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Spatiotemporal Dynamics of Intracellular [Ca2+] I Oscillations During the Growth and Meiotic Maturation of Mouse Oocytes

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Spatiotemporal Dynamics of Intracellular [Ca2+] I Oscillations During the Growth and Meiotic Maturation of Mouse Oocytes
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  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 µ m to 70 µ m in size, duringwhich it remains arrested in the dictyate stage of meioticprophase, with a prominent germinal vesicle (GV). During thegrowth phase, at about 60 µ m 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 of gonadotrophins 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 IIwhere 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 of calcium homeostasis is also modified during the maturation of oocytes from several different species (Bement, 1992; Chibaet al., 1990). In mammalian oocytes, there is a decrease in theoccurrence of spontaneous InsP 3 -mediated calcium oscillations(Carroll and Swann, 1992), an increase in the amount of  3507 Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization.We have monitored [Ca 2+ ] i in oocytes at different stages of growth 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 of global synchronous [Ca 2+ ] 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 [Ca 2+ ] i signal. Calcium appears to reachhighest levels in the germinal vesicle but this apparent dif-ference of [Ca 2+ ] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are lesscompetent: about 40% are able to resume meiosis and asimilar proportion of these oocytes show spontaneouscalcium oscillations. [Ca 2+ ] i transients are not seen inoocytes that do not resume meiosis spontaneously in vitro.Nonetheless, these oocytes are capable of [Ca 2+ ] i oscilla-tions since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium 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 µ m/second weregenerated in mature oocytes, but not in immature oocytes.In some mature oocytes, successive calcium waves haddifferent sites of srcin. 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 [Ca 2+ ] i oscillations during thegrowth and meiotic maturation of mouse oocytes John Carroll 1, *, Karl Swann 2 , David Whittingham 1 and Michael Whitaker 3 1 MRC Experimental Embryology and Teratology Unit, St George’s Hospital Medical School, Cranmer Terrace, London, SW170RE, UK 2 Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK 3 Department 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  3508 calcium released in response to ionomycin (Tombes et al.,1992) and an increase in the sensitivity of the oocyte to InsP 3 -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 [Ca 2+ ] 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 of different 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. [Ca 2+ ] 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 of a [Ca 2+ ] i wave is at fertilization, where a wave of [Ca 2+ ] i prop-agates across the oocyte from the point of sperm-egg fusion(Miyazaki et al., 1986; reviewed in Whitaker and Swann,1993). [Ca 2+ ] i transients induced by agonists also trigger[Ca 2+ ] 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, if unphysiological, 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 [Ca 2+ ] 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 Oocytes Fully grown oocytes were collected from 21- to 23-day-old B6CB(C57Bl/6JLac × CBA/CaLac)F 1 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 B6CBF 1 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 thatthe 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 culture Denuded 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% CO 2 in 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 measurements Intracellular 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 µ M 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 (  M  r =10 000; Molecular Probes) was microinjected asdescribed previously (Carroll and Swann, 1992). The oocytes wereplaced in a 200-500 µ l drop of M2 under paraffin oil in a dish with apolylysine (100 µ g/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 of the 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 [Ca 2+ ] 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 [Ca 2+ ] i . Microinjection of sperm extracts Sperm 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 [Ca 2+ ] i oscil-lations. Mature oocytes were collected and microinjected about 15-16hours post hCG. RESULTSThe evolution of spontaneous [Ca 2+ ] i oscillationsduring oocyte growth and meiotic maturation To find out when oocytes begin to generate spontaneouscalcium oscillations, we recorded [Ca 2+ ] i in oocytes at differentstages of oocyte growth. Growing oocytes from 10- to 13-day- J. Carroll and others  3509Calcium oscillations in mouse oocytes old mice are about 50 µ m in diameter and do not resumemeiosis when released from their follicle cells. These meioti-cally incompetent oocytes showed no evidence of spontaneous[Ca 2+ ] 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 [Ca 2+ ] i transients wereseen in a similar proportion of oocytes (5 of 12). In two of theseoocytes, we recorded repetitive [Ca 2+ ] 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 [Ca 2+ ] 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 of different 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 [Ca 2+ ] i oscillations could begenerated, we added carbachol, which increases InsP 3 produc-tion. Carbachol addition triggered a series of small [Ca 2+ ] i oscillations (Fig. 2B), suggesting the presence of a muscarinicreceptor coupled to phosphoinositide turnover in meioticallyincompetent 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 [Ca 2+ ] i transients (Fig. 2C). So,while meiotically incompetent oocytes show no sign of spon-taneous [Ca 2+ ] i oscillations, they respond with [Ca 2+ ] i oscilla-tions to exogenous agonists.We measured [Ca 2+ ] i in fully grown oocytes at differenttimes after release from the follicle to pinpoint when sponta-neous [Ca 2+ ] i transients petered out: within 2 hours, 2-4 hoursafter release or 6 hours after release. The overall pattern of [Ca 2+ ] 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 [Ca 2+ ] i oscillations. The proportion decreases to about 33% foroocytes 2-4 hours after release, with a further 20% showingsome irregular [Ca 2+ ] i oscillations. By 6 hours after releasefrom the follicle, no [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i transients are dependent on the oocyte’s stage of growth.Fluorescence was recorded from oocytes loaded with the Ca 2+ -sensitive dye fluo-3 AM. Meiotically incompetent oocytes isolatedfrom 13-day-old mice showed no sign of Ca 2+ 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 Ca 2+ transients (B). In fully grown oocytes recovered from mice olderthan 23 days, repetitive Ca 2+ 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 spontaneousCa 2+ oscillations do show a transient calcium increase in response to1 µ M ionomycin (A) and are able to generate repetitive Ca 2+ transients in response to 100 µ M carbachol (B) and thimerosal (C).  3510 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 [Ca 2+ ] i oscillations while a further 33% (9 of 26) showed some spon-taneous [Ca 2+ ] i activity (Table 1). The remainder (14 of 26)showed no sign of [Ca 2+ ] i transients. Oocytes monitored 6hours after release showed no spontaneous [Ca 2+ ] i oscillationsexcept for one oocyte that generated one small [Ca 2+ ] i transient(Table 1). So the pattern of [Ca 2+ ] i oscillations in oocytes stim-ulated to resume meiosis in vivo appears similar to thoseundergoing in vitro maturation. Spontaneous [Ca 2+ ] i oscillations during the onset ofmeiotic maturation occur synchronously throughoutthe cytoplasm We used confocal calcium imaging microscopy to examine thespatial organization of [Ca 2+ ] i oscillations in oocytes undergo-ing meiotic maturation. All spontaneously occurring [Ca 2+ ] i oscillations ( n =21) consisted of a synchronous increase in thefluorescence signal throughout the cytoplasm (Fig. 4). We con-sistently found that the peak nuclear [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i wave was responsible for theentirety of the [Ca 2+ ] 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 [Ca 2+ ] i transientalthough the [Ca 2+ ] i increases observed at low temperaturewere, as expected, slower to peak (not shown). Finally, todetermine that a small, local [Ca 2+ ] 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 [Ca 2+ ] 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 gradients In immature oocytes loaded with fluo-3 AM, we consistentlyfound that during the course of a Ca 2+ 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 Ca 2+ -green dextran (  M  r =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 J. Carroll and others Fig. 3. Different patterns of [Ca 2+ ] 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 Ca 2+ oscillationsof a regular frequency and amplitude (top panel). Between 2 and 5hours after release, the Ca 2+ 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 [Ca 2+ ] i oscillations in oocytes stimulated to resume meiosis in vivoor in vitro Hours after stimulatingRegularIrregularNo.resumption of meiosis n oscillationsoscillationschangesin vitro03123262-418648in vivo32639146230122  3511Calcium oscillations in mouse oocytes 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 isaccessible to [Ca 2+ ] 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 [Ca 2+ ] i oscillations 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 peak values in a region thatcorresponds to the position of the germinal vesicle (A). Todemonstrate that the Ca 2+ 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 Ca 2+ transient comparingdiametrically opposite pointson each plot (C,D). Note thesimilar rate of increase at thedifferent regions of cytoplasm. A
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