INTRODUCTION

The end-point of mammalian oocyte development is in the pro-duction of a mature fertile oocyte. The mouse oocyte growsfrom an initial diameter of 20

µm to 70 µm in size, duringwhich it remains arrested in the dictyate stage of meioticprophase, with a prominent germinal vesicle (GV). During thegrowth phase, at about 60 µm diameter, the oocyte firstbecomes competent to resume meiosis but is maintained in thegerminal vesicle stage by the follicular environment(Wassarman, 1988, for review). Meiotic resumption isnormally stimulated in vivo by the preovulatory surge ofgonadotrophins but can also be stimulated in vitro by releasingthe oocyte from the follicle into a suitable culture medium(Pincus and Enzman, 1935; Edwards, 1965). About 2 hoursafter the resumption of meiosis in either case, the oocyteundergoes germinal vesicle breakdown (GVBD) and entersmetaphase I. By 12 hours, the oocyte reaches metaphase II

where it arrests, awaiting fertilization. At fertilization thesperm triggers a series of repetitive calcium oscillations thatare responsible for all the events of oocyte activation includingrelease of the cortical granules, the completion of the secondmeiotic division and entry into the first mitotic cell cycle (Klineand Kline, 1992; Whitaker and Swann, 1993).

During meiotic maturation of the mammalian oocyte, anumber of changes occur within the oocyte that ensure normalfertilization and development take place. These include, theacquisition of the ability to release cortical granules (Ducibellaet al., 1990, 1993) and development of the competence todecondense the sperm nucleus (Usui and Yanagamachi, 1976).More recently it has become evident that the mechanism ofcalcium homeostasis is also modified during the maturation ofoocytes from several different species (Bement, 1992; Chibaet al., 1990). In mammalian oocytes, there is a decrease in theoccurrence of spontaneous InsP3-mediated calcium oscillations(Carroll and Swann, 1992), an increase in the amount of

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Calcium oscillations occur during meiotic maturation ofmouse oocytes. They also trigger activation at fertilization.We have monitored [Ca2+]i in oocytes at different stages ofgrowth and maturation to examine how the calcium releasemechanisms alter during oogenesis. Spontaneous calciumoscillations occur every 2-3 minutes in the majority of fullygrown (but immature) mouse oocytes released from antralfollicles and resuming meiosis. The oscillations last for 2-4hours after release from the follicle and take the form ofglobal synchronous [Ca2+]i increases throughout the cell.Rapid image acquisition or cooling the bath temperaturefrom 28°C to 16°C did not reveal any wave-like spatial het-erogeneity in the [Ca2+]i signal. Calcium appears to reachhighest levels in the germinal vesicle but this apparent dif-ference of [Ca2+] in nucleus and cytoplasm is an artifact ofdye loading. Smaller, growing immature oocytes are lesscompetent: about 40% are able to resume meiosis and asimilar proportion of these oocytes show spontaneouscalcium oscillations. [Ca2+]i transients are not seen in

oocytes that do not resume meiosis spontaneously in vitro.Nonetheless, these oocytes are capable of [Ca2+]i oscilla-tions since they show them in response to the addition ofcarbachol or thimerosal. To examine how the properties ofcalcium release change during meiotic maturation, acalcium-releasing factor from sperm was microinjectedinto fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger andlonger in mature oocytes compared to immature oocytes.Calcium waves travelling at 40-60 µm/second weregenerated in mature oocytes, but not in immature oocytes.In some mature oocytes, successive calcium waves haddifferent sites of origin. The modifications in the size andspatial organization of calcium transients during oocytematuration may be a necessary prerequisite for normal fer-tilization.

Key words: oocyte, mouse, meiotic maturation, calcium, confocal

SUMMARY

Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the

growth and meiotic maturation of mouse oocytes

John Carroll1,*, Karl Swann2, David Whittingham1 and Michael Whitaker3

1MRC Experimental Embryology and Teratology Unit, St George’s Hospital Medical School, Cranmer Terrace, London, SW170RE, UK 2Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK3Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK

*Author for correspondence

Development 120, 3507-3517 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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calcium released in response to ionomycin (Tombes et al.,1992) and an increase in the sensitivity of the oocyte to InsP3-induced calcium release (Fujiwara et al., 1993). These studiessuggest that the increased capacity of the calcium stores maybe a prerequisite for fertilization to occur.

There is less information about the changes that occur priorto the resumption of meiosis. A number of modifications occurduring oocyte growth that may be necessary for the acquisitionof meiotic competence. These include microtubule re-organiz-ation and chromatin condensation (Albertini, 1993 for review).In addition to these presumably cell-cycle-related events, thereis an increase in the number of calcium channels (Murnane andDeFelice, 1993) and an increase in sensitivity to exposure tocalcium-free medium (DeFelici and Siracusa, 1982) around thetime of meiotic competence. No measurements of [Ca2+]i havebeen made in oocytes around the time that they acquire com-petence and it is not known when spontaneous calcium oscil-lations first appear.

Calcium oscillations are thought to control a number ofdifferent cellular activities including, cell cycle progressionand differentiation (Berridge, 1993). Calcium imaging tech-niques have revealed that calcium transients in cells areorganized both spatially and temporally. [Ca2+]i transientsgenerally occur in the form of waves that propagate from theregion of stimulation to the rest of the cell (Jaffe, 1991;Berridge, 1993 for review). The best documented example ofa [Ca2+]i wave is at fertilization, where a wave of [Ca2+]i prop-agates across the oocyte from the point of sperm-egg fusion(Miyazaki et al., 1986; reviewed in Whitaker and Swann,1993). [Ca2+]i transients induced by agonists also trigger[Ca2+]i waves in other cell types including hepatocytes(Rooney et al., 1990), pancreatic acinar cells (Kasai et al.,1993; Thorn et al., 1993; Nathanson et al., 1992) and adrenalchromaffin cells (O’Sullivan et al., 1989). Also, striking, ifunphysiological, spiral calcium waves can also be triggered inimmature Xenopus oocytes (Lechleiter et al., 1991).

Here we use calcium imaging techniques to study theevolution of the calcium response as the oocyte grows andmatures. We find that spatially homogeneous [Ca2+]i oscilla-tions appear as the oocyte acquires meiotic competence anddisappear as the oocyte matures. In addition, as meiotic matu-ration proceeds the oocyte acquires the ability to propagatecalcium waves.

MATERIALS AND METHODS

OocytesFully grown oocytes were collected from 21- to 23-day-old B6CB(C57Bl/6JLac × CBA/CaLac)F1 hybrid mice that were given 5 i.u.pregnant mares’ serum gonadotrophin 48 hours previously. Ovarieswere removed and placed in Medium M2 (Fulton and Whittingham,1978). Antral follicles were ruptured with a sterile needle and oocytessurrounded by cumulus cells were collected. The cumulus cells wereremoved from the oocyte using a narrow bore pipette. Growingoocytes were recovered from 10- to 16-day-old B6CBF1 mice by dis-aggregating the ovary using sterile needles. The oocytes were denudedof cumulus cells using fine bore pipettes. To recover oocytes that werestimulated to resume meiosis in vivo, 5 i.u. human chorionicgonadotrophin (hCG) was administered 48 hours after PMSG and thecumulus cells were removed by a brief incubation (3 minutes) in M2medium containing hyaluronidase (150 units/ml). To be certain that

the oocytes had been stimulated to resume meiosis in response tohCG, oocytes with partially expanded cumulus cells and no cleargerminal vesicle were collected at 3 and 6 hours post hCG. Matureovulated oocytes arrested at metaphase II were recovered from theoviducts of mice 14-15 hours post hCG. Cumulus cells were removedusing hyaluronidase as described above. The cumulus-free oocyteswere washed in M2 before culture or recording intracellular calcium.

Oocyte cultureDenuded oocytes were washed three times in bicarbonate-bufferedM16 medium (Whittingham, 1971) and placed in microdrops of thesame medium under oil in a plastic culture dish (Falcon). The dishwas maintained in an incubator at 37°C with a gas phase of 5% CO2in air. The oocytes were examined at regular intervals for up to 6 hoursafter the release from their follicles to determine whether germinalvesicle breakdown had taken place.

Calcium measurementsIntracellular calcium was monitored using the calcium-sensitive dyefluo-3 or indo-1. To load the dyes intracellularly, oocytes wereincubated for 15 minutes at 37°C in 50 µM of the acetoxymethyl ester(AM) form of the dye made up in M2 containing 0.02% pluronic. Flu-orescence was monitored using photomultipliers as described previ-ously (Carroll and Swann, 1992).

For confocal microscopy, oocytes were loaded with fluo-3 asdescribed above. In some experiments fluo-3 potassium salt or Ca-Green dextran (

Mr=10 000; Molecular Probes) was microinjected asdescribed previously (Carroll and Swann, 1992). The oocytes wereplaced in a 200-500 µl drop of M2 under paraffin oil in a dish with apolylysine (100 µg/ml)-coated cover slip as the base. The preparationwas maintained at a temperature of 27-34°C except in some experi-ments that were performed at 16°C. The dish containing the oocyteswas placed on the stage of a Leica laser-scanning confocal microscopeand oocytes were observed using a 40× oil objective (1.2 NA). Anargon laser was used for excitation at 488 nm and signals emittedabove 515 nm were collected. The laser power was the minimumrequired to provide an adequate signal-to-noise ratio. Images wereacquired over a 70 second period in a 128×128 pixel format (oneimage every 0.56 seconds). To improve the signal-to-noise ratio ofthe confocal image each of the 128 lines was scanned four times andaveraged. Other scanning procedures were also used to obtain higherresolution formats or faster image acquisition. Most of the imagespresented are ratio images obtained by dividing the experimentalimages pixel by pixel by a control image where [Ca2+]i is at restinglevels (Gillot and Whitaker, 1993). This method of normalizationcontrols for uneven dye distribution and differences in scatteringalong the laser light path, but cannot eliminate artefacts due to com-partmentalization of indicator dye into compartments where calciumdoes not track [Ca2+]i.

Microinjection of sperm extractsSperm extracts were prepared and microinjected (Swann, 1990; 1994)into immature and mature oocytes. Immature oocytes were incubatedfor 2-3 hours after release from the follicle before loading andmicroinjection to avoid the occurrence of spontaneous [Ca2+]i oscil-lations. Mature oocytes were collected and microinjected about 15-16hours post hCG.

RESULTS

The evolution of spontaneous [Ca2+]i oscillationsduring oocyte growth and meiotic maturationTo find out when oocytes begin to generate spontaneouscalcium oscillations, we recorded [Ca2+]i in oocytes at differentstages of oocyte growth. Growing oocytes from 10- to 13-day-

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old mice are about 50 µm in diameter and do not resumemeiosis when released from their follicle cells. These meioti-cally incompetent oocytes showed no evidence of spontaneous[Ca2+]i oscillations during recordings for periods of 20 to 90minutes after release from the follicle (n=15) (Fig. 1). Abouthalf (27 of 54) the oocytes isolated from 16-day-old mice aremeiotically competent. Spontaneous [Ca2+]i transients wereseen in a similar proportion of oocytes (5 of 12). In two of theseoocytes, we recorded repetitive [Ca2+]i oscillations with aninterspike interval of about 10-12 minutes (Fig. 1B). Themajority of fully grown oocytes (isolated from mice aged 21-25 days) resume meiosis in vitro (95%: 45 of 54) and werecorded [Ca2+]i oscillation in about 75% (25 of 31) of thesefully grown but immature oocytes, with an interspike intervalof 1-3 minutes (Fig. 1C).

To determine whether meiotically incompetent oocytespossess a calcium signalling system, we applied a number ofdifferent calcium-releasing agonists. Addition of the calciumionophore ionomycin caused a large increase in the fluo-3signal, presumably by releasing calcium from intracellularstores (Fig. 2A). To confirm that [Ca2+]i oscillations could begenerated, we added carbachol, which increases InsP3 produc-tion. Carbachol addition triggered a series of small [Ca2+]ioscillations (Fig. 2B), suggesting the presence of a muscarinicreceptor coupled to phosphoinositide turnover in meiotically

incompetent oocytes. Application of thimerosal, which sensi-tizes calcium release mechanisms in immature and matureoocytes (Carroll and Swann, 1992; Swann, 1991; Miyazaki etal., 1992a) triggered a series of [Ca2+]i transients (Fig. 2C). So,while meiotically incompetent oocytes show no sign of spon-taneous [Ca2+]i oscillations, they respond with [Ca2+]i oscilla-tions to exogenous agonists.

We measured [Ca2+]i in fully grown oocytes at differenttimes after release from the follicle to pinpoint when sponta-neous [Ca2+]i transients petered out: within 2 hours, 2-4 hoursafter release or 6 hours after release. The overall pattern of[Ca2+]i oscillations generated in different oocytes varied andoscillations were classified as regular or irregular in frequencyand amplitude, while in other oocytes no transients were seen.Representative examples are shown in Fig. 3. The results areshown in Table 1. In oocytes monitored during the first 2 hoursafter release, about 75% show regular spontaneous [Ca2+]ioscillations. The proportion decreases to about 33% foroocytes 2-4 hours after release, with a further 20% showingsome irregular [Ca2+]i oscillations. By 6 hours after releasefrom the follicle, no [Ca2+]i oscillations were seen in 6 oocytes.

In the experiments that we have described so far, oocyteswere encouraged to resume meiosis by releasing them fromtheir follicular environment. It seemed possible that either thespontaneous [Ca2+]i oscillations or their decline were due tothis artificial procedure. We therefore stimulated maturation invivo. hCG was administered to mice and oocytes that hadundergone GVBD were isolated from the ovary at 3 and 6hours after injection. Three hours after hCG injection was the

Fig. 1. The occurrence and characteristics of spontaneous [Ca2+]itransients are dependent on the oocyte’s stage of growth.Fluorescence was recorded from oocytes loaded with the Ca2+-sensitive dye fluo-3 AM. Meiotically incompetent oocytes isolatedfrom 13-day-old mice showed no sign of Ca2+ transients duringrecordings that lasted for 20 to 90 minutes. Part of one of theserecords is shown (A). Increases in fluorescence were detected inabout half of the growing oocytes recovered from 16-day-old mice.Some of these oocytes showed repetitive low frequency Ca2+

transients (B). In fully grown oocytes recovered from mice olderthan 23 days, repetitive Ca2+ oscillations were recorded in themajority of oocytes (C).

Fig. 2. Calcium changes can be generated in small meioticallyincompetent oocytes using a number of different agonists.Meiotically incompetent oocytes that do not undergo spontaneousCa2+ oscillations do show a transient calcium increase in response to1 µM ionomycin (A) and are able to generate repetitive Ca2+

transients in response to 100 µM carbachol (B) and thimerosal (C).

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earliest time at which oocytes stimulated to resume meiosis inresponse to hCG could be positively identified. At this time,12% of oocytes (3 of 26) showed regular frequency [Ca2+]ioscillations while a further 33% (9 of 26) showed some spon-taneous [Ca2+]i activity (Table 1). The remainder (14 of 26)showed no sign of [Ca2+]i transients. Oocytes monitored 6hours after release showed no spontaneous [Ca2+]i oscillationsexcept for one oocyte that generated one small [Ca2+]i transient(Table 1). So the pattern of [Ca2+]i oscillations in oocytes stim-ulated to resume meiosis in vivo appears similar to thoseundergoing in vitro maturation.

Spontaneous [Ca2+]i oscillations during the onset ofmeiotic maturation occur synchronously throughoutthe cytoplasmWe used confocal calcium imaging microscopy to examine thespatial organization of [Ca2+]i oscillations in oocytes undergo-ing meiotic maturation. All spontaneously occurring [Ca2+]ioscillations (n=21) consisted of a synchronous increase in thefluorescence signal throughout the cytoplasm (Fig. 4). We con-sistently found that the peak nuclear [Ca2+] level exceeded thatin the cytosol. This observation is discussed in more detailbelow. The oscillations comprised a pacemaker rise in the flu-orescence ratio signal, followed by a rapid upstroke (Fig. 4B).We verified that spontaneous oscillations were synchronous bymeasuring the fluorescence intensity ratio throughout the risingphase of the [Ca2+]i transient in four regions in the oocytecytoplasm. This is shown in the first confocal image of Fig.

4A. Six oocytes were analysed thus and in all cases weconfirmed that the ratio increased synchronously in the oocyte(Fig. 4C,D).

If a cytoplasmic [Ca2+]i wave was responsible for theentirety of the [Ca2+]i transient then it would be expected tocross the egg in 3-4 seconds (the rise time of the transient). Awave of this sort is clearly absent as we would have detectedit by sampling at 0.56 second intervals. To confirm that norapid waves were present, we performed two experiments. Inthe first, the bath temperature was reduced from about 27°C to16°C, which might be expected to decrease the wave velocity1.5-fold (Lechleiter and Clapham, 1992). In the second, thescan speed was increased from 540 msec/scan to 130msec/scan. Lower temperatures or faster scanning speedsfailed to reveal any spatial heterogeneity in the [Ca2+]i transientalthough the [Ca2+]i increases observed at low temperaturewere, as expected, slower to peak (not shown). Finally, todetermine that a small, local [Ca2+]i event faster than the speedof sampling was detectable we fired action potentials inoocytes using the rising edge of hyperpolarizing current pulses.We saw a cortical increase in [Ca2+]i that rapidly reached thecentre of the oocyte (Fig. 5A). Triggering an action potentialafter the scan was initiated resulted in an apparent increase influorescence only in the cortex of the bottom half of the oocytewhile the fluorescence in the top half of the oocyte remainedat resting levels (Fig. 5B). In the following scan, the fluores-cence was increased uniformly throughout the cortex (Fig. 5B).Such discontinuous patterns of fluorescence were never seenin the course of a spontaneous oscillation.

Nuclear-cytosolic calcium gradientsIn immature oocytes loaded with fluo-3 AM, we consistentlyfound that during the course of a Ca2+ oscillation the nuclearfluorescence ratio peaked at a higher value than that in thecytoplasm (see Fig. 4A for example). We also noted that theresting fluorescence intensity was consistently lower in thenucleus compared to the cytoplasm (Fig. 6A). To determinewhether this pattern of fluorescence was peculiar to the fluo-3AM loading method, we microinjected oocytes with the freeacid form of fluo-3 or Ca2+-green dextran (Mr=10 000). Inoocytes microinjected with fluo-3, the resting levels of fluo-rescence were more uniform than after loading using the AMester technique, while the calcium-green injected oocytesshowed a higher resting nuclear signal (compare Fig. 6A,C,E).The fluorescence ratio images at the peak of a calcium transientwere also markedly different using the three different tech-niques (Fig. 6B,D,F). We saw a nuclear/cytoplasm disparity inall fluo-3 AM-loaded oocytes imaged through the plane of thenucleus (n=12). When we microinjected fluo-3, the disparity

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Fig. 3. Different patterns of [Ca2+]i oscillations are seen at differenttimes after the resumption of meiosis. Fluorescence recordings fromfluo-3 AM loaded fully grown oocytes undergoing meioticmaturation showed three basic patterns. In the first 2 hours aftermeiotic resumption, the majority of oocytes showed Ca2+ oscillationsof a regular frequency and amplitude (top panel). Between 2 and 5hours after release, the Ca2+ oscillations become increasinglyirregular in frequency and amplitude (middle panel) and finally, afterabout 6 hours no changes in fluorescence were recorded (bottompanel).

Table 1. Time course of the occurrence of [Ca2+]ioscillations in oocytes stimulated to resume meiosis in vivo

or in vitroHours after stimulating Regular Irregular No.resumption of meiosis n oscillations oscillations changes

in vitro0 31 23 2 62-4 18 6 4 8

in vivo3 26 3 9 146 23 0 1 22

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was much less, though a small difference was seen in two outof three oocytes (Fig. 6D). Calcium-green dextran-injectedoocytes showed a uniform ratio increase in the nucleus andcytoplasm (n=2) (Fig. 6F). The nuclear/cytoplasm disparityclearly depended on the dye used and the method of dyeloading, suggesting that it is artifactual. Since ratio imagesshould not be subject to artifacts caused by different dye con-centrations or optical pathlengths, provided that all the dye is

accessible to [Ca2+]i, these experiments imply that dye hasbecome compartmentalized. We imaged an oocyte loaded withfluo-3 AM using high resolution averaging of 32 consecutivescans. The resting fluorescence pattern showed intense regionsof fluorescence particularly in the perinuclear region and in asurrounding reticular network. In addition, some areas of highfluorescence intensity were seen in the cortex of the oocyte(Fig. 6G). This pattern of fluorescence is consistent with the

Fig. 4. Spontaneous [Ca2+]ioscillations in maturingoocytes are synchronousthroughout the cytosol.Sequential confocal ratioimages sampled at 0.56 secondintervals. Note thefluorescence ratio increaseshomogeneously throughout thecytosol during the first 5images and reaches peakvalues in a region thatcorresponds to the position ofthe germinal vesicle (A). Todemonstrate that the Ca2+

increase is homogeneous, thefluorescence ratio intensities atthe four positions marked onthe first confocal image wereplotted against time. Note thatthe four points increase inunison (B). To show this moreclearly, the rising phase of thesame Ca2+ transient comparingdiametrically opposite pointson each plot (C,D). Note thesimilar rate of increase at thedifferent regions of cytoplasm.

A

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idea that when oocytes are loaded with fluo-3 AM, some of thedye is sequestered in cytoplasmic organelles (cf Gillot andWhitaker, 1993).

Since cytoplasmic organelles are absent from the nucleus,we can use our measurement of the nuclear/cytoplasm ratiodisparity to estimate the error in the resting signal due to dyecompartmentalization. About 25% of the resting signal is dueto sequestered dye. This, and the fact that we observed spatiallyhomogeneous [Ca2+]i increases with both fluo-3- and calciumgreen dextran-injected oocytes, makes us confident that uptakeof dye into cytoplasmic organelles is not masking a spatiallyinhomogeneous component of the [Ca2+]i response.

Spatial organization of calcium transients inimmature and mature oocytes in response to acalcium-releasing factor from spermWe have previously shown that the spontaneous [Ca2+]i oscil-lations in maturing oocytes involve InsP3 and the InsP3receptor (Carroll and Swann, 1992). The oscillations in thematuring oocyte stop some time after GVBD (Table 1) yet itis apparent that the sensitivity to InsP3 increases during meioticmaturation (Fujiwara et al., 1993). While the decreased occur-rence of Ca2+ oscillations may be explained by a decrease inInsP3 levels during maturation, this paradox suggests that someexplanation other than a simple change in receptor number oraffinity may underlie the increased sensitivity of the matureoocyte to InsP3. To examine differences in the response of thecalcium-releasing store in mature and immature oocytes, wemicroinjected a sperm extract known to cause [Ca2+]i oscilla-tions similar to those seen at fertilization (Swann, 1990, 1994).[Ca2+]i increased rapidly immediately after microinjection,then decreased over 3-10 minutes (not shown). Once [Ca2+]ihad returned to resting values, both immature and matureoocytes began to show repetitive calcium oscillations. The timecourse of one of these Ca2+ oscillations in mature and maturingoocytes is shown in Fig. 7. There were a number of clear dif-ferences between the oscillations that occurred in immatureand mature oocytes (Table 2). These differences were not aresult of artifacts of dye loading. Permeabilization of oocyteswith 20 µM digitonin releases dye from the cytosol but not

from intracellular organelles (Gillot and Whitaker, 1993).Treatment of maturing and mature oocytes with digitonin ledto the loss of similar proportion of the fluorescence signal(about 80%). In response to injection of the sperm factor, theaverage peak increase in fluorescence ratio in mature eggs wasabout double that of the immature oocyte and, once elevated,the calcium stayed higher for longer (Table 2; Fig. 7). Also,the rate of rise of calcium was about 4-fold faster in the maturecompared to the immature oocyte (Table 2). In addition, thespatial organization of the calcium increase was markedlydifferent. In immature oocytes the calcium increase wasspatially homogeneous in all of the 15 transients examined(Fig. 7B) while, in mature eggs, the calcium increase normallytook the form of a wave that propagated throughout the cytosol(Figs 7C, 8). One of the mature oocytes showed a singlecalcium wave that was followed by five synchronous increasesin [Ca2+]i, while nine oscillations from two other matureoocytes were in the form of calcium waves. During therecovery phase of the Ca2+ transient, no spatial inhomogene-ity in the fluorescence signal was seen.

We noted an interesting feature of the calcium waves inmature oocytes. The site of origin of the calcium wave variedin successive oscillations (Fig. 8A,C). The calcium wavetravels across the oocyte cytoplasm in 1-2 seconds (Fig. 8B,D)corresponding to a wave speed of about 40-80 µm/sec.

DISCUSSION

We show here that the calcium signalling system evolvesduring the growth and meiotic maturation of mouse oocytes.Small, meiotically incompetent oocytes are capable of gener-ating [Ca2+]i oscillations in response to carbachol orthimerosal, but do not do so spontaneously. Meioticallycompetent maturing oocytes show spontaneous [Ca2+]i oscil-lations whose frequency is highest in oocytes that are fullygrown. As the oocyte approaches GVBD of first meioticmetaphase, the oscillations decrease in frequency, then dieaway. However, the oocytes remain competent to respond with[Ca2+]i oscillations. Indeed, using a calcium-releasing factor

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Fig. 5. Rapid calcium events fasterthan the scan speed can bedetected. Firing of an actionpotential before the start of a scancauses a cortical increase in theoocyte that rapidly reaches thecentre of the oocyte (A). When theaction potential is fired after thescan has started, the top half of theegg has been scanned at the timethe Ca2+ begins to increase. Suchdistributions of Ca2+ were neverseen during the course of aspontaneous [Ca2+]i oscillation.

A

B

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isolated from sperm that mimics the fertilization response tocompare immature and mature oocytes, we find that a calciumwave mechanism develops in maturing oocytes, where the[Ca2+]i response increases more rapidly, reaches higher [Ca2+]iand is longer lasting than in immature oocytes.

Spontaneous calcium oscillations are correlatedwith the meiotic status of the oocyteSpontaneous calcium transients are first seen in growingoocytes around the time that the ability to resume meiosis isfirst apparent and not in oocytes that are meiotically incompe-tent. Also after meiosis has been stimulated either in vitro orin vivo, the occurrence of spontaneous calcium oscillationsdecreases dramatically between 2 and 5 hours after releasefrom the follicle. Thus the generation of spontaneous calciumoscillations appears to occur first at the time of meiotic com-petence and stop as the oocyte approaches metaphase of thefirst meiotic division. The acquisition of meiotic competenceshows many of the structural and biochemical hallmarks oftransition from G2 into metaphase (Albertini, 1993). The cor-relation between the two events, meiotic competence and thegeneration of spontaneous calcium transients, raises the possi-bility that an underlying change in [Ca2+]i homeostasis is asso-ciated with the G2-M transition.

During oocyte growth, there is an increase in the peakcalcium-dependent inward current suggesting the number ofcalcium channels increase as the oocyte grows in the follicle(Murname and DeFelice, 1993). This may provide the stimulusfor spontaneous [Ca2+]i oscillations through increased [Ca2+]iinflux (Missiaen et al., 1991). This cannot be a complete expla-nation, since it is known that the oscillations stop after GVBD(see below) and maturation-associated changes in calciuminflux through calcium channels would also need to be invoked(Preston et al., 1991). Since [Ca2+]i oscillations in mouseoocytes are generated through the InsP3 receptor and requirecalcium influx (Carroll and Swann, 1992), it is likely that mod-ifications in the turnover of the phosphoinositide messengersystem (Carrasco et al., 1990; Homa et al., 1991; Ciapa et al.,1994), the InsP3 receptor, or plasma membrane permeability tocalcium (Clothier and Timourian, 1972; Preston et al., 1991)explain the changes in calcium homeostasis.

Calcium release mechanisms are modified duringmeiotic maturationMicroinjection of a partially purified calcium-releasing factorfrom sperm triggered calcium oscillations in both mature andimmature oocytes. The peak calcium levels in mature oocyteswere about double that seen in immature oocytes and the

Fig. 6. The nuclear cytoplasmic calcium gradient is dependent uponthe method of dye loading. Confocal images of resting fluorescence(A,C.E) and peak fluorescence ratio (B,D,F) in oocytes loaded withfluo-3AM (A,B), or after microinjection of fluo-3 (C,D) or calcium-Green linked to dextran (Mr=10000) (E,F). Note the low restingfluorescence of the nucleus after loading with the fluo-3 AM and thehigher resting fluorescence in oocytes loaded with calcium-greendextran. At the peak of the oscillation a marked nuclear-cytosoliccalcium gradient is present in the AM loaded oocyte (B) but not inthe oocyte loaded with fluo-3 or calcium-green by microinjection(D,F). At high resolution a perinuclear reticular network is apparentin oocytes loaded with fluo-3 AM (G).

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calcium remained elevated for three times as long. A plausiblereason for this difference may be that cytoplasmic calciumstores increase as meiotic maturation progresses (Tombes etal., 1992; Jones and Carroll, unpublished data).

The maturation-associated changes in the properties ofcalcium release in response to sperm factor were similar tothose described for fertilization of immature and maturehamster oocytes (Fujiwara et al., 1993). This study in hamsteroocytes also demonstrated that maturation led to an increasedsensitivity to InsP3, in that mature oocytes generated an all-or-none [Ca2+]i response at lower InsP3 concentrations thanimmature oocytes. One explanation for the enhanced [Ca2+]iresponse of the mature oocyte may be that the sensitivity ofthe InsP3 receptor to InsP3 and [Ca2+]i increases, as Fujiwaraand his co-workers suggest. In smooth muscle, the state offilling of the calcium stores significantly affects the positiveco-operation provided by calcium on calcium release throughthe InsP3 receptor (Iino and Endo, 1992). In hamster eggs,InsP3 at very low concentrations, causes regenerativeresponses (Miyazaki et al., 1992b; Galione et al., 1994). It isimpossible to say whether this is due to an elevation of InsP3sensitivity at the InsP3 receptor or to sensitization of another

element of the positive feedback loop. Since the size of calciumstores during meiotic maturation has been reported to increase(Tombes et al., 1992), it seems to us that this may be primarilyresponsible for the enhanced calcium release seen in matureoocytes. Whether larger calcium stores in mature mouseoocytes are gated solely through the InsP3 or involve theryanodine receptor is unknown (Whitaker and Swann, 1993).

[Ca2+]i oscillations in maturing oocytes aresynchronous throughout the cytoplasmConfocal calcium imaging microscopy shows that the sponta-neous [Ca2+]i oscillations in maturing oocytes occur relativelyhomogeneously throughout the cytoplasm of the oocyte. Laserscanning confocal imaging is anisotropic: the temporal resolu-tion in the z plane along the x axis is ten or more times greaterthan along the y axis, depending on the size of the inhomo-geneity. We have shown here that events of tens of millisec-ond duration can be resolved across the oocyte. Since theoocyte diameter is 80 µm and the time taken to scan ten linesof pixels in the x axis is about 20 ms, we should be able toresolve waves travelling across the oocyte diameter along thex axis with a velocity of, say, 80 µm/20 ms (4000 µm/second).

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A B

CFig. 7. The dynamics of calcium releasein response to microinjection of acytosolic sperm extract are different inmature and immature oocytes. Graph offluorescence ratio intensities of animmature and a mature oocyteundergoing a typical sperm extractinduced Ca2+ response (A). Note that theamplitude and duration of the calciumtransient are less in the immature oocyte.The rate of rise is also less in theimmature oocyte. The spatial organizationof the Ca2+ transient in immature (B) andmature (C) oocytes is shown in two seriesof eight confocal images sampled at 0.56second intervals. In the immature oocytethe Ca2+ increases homogeneouslythroughout the cytoplasm inapproximately 3 seconds (B). In themature oocyte, the fluorescence increaseshows a wavelike heterogeneity thatrapidly propagates across the oocyte inabout 1 second (C, see also Fig. 8).

3515Calcium oscillations in mouse oocytes

However, though our spatial and temporal resolution shouldhave permitted us to resolve unitary calcium release events(puffs, sparks) of the size and duration of those recorded inXenopus oocytes (Yao and Parker, 1994), it is unlikely that weshould have detected those recorded in cardiac cells (Cheng etal., 1993). In fact, our temporal and spatial resolution is far inexcess of that necessary to detect a wave with the slow risetime of the [Ca2+]i transients that we measured in maturingoocytes.

It has been suggested that synchronous [Ca2+]i oscillationsare an unlikely phenomenon on theoretical grounds (Jaffe,1991). Several observations in this study suggest that syn-chronous [Ca2+]i oscillations occur in mouse oocytes. From thediscussion above, it is clear that we had sufficient temporal res-olution to detect inhomogeneities and the fact that the fluores-cence increases homogeneously at the four poles of a confocalsection over several images is further evidence that a [Ca2+]iwave faster than the vertical scan speed was not responsiblefor the [Ca2+]i increase. This is supported by the fact that wedid not observe any heterogeneities at faster scan speeds or

lower temperatures. Further, calcium increases that occurredfaster than the scan speed can be recognised as our experimentwith voltage gated calcium influx shows. These experimentsindicate that calcium oscillations in immature oocytes arehomogeneous at a 5-10 µm length scale.

The only other reported cases of synchronous [Ca2+]i oscil-lations in cells is (1) at the fertilization of immature hamsteroocytes where the rise time of the calcium increase is about 4-5 seconds, similar to our sperm extract injection data (Fujiwaraet al., 1993) and (2) at fertilization of mature oocytes where,although the first few transients originate from the point ofsperm-egg fusion, subsequent oscillations have no clear site oforigin (Miyazaki et al., 1986). However, the rise time of theseoscillations is rapid, about 1 second, and the temporal resolu-tion used (about 1-2 seconds) may not have been sufficient toresolve fast waves of the sort that we have measured here. Wehave found that after injection of sperm factor the [Ca2+]i oscil-lations often continue in the form of waves and that the originof the wave can shift from one pacemaker region to another.This has not been observed to occur at fertilization. Whether[Ca2+]i oscillations in mature eggs occur as fast waves or syn-chronous oscillations requires clarification. We have foundwith the injection of sperm extracts that Ca2+ waves aregenerated in mature oocytes, but spatially homogeneous oscil-lations in maturing oocytes. Why might this be?

[Ca2+]i waves and synchronous [Ca2+]i oscillationsCalcium waves that behave in a similar manner to reaction-diffusion systems such as the Belousov-Zhabotinskii (BZ)reaction (Lechleiter et al., 1991) are a common feature of

Table 2. Characteristics of [Ca2+]i oscillations in immatureand mature mouse oocytes microinjected with sperm

extractPeak fluorescence Rate of rise Time from peak

ratio (ratio units/sec) to rest (sec)Immature oocyte 2.0±0.1 0.4±0.06 12.7±0.4Mature oocyte 3.0±0.1* 1.5±0.14* 34.5±4.4*

*All values are significant to P<0.001 as determined by Student’s t-test.

Fig. 8. Calcium waves in a mature oocyte injected with sperm factor. Confocal images sampled at 0.56 second intervals of the rising phase oftwo consecutive sperm extract induced Ca2+ oscillations in a mature oocyte (A,C). Note that the origin of the calcium waves changes by about90 degrees in the sequential oscillations. In B and D, fluorescence ratio values from the region of the square and the circle marked on the firstconfocal images have been plotted against time to illustrate the speed of the Ca2+ wave. The calcium wave takes 1-2 seconds to cross theoocyte.

A

C

3516

calcium signalling. Synchronous oscillations are rarer.However, BZ systems normally generate synchronous oscilla-tions in their simplest form and only begin to generate wavesif gradients of concentration or temperature are present in thesolution (phase waves) or if a very small scale inhomogeneitygenerates a local pacemaker that will initiate a reactiondiffusion wave (Winfree, 1971; Ross et al., 1988). Thus, thehomogeneous oscillations observed in mouse oocytes areanalogous to the synchronous chemical oscillations seen in theB-Z reaction, just as calcium waves are analogous to the B-Zreaction diffusion waves (Lechleiter et al., 1991; Lechleiter andClapham, 1992).

A reaction diffusion wave will propagate only if the rate ofrise at the wavefront exceeds the characteristic rate of rise ofthe bulk solution; otherwise a homogeneous rate of riseoutpaces the wave (Winfree, 1971; Ross et al., 1988). If therate of calcium release is slow relative to diffusion processes,such as diffusion of calcium away from the site of calciumrelease through the cytoplasm or towards the site of releasewithin the lumen of the ER, then spatial inhomogeneities willbe hard to sustain and a synchronous calcium oscillation islikely. If, conversely, a more rapid rate of calcium release canlead to marked local increases, then a wave may be generated.

Based on this model, there are a number of potential expla-nations for the increased excitability of the cytoplasm ofmature oocytes. Since the rate of calcium release in responseto the sperm factor is four-fold greater in mature than inmaturing oocytes, there may be a simple basis for the switchfrom synchronous oscillation to wave propagation. As wementioned earlier, the larger calcium store may increase thepositive feedback by calcium on further calcium releasethrough the InsP3 receptor (Iino and Endo, 1992; Missiaen etal., 1991). This would lead to local regions of high calcium anda subsequent Ca2+ wave. Alternatively, expression of a calciumrelease channel with a large conductance, such as a ryanodinereceptor, may occur during maturation. There would appear tobe a precedent for this idea in that immunoreactive protein hasbeen detected in mature but not immature sea-urchin oocytes(McPherson et al., 1992). Also, a change in the density of thecalcium channels in the ER, or a decrease in the Ca2+ bufferingcapacity of the cytosol may also fulfil the predictions of themodel and contribute to a transition from a cell that generatessynchronous oscillations to one that produces waves.

The spatial distribution of [Ca2+]i in many cell types hasbeen shown to be organized in a manner that reflects function;cortical increases are associated with secretion (O’Sullivan etal., 1989; Cheek et al., 1989; Toescu et al., 1992; Kasai et al.,1993; Thorn et al., 1993) and at fertilization a wave of [Ca2+]ipropagates from the point of sperm-egg fusion across the entireoocyte thereby ensuring complete egg activation (Jaffe 1991;Whitaker and Swann, 1993). The functional significance ofsynchronous Ca2+ oscillations remains unclear. However,cellular differentiation is often associated with the generationof spontaneous oscillations (Holliday et al., 1991).

Nuclear calcium transients - real or artifactual? Apparent nuclear-cytosolic [Ca2+]i gradients were consistentlyseen in oocytes. Similar observations have been reported in anumber of other cell types (Hernandez-Cruz et al., 1990;Holliday et al., 1991; Birch et al., 1992; Przywara et al., 1991;Himpens et al., 1992; Shen and Buck, 1993) and have led to

suggestions that calcium in the nucleus may be separatelyregulated for the purpose of altering gene expression and soaffecting cellular differentiation. The nuclear calciumincreases that we observed in immature oocytes were largelydependent on the method of dye loading; large nuclearincreases in oocytes loaded with fluo-3 AM, while aftermicroinjection of dye, the gradients were not seen or weremuch reduced. The reason why nuclear calcium increasesabove cytosolic after loading with fluo-3 AM but not fluo-3 orcalcium-Green can be explained by the compartmentalizationof the AM ester into calcium-containing cytoplasmicorganelles (Connor, 1993; Gillot and Whitaker, 1993; Al-Mohanna et al., 1994). Trapped indicator dye provides a highresting level of fluorescence in the cytoplasm relative to theorganelle-free regions of the cytoplasm and the nucleus. As aresult, when [Ca2+]i increases, the proportional change in thecytoplasm is less than that in the nucleus (Connor, 1993; Gillotand Whitaker, 1993). While our observations offer no evidencefor a differentially regulated nuclear calcium store in oocytes,it is nonetheless clear that when calcium is released into thecytosol it rapidly enters the germinal vesicle.

Supported by grants from the Royal Society, the Medical ResearchCouncil and the Wellcome Trust. Thanks to Isabelle Gillot, AlexMcDougall and Martin Wilding for discussions and assistance withconfocal microscopy.

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(Accepted 17 August 1994)