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16 (2008) 431–440www.elsevier.com/developmentalbiology

Developmental Biology 3

Regulation of cytosolic and mitochondrial ATP levels inmouse eggs and zygotes

Rémi Dumollard a,c,⁎,1, Karen Campbell b,1, Guillaume Halet c, John Carroll c, Karl Swann b,⁎

a Laboratoire de Biologie du Développement UMR 7009 CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230, Franceb Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Heath Park CF14 4XN, UK

c Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK

Received for publication 2 November 2007; revised 23 January 2008; accepted 5 February 2008Available online 15 February 2008

Abstract

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation ofenergy production in the zygote. Sperm-triggered Ca2+ oscillations are known to be responsible for the stimulation of both ATP consumption andATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca2+] and [ATP] in themitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca2+ oscillations in the cytosol that are transduced intomitochondrial Ca2+ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]mito and[ATP]cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca2+]cyto and [ATP]cyto duringfertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote.Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Togetherwith our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset ofdevelopment, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.© 2008 Elsevier Inc. All rights reserved.

Keywords: Calcium; ATP; Mitochondria; Fertilization; Pyruvate; Lactate

Introduction

At fertilization, a Ca2+ increase in the egg is the universaltrigger for the activation of embryo development. In mammals,the sperm-triggered Ca2+ increase takes the form of oscillations(termed “Ca2+ oscillations”) lasting from 4 to 6 h (Carroll, 2001).These Ca2+ oscillations stimulate the block to polyspermy,

Abbreviations: ATP, adenosine trisphosphate; [Ca2+]cyto, cytosolic Ca2+

concentration; [Ca2+]mito, mitochondrial Ca2+ concentration; GFP, green fluo-rescent protein; GV, germinal vesicle; NADH, reduced nicotinamide adeninedinucleotide; coxVIII, cytochrome oxidase subunit VIII.⁎ Corresponding authors. R. Dumollard is to be contacted at Laboratoire de

Biologie du Development, Station Zoologique, Villefranche sur Mer, 06230,France. Fax: +33 493 76 37 92. K. Swann, Department of Obstetrics andGynaecology, School of Medicine, Cardiff University, Heath Park CF14 4XN,UK. Fax: +44 2920 744399.

E-mail addresses: remi.dumollard@obs-vlfr.fr (R. Dumollard),swannk1@Cardiff.ac.uk (K. Swann).1 These authors contributed equally to this work.

0012-1606/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2008.02.004

cytoskeletal remodelling, meiotic resumption and recruitment ofmaternal RNAs and are absolutely necessary for the egg toembryo transition (Ducibella et al., 2006). The many processesstimulated at fertilization are all energy consuming and energeticmetabolism must be up regulated at fertilization to supportactivation of development (Dumollard et al., 2006, 2007b). Up-regulation of energetic metabolism at fertilization is illustratedby the increase in the cytosolic ATP concentration ([ATP]cyto)observed in mouse eggs during sperm-triggered Ca2+ oscilla-tions (Campbell and Swann, 2006).

In the mammalian egg and zygote, several observationssuggest that ATP is mainly supplied by mitochondrialoxidative phosphorylation while glycolysis contributes poorly(Dumollard et al., 2007a,b). Mitochondrial poisons provoke arapid decline of intracellular ATP levels resulting in disruptionof Ca2+ homeostasis (Liu et al., 2001; Dumollard et al., 2004;Campbell and Swann, 2006) while inactivating pyruvatedehydrogenase (a mitochondrial enzyme feeding the Krebscycle) in murine oocytes at the beginning of follicular growth

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phase impairs development beyond the one cell stage (Johnsonet al., 2007). Imaging of mitochondrial redox state duringsperm-triggered Ca2+ oscillations in mouse eggs revealed thatthese cytosolic Ca2+ oscillations stimulate repetitive cycles ofoxidation and reduction in the mitochondria (Dumollard et al.,2004). It was thus hypothesised that up-regulation of energeticmetabolism at fertilization is due to a paced stimulation ofmitochondrial oxidative phosphorylation by the sperm-trig-gered Ca2+ oscillations (Dumollard et al., 2006, 2007b).However, no direct measurement of mitochondrial ATPconcentration ([ATP]mito) in a developing embryo has everbeen published to substantiate this hypothesis. Moreover, suchhypothesis implies that cytosolic Ca2+ oscillations aretransduced into mitochondrial Ca2+ signals, and to date nomeasurement of [Ca2+]mito in an egg has ever confirmed such apossibility.

Pyruvate is the only metabolite present in oviductal fluid orculture media that is absolutely required for the embryo toproceed from fertilization through the early cleavage divisions (2cell stage in mice) (Summers and Biggers, 2003; Leese, 1995).Pyruvate is, therefore, thought to be the main metaboliteoxidised for energy production in the mammalian zygote(Summers and Biggers, 2003; Dumollard et al., 2007a).However, pyruvate (b0.5 mM) is not the most abundantmetabolite in oviductal fluid and culture media which containhigher levels of lactate (10–25 mM), glucose (∼0.5 to 5 mM)and glutamine (1 mM) (Summers and Biggers, 2003). Lactatemust be first oxidised to pyruvate before further metabolism(Stryer, 1970) and it is rapidly oxidised by lactate dehydrogenasein the cytosol of mouse eggs and zygotes (Dumollard et al.,2007a). However lactate cannot support development fromfertilization suggesting that it can not be metabolised in the egg(Leese, 1995, Biggers et al., 1967). More recently, it washypothesised that lactate is not oxidised to pyruvate in mouseeggs and zygotes because the intracellular redox potential isreduced and the cytosolic level of NAD+ (the oxidised cofactornecessary for lactate oxidation) is too low (Lane and Gardner,2005). This hypothesis contradicts our previous observation thatlactate addition increases NADH level in mouse eggs (Dumol-lard et al., 2007a,b) and illustrates that the metabolism and rolesof lactate are unresolved. Even though it is long known thatglycolysis is repressed in the mouse zygote (Barbehenn et al.,1974), it has been shown that glucose is required for sperm–eggfusion, and that some glucose is taken up and metabolised byglycolysis and pentose phosphate pathway in fertilizingmammalian eggs (Comizzoli et al., 2003; Urner and Sakkas,2005). Here again, the actual contribution of glucose to energyproduction during the activation of development is unclear.

In this study, we have monitored directly [ATP]cyto or[ATP]mito at the same time as [Ca2+]cyto, and we have measuredsimultaneously [Ca2+]cyto and [Ca2+]mito in living mouse eggsbefore fertilization and during sperm-triggered Ca2+ oscilla-tions. We have also sought to observe the increase in ATPconsumption accompanying egg activation more directly byinhibiting pyruvate entry into the mitochondria. Finally, weassessed the contribution of exogenous pyruvate in the supplyof energy in the mouse zygote and compared it to the

contribution of glucose, lactate and glutamine which are thethree other major metabolic substrates promoting pre implanta-tion development.

Materials and methods

Eggs

Mature (MII) eggs were recovered from superovulated MF1 female mice asdescribed previously (Dumollard et al., 2004; Campbell and Swann, 2006).Mature eggs at metaphase II (MII) were maintained in Hepes-buffered KSOM(H-KSOM, Biggers et al., 2000) containing 1 mg/ml BSA. The H-KSOM wasmade in the laboratory as described previously and contains glucose, lactate,pyruvate and glutamine but no amino acids (Dumollard et al., 2004, 2007a). Thecumulus cells were removed with hyaluronidase (150 i.u. ml−1) added to the H-KSOM. Cumulus-eggs were collected and washed in H-KSOM three times andplaced in a drop of the same medium under mineral oil. Germinal Vesicle (GV)oocytes were recovered by direct dissection of ovaries removed from miceprimed with 7 i.u. of PMSG 48 h before collection. GVoocytes were maintainedin M2 media (Sigma Aldrich) containing 250 μM dibutryl cAMP and they werethen injected with mRNAs and cultured in until imaging (typically 2–3 h), orallowed to mature to MII for ∼15 h.

[Ca2+]cyto and mitochondrial electrical potential measurements

Intracellular Ca2+ was measured using Rhod2 AM (Molecular Probes) orOregon green BAPTA dextran as described previously (Dumollard et al., 2004;Campbell and Swann, 2006). Rhod2 was excited by the 543 nm laser line whileemission was collected using a 580 nm longpass filter. Measurements wereobtained by averaging the signal collected in the whole egg. Oregon greenBAPTAdextranwas excited using a halogen lampwith a 490 nm band-pass filter.

Mitochondrial electrical potential was measured as described previously(Dumollard et al., 2004). Briefly, eggs were incubated for 15 min in H-KSOMcontaining 10 μg/ml of the lipophilic cationic dye Rhod 123 (excitation: 488 nm,emission: 520 nm). With this loading protocol, internalisation of the dye in themitochondria quenches the Rhod123 fluorescence and depolarisation induces a“dequench” of Rhod 123 fluorescence seen as an increase in green fluorescence.

Microinjection procedure

Microinjection procedures were carried out as previously described(Dumollard et al., 2004; Campbell and Swann, 2006). Briefly, eggs weremicroinjected using pressure pulses applied to the back of micropipettes insertedinto eggs using overcompensation of the negative capacitance of a seriallyconnected electrical amplifier. The volume of solution injected was estimated bythe diameter of cytoplasmic displacement caused by the injection; which wasapproximately 3–5%. For cytosolic ATP measurements eggs were injected witha solution containing 7.5 mg/ml firefly luciferase and 0.5 mM Oregon greenBAPTA dextran (pipette concentrations). For mitochondrial ATP or mitochon-drial Ca2+ measurements, eggs were injected with the appropriate cRNA and,where applicable, 0.5 mM Oregon green BAPTA dextran. After microinjectionthe zona pellucidae were removed via treatment with acidic Tyrode's solutionand then the eggs were attached to a polylysine-coated coverslip that formed thebottom of a heated (37 °C) chamber on an inverted microscope. For allexperiments using a luciferase the eggs were maintained in Hepes bufferedKSOM media containing 100 μM luciferin. The luminescence signal wasallowed to stabilize for approximately 1 h before the addition of sperm orchemical stimuli.

Imaging of fluorescence and luminescence

Fluorescence and luminescence were both imaged in the same sets of eggsusing a Nikon TE2000 microscope equipped with a 20×0.75 NA Fluorobjective lens. The light (100%) was directed via a sideport to a cooledintensified CCD (ICCD) camera equipped with an intensifier with a S20 typephotocathode that was cooled to 0 °C. The ICCD camera, a peltier cooler, and

Fig. 1. Both [ATP]cyto and [ATP]mito show a biphasic increase at fertilization. (A)Simultaneousmeasurement of [Ca2+]cyto by fluorescence imaging (F=[Ca2+]cyto)and of [ATP]cyto by luminescence imaging of untargeted luciferase (L=[ATP]cyto) from a single egg. The graph shows an initial and secondary changein [ATP]cyto occurring during Ca

2+ oscillations after the addition of sperm, whichis indicated by the arrow. (B) Simultaneous measurement of [Ca2+]cyto byfluorescence imaging (F=[Ca2+]cyto) and of [ATP]mito by luminescence imagingof mito-luc (L=[ATP]mito) from a single egg. Strikingly, the changes in [ATP]mito

induced at fertilization are very similar to the observed changes in [ATP]cyto. (C)Continuous measurement of [ATP]mito in a control egg where no sperm wasadded shows that the [ATP]mito luminescence signals was stable over the timecourse of experiments.

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software for control and analysis were supplied by Photek Ltd (www.photek.co.uk). The emitted light passed through a fluorescence filter block and wasused with a 505 nm dichroic mirror and 500 nm longpass filter. Thefluorescence of eggs was measured by intermittent opening of a shutter thatwas placed in the path of fluorescent excitation light (490 nm) from a halogenlamp. The fluorescence and luminescence were then measured in a repeatedcycle (10 s). In all recordings the fluorescence was recorded with the camerasensitivity on 10% and the fluorescence was 10–100 times greater thanluminescence (Campbell and Swann, 2006). The luminescence values inexperiments represent the absolute number of measured photon counts persecond whereas the intensity of fluorescence is displayed in arbitrary units andis offset in the y-axis for the sake of presentation. The traces shown wereconsidered to be representative for each set of experiments. The cited changesin luminescence for Figs. 4 and 5 were measured 1 h after the addition ofinhibitor or metabolite. The values cited are the means and standard errors ofthe means.

[ATP]mito and [Ca2+]mito measurements using mito-pericam andmito-luc

A vector (pRN3) containing mito-pericam was generated from 2mt-pericam (Filippin et al. 2005), kindly provided by Rosario Rizzuto in vectorpMITO. The 2mt-pericam (which bears a duplicated COX VIII signal peptide)was digested with BamHI and NotI to isolate the ratiometric pericam precededby a single COX VIII signal peptide that we term mt-pericam. The BamHI andNotI fragment was then cloned into the BglII/NotI sites of pRN3 (Lemaire etal. 1995), kindly provided by Alex McDougall) in order to generate cRNAfrom the T3 promoter. A vector (pRN3) containing mito-luciferase (mito-luc)was made by taking the firefly luciferase coding sequence obtained from pBS-Luc (by HindIII and NotI digestion), a kind gift from Kathy Tamai. Themitochondria-targeting sequence from coxVIII was obtained by EcoRI andHindIII digestion of a mito-GFP construct kindly provided by RosarioRizzuto (Rizzuto et al., 1995). To generate mito-luc, coxVIII targetingsequence was cloned at the N-terminus of luciferase into the EcoRI/NotI sitesof pRN3. For each of these two constructs, polyadenylated capped cRNA wasproduced using mMessage mMachine kits and Poly (A) Tailing Kits(Ambion). In vitro matured oocytes expressing mito-pericam were imagedon a Zeiss LSM 510 meta confocal microscope. Dual excitation at 408 and488 nm (emission: 505–550 nm) was realised before dividing the 488 nmimage (shown in Fig. 2D) by the 408 nm image to obtain ratiometricmeasurements of [Ca2+]mito.

Chemicals and perfusions

D(+) Glucose, DL-lactic acid, pyruvate, glutamine, and α-cyano-4-hydroxycinnamate were all obtained from Sigma-Aldrich. During imaging experimentsthese chemicals were added as 10× solution of their final concentration(indicated on the graphs) in the appropriate HKSOM buffer with the pH adjustedto 7.4.

Results

Sperm-triggered Ca2+ oscillations induce a biphasic increasein cytosolic as well as mitochondrial ATP levels

In a previous study (Campbell and Swann, 2006), werecorded a biphasic increase in [ATP]cyto during sperm-triggered Ca2+ oscillations in mouse zygotes. We confirmedthese observations by recording the luminescence emanatingfrom a non-targeted luciferase which diffuses throughout thewhole egg cytoplasm after injection of the purified protein (Fig.2E) while imaging fluorescently [Ca2+]cyto (Fig. 1A, and Table1). In order to understand how such an increase in cytosolic ATPlevels is generated we used a recombinant luciferase targeted to

the mitochondrial matrix (called mito-luc, see Materials andmethods section). Mature MII eggs were co-injected withcapped RNAs coding for mito-luc and the Oregon GreenBAPTA dextran and left to express for 3–4 h before addingsperm. Fig. 1B shows the luminescence (indicating [ATP]mito)and fluorescence (indicating [Ca2+]cyto) changes occurring uponfertilization of an egg expressing mito-luc. As with [ATP]cyto,the [ATP]mito was consistently seen to increase in a biphasicmanner with the first increase starting during the initial sperm-induced Ca2+ transient followed after 1 h by a second [ATP]mito

increase (Fig. 1B). As illustrated in Figs. 1A and B anddocumented in Table 1, the relative increase in luminescencesignal from the mito-luciferase was about two fold which issignificantly larger than that the relative increase in lumines-cence from the cytosolic luciferase (∼60%). Fig. 1C shows thatno significant increase in signal was seen in controls wheremito-luciferase was expressed and eggs were not fertilized since

Fig. 2. Mito-luc and mito-pericam localise to mitochondria in mouse eggs. (A)Confocal imaging of FAD+ autofluorescence in a GV-arrested oocyte showingthe distribution of mitochondria. GV: germinal vesicle; scale bar=15 μm. (B)Confocal image of a GV-arrested oocyte expressing mito-luc and stained withanti-luciferase antibody. Luciferase distribution in this egg most probablyreflects the mitochondrial localisation of mito-luc. GV: germinal vesicle; scalebar=25 μm. (C, D) Simultaneous confocal imaging of Rhod2 in the cytosol (C)and of mito-pericam (D) in an in vitro matured egg expressing mito-pericam.ms: meiotic spindle, scale bar=15 μm. (E) Confocal image of a mature MII egginjected with untargeted luciferase (cytoluc) and stained with anti-luciferaseantibody showing a very diffuse pattern of luciferase distribution. Scalebar=25 μm. (F) Confocal image of a mature MII egg expressing mito-luc andstained with an anti-luciferase antibody. The punctate pattern and enrichmentaround the meiotic spindle (ms) is reminiscent of mitochondrial distribution.Scale bar=25 μm.

Table 1Changes in luciferase luminescence from mouse eggs at fertilization

Experiment Initial increase inluminescence (%)

Final increase inluminescence (%)

IVF [ATP]cyto(cytosolic luciferase)

136±3.23(S.E.M. n=9)

160±5.3*(S.E.M. n=10)

IVF [ATP]mito

(mitochondrial luciferase)159.1±8.7(S.E.M. n=18)

197.4±10.3*(S.E.M. n=22)

In most eggs the increase in luminescence could be resolved into an initialincrease and a secondary or final increase. The size of the initial increase istabulated for eggs where an initial increase could be resolved. The * indicates asignificant difference (Pb0.05) between [ATP]cyto and [ATP]mito.

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luminescence values only changed by 103±1.4% (S.E.M.,n=6) over 6 h.

The mitochondrial location of mito-luc was confirmed byimmunocytochemistry using an anti-luciferase antibody (Fig.2). Fig. 2B shows the distribution of mito-luc in a germinalvesicle (GV)-arrested oocyte 3 h after injection of the relevantRNA. The mitochondrial distribution in such GV oocytes (asobserved by imaging endogenous mitochondrial autofluores-cence, Fig. 2A; Dumollard et al., 2006, 2007b) shows anaccumulation of mitochondria around the GV that is verysimilar to the distribution of mito-luc. This strongly suggeststhat mito-luc was correctly targeted to the mitochondrial matrix.The distribution of mito-luc in a mature MII egg (Fig. 2F) wasalso reminiscent of mitochondrial distribution at this stage(compare with Fig. 2D and see Dumollard et al., 2004) but verydifferent from the distribution of cytosolic luciferase (Fig. 2E)further indicating that our mito-luc indicator was targetedsuitably to measure [ATP]mito. Therefore, the observationsdepicted in Fig. 1 reveal that changes in [ATP]cyto strictly reflectchanges in [ATP]mito and suggest that ATP synthesised in themitochondria is rapidly exported into the cytosol where it willbe consumed.

We demonstrated previously that the changes in mitochon-drial redox metabolism and the increase in [ATP]cyto observed atfertilization require the sperm-triggered Ca2+ oscillations(Dumollard et al., 2004; Campbell and Swann, 2006). Wehypothesised that cytosolic Ca2+ oscillations are transduced intomitochondrial Ca2+ signals. However, the conventional inhibi-tors of mitochondrial Ca2+ influx, such as ruthenium red, haveproved to be ineffective in mouse eggs (R.D. and K.S. personalobservations), so the requirement for mitochondrial Ca2+ signalsat fertilization is still unclear. In order to substantiate a role formitochondrial Ca2+ signals at fertilization we measured directly[Ca2+]mito by expressing a GFP-based Ca2+ indicator (calledpericam; Miyawaki, 2003) into the mitochondria of a mouseegg. The distribution of mito-pericam in a MII egg matured invitro is depicted in Fig. 2D. It shows a punctate pattern and anenrichment around the meiotic spindle that is typical formitochondrial distribution at this stage (Dumollard et al.,2006, 2007b). The distribution of mito-pericam resembles thatpreviously reported for mitochondrially targeted GFP (Aida etal. 2001; Nagai et al. 2006). Fig. 3 shows three examples of thetypical variations of [Ca2+]cyto and of [Ca

2+]mito (measured withmito-pericam) simultaneously imaged at fertilization (n=15

eggs). We consistently observed that changes in [Ca2+]mito

occurred following the first [Ca2+]cyto increase at fertilization. Insome cases an increase in [Ca2+]mito was seen with eachsubsequent increase in [Ca2+]cyto (Figs. 3A and C) whereas inother case there was a large [Ca2+]mito increase at the time of theinitial [Ca2+]cyto increase at fertilization, such that [Ca2+]mito

response remained elevated for several subsequent [Ca2+]cytooscillations (Fig. 3B). With lower frequency [Ca2+]cyto oscilla-tions (Fig. 3C) the increase in [Ca2+]mito could be seen to outlastthe [Ca2+]cyto by several minutes. These observations directlysuggest that cytosolic Ca2+ oscillations are efficiently trans-duced into mitochondrial Ca2+ oscillations.

Fig. 4. Mitochondrial metabolism of pyruvate is critical for the maintenance ofATP levels at rest and for up-regulation of ATP production by sperm-triggeredCa2+ oscillations. (A, B) Simultaneous measurement of [Ca2+]cyto (F=[Ca2+]cyto) and of [ATP]cyto (in panel A) and [ATP]mito (in panel B) by luminescenceimaging (L=[ATP]mito) from a single MII egg cultured in complete H-KSOMmedium. Where indicated, 0. 5 mM CIN was added to block pyruvate entry intomitochondria before FCCP (1 μM) was added to deplete intracellular ATP. (C)Measurement of the mitochondrial electrical potential (ΔΨmito) by imaging ofRhod 123 fluorescence from a single MII eggs cultured in complete H-KSOMmedium. Where indicated, 0.5 mM CIN and FCCP (1 μM), to collapse ΔΨmito

was added.

Fig. 3. Sperm-triggered [Ca2+]cyto oscillations are mirrored by [Ca2+]mito

oscillations. Simultaneous measurement of [Ca2+]cyto (Rhod2 fluorescence) andof [Ca2+]mito (displayed as the normalised 488/408 ratio) in individual eggsduring fertilization. In panels A–C are shown three examples showing the rangeof responses. In panel A the egg displayed a discernable increase in [Ca2+]mito inresponse to most increases in [Ca2+]cyto. In panel B the egg showed a largerinitial [Ca2+]mito that persisted during a series of initial [Ca2+]cyto increases. Inpanel C the egg displayed lower frequency [Ca2+]cyto oscillations and each[Ca2+]mito increase can be seen to outlast the [Ca2+]cyto increase.

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Inhibiting pyruvate entry into mitochondria before fertilizationblocks the up-regulation of energetic metabolism

Although the mature MII egg is said to be “quiescent” beforefertilization, mouse eggs at this stage show a high turnover ofcytosolic ATP supplied by mitochondrial ATP production(Dumollard et al., 2004). However these data were obtained byusing mitochondrial poisons and the metabolic substrates usedto produce ATP could not be determined. We have previouslyshown that mitochondrial metabolism of pyruvate sets thecytosolic and mitochondrial redox potential in mouse eggs(Dumollard et al., 2007a). In order to assess the importance ofmitochondrial metabolism of pyruvate in maintaining intracel-

lular ATP levels, we measured the changes in [ATP]cyto and[ATP]mito taking place upon inhibition of pyruvate import intomitochondria (Figs. 4A, B). Addition of 0.5 mM of α-cyano-4-hydroxy cinnamate (CIN) (Del Prete et al., 2004) is able toblock pyruvate import into the mitochondria without affectingpyruvate transport through the plasma membrane (Dumollard etal., 2007a). As observed earlier, addition of CIN provoked asmall amplitude and long lasting increase in [Ca2+]cyto whilesubsequent addition of the mitochondrial uncoupler FCCP (toconsume all intracellular ATP) provoked a large and sustained[Ca2+]cyto increase preceding cell death (Figs. 4A, B Ca2+

traces; Liu et al., 2001). Measurement of [ATP]cyto during CINaddition showed a rapid fall of cytosolic ATP levels until alower steady state [ATP]cyto was reached (Fig. 4A, a change of−25±2.3%, mean and S.E.M., n=18 eggs). In contrast,measurement of [ATP]mito during CIN addition showed amuch slower but sustained decrease in mitochondrial ATP

Fig. 5. Fertilization provokes ATP depletion in eggs cultured in a mediumdevoid of any metabolic substrate. (A) Simultaneous measurement of [Ca2+]cyto(F=[Ca2+]cyto) and of [ATP]cyto (L=[ATP]cyto) in a single mouse egg culturedin complete H-KSOM in the presence 0. 5 mM CIN before sperm was added(as indicated in the graph). (B, C) Simultaneous measurement of [Ca2+]cyto (F=[Ca2+]cyto) and of [ATP]cyto (L=[ATP]cyto) from a single MII egg cultured in M2medium devoid of any metabolic substrate for 2 h before sperm was added(indicated by an arrow). After Ca2+ oscillations were initiated, 2 mM pyruvatewas added (in panel A) or 0. 5 mM CIN then 2 mM pyruvate were added (inpanel B).

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levels (Fig. 4B, a change of −36±2.8%, mean and S.E.M.,n=15 eggs). The fact that the initial fall in ATP levels was fasterin the cytosol than in mitochondria indicates that ATP turnoverin the cytosol is much higher than in the mitochondria, but it alsosuggests that export of mitochondrial ATP is rather slow after theaddition of CIN. This contrasts with the rapid equilibriumbetween mitochondrial and cytosolic ATP observed duringsperm-triggered Ca2+ oscillations (Fig. 1). As ATP export frommitochondria (coupled to import of ADP) is electrogenic it canbe inhibited by a decrease in mitochondrial electrical potential(Klingenberg and Rottenberg, 1977). The apparent slowequilibration of mitochondrial and cytosolic ATP after CINaddition could, therefore, be due to an effect on mitochondrialpotential. The effect of CIN addition on mitochondrial potentialas measured with Rhod 123 using a “dequench” protocol (seeMaterials and methods and Dumollard et al., 2004) is shown inFig. 4C. Strikingly, CIN induced a small increase in Rhod123fluorescence indicating a small depolarisation of mitochondriacompared to the full depolarisation provoked by FCCP addition(Fig. 4C, n=25 eggs). These observations show that mitochon-drial metabolism of pyruvate contributes to the build up ofmitochondrial electrical potential and explain why [ATP]cytoequilibrates slowly with [ATP]mito after CIN addition.

The impact of the mitochondrial metabolism of pyruvate onthe up-regulation of mitochondrial ATP synthesis during sperm-triggered Ca2+ oscillations was then assessed by measuring[ATP]cyto during fertilization of an egg incubated in 0. 5 mMCIN. Fig. 5A shows that eggs incubated in CIN before spermaddition could not trigger a complete pattern of Ca2+

oscillations since [Ca2+]cyto remained elevated after a varyingnumber of Ca2+ oscillations (n=18 eggs). In addition, thefrequency of Ca2+ oscillations in eggs incubated in CIN wasstrongly increased (25.30±3.04 spikes/h, S.E.M., n=18 eggs)compared to control eggs (7.85±0.68 spikes/h, S.E.M., n=10eggs, pb0.005). These observations suggest that ATP produc-tion is compromised in CIN-treated eggs (Dumollard et al.,2004). Furthermore, in eggs treated with CIN, no increase in[ATP]cyto could be observed after the onset of Ca2+ oscillationsand [ATP]cyto rather decreased during the course of Ca2+ oscil-lations (the luminescence change was −44±5.2%, n=18 eggs).Together these observations demonstrate that mitochondrialmetabolism of pyruvate is responsible for the maintenance ofcytosolic ATP levels in the unfertilized egg as well as for theincrease in ATP levels induced by sperm-triggered Ca2+

oscillations.

Fertilization stimulates ATP consumption that is provided forby mitochondrial metabolism of exogenous pyruvate

The decrease in ATP levels seen during Ca2+ oscillations ineggs pre-incubated in CIN was rather delayed with regards tothe onset of Ca2+ oscillations and was also relatively slow.Together with the fact that several other metabolites can bemetabolised by mitochondria even when mitochondrial pyr-uvate import is inhibited (Dumollard et al., 2007b), thisobservation suggests that eggs treated with CIN may maintainATP level by oxidising exogenous and endogenous amino acids.

To test this hypothesis we measured [ATP]cyto in eggs incubatedfor several hours in a culture medium devoid of any substrate inorder to “starve” them (Dumollard et al., 2004) before addingsperm. The starvation medium consisted of H-KSOM withoutglucose, lactate, pyruvate or glutamine. Fig. 5B shows anexample of such an egg displaying high frequency Ca2+

oscillations followed by a sustained Ca2+ increase. Addition of2 mM pyruvate to such an egg abolished the sustained Ca2+

increase and allowed resumption of Ca2+ oscillations as wasobserved earlier (Dumollard et al., 2004). Measurement of[ATP]cyto in these eggs reveals that sperm-triggered Ca2+

oscillations induce a decrease in ATP levels that is acceleratedduring the high Ca2+ plateau (Fig. 5B, n=28 eggs). Strikingly,the sole addition of pyruvate could restore pre-fertilization ATPlevels after which low frequency Ca2+ oscillations couldresume. The luminescence change after pruvate addition was a237±12% increase (n=28). That the effect of added pyruvate on[ATP]cyto and Ca2+ oscillations is due to mitochondrial meta-

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bolism of pyruvate was established by undertaking the sameexperiment as in Fig. 5B but in the presence of CIN (Fig. 5C).We found that pyruvate addition could not restore [ATP]cyto orlow frequency Ca2+ oscillations when CIN was present (Fig.5C) and the luminescence decreased further by 44±5.4% (n=14 eggs). Together these observations demonstrate that fertili-zation does increase ATP consumption in the egg and that thesole metabolism of pyruvate by mitochondria can supply all theATP consumed in the cytosol.

Neither glucose, nor lactate, nor glutamine metabolism cansupport ATP production in the fertilized egg

After establishing the primordial role for pyruvate metabo-lism in the up-regulation of ATP production at fertilization wesought to investigate potential roles for glucose, lactate andglutamine in ATP supply during sperm-triggered Ca2+ oscilla-tions. A potential role for glucose metabolism was assessedusing the same protocol as for pyruvate and changes in[ATP]cyto and [Ca2+]cyto were monitored during fertilizationand subsequent addition of glucose (10 mM) in starved eggs(Fig. 6A). Fig. 6A shows that fertilization triggered high

Fig. 6. Neither glucose, lactate nor glutamine can fuel mitochondrial ATPproduction in the zygote. (A–C) Same experiments as in Fig. 5 except thatglucose (A), lactate (B) and glutamine (C) were added (as indicated by arrows)after the onset of Ca2+ oscillations.

frequency Ca2+ oscillations followed by a sustained high[Ca2+]cyto with cytosolic ATP levels falling drastically at theonset of the sustained [Ca2+] rise. Remarkably, addition of10 mM glucose never restored Ca2+ oscillations or ATP levels.In fact the addition of glucose lead to a further decrease inluminescence (by 64±3.6%, n=11). This suggests that glucosemetabolism does not contribute to ATP generation in fertilizedeggs even though no other metabolic route can operate (i. e. in astarved zygote).

By imaging cytosolic NAD(P)H in mouse eggs, wepreviously demonstrated that lactate is oxidised to pyruvate inthe cytosol of a starved eggs (Dumollard et al., 2007a). Toestablish whether lactate-derived pyruvate can support ATPproduction in the zygote we fertilized starved eggs beforeadding 20 mM lactate (Fig. 6B). To our surprise, we observedthat lactate could never restore cytosolic ATP levels or Ca2+

oscillations (Fig. 6B), and luminescence values continued todecrease by 41±5.8% (n=11). Even in the presence of 20 mMaspartate (to stimulate NADH and NAD+ shuttling betweenmitochondria and the cytosol and allow for a better oxidation oflactate to pyruvate (Lane and Gardner, 2005)), lactate was notable to restore ATP levels (data not shown, n=9 eggs). Theseobservations suggest that, somehow, lactate-derived pyruvatecannot supply mitochondrial ATP production even in a starvedzygote.

Finally, glutamine was added to starved zygotes as 1 mMglutamine, present in the culture medium, promotes develop-ment of the pre implantation embryo (Summers and Biggers,2003; Biggers et al., 2000). Fig. 6C shows that addition of10 mM glutamine to starved zygotes could not restore cytosolicATP levels or Ca2+ oscillations. In the case of glutamine theluminescence values were not increase but they only decreasedby only 5±5.9% (n=8 eggs) which was not significant.Together these observations demonstrate that while exogenouspyruvate is efficiently metabolised by mitochondria to supplyATP production and support sperm-triggered Ca2+ oscillations,neither glucose, nor lactate, or, glutamine seem to supportmitochondrial ATP production.

Discussion

This work was aimed at studying the changes in energeticmetabolism accompanying mammalian egg activation. Weanalysed mitochondrial and cytosolic ATP turnover bydirectly measuring [ATP]cyto and [ATP]mito in unfertilized aswell as zygotes undergoing meiotic Ca2+ oscillations. Thiswork, together with our earlier work (Dumollard et al., 2004,2007a; Campbell and Swann, 2006), demonstrates thatcytosolic Ca2+ oscillations triggered by sperm entry areefficiently transduced into mitochondrial Ca2+ oscillations thatpace oxidative phosphorylation. In addition, we characterisedthe primordial role of pyruvate metabolism in supplyingmitochondrial ATP production during egg activation. We alsoobserved directly, for the first time, the stimulation of ATPconsumption earlier hypothesised to accompany egg activa-tion. Finally, comparison of the ability of different exogenoussubstrates to fuel mitochondrial ATP production suggests that

438 R. Dumollard et al. / Developmental Biology 316 (2008) 431–440

exogenous pyruvate but not lactate-derived pyruvate can bemetabolised by mitochondria.

The egg to embryo transition activated at fertilizationrequires stimulation of numerous ATP consuming processes(Ducibella et al., 2006). Translation of maternal RNAs, proteindegradation, ionic homeostasis, exocytosis/endocytosis andphosphorylation reactions are the main ATP consumingprocesses stimulated during egg activation (Ducibella et al.,2006). For these reasons it was hypothesised some time ago thatthe overwhelming increase in ATP demand triggered byfertilization must be accompanied by stimulation of energyproduction in the developing zygote. Recordings of increases inmitochondrial-dependant oxygen consumption during fertiliza-tion of invertebrates oocytes has supported this theory (D'Anna,1973; Schomer and Epel, 1998; Dumollard et al., 2003) butsuch evidence is lacking in mouse embryos (Houghton et al.,1996; Trimarchi et al., 2000). An indicator of an activation ofmitochondrial energy production at fertilization in mammalswas the discovery that sperm-triggered Ca2+ oscillationsstimulate repetitive cycles of reduction and oxidation in themitochondria (Dumollard et al., 2004). The rapid reduction ofmitochondrial redox state reflects stimulation of the Kreb'scycle while the ensuing slow oxidation reflects functioning ofthe mitochondrial respiratory chain, both of which lead togeneration of ATP (Dumollard et al., 2004, 2006). Directmeasurement of cytosolic ATP levels during fertilization of amouse egg revealed that cytosolic ATP levels do not decreaseupon fertilization but rather increase thus suggesting that energyproduction is up-regulated at fertilization (Campbell andSwann, 2006).

Most remarkably, the concomitant stimulation of ATP-dependant reactions and ATP production at fertilization reliesalmost exclusively on Ca2+ oscillations as Ca2+ oscillationsper se can stimulate embryonic development and are requiredduring egg activation (Ozil and Swann, 1994; Carroll, 2001).Our earlier observations indirectly suggested that sperm-triggered Ca2+ oscillations in the cytosol might producemitochondrial Ca2+ signals to keep pace with mitochondrialATP production (Dumollard et al., 2004, 2006). Howeverevidence of any requirement for mitochondrial Ca2+ import orof any occurrence of mitochondrial Ca2+ signals during sperm-triggered Ca2+ oscillations in mammals was still lacking. Bydirectly imaging [ATP]mito and [Ca2+]mito using mito-luciferaseand mito-pericam we now have evidence that both [ATP]mito

and [Ca2+]mito are increased at fertilization. The images of eggsand oocytes injected with mito-luciferase and mito-pericamsuggest that these probes were correctly targeted to mitochon-dria. It is, of course, still possible that a small amount of theseprobes were not correctly targeted to mitochondria. However,if this were the case then the contribution of untargeted probewas likely to be insignificant in either case. For luminescencemeasurements of ATP it was evident that the relative, orproportionate, increase in signal was somewhat greater formitochondrial luciferase than for cytosolic luciferase (Table 1).Hence any ‘untargeted’mito luciferase would make a negligiblecontribution to the increase in luminescence we observed. Forthe fluorescence measurements of Ca2+ it is clear from the traces

in Figs. 3B and C in particular that our [Ca2+]mito signals do notsimply track changes in [Ca2+]cyto which would otherwise beexpected if there were significant contributions of cytosolicpericam to our mitochondrial pericam based probe. In fact ourmeasurements with mitopericam suggest that [Ca2+]mito infertilizing eggs can sometimes outlast the increase in [Ca2+]cyto.This has previously been noted for [Ca2+]mito in various somaticcells (Bruce et al. 2004; Collins et al. 2001; Jaconi et al. 2000;Pacher et al. 2000; Voronina et al. 2002). Strikingly cytosolicCa2+ oscillations were reflected in mitochondrial Ca2+ oscilla-tions that generated a sustained increase in mitochondrial andcytosolic ATP levels. Such mitochondrial Ca2+ oscillations aremost probably responsible for the cyclic stimulation of theKreb's cycle and respiratory transport chain observed in aprevious study (Dumollard et al., 2004).

The dynamics of the changes in [ATP]mito observed atfertilization were similar to the changes in [ATP]cyto, bothshowing a biphasic increase in ATP levels. Therefore, the ATPproduced in the mitochondria is rapidly exported from themitochondria to be consumed in the cytosol. Such rapidequilibrium between mitochondrial and cytosolic ATP levels issupported by the enormous mitochondrial complement presentin the zygote (more than 100000 mitochondria per egg;Dumollard et al., 2006) and ensures that up-regulation ofmitochondrial ATP production rapidly increases ATP avail-ability in the cytosol to provide for the increased ATP demand.The reason why eggs show an initial increase in ATP productioncan be explained by the increase in mitochondrial Ca2+ that islikely to stimulate pyruvate dehydrogenase and several otherenzymes of the Kreb's cycle (Denton andMcCormack, 1990). Itis less obvious why there is a second increase in ATP productionsince a biphasic increase in mitochondrial redox state or Ca2+ isnot evident (Dumollard et al., 2004; and Fig. 3). This secondaryincrease in ATP could be either due to an increase in ATPproduction or a decrease in ATP consumption. We are nowinvestigating which factor(s) affect this secondary ATP increaseas a variety of post translational modifications of enzymesinvolved in both ATP synthesis and consumption could occurduring sperm-triggered Ca2+ oscillations.

Pyruvate is long known to be an important metabolicsubstrate for mouse eggs as glycolysis is inhibited during thefirst stages of embryonic development (Dumollard et al., 2006,2007b). Our observations support the previous studies since weshow that mitochondrial metabolism of pyruvate is essential formaintaining cytosolic ATP levels in the unfertilized egg, as wellas for the up-regulation of energy production at fertilization.Indeed, inhibiting mitochondrial import of pyruvate (with CIN)caused [ATP]cyto to fall in the egg and abolished the up-regulation of ATP production by sperm-triggered Ca2+ oscilla-tions. Nevertheless, cytosolic ATP levels could still bemaintained (albeit to a lower level) after inhibition ofmitochondrial pyruvate import in the unfertilized egg. Further-more, in the presence of CIN, [ATP]cyto seemed to be transientlymaintained during Ca2+ oscillations before disruption of Ca2+

homeostasis and complete ATP depletion. This suggested thatother metabolic substrates might fuel the mitochondrial Kreb'scycle.

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We therefore fertilized eggs that had been pre-incubated for2 h in a medium devoid of any metabolic substrate (to starvethe egg), and then added each metabolite separately todetermine the ability of each substrate to fuel mitochondrialATP production. It was noticeable that fertilization of suchstarved eggs consistently induced high frequency Ca2+

oscillations, indicating that low ATP levels lead to aderegulation of Ca2+ homeostasis. Moreover, sperm-triggeredCa2+ oscillations in these eggs provoked a decrease in[ATP]cyto that was accelerated during deregulation of Ca2+

homeostasis. These observations are the first direct evidence ofan increase in ATP demand at fertilization. This substantiatesthe hypothesis that activation of development is accompaniedby activation of energy demand and matching energyproduction. We also found that the addition of pyruvate tostarved eggs undergoing Ca2+ oscillations could restore bothATP levels and low frequency Ca2+ oscillations provided thatpyruvate import into mitochondria is not inhibited. This showsthat the sole mitochondrial metabolism of pyruvate is able tosupply all the required energy production. In contrast, glucosecould not restore ATP levels or Ca2+ oscillations even though,in these starved eggs, glycolysis should be depressed to alower extent than in eggs cultured control medium (seeBarbehenn et al., 1974). This observation indicates that, eventhough glucose transport might be stimulated after fertilization(Comizzoli et al., 2003, Urner and Sakkas, 2005), glucosemetabolism is poorly active in supplying ATP in the mousezygote.

Lactate was also unable to restore ATP levels or normal Ca2+

oscillations in starved eggs undergoing Ca2+ oscillations. Thislack of effect of lactate on mitochondrial ATP production wassurprising as lactate is by far the most abundant metabolicsubstrate in oviductal fluid and in embryo culture media andthe first and obligatory route for lactate metabolism in the cellis oxidation by lactate dehydrogenase to produce pyruvate(Stryer, 1970). Therefore lactate-derived pyruvate appears to bediverted from mitochondrial oxidation while exogenouspyruvate is rapidly oxidised by mitochondria. The inabilityfor exogenous lactate to fuel mitochondrial production is ratherintriguing even though it is consistent with the fact that lactatecannot support development from fertilization (Leese, 1995;Biggers et al., 1967). A recent study by Lane and Gardner(2005) suggested that the inability of lactate to fuelmitochondrial ATP production is because the low levels ofNAD+ in the cytosol does not allow lactate to be oxidised topyruvate by the cytosolic lactate dehydrogenase (LDH) (Laneand Gardner, 2005). However we have previously shown thatstarving eggs in a medium containing no metabolite provokesan oxidation of the intracellular redox potential (visualised by adecrease in NAD(P)H fluorescence) and that lactate addition insuch eggs increases cytosolic NADH levels in a LDH-dependant manner thus proving that pyruvate is made fromlactate by LDH in such eggs (Dumollard et al., 2007a). Ifpyruvate is made from lactate, such pyruvate could bemetabolised by mitochondria to produce NADH and ATP inthe mitochondria. By imaging mitochondrial redox state wepreviously found that lactate-derived pyruvate cannot reduce

mitochondrial redox state as opposed to exogenous pyruvate(Dumollard et al., 2007a). We now show that lactate-derivedpyruvate does not supply mitochondrial ATP production evenin the absence of exogenous pyruvate. These two independentobservations strongly suggest that while exogenous pyruvaterapidly enters mitochondria, pyruvate generated intracellularlyby LDH is diverted from mitochondrial oxidation. Suchcompartmentalization of pyruvate has been observed inneurons and astrocysts where glycolytic pyruvate is preferen-tially oxidised by mitochondria, while pyruvate derived fromcytosolic alanine is channeled towards reduction into lactate(Zwingmann et al., 2001; Cruz et al., 2001). We cannot explainthe lack of mitochondrial oxidation of lactate-derived pyruvate.However, it is plausible to speculate that lactate-derivedpyruvate is converted to alanine by the cytosolic alanineaminotransferase (Dumollard et al., 2007a,b), since alanine isproduced by mammalian embryos and mammalian eggspossess the cytosolic alanine aminotransferase (Gopichandranand Leese, 2003; Cetica et al., 2002).

Acknowledgments

This work was supported by the BBSRC and by CardiffUniversity School of Medicine funds awarded to KS. JC wassupported by the MRC. RD was supported by CNRS and anEMBO short term fellowship. The authors would like to thankKathy Tamai, Rosario Rizzuto and Alex McDougall forsupplying plasmids.

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