 channels, and supportsoscillations by promoting Ca2+ influx. To ascertain the presenceof a constitutive Ca2+ leak in GV oocytes, we took advantage of theknowledge that the plasma membrane Ca2+ ATPase (PMCA, alsoknown as ATP2B) family of proteins, which remove Ca2+ from thecell, are inhibited by millimolar (mM) concentrations of lanthanum(La3+). Further, at these concentrations La3+ generates a Ca2+

insulation system, because both Ca2+ influx and efflux are inhibited,allowing the leaking Ca2+ to build up in the cytosol and induceCICR (Bird and Putney, 2005). Using this experimental system, wefound that, indeed, GV oocytes (n=21/21) cultured in the absence ofexternal Ca2+ but in the presence of 1 mMLa3+ show a sharp, singleCa2+ transient a few minutes after addition of La3+ (Fig. 1A).Significantly, the same procedure in GVBD oocytes (n=4/10)generates a smaller Ca2+ response with a shallow sloping rise(Fig. 1B), and Ca2+ responses are no longer induced in MII oocytes(n=0/14) (Fig. 1C). Quantification of the response showed that theamplitude of the Ca2+ peak induced by La3+ progressivelydecreased during maturation (Fig. 1D). We next examined theremaining Ca2+ content in the ER, as the expectation would be thatthe leaked Ca2+ would empty the [Ca2+]ER store, which should thendisplay a decreased Ca2+ response following addition of the SERCAinhibitor thapsigargin (10 μM). This was the case, as whereasaddition of thapsigargin failed to cause a Ca2+ rise in GV oocytes, itinduced greater responses in GVBD oocytes and even greater inMIIoocytes (Fig. 1E). Taken together, these results show that GVoocytes spontaneously leak Ca2+ out of the [Ca2+]ER store, and thisleak is progressively inactivated duringmaturation starting at aroundthe GVBD stage.

IP3R acts as the [Ca2+]ER leak channel in GV oocytesTo gain insight into the mechanism(s) underlying the constitutiveCa2+ leak from the [Ca2+]ER store in GV oocytes, we assessed therole of IP3R1 channels. To accomplish this we used a ligand-induced knockdown approach. The type 1 IP3R (ITPR1) is the mostabundantly expressed IP3R isoform in mammalian oocytes (Fissoreet al., 1999; Parrington et al., 1998), and adenophostin A (AdA) is apotent agonist of IP3Rs capable of promoting long-lasting Ca

2+

Fig. 1. GV oocytes display aconstitutive Ca2+ leak out of the ERstore. (A–C) Representative traces ofCa2+ responses induced by La3+ (La)Ca2+ insulation in GV (A), GVBD (B) andMII (C) oocytes (GV, n=21; GVBD, n=10;MII, n=14). Ca2+ measurements wereperformed in Ca2+-free mediumcontaining 1 mM LaCl3. When the Ca2+

increase returned to baseline, 10 μMthapsigargin (Tg) was applied to confirmthe remaining Ca2+ in the ER.(D,E) Comparisons of the amplitude ofthe Ca2+ peak induced by La3+ (D) andthe area under each curve (arbitrary units,A.U.) of thapsigargin-induced Ca2+ rise(E) between the different stages of oocytematuration. Error bars represent s.d., andbars with different superscripts aresignificantly different from each other(P

release through this receptor, while at the time inducing powerfulIP3R1 degradation (Brind et al., 2000; He et al., 1999). Consistentwith these previous results, we observed that injection of 10 μMAdAinto GV oocytes stimulated Ca2+ release and oscillations (Fig. 2A),and reduced IP3R1 protein to nearly undetectable levels by 6 h post-injection (Fig. 2B). Using the Ca2+ insulation approach we examinedwhether the Ca2+ leak was present in AdA-injected GV oocytes.Addition of La3+ failed to induce a Ca2+ rise in just over half ofthe oocytes (16/30), and the Ca2+ rise was severely disturbed inthe remaining oocytes (14/30) (Fig. 2B). Moreover, the spontaneousCa2+ oscillations present in GV oocytes at physiologicalconcentrations of extracellular Ca2+ (1.7 mM) were inhibited inAdA-injected oocytes (Fig. 2C). Consistent with above results, the[Ca2+]ER content estimated from the Ca

2+ response to ionomycinwas greatly increased in AdA-injected GV oocytes, reaching levelssimilar to those observed in MII oocytes (Fig. 2D). These resultsdemonstrate that IP3R1 channels are largely responsible for theconstitutive Ca2+ leak that occurs in GV oocytes and maintains lowlevels of Ca2+ in [Ca2+]ER. Consistent with these results, inhibitionof the oocyte family of PLC proteins with 10 μM of the PLCinhibitor U73122 terminated the oscillations of GV oocytes (Fig.S3A), whereas addition of the same concentration of inactive analogU73433 did not block oscillations (Fig. S3B).

Ca2+ influx is required for the spontaneous Ca2+ oscillationsin GV oocytesAlthough the constitutive Ca2+ release and oscillations in GVoocytes are seemingly autonomous and independent of a knownagonist, the oscillatory Ca2+ responses suggest participation of theIP3R1 channels. We hypothesize that in GV oocytes the incoming

Ca2+ modulates the function of IP3R1 channels. GV oocytes expressseveral divalent cation permeable plasma membrane channels forCa2+ influx, although the individual contribution of each of thesechannels to the influx and oscillations has not yet been fullyascertained. One possible mechanism of Ca2+ influx is the store-operated Ca2+ entry (SOCE) mechanism, which is ubiquitous innon-excitable cells and is activated by the depletion of [Ca2+]ER(Parekh and Putney, 2005). We and others have previouslydemonstrated that Ca2+ influx, and SOCE activity following over-expression of SOCE mechanism components, is highest in GVoocytes and progressively decreases during oocyte maturation(Cheon et al., 2013; Lee et al., 2013). Thus, this temporalcoincidence of downregulation of Ca2+ influx and termination ofCa2+ oscillations at around the GVBD stage strengthens theassociation of Ca2+ influx and the presence of oscillations in GVoocytes. To decipher the role of Ca2+ influx on the oscillations atthis stage, we modulated the strength of Ca2+ influx byoverexpressing STIM1 and ORAI1, which are the molecularcomponents of SOCE. We observed that under the regularexternal Ca2+ concentrations of 1.7 mM, control GV oocytesdisplayed the customary Ca2+ oscillations, whereas oocytesoverexpressing STIM1 and ORAI1 showed permanently increasedbasal Ca2+ levels, indicative of persistent Ca2+ influx (Fig. 3A,D).We also found that these changes in intracellular Ca2+ were due toCa2+ influx, as addition of 100 µM 2-APB, a broad-spectrum Ca2+

channel inhibitor (DeHaven et al., 2008), terminated the oscillationsand/or returned the levels of Ca2+ to basal values. We next exposedoocytes to medium containing lower external Ca2+ concentrations,0.5 mM, which prevented oscillations in control oocytes, althoughoocytes overexpressing STIM1 and ORAI1 now displayed

Fig. 2. IP3R1 channels are responsible for the low [Ca2+]ER content of GVoocytes. (A) Representative Ca2+ responses induced by the potent agonist of IP3Rchannels, adenophostin A (AdA), in GV oocytes is shown. Loss of IP3R1 expression was confirmed by western blot analysis using antibodies specific to IP3R1and α-tubulin 6 h after injection of 10 μM AdA. Oocytes undergoing microinjection of AdA (GV-AdA) or buffer (GV) were cultured in the presence of IBMX tomaintain the arrest at the GV stage. (B) Representative traces of Ca2+ responses induced by La3+ (La) Ca2+ insulation in AdA-injecting GV oocytes. Ca2+

measurements were performed in Ca2+-free medium containing 1 mM LaCl3. The number of oocytes responding to lanthanide is shown. (C) Representativetraces of Ca2+ responses in GV oocytes (black trace) and AdA-injected GV oocytes (red trace) are shown. Ca2+ measurement were performed under normalexternal Ca2+ concentration (1.7 mM). The percentages of oocytes showing spontaneous Ca2+ oscillations (oocytes showing more than 3 repetitive Ca2+ rises)were compared. Numbers of oocytes showing oscillations/total number of oocytes are indicated. (D) ER Ca2+ store content in in GV oocytes (black trace; n=20),AdA-injecting GV oocytes (red trace; n=14) and MII oocytes (blue trace; n=10), which was estimated by the mean fluorescent (Fura-2) peak after addition of 1 μMionomycin. Amplitudes of the Ca2+ peak were compared. Error bars represent s.d., and bars with different superscripts are significantly different from each other(P

oscillations rather than elevated basal Ca2+ levels, confirmingthe association of influx and oscillations (Fig. 3B,D). Finally,under nominal Ca2+-free conditions, both groups of oocytes wereno longer able to mount changes in intracellular Ca2+ levels(Fig. 3C,D). Collectively, these results suggest that in GV oocytes,Ca2+ influx regulates intracellular Ca2+ responses, and thereforeCa2+ influx not only replenishes the [Ca2+]ER content but alsoactuates the spontaneous oscillations at this stage.

Decline in the number of channels at the plasma membranecoincides with downregulation of spontaneous Ca2+oscillationsGiven that in most oocytes the spontaneous Ca2+ oscillations cease2 to 4 h after the initiation of maturation, at the GVBD stage (Carrolland Swann, 1992), the downregulation of Ca2+ influx is likely tooccur during this time period, which curiously precedes the increasein [Ca2+]ER store content that takes place mostly after GVBD.Previous studies have noticed downregulation of influx and SOCEduring mouse oocyte maturation (Cheon et al., 2013; Lee et al.,2013), although the precise timing and mechanism(s) remainunknown. Because expression of ORAI1 in mouse GV oocytesresults in specific plasma membrane localization, and whenexpressed together with STIM1 they stimulate Ca2+ influx inthese cells, we expressed fluorescently labeled human ORAI1 toexamine its fate during maturation. Following injection of ORAI1cRNA, we examined the distribution of ORAI1–RFP at 0, 2, 4, 8and 12 h after the initiation of in vitro maturation, which broadlycorresponded with GV, early GVBD, late GVBD, MI and MIIstages of meiotic progression, respectively.We found that there is anabrupt decrease in the presence of ORAI1–RFP in the plasmamembrane at 2 h of maturation (Fig. 4A–C), suggesting that

internalization of the putative active channel(s) might be one of themechanisms that bring about the inactivation of Ca2+ influx andtermination of the Ca2+ oscillations.

To further demonstrate the association between the expression ofCa2+ channels in the plasma membrane and the spontaneous Ca2+

oscillations, we interfered with the expected distribution of ORAI1–RFP in GVoocytes. Synaptosomal-associated protein 25 (SNAP25)is a t-SNARE protein component of the trans-SNARE complexinvolved in membrane fusion between vesicles and plasmamembrane. In Xenopus oocytes, expression of a dominant-negative SNAP25 mutant (SNAP25Δ20) was shown to effectivelyblock exocytosis and inhibit ORAI1 trafficking between endosomesand the plasma membrane (Yu et al., 2009). Expression ofSNAP25Δ20 or wild-type SNAP25 (SNAP25WT) in mouse GVoocytes (Fig. S2A) did not relieve meiotic arrest, contrary to what isobserved in Xenopus oocytes, although SNAP25Δ20 compromisedthe distribution of ORAI1, which appeared discontinuous, incontrast to the continuous distribution displayed in control oocytes(Fig. S2B). Furthermore, the spontaneous Ca2+ oscillations inoocytes expressing SNAP25Δ20 were severely disturbed (Fig. S2C).Taken together, these results support the notion that the presence ofactive channels in the plasma membrane is required for spontaneousCa2+ oscillations in GV oocytes.

Spontaneous cytoplasmic Ca2+ oscillations cause Ca2+oscillations and stimulate metabolism in mitochondriaThe physiological significance of the spontaneous Ca2+ oscillationsin GV oocytes is poorly understood. A recent study in somatic cellsuncovered a novel function for this constitutive, IP3R-mediated Ca2+

release in maintaining cellular bioenergetics by transferring Ca2+ tothe mitochondria (Cárdenas et al., 2010). The role of Ca2+ transferbetween the ER and mitochondria has also been documented inmammalian MII oocytes, as the sperm-triggered Ca2+ oscillationsduring fertilization were shown to be sustained by Ca2+-driven ATPproduction (Dumollard et al., 2004; Wakai et al., 2013), which verylikely was required for refilling of the [Ca2+]ER by SERCA proteins.To address the roles of the spontaneous Ca2+ oscillations, wemeasured mitochondrial Ca2+ ([Ca2+]mt) levels in GV oocytes.Radiometric chimeric fluorescent protein pericam harboring amitochondrial targeting sequence (pericam-mito) has beensuccessfully used to detect Ca2+ changes in the mitochondria ofsomatic cells (Nagai et al., 2001) and to show Ca2+ increases inmitochondria in fertilized mouse eggs (Dumollard et al., 2008).Pericam-mito cRNA was injected into GV oocytes and confocalimages of pericam-mito fluorescence demonstrated it wassuccessfully targeted to the mitochondria of GV oocytes, as it co-localized with mitochondrial staining provided by Mitotracker(Fig. 5A). To determine whether pericam-mito in GV oocytescould detect intra-mitochondrial oscillations, we examined Ca2+

responses in oocytes displaying spontaneous oscillations. Asexpected, the fluorescence intensities of the excitation wavelengthsshifted in opposite directions with fluorescence intensities increasingat 480 nm (F480) with Ca2+ rises, whereas at the same timeintensities at 410 nm (F410) decreasing (Fig. 5B). Importantly, theseratiometric measurements of [Ca2+]mt (F480/F410) revealed that inGVoocytes, mitochondrial Ca2+ responses were oscillatory, which isconsistent with the spontaneous cytoplasmic Ca2+ oscillationsobserved at this stage. In MII-arrested oocytes, contrarily, changesin [Ca2+]mt levels were not observed, which is consistent with thelack of Ca2+ oscillations in unfertilized oocytes (Fig. 5C).

A method to demonstrate how [Ca2+]mt oscillations affectmitochondrial function is to evaluate changes in the oxidation

Fig. 3. The magnitude of Ca2+ influx impacts the strength of spontaneousCa2+ oscillations in GV oocytes. (A–C) Representative traces of Ca2+

responses in control GV oocytes (black traces) and in GV oocytesoverexpressing STIM1 and ORAI1 (Stim1/Orai1-OVE, red traces) are shown.Oocytes were arrested at GV stage throughout the imaging by the addition of100 mM IBMX. Ca2+ measurements were performed under 1.7 mM (A),0.5 mM (B) and 0 mM (C) external Ca2+ concentrations. Under normal externalCa2+ concentrations, 1.7 mM, 2-APB, a broad-spectrum SOCE inhibitor, wasadded at concentrations of 100 µM. (D) The percentages of oocytes showingspontaneous Ca2+ oscillations (oocytes showing more than 3 repetitive Ca2+

rises were counted) were compared. Numbers of oocytes showing oscillations/total number of oocytes are indicated.

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status of nicotinamide adenine dinucleotide (NAD) and flavinadenine dinucleotide (FAD). The reduced form of NAD, NADH,and oxidized FAD display autofluorescence, which can be used toestimate the mitochondrial redox state (Duchen et al., 2003;Dumollard et al., 2004). NADH and FADH2 are products of theTCA cycle that undergo oxidation during the process of oxidativephosphorylation, which occurs in the mitochondria and isstimulated by Ca2+ uptake. Time-lapse imaging of NADH(360 nm wavelengths) and FAD (480 nm wavelengths)demonstrates that, consistent with the oscillatory response in[Ca2+]mt, oscillations in autofluorescence are detected in GVoocytes, whereas changes in autofluorescence are not observed inMII-arrested oocytes (Fig. 5D,E). These results suggest that at thesteady state in GV oocytes, the Ca2+-sensitive mitochondrialdehydrogenases are activated by spontaneous, cytoplasmic andmitochondrial Ca2+ oscillations.To directly assess the contribution of [Ca2+]mt oscillations to

cellular ATP levels, we expressed a fluorescence resonance energytransfer (FRET)-based ATP biosensor, ATeam AT1.03, whichreports intracellular ATP concentrations (Imamura et al., 2009),with the emission ratio of AT1.03 fluorescence (YFP:CFP) used toestimate ATP levels. A conventional approach for real-time imagingof intracellular ATP levels in a single live cell is luminescenceby luciferase, which has been used in attempts to show thedynamic changes in ATP levels during mouse oocyte maturation(Yu et al., 2010). However, the luciferase luminescence depends notonly on the ATP level but also on multiple other parametersincluding oxygen, pH and luciferin. The expression of AT1.03persists stably in the oocyte and has been successfully used to report

intracellular ATP levels during maturation (Dalton et al., 2014;Wakai et al., 2015).

We compared ATP concentrations in uninjected oocytes vsoocytes injected with 10 μM AdA, which do not display Ca2+

oscillations. Accordingly, 6 h after injection of AdA and AT1.03cRNA, we first monitored Ca2+ responses and confirmed that [Ca2+]mtoscillations were inhibited in AdA-injected oocytes, whereas theywere undisturbed in uninjected oocytes (Fig. 5F). We then found thatin AdA-injected oocytes and without [Ca2+]mt oscillations the ATPlevels were significantly decreased (Fig. 5G). Taken together, weinterpret these results to mean that spontaneous Ca2+ oscillationsinduce corresponding Ca2+ changes in the mitochondria, whichsubsequently stimulate ATP synthesis.

DISCUSSIONDuring maturation, mammalian oocytes undergo marked changes inCa2+ homeostasis. The underlying mechanism(s) and function ofthese changes are not known. In the present study we report uniquefeatures of Ca2+ regulation in mouse GV oocytes: (1) they displayconstitutive Ca2+ release through IP3R1 that mediates spontaneousCa2+ oscillations and maintains the [Ca2+]ER levels persistently low;(2) they show continuous Ca2+ influx from extracellular media thatsustains the oscillations; (3) the spontaneous Ca2+ oscillationspropagate into the neighboring mitochondria, activatemitochondrial metabolism and increase the production of ATPand ATP content. These results suggest that the Ca2+ changesobserved in GV oocytes are associated with cellular homeostasisand may be a requirement for the acquisition of maturation anddevelopmental competence.

Fig. 4. Progression of oocyte maturation causes reduced presence of ORAI1 at the plasma membrane. (A) The subcellular distribution of ORAI1 wasanalyzed using an mRFP-tagged (top panel) version of the protein. The observations were performed at 0, 2, 4, 8 and 12 h after initiation of in vitro maturation,which corresponded with GV, early GVBD, late GVBD, MI and MII stages, respectively. Square area on the top panels indicate areas magnified to observeORAI1 plasma membrane presence at the different stages of maturation. Higher magnification views of the selected area (inset panels) are shown to the left ofeach image where differences in ORAI1 distribution between different maturation stages can be observed. Corresponding DIC images are shown in the bottompanel. Scale bar: 20 µm. (B) Intensity profiles of the line scans drawn in oocytes in A (white lines in insets), representing the distribution of ORAI1–RFPfluorescence from the cytoplasm (Cyto) and across the plasmamembrane (PM) at different stages of maturation. (C) Mean±s.d. relative intensity of ORAI1 signalbetween plasma membrane and cytoplasm is shown.

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[Ca2+]ER levels and the ER Ca2+ leak through IP3R1This spatiotemporal complexity of intracellular Ca2+ responses is inpart a consequence of the requirement for Ca2+ from two sources,extracellular medium and intracellular stores, to be integrated toproduce stereotypical responses. To accomplish this, cells possess avariety of pumps, channels and buffering mechanisms in the plasmamembrane and intracellular organelles, the ‘Ca2+ toolkit’, whichmake possible the generation of precise Ca2+ rises (Berridge et al.,

2003). The ER is the main intracellular Ca2+ store ([Ca2+]ER), andserves as the main source of Ca2+ in oocytes of most species. Themajority of the uptake of Ca2+ into the ER is mediated by SERCAproteins, and we have previously demonstrated that mouse oocytesconstitutively express the housekeeping SERCA2b protein (alsoknown as ATP2A2) (Wakai et al., 2013). Therefore, given thesteady expression SERCA2b in oocytes, it is unlikely that functionalchanges in this molecule can explain the increase in [Ca2+]ER levels

Fig. 5. Mitochondrial Ca2+ uptake of IP3R1-mediatedCa2+ release stimulatesmitochondrialmetabolism inGVoocytes. (A) Confocal images of radiometricpericam-mito fluorescence at 480 nm (F480, green) in GV oocytes, to detect mitochondrial Ca2+. To visualize colocalization with mitochondria, oocytes werestained with MitoTracker (red). Brightfield, BF. Scale bar: 20 µm. (B) The fluorescence intensities of pericam-mito in GV oocytes shifted in oppositedirections between the excitation at 405 nm (left axis) and 480 nm (right axis) coinciding with Ca2+ oscillations. (C) Emission ratios (F480:F405) of pericam-mito,which allows estimation of the relative changes in mitochondrial Ca2+ ([Ca2+]mt), were analyzed in GV (black trace; n=9/12) and MII oocytes (red trace; n=0/10)under normal external Ca2+ concentrations (1.7 mM). (D,E) Changes in NADH (left axis) and FAD (right axis) autofluorecence in GV (D; n=8/10) and MIIoocytes (E; n=0/9) are reported. The intensities of NADH (360 nmwavelengths) and FAD (480 nmwavelengths) autofluorescence shifted in opposite directions inGV oocytes but not in MII oocytes, reflecting the stimulation of mitochondrial metabolism by the Ca2+ oscillations. (F) Representative traces of [Ca2+]mt in GVoocytes (black trace; n=8/9) and adenophostin A (AdA)-injected GV oocytes (red trace; n=0/9) with reduced IP3R1 expression (Fig. 2) are shown. [Ca2+]mtmeasurements were performed under normal external Ca2+ concentrations, 1.7 mM, and 6 h after injection of AdA. (G) Comparison of ATP levels between GVoocytes and AdA-injected GV oocytes. The levels of ATP were estimated using the emission ratio of AT1.03 (YFP:CFP). Error bars represent s.d. Oocytes wereinjected at the GV stage and remained at this stage in media supplemented with IBMX.

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that begin around the GVBD stage and continue during maturation.We found instead that GVoocytes display a constitutive Ca2+ leak outof the ER whose regulation may more effectively impact [Ca2+]ERlevels. To uncover this leak, we used high concentrations of La3+ toinsulate Ca2+ traffic in and out of the cell. We found that the activeCa2+ leak out of the ER in GV oocytes ceases around the GVBDstage, which is when [Ca2+]ER starts to increase.We thus propose thatregulation of this leak may control [Ca2+]ER levels during oocytematuration.Although the molecular mechanism(s) underlying this Ca2+ leak

remain undetermined, IP3R1 may act as an ER leak channel in GVoocytes. Under basal conditions, several regulatory mechanismscan sensitize IP3R1 including phosphorylation and thiolgroup modifications (Ivanova et al., 2014). With regards tophosphorylation, a candidate kinase is the protein kinase A (PKA)enzyme family, which phosphorylate IP3R1 in somatic cells andincreases the activity of the channel (DeSouza et al., 2002). Asimilar mechanism may operate in oocytes, as we have previouslydescribed PKA phosphorylation of IP3R1 in GV oocytes, whichshows a consistent and significant decrease at the time of GVBD(Wakai et al., 2012) coinciding with termination of the oscillations.Further, high levels of cAMP and PKA activity are needed tomaintain arrest at the GV stage, and their reduction by the LH surgethat induces the resumption of meiosis is also well established(Norris et al., 2009). It is therefore possible that IP3R1phosphorylation by PKA might be a mechanism involved inregulation of the ER Ca2+ leak and Ca2+ homeostasis during oocytematuration. Whether modifications of IP3R1 phosphorylationassociated with anti-apoptotic effects by some members of theBcl-2 family of proteins (Oakes et al., 2005) play any role in GVoocytes is unknown.Changes in oxidation/reduction status during maturation may also

modulate the ER Ca2+ leak. Thimerosal, a thiol oxidizing agent, isknown to induce oscillations in mammalian GV and MII oocytes(Swann, 1991; Wakai et al., 2012). It is proposed that thimerosalaccomplishes this by thiol modification and sensitization of IP3Rs1.In fact, intracellular levels of reduced glutathione, which is the majorantioxidant in the cell, increase during maturation (Dumollard et al.,2007), which may render GV oocytes more susceptible to oxidativestimuli. Lastly, besides IP3R1, cells have at their disposal otherpassive Ca2+ leak mechanisms out of the ER, although their identityand regulation remain largely unknown (Ivanova et al., 2014).

The mechanism(s) of spontaneous Ca2+ oscillationsin GV oocytesIn most cell types, especially in non-excitable cells, Ca2+ releasefrom stores activates Ca2+ influx through plasma membranechannels, an observation that led to the discovery of SOCE(Parekh and Putney, 2005). We propose that persistently low[Ca2+]ER levels and spontaneous Ca2+ oscillations in GV oocytesserve as the natural trigger for Ca2+ influx, which is required tosustain the oscillations. To confirm the role of Ca2+ influx, wemodulated it by overexpressing STIM1 and ORAI1, thecomponents of SOCE, in the presence of different concentrationsof extracellular Ca2+. We found that high rates of Ca2+ influxpersistently elevate basal Ca2+, whereas moderate levels of Ca2+

influx lead to generation of oscillations, and the absence of influxcaused by a lack of Ca2+ in the extracellular media terminates theoscillations. These results demonstrate that Ca2+ influx is sufficientto stimulate oscillations in GV oocytes.The underlying signaling pathway(s) whereby Ca2+ influx

promotes these Ca2+ oscillations is not established. Oocytes are

known to express PLC family proteins, especially PLCB1, whichhas been associated with oscillations in GV oocytes (Avazeri et al.,2000; Igarashi et al., 2007). Addition of the PLC inhibitor U73122to oscillating GV oocytes terminated oscillations (Fig. S3A).Conversely, addition of the same concentration of U73433, which isthe inactive analogue of U73122, did not block oscillations(Fig. S3B). It could be argued that increased cytosolic levels ofCa2+ caused by the enhanced influx stimulate inositol 1,4,5-trisphosphate (IP3) synthesis, which induces Ca

2+ release throughIP3R1. In the presence of basal IP3 levels and given the biphasicregulation of the open probability of IP3R1 by Ca

2+ concentrations,Ca2+ release is amplified through CICR, maintaining spontaneousoscillations (Peres et al., 1991).

The channel(s) that underlie Ca2+ influx in these cells remains tobe fully established. We promoted Ca2+ influx by overexpressingSTIM1 and ORAI1, although recent genetic studies seem to suggestthat SOCE is not involved in Ca2+ influx in mouse GV oocytes(Bernhardt et al., 2015). Importantly, a number of other plasmamembrane permeable Ca2+ channels are expressed in mammalianGV oocytes, including the T-type voltage-gated Ca2+ channelsCaV3.2, which might underlie the increase in [Ca2+]ER levels duringmaturation (Bernhardt et al., 2015). In this context, Ca2+ influxthrough voltage-gated Ca2+ channels is known to be the main triggerof CICR in excitable cells (Bers, 2002), and it could play a similarrole in GV oocytes. Other channels expressed in GV oocytes aremembers of the family of transient receptor potential (TRP)channels, which are widely distributed in mammalian tissues.Mouse MII oocytes functionally express TRPV3 and although itsexpression is reduced in GV oocytes, it becomes progressivelyactive during maturation (Carvacho et al., 2013). Another TRPchannel, a TRPM7-like channel, is active in GV oocytes andspontaneous Ca2+ oscillations were reduced by the TRPM7antagonist NS8593 (Carvacho et al., 2016). Therefore, there areseveral channels capable of mediating Ca2+ influx in GV oocytes.Future studies should determine which one(s) are active during theoscillations and how they are regulated.

The roles of mitochondrial Ca2+ oscillations in GV oocytesThe functional significance of Ca2+ oscillations in many systemsremains to be fully elucidated, although it is generally accepted thattheir periodical behavior provides a digital signal to downstreameffectors (Dupont et al., 2011). Owing to their proximity to the ERand IP3R1 channels, mitochondria are a common downstream targetof Ca2+ increases (Rizzuto et al., 1998; Rizzuto and Pozzan, 2006).Cytosolic Ca2+ reaches the mitochondrial matrix by permeating theouter mitochondrial membrane through the voltage-dependentanion-selective channel (VDAC) (Blachly-Dyson et al., 1993) andthe inner mitochondrial membrane (IMM) via the Ca2+ selectivemitochondrial calcium uniporter (MCU) (Baughman et al., 2011;De Stefani et al., 2011); the contact sites between the ER andmitochondria create localized sites of high Ca2+ concentrationrequired for influx into the matrix. Besides contributing to Ca2+

homeostasis, the propagation of Ca2+ increases into themitochondria is important for a variety of cell functions, rangingfrom ATP production to cell death (Rizzuto and Pozzan, 2006). Inparticular, basal uptake of Ca2+ by the mitochondria in resting cellsis important to maintain NADH production to support oxidativephosphorylation (Cárdenas et al., 2010). Our data here are in linewith this concept, as ligand-mediated degradation of IP3R1, whichinhibits the constitutive [Ca2+]ER leak and eliminates Ca2+ uptakeinto the mitochondria, also significantly decreases ATP levels.Future studies should identify the molecular presence of these

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mitochondrial Ca2+ transporters and how they are regulated inmammalian GV oocytes.

Distinct regulation of Ca2+ homeostasis during oocytematurationThe roles and regulation of Ca2+ homeostasis and mitochondrialoutput are seemingly different in GV andMII oocytes (Fig. 6). Afterfertilization, the function of the mitochondria is indispensable forCa2+ oscillations because it supplies the ATP necessary for the Ca2+

pumps required to maintain Ca2+ levels in both the cytosol and inthe ER (Dumollard et al., 2004; Wakai et al., 2013); a high [Ca2+]ERlevel in MII oocytes is important for robust and long-lasting Ca2+

oscillations responsible for completing all events of egg activationand initiation of embryo development. Conversely, the spontaneousCa2+ oscillations in GV oocytes and their counterpart oscillations inthe mitochondria seem mostly important for cell bioenergetics tomaintain the steady state that the meiotic arrest represents. Further,the low levels of [Ca2+]ER in GV oocytes may lower the risk ofapoptosis, as high [Ca2+]ER levels render cells prone to apoptosis(Rong and Distelhorst, 2008). Namely, loss of the high [Ca2+]ERcontent in ovulated MII oocytes appears to underlie the time-dependent aging process that sets in, which compromises theseoocytes’ developmental competence (Gordo et al., 2002). Lastly, itcannot be discounted that Ca2+ from the surrounding cumulus andgranulosa cells enter GV oocytes through gap junctions, especiallyafter stimulation with gonadotropins, and modify Ca2+ homeostasisin these cells (Homa, 1995; Flores et al., 1990), although thecontributions of this regulation in GVs requires additionalexamination.In summary, the present study provides new insights into the

mechanisms of Ca2+ homeostasis in GV oocytes. We found thatcytosolic Ca2+ oscillations are propagated into the mitochondria andcontribute to their metabolism. We also show that finely controlledCa2+ influx regulates oscillations in GV oocytes. Lastly, we point tothe distinct regulatory mechanisms of Ca2+ homeostasis in GV andMII oocytes that may fulfill stage-specific cellular functions. Future

studies should define the channel(s) and the regulatory mechanismsthat underpin Ca2+ oscillations in GVs, as well as the mitochondrialCa2+ transporter(s) and their regulation during maturation andfertilization. Understanding and modulation of these molecularprocesses may lead to improvements in in vitro oocyte maturationand embryo development.

MATERIALS AND METHODSChemical reagentsIonomycin and thapsigargin were purchased from Calbiochem (San Diego,CA). Other all chemicals were from Sigma-Aldrich (St Louis, MO) unlessotherwise specified.

Collection of oocytesGV oocytes were collected from the ovaries of 8- to 12-week-old CD-1female mice. Females were injected with 5 IU of pregnant mare serumgonadotrophin (PMSG). Cumulus cell-enclosed GVoocytes were recovered42–46 h post-PMSG administration, and the cumulus cells were removed byrepeated pipetting. GV arrest was maintained where necessary by theaddition of 100 mM 3-isobutyl-1-methylxanthine (IBMX) to the medium.Oocyte maturation was induced by removing IBMX, and oocytes werematured in vitro for 12–15 h in CZBmedium under paraffin oil, at 37°C in ahumidified atmosphere containing 5% CO2. All animal procedures wereperformed according to research animal protocols approved by theUniversity of Massachusetts Institutional Animal Care and Use Committee.

PlasmidsHuman STIM1–YFP and ORAI1 were generously provided by TobiasMeyer (Stanford University, Stanford, CA) and Mohamed Trebak(Albany Medical College, Albany, NY), respectively. STIM1–YFP wassubcloned into a pcDNA6/Myc-His B vector (Invitrogen) between therestriction sites AgeI and XbaI. The ORAI1 insert was amplified byPCR and ligated to the N-terminus of the mRFP-bearing pcDNA6/Myc-His B vector (Dominique Alfandari, University of Massachusetts,Amherst, MA) between EcoRI and XhoI restriction sites. The SNAP25mutant (SNAP25Δ20) sequence was amplified by PCR from themouse SNAP25 expression vector (Origene) and subcloned into apcDNA6/Myc-His B vector between the EcoRI and XhoI restrictionsites. The sequences of the forward and reverse primers were, respectively,

Fig. 6. Schematic diagrams of the distinct regulation of Ca2+ homeostasis between GV and MII oocytes. In GV oocytes (left), Ca2+ influx stimulates Ca2+

oscillations by stimulating IP3R1 and triggering CICR and/or activating PLC family proteins, leading to IP3 synthesis. Note that a variety of Ca2+ channels in theplasma membrane including Cav3.2 T-type, TRP and/or ORAI1 channels are proposed to mediate the majority of this influx, although the plasma membranechannels that mediate Ca2+ influx during maturation and fertilization remain to be fully identified. The persistently low [Ca2+]ER levels and spontaneous Ca2+

oscillations in GV oocytes serve as the natural trigger for Ca2+ influx through a store-operated Ca2+ influx pathway that is normally associated with expression ofSTIM1 and ORAI1, which is required to sustain the oscillations. Spontaneous Ca2+ oscillations are propagated into the neighboring mitochondria, activatingmitochondrial metabolism and increasing the production of ATP. During oocyte maturation, around the GVBD stage, the putative active channel(s) areinternalized, which causes downregulation of Ca2+ influx and termination of spontaneous Ca2+ oscillations, thereby allowing the increase in [Ca2+]ER contentduring maturation. A high [Ca2+]ER level in unfertilized (arrested) MII oocytes (middle) is important for robust and long-lasting Ca2+ oscillations responsible for thecompletion of all events of egg activation. After fertilization, the sperm-derived PLCζ (PLCZ1) triggers Ca2+ oscillations inMII oocytes (right). Mitochondria take uppart of IP3R1-mediated Ca2+ release, which is required to maintain [Ca2+]ER levels and long-lasting Ca2+ oscillations to maintain ATP output that is necessary tosupport SERCA activity that in turn replenishes [Ca2+]ER and sustains the oscillations.

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5′-CGGAATTCGCCACCATGGCCGAAGACGCAGACATG-3′, 5′-GCT-CGAGTTAATCAGCCTTCTCCATGAT-3′. ATP biosensor, AT1.03 andmitochondrial Ca2+ probe, pericam-mito, in pcDNA3.1 vector were kindlyprovided by Dr Hiromi Imamura (Kyoto University, Japan) and Dr AtsushiMiyawaki (RIKEN Center for Brain Science, Japan).

Preparation and microinjection of cRNAPlasmids were linearized with a restriction enzyme downstream of the insertto be transcribed. Capped DNA was transcribed in vitro using the T7mMESSAGE mMACHINE Kit (Themo Fisher Scientific, MA) using thepromoter that was contained in the constructs. A poly(A)-tail was added tothe transcribed RNAs using a Tailing Kit (Themo Fisher Scientific), andpoly(A)-tailed RNAs were eluted with RNase-free water and stored inaliquots at −20°C. For microinjection, cRNA solution was loaded into glassmicropipettes at a concentration of 500 ng/l and delivered into oocytes bypneumatic pressure (PLI-100, Harvard Apparatus, Cambridge, MA). Thevolumes injected typically ranged from 2 to 10 pl, which is 1–5% of oocytevolume (250–300 pl).

Live cell imagingTo measure cytoplasmic Ca2+, oocytes were incubated with 1.25 mMFura-2 (Themo Fisher Scientific) supplemented with 0.02% pluronic acid(Themo Fisher Scientific) for 20 min at room temperature. Fura-2-loadedoocytes were attached to glass-bottomed dishes (MatTek Corp., Ashland,MA) and placed on the stage of an inverted microscope. Fura-2 fluorescencewas excited with 340 nm and 380 nm wavelengths and emitted light wascollected at wavelengths above 510 nm. Radiometric measurement ofpericam-mito is efficiently used as a mitochondrial-specific Ca2+ probe(Nagai et al., 2001). To estimate [Ca2+]mt levels, emitted light was collectedafter dual excitation at 410 nm and 480 nm wavelengths. The confocalfluorescence images of pericam-mito were obtained using a laser-scanningconfocal microscope (LSM 510 META, Carl Zeiss Microimaging Inc.,Germany) fitted with a 63×1.4 NA oil-immersion objective lens. Imageswere acquired with LSM software (Carl Zeiss). ATeam AT1.03, afluorescence resonance energy transfer-based ATP indicator, has beenused successfully to measure cellular ATP levels in live somatic cells(Imamura et al., 2009). To estimate the relative changes in ATP levels, theemission ratio of AT1.03 (YFP:CFP) was imaged using a CFP excitationfilter, dichroic beam splitter, and CFP and YFP emission filters. CFP andYFP intensities were collected every 20 s by a cooled Photometrics SenSysCCD camera (Roper Scientific, Tucson, AZ).

Western blot analysisCell lysates from mouse oocytes were prepared by adding 2× sample buffer[0.125MTris-HCl, pH6.8; 4% (w/v) SDS; 20% (w/v) glycerol; 0.01% (w/v)Bromophenol Blue; 10% (w/v) β-mercaptoethanol]. Proteins were separatedby SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford,MA). After blocking, membranes were probed with the rabbit polyclonalantibody specific to IP3R1 (1:1000; a generous gift from Dr Jan BaptisteParys, Katholieke Universiteit, Leuven, Belgium; Parys et al., 1995). Goatanti-rabbit antibody conjugated to horseradish peroxidase (HRP)was used asa secondary antibody (1:2000; STAR124P; Bio-Rad, Hercules, CA) fordetection of chemiluminescence using a Western Lightning Plus-ECL kit(NEN Life Science Products, Boston, MA) according to the manufacturer’sinstructions. The signal was digitally captured using a Kodak 440 ImagingStation (Rochester, NY). The same membranes were stripped at 50°C for30 min (62.5 mM Tris, 2% SDS and 100 mM 2-beta mercaptoethanol) andre-probed with anti-α-tubulin monoclonal antibody (1:1000; T-9026;Sigma-Aldrich, St Louis, MO) to detect tubulin.

Statistical analysisValues from three or more experiments performed on different batches ofoocytes were analyzed by Student’s t-test or one-way ANOVA followed byFisher’s protected least significant difference test as appropriate. Differenceswere considered significant at P

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