Molecular Human Reproduction vol.2 no.12 pp. 967-977, 1996

Developmental changes in calcium dynamics, protein kinase Cdistribution and endoplasmic reticulum organization in humanpreimplantation embryos

Mario Sousa1, Alberto Barros2 and Jan Tesarik3 4 5

laboratory of Cell Biology, Institute of Biomedical Sciences, laboratory of Medical Genetics, Faculty of Medicine,University of Porto, Porto, Portugal, 3Department of Biochemistry and Molecular Biology, University of Granada Facultyof Sciences, Granada, Spain, and 4Laboratoire d'Eylau, 55 Rue Saint-Didier, 75116 Paris, France5To whom correspondence should be addressed at the laboratoire d'Eylau, 55 Rue Saint-Didier, 75116 Paris, France

Developmental changes in the Ca2+ dynamics of human zygotes and preimplantation embryos were relatedto changes in the distribution of endoplasmic reticulum (ER) and protein kinase C (PKC). The fertilization-induced Ca2+ oscillations were typically observed over >5 h, were ryanodine-sensitive and showed aperiphery-to-centre propagation of Ca2+ waves. At the same time, ER and PKC were accumulated in the cellperiphery. After the appearance of pronuclei, ryanodine-sensitive Ca2+ oscillations of lower amplitude andfrequency were observed until the pronuclear breakdown. However, Ca2+ waves then began in the perinuclearregion, in the area of ER and PKC accumulation and spread towards the cell periphery. During the second tofourth cell cycle, small sinusoidal Ca2+ fluctuations were observed; sparse higher-amplitude Ca2+ spikes,superimposed on these basal fluctuations, appeared shortly before cell division. The sinusoidal Ca2+

fluctuations were asynchronous in individual blastomeres and disappeared progressively in arrested embryos.The direction of Ca2+ wave propagation and the distribution of ER and PKC were similar to the situationobserved in pronuclear zygotes. In contrast to the zygotes, ryanodine did not arrest the Ca2+ oscillations butaugmented their amplitude and frequency. These data suggest that human pre-embryos use differentmechanisms of Ca2+ signalling in the early post-fertilization period, during the pronuclear development andduring cleavage.

Key words: calcium dynamics/endoplasmic reticulum/human preimplantation embryos/protein kinase C/ryanodine

IntroductionCa2+ signals play an essential role both at the time ofconception, when they transduce a sperm-generated epigeneticmessage required for launching embryonic development(reviewed in Swann and Ozil, 1994), and during subsequentlife when they modulate various cellular processes relatedto cell division and cell cycle control (e.g. Poenie et ai,1985; Whitaker and Patel, 1990; Lu and Means, 1993;Ciapa et ai, 1994; Hepler, 1994). The nature of theCa2+ signals employed at fertilization and during furtherdevelopment may be different; however, studies reportinglong-term Ca2+ recordings of embryos, going on beyondthe first cell cycle, are sparse and limited to non-mammalianspecies (Shantz, 1985; Yoshimoto et ai, 1985; Speksnijderet ai, 1989; Fluck et ai, 1991; Grandin and Charbonneau,1991; Kubota et ai, 1993; Keating et ai, 1994; Strieker,1995). The only available information concerning the humanspecies refers to the early sperm-induced series of Ca2+

oscillations occurring during the first hours followingfertilization (Taylor et ai, 1993; Tesarik et ai, 1994; Tesarikand Sousa, 1994; Tesarik et ai, 1995; Sousa et ai, 1996a,b),whereas information about the Ca2+ dynamics during laterphases of the first cell cycle and during the subsequent cellcycles of the early human development is lacking.

© European Society for Human Reproduction and Embryology

In spite of the current debate about the mechanism ofCa2+ oscillations in oocytes and other cell types and aboutthe significance of this particular Ca2+ signal in earlymammalian development (Berridge, 1996; Swann and Lawr-ence, 1996; Tesarik and Sousa, 1996), there is a generalagreement as to the central role played by the endoplasmicreticulum (ER), which serves as the principal intracellularstore of releasable Ca2+ signals (Streb et ai, 1984; Clapham,1995). ER is also believed to be the main source of Ca2+

released to the cytosol during the sperm-induced Ca2+

oscillations of mammalian oocytes (Homa et ai, 1993;Whitaker and Swann, 1993; Swann and Ozil, 1994). Themechanism of the sperm-induced Ca2+ oscillations in oocytesis believed to involve a specific action of protein kinase C(PKC) (Swann et ai, 1989; Miyazaki et ai, 1990; Birdet ai, 1993; Gallicano et ai, 1993, 1995; Petersen andBerridge, 1994). Developmental changes in the Ca2+

dynamics may thus be accompanied by changes in the ERorganization and PKC intracellular distribution.

In this study we examined the Ca2+ dynamics of humanpreimplantation embryos from the 1-cell zygote to the fourthcell cycle of embryonic development. These findings wererelated to the pattern of the intracellular distribution of ERand PKC at the corresponding developmental stages.

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Figure 1. Sperm-induced Ca2+ responses of freshly fertilizedoocytes. (A) Long-term follow-up of Ca2+ oscillations developingafter the addition of spermatozoa (arrow) to an oocyte(representative of data obtained with 12 oocytes). Interruptions ofthe record correspond to times during which the oocyte wasremoved from the confocal microscopy system, washed in freshmedium and kept in a CO2 incubator. (B) Effect of the addition of4 mM ryanodine (Ry) during a late phase of sperm-induced Ca2+

oscillations (representative of data obtained with six oocytes). Therecord begins 5.5 h after the onset of the sperm-induced Ca2+

oscillations. Fluo-3 fluorescence emitted from the equatorial planeof the oocytes was recorded at intervals of 2 s.

Materials and methods

Source and preparation of gametes and embryosAll gametes and embryos used in this study were obtained from thein-vitro fertilization (TVF) programme of the Laboratory of MedicalGenetics, Faculty of Medicine, University of Porto, Portugal. Semensamples were obtained from men showing normal values of spermcount, concentration, motility and morphology (World HealthOrganization, 1992). After liquefaction at room temperature (30 min),semen samples were diluted in sperm preparation medium (SPM;Medi-Cult, Copenhagen, Denmark) and centrifuged for 10 min at600 g to wash out the seminal plasma. This dilution-centrifugationstep was repeated once more, and the resulting sperm pellets wereresuspended in SPM equilibrated with 5% CO2 in air, and incubatedat 37°C for 3 h to achieve capacitation.

Oocytes were recovered from large ovarian follicles by ultra-sonically-guided follicular aspiration in cycles stimulated with humanmenopausal gonadotrophin (HMG) and human chorionic gonado-

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trophin (HCG) after pituitary desensitization with a gonadotrophin-releasing hormone (GnRH) agonist; these oocytes were used intherapeutic IVF or intracytoplasmic sperm injection (ICSI) attempts.Details of the ovarian stimulation and of the IVF and ICSI techniquesused have been described previously (Sousa and Tesarik, 1994;Tesarik et al., 1994; Tesarik and Sousa, 1995).

This study involves supernumerary oocytes from cases in whichembryo freezing was not available. These oocytes were fertilizedwith husbands' spermatozoa and used for the study of events occurringduring the early post-fertilization period and at the pronuclear stage.They were discarded after the breakdown of the pronuclear membrane.

Surplus embryos from IVF and ICSI treatment cycles which couldnot be frozen because of unavailability of the embryo freezing facilitywere also allocated to this study. Only normally fertilized (twopronucleated) zygotes and morphologically good embryos, classifiedas grade 4 (equal sized symmetrical blastomeres) or grade 3 (unevenblastomeres with <10% fragmentation) according to Steer et al.(1992), whose grade did not change during the time-course ofexperimentation, were included. The embryos were discarded immedi-ately after the termination of each experiment. All of the gametesand embryos allocated to this study were used with the informedconsent from the donating patients.

Evaluation of the intracellular free Ca*+ concentrationRelative changes in the intracellular free Ca2+ concentration ([Ca2+]j)were analysed using Fluo-3, a fluorescent probe for visualizing Ca2+

in living cells (Minta et al., 1989) which was loaded into the embryosin the form of the cell-permeant acetoxymethyl ester Fluo-3-AM(Molecular Probes, Eugene, OR, USA). The advantages and disadvant-ages of the use of this non-ratiometric probe for the study of Ca2+

dynamics in human oocytes have been discussed previously (Tesariket al., 1995).

Oocytes, pronuclear zygotes and cleaving embryos were incubatedwith 9 jiM Fluo-3-AM in SPM [prepared from a stock solution of890 |iM Fluo-3-AM in dimethylsulphoxide (DMSO)] for 30 min at37°C under a gas phase of 5% CO2 in air (the final concentration ofDMSO was \%). They were then washed, placed in SPM and allowedto adhere to culture dishes coated with poly-L-lysine (Sigma, StLouis, MO, USA). The specimens were examined with a BioradMRC 600 (Richmond, CA, USA) confocal laser scanning microscopyunit. The fluorescence emitted from the equatorial plane of the zygotesor of individual blastomeres of the cleaving embryos was monitoredat intervals of 2 or 5 s depending on the experiment. Images wereanalysed using the Biorad Time Course Ratiometric Software Module.Specimens were scanned simultaneously in two optical channels. Onechannel served for the acquisition of the Fluo-3 fluorescence signaland the other was used for the visualization of a phase-contrast imageof each specimen; die latter was required for concomitant evaluationof cytological changes, such as pronuclear breakdown or cell division,occurring in the specimens in the course of each experiment. At theend of each experiment, oocytes were treated with 10 (iM ionophoreA23187 (Sigma) which produced an increase in [Ca2+], that wasused for calibration of the whole record. Relative fluorescenceintensity was thus expressed as percentage of this ionophore-induced[Ca2+]i increase.

Some Fluo-3-loaded oocytes were inseminated in vitro in the courseof the Ca2+ monitoring procedure. For in-vitro insemination, oocyteswere freed from the zona pellucida with 1% pronase (Sigma) inTyrode's salt solution (Sigma, cell culture grade) as described byTesarik and Kopecny (1989a,b). After incubation for in-vitro capacit-ation, spermatozoa were treated with ionophore A23187 to inducethe acrosome reaction and thus shorten the time between spermaddition and sperm-oocyte fusion. Ionophore A23187 was added at

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Figure 2. Spatial characteristics of Ca2+ waves, protein kinase C (PKC) distribution and endoplasmic reticulum (ER) organization in thelate pre-pronuclear period (5 h after the beginning of the fertilization-induced Ca2+ response). (A) Sequential confocal images of a Fluo-3-loaded oocyte showing a late fertilization-induced [Ca2+]j increase (representative of data obtained with 12 oocytes). Fluo-3 fluorescenceemitted from the oocyte equatorial region was recorded at intervals of 5 s. The numbers indicate the time (s) after the recording of the firstimage of the series presented. (B) Confocal imaging of PKC (visualized with the use of Fim-1) in an oocyte undergoing late fertilization-induced Ca2+ oscillations (representative of data obtained with three oocytes). The same data were also obtained with three other oocytesfixed during the first h of the fertilization-induced Ca2+ oscillations. (C) Confocal imaging of ER visualized with the use of DiOC5(3) in anoocyte undergoing late fertilization-induced Ca2+ oscillations (representative of data obtained with three oocytes). The same data were alsoobtained with three other oocytes fixed during the first hour of the fertilization-induced Ca2+ oscillations. In all parts of this compositefigure, fluorescence intensity is expressed in pseudocolour according to the scale bar where the lowest values are coded black.

a final concentration of 10 uM in SPM and was prepared from astock solution of 4 mM ionophore A23187 in DMSO (Sigma). Theincubation of spermatozoa with the ionophore took place at 37°C for15 min, after which time spermatozoa were washed by four cyclesof centrifugation (600 g; 5 min) and resuspended in fresh SPM. Inpreliminary experiments the supernatant from the final washing cyclewas tested for its ability to induce oocyte activation (through theaction of residual ionophore), but no activity was detected in anycase. Insemination was carried out by addition of lxiO5/ml motileionophore-treated spermatozoa to zona-free oocytes.

Pharmacological experimentsRyanodine (Sigma) was used at a final concentration of 4 mM. Thisconcentration of ryanodine has previously been shown to be mosteffective in producing a Ca2+ discharge from the ryanodine-sensitiveCa2+ stores of human oocytes without apparent toxic effects (Sousaet ai, 1996a). The stock solution of 160 mM ryanodine was preparedin 40% DMSO in water and then diluted in SPM to double the finalconcentration desired. Ryanodine addition to cells was performed bymixing equal volumes of this solution with the cell-containingmedium, resulting in final concentrations of 4 mM ryanodine and1% DMSO. These concentrations were previously shown to givereproducible specific Ca2+ responses in human oocytes withoutapparent toxicity (Sousa et al., 1996a,b), and similar concentrationswere also optimal for mouse oocytes (Swann, 1992).

Visualization of PKC and ERFor PKC staining, cells were fixed for 10 min in phosphate-bufferedsaline (PBS; Sigma, cell culture grade; pH 6.8) containing 3.75%depolymerized paraformaldehyde (Biorad). After fixation, cells werepermeabilized with 100% methanol (-20°C, 10 min) and washedtwice in PBS. Cells were then incubated in PBS containing 200 nMFim-1 (Teflabs, Austin, TX, USA) prepared from a 0.297 mM stock

solution in DMSO. Fim-1 is the fluorescein-conjugated K+ salt of anATP-competitive catalytic site inhibitor of PKC-p\ and is used hereas a PKC reporter dye (Chen and Poenie, 1993).

For ER staining, cells were fixed with 0.25% glutaraldehyde in0.1 M sodium cacodylate buffer (pH 7.4) for 3 min, stained for 30 sin the same buffer containing 2.5 ug/ml of the fluorescein-conjugateddye DiOC5(3) (Molecular Probes) prepared from a stock solutionof 0.5 mg/ml DiOC5(3) in ethanol, and washed in buffer beforeobservation.

Cells stained for PKC and ER were scanned by confocal microscopyfrom pole to pole with a 5 urn distance between sequential opticalsections.

Results

Pre-pronuclear period

The sperm-induced Ca2+ oscillations were monitored in freshlyfertilized human oocytes for ~6 h from their onset. To avoida prolonged uninterrupted stay of oocytes outside the CO2incubator and to limit the possibility of oocyte damage bylaser light, periods of measurement by confocal microscopywere alternated with periods during which oocytes were washedin SPM and returned to the incubator. Of 12 fertilized oocytesthat survived this long measurement procedure, all still exhib-ited Ca2+ oscillations at its end, i.e. 5-6 h after sperm-oocytefusion (Figure 1A). In six of these oocytes, the sensitivity ofthe late fertilization-induced Ca2+ oscillations to ryanodinewas tested; the addition of ryanodine inhibited the Ca2+

oscillations in all six cases (Figure IB).Spatial image analysis (12 oocytes) showed that each of the

late fertilization-induced [Ca2+]j rises was introduced by a

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Figure 3. Ca2+ signals of pronuclear zygotes. (A) Ca2+ oscillationsrecorded in a two-pronuclear (2PN) zygote (representative of dataobtained with 11 zygotes). (B) Effect of the addition of 4 mMryanodine (Ry) on Ca2+ oscillations of a 2PN zygote(representative of data obtained with three zygotes).(C) Spontaneous arrest of Ca2+ oscillations following pronuclearbreakdown (arrow) (representative of data obtained with twozygotes). Fluo-3 fluorescence emitted from the equatorial plane ofthe zygotes was recorded at intervals of 2 s.

focal release of Ca2+ at a variable site in the cortex of theoocyte followed by a global Ca2+ discharge, after which[Ca2+]i returned relatively rapidly to basal values in the oocyte

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cortex and subcortex, whereas it remained elevated for a longertime in the oocyte central region (Figure 2A).

The six fertilized oocytes that still showed ongoing Ca2+

oscillations at the end of the [Ca2+]j monitoring period (5-6h after sperm-oocyte fusion) and that were not treated withryanodine (see above) were subsequently processed either forPKC (three oocytes) or ER (three oocytes) visualization. Inboth cases, three other oocytes fixed in the course of the firsthour of the fertilization-induced Ca2+ oscillations were alsoincluded. Irrespective of the time after fertilization, both PKC(Figure 2B) and ER (Figure 2C) were accumulated in the cellperiphery.

Pronuclear period

Pronuclear zygotes (11 cases) also showed periodical [Ca2+]jincreases (Figure 3A); however, these occurred with a lowerfrequency as compared to the pre-pronuclear stage. In threecases, the sensitivity of these Ca2+ signals to ryanodine wastested, and a clear inhibitory effect (Figure 3B) was apparentin all of them. In two cases, it was possible to monitor [Ca2+],shortly before, during, and after the process of pronuclearenvelope breakdown (Figure 3C). In both cases, the Ca2+

oscillations did not continue beyond the pronuclear stage.The analysis of the spatial propagation of Ca2+ waves in

pronuclear zygotes (11 cases) showed that the initial [Ca2+],increase of each Ca2+ spike invariably began from the centralregion of the cell (where the pronuclei were also located) andthen spread towards the cell periphery (Figure 4A). It was alsothe central region in which [Ca2+]; remained elevated for thelongest time during each Ca2+ transient. Six of the pronuclearzygotes in which [Ca2+][ was monitored were subsequentlyprocessed for PKC (three zygotes) and ER (three zygotes)visualization. Both PKC (Figure 4B) and ER (Figure 4C) wereaccumulated in the zygote central region, around the pronuclei.

Cleavage period

Only a limited number of supernumerary cleaving embryoswas available for this study: three embryos at the 2-cell stageand seven embryos at the 4-cell stage. The 2-cell embryoswere allocated to long-term observations in an attempt tovisualize changes in [Ca2+]| occurring during cell division,which proved possible in one case only. In this case, the embryowas subsequently treated with ryanodine. The remaining 2-cell embryos, in which we did not succeed in monitoring[Ca2+]i at the time of cell division, were used for PKC (oneembryo) and ER (one embryo) visualization. Six of theoriginally 4-cell embryos were used in experiments to evaluatethe effect of ryanodine on Ca2+ dynamics (two embryos) andPKC (two embryos) or ER (two embryos) distribution. In oneof the originally 4-cell embryos it proved possible to monitor[Ca2+]i during cell division occurring simultaneously in twoblastomeres and leading to the formation of a 6-cell embryo;this embryo was subsequently used to study the effect ofryanodine on Ca2+ dynamics.

Similar to the preceding developmental stages, cleavingembryos exhibited repetitive [Ca2+]i increases; however, theircharacteristics were completely different. Unlike the first cellcycle, during which [Ca2+]j baseline values were stable between

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Figure 4. Spatial characteristics of Ca2+ waves, protein kinase C (PKC) distribution and endoplasmic reticulum (ER) organization in thepronuclear period. (A) Sequential confocal images of a Fluo-3-loaded two-pronuclear zygote snowing a [Ca2+]j increase (representative ofdata obtained with 11 zygotes). Fluo-3 fluorescence emitted from the equatorial region was recorded at intervals of S s. The numbersindicate the time (s) after the recording of the first image of the series presented. (B) Confocal imaging of PKC in a pronuclear zygoteundergoing Ca2+ oscillations (representative of data obtained with three zygotes). (C) Confocal imaging of ER in a pronuclear zygoteundergoing Ca2+ oscillations (representative of data obtained with three zygotes). The dyes used and the mode of image presentation arethose described in Figure 2.

individual Ca2+ spikes, cleaving embryos exhibited continuoussinusoidal changes in [Ca2+]j upon which higher-amplitudeCa2+ spikes were sometimes superimposed (Figures 5 and 6).These superimposed Ca2+ spikes were seen only in blastomeresof one 2-cell and one 4-cell embryo shortly before cell division(Figures 5 and 6). After the accomplishment of cell division,only the background sinusoidal Ca2+ fluctuations were detected(Figure 7), which represented the only Ca2+ changes seen inthe case of embryos that did not cleave during the monitoringperiod (Figure 8).

The [Ca2+]j changes observed in cleaving embryos wereasynchronous in individual blastomeres of the same embryos;this asynchrony became even more apparent when individualblastomeres began to display the cell-division-related Ca2+

spikes (Figures 5 and 6). Embryos that underwent spontaneousarrest of development between the 4-cell and 6-cell stagesshowed a progressive disappearance of the sinusoidal Ca2+

fluctuations, resulting in a straight-line [Ca2+], record (datanot shown).

Unlike the first cell cycle, the addition of ryanodine did notinhibit the Ca2+ signals of cleaving embryos. Instead, ryanodineelicited a marked increase in the amplitude and a moderateincrease in the frequency of the sinusoidal Ca2+ fluctuations(Figure 9). The eventual effect of ryanodine on the cell-division-related Ca2+ spikes was not addressed.

Similar to pronuclear zygotes, cleaving embryos showed acentre-to-periphery propagation of Ca2+ waves (Figure 10A-C) and a central accumulation of PKC (Figure 10D) and ER(Figure 10E).

DiscussionThe results of this study have shown that the fertilization-induced series of Ca2+ oscillations seen in human zygotes is

followed by further temporal and spatial changes in [Ca2+]|during the first cell cycle, as well as during the subsequent cellcycles occurring during preimplantation embryo development.Based on evaluations of the frequency and amplitude of theseCa2+ transients and their sensitivity to ryanodine, the spatialpropagation of Ca2+ waves and the observed intracellulardistribution of PKC and ER, three developmental^ relatedpatterns of Ca2+ dynamics were distinguished. The distinctivefeatures of each of the three patterns are summarized in Table I.

Pre-pronuclear period

The period of zygote development between sperm-oocytefusion and the appearance of pronuclei is characterized by theoccurrence of sperm-induced Ca2+ oscillations. The character-istics of these Ca2+ oscillations, as observed in this study,were similar to those described previously in human fertilizedoocytes by ourselves (Tesarik et al., 1994, 1995; Tesarik andSousa, 1994; Sousa et al, 1996a,b) and others (Taylor et al.,1993) and to those induced in human oocytes by injection ofa cytosolic sperm fraction (Homa and Swann, 1994). However,in comparison with our earlier studies in which the overallduration of the period of Ca2+ oscillations rarely exceeded2 h, oocytes usually showed this kind of Ca2+ response for>5 h in this study. This difference may be due to the fact thatthe Ca2+ monitoring was intermittent in this study, with periodsof measurement alternating with periods of rest in a CO2

incubator, whereas the monitoring was continuous in our earlierstudies. The continuous Ca2+ monitoring may have led to ahigher degree of laser-light damage of embryos as compared tothe present experiments. Fertilization-induced Ca2+ oscillationslasting for 4-5 h were reported by Taylor et al. (1993) whomonitored oocytes in a perfusion chamber, which may alsohave modulated the adverse effect of light exposure.

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Figure 5. Ca2+ signals of a 2-cell embryo shortly before celldivision. (A) Record of the whole embryo. (B) Record ofblastomere 1 (arrow and double arrow indicate low-intensity Ca2+

spikes corresponding to confocal images shown in Figure 10A andB respectively). (C) Record of blastomere 2 (arrow and doublearrow indicate a high-intensity Ca2+ spike and an intermediate-intensity Ca2+ spike corresponding to confocal images shown inFigure 10A and B respectively). Fluo-3 fluorescence was recordedat intervals of 2 s.

Both early (Sousa et ai, 1996a) and late (this study) sperm-induced Ca2+ oscillations can be inhibited by ryanodine andcoincide with an accumulation of PKC and ER in the zygoteperiphery. Earlier confocal studies, performed with mouse

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(Mehlmann et ai, 1995) and hamster oocytes (Shiraishi et ai,1995), also demonstrated a peripheral accumulation of ER. Anaccumulation of PKC in the cortical region of freshly fertilizedoocytes has also been described in the sea urchin (Olds et ai,1995) and hamster (Gallicano et ai, 1995) and may reflect anassociation of the activated enzyme with the plasma membraneor with cortical granules (Olds et ai, 1995). However, theperipheral accumulation of ER and PKC in human oocytes isnot a direct sequel of oocyte activation because a similardistribution of ER and PKC has also been observed inmature unfertilized human oocytes (M.Sousa and J.Tesarik,unpublished results). The Ca2+ stores present in the corticaland subcortical regions of human oocytes are essentiallyinsensitive to ryanodine and presumably sensitive to inositol1,4,5-trisphosphate (InsP3) (Sousa et ai, 1996a). At the begin-ning of each Ca2+ spike during the steady-state phase of thesperm-induced Ca2+ oscillations, these peripheral Ca2+ storesare responsible for the initial Ca2+ discharge which acts as adetonator for a global Ca2+-induced Ca2+ release (CICR)involving the ryanodine-sensitive stores which are abundantin the rest of the oocyte cytoplasm (Tesarik et ai, 1995; Sousaet ai, 1996a). Accordingly, Ca2+ waves observed during eachCa2+ spike during this period show a periphery-to-centredirection of propagation.

Pronuclear periodA unique type of Ca2+ signal has been detected in pronucleatedzygotes. Like the earlier fertilization-induced Ca2+ signal, thissignal also consisted of repetitive Ca2+ spikes which were,however, of lower frequency and amplitude than those observedin the pre-pronuclear period. Furthermore, contrary to thefertilization-induced Ca2+ oscillations, the Ca2+ waves devel-oping in the pronuclear period showed a centre-to-peripherydirection of spatial propagation. This reversal of the directionof Ca2+ wave propagation coincided with a redistribution ofPKC and ER, both of which became accumulated in the centralcytoplasm, around the pronuclei. Notwithstanding, as in thepre-pronuclear period, the Ca2+ oscillations occurring in thepronuclear period were sensitive to ryanodine (Table I). Thisstudy did not involve a continuous Ca2+ monitoring fromfertilization through pronuclear development. Thus, it remainsunknown what happens with [Ca2+]; between the dissolutionof the fertilization-induced series of Ca2+ oscillations and theappearance of the Ca2+ oscillations typical of the pronuclearstage. It cannot be excluded that, under physiological condi-tions, the series of fertilization-induced Ca2+ oscillations isnot interrupted but transforms gradually into the pronuclear-type of Ca2+ oscillation. The arrest of Ca2+ oscillations inmonitored oocytes may be artificial and due to the unphysio-logical environmental conditions including exposure to laserlight (see above) and the action of the Ca2+ chelating dyesused for intracellular Ca2+ visualization.

Information about the Ca2+ dynamics of pronuclear zygotesof other mammalian species is scarce. Fissore and Robl(1993) demonstrated transient [Ca2+]j rises in rabbit pronuclearzygotes; these Ca2+ transients decreased shortly before nuclearenvelope breakdown. In the mouse, Ca2+ transients were alsoobserved at the time of pronuclear breakdown and, in addition,

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Figure 6. Ca2+ signals of a 4-cell embryo shortly before cell division. (A) Record of the whole embryo. (B) Record of blastomere 1 (arrowindicates a low-intensity Ca2+ spike corresponding to confocal images shown in Figure IOC). (C) Record of blastomere 2. (D) Record ofblastomere 3. (E) Record of blastomere 4 (arrow indicates an intermediate-intensity Ca2+ spike corresponding to confocal images shown inFigure 10C). Fluo-3 fluorescence was recorded at intervals of 2 s.

at the time of the zygote's entry into the first embryonicmitosis (Tombes et al., 1992; Kono et al, 1996). Studiescarried out in several non-mammalian species, in whichfertilization induces a single [Ca2+]j rise rather than a seriesof Ca2+ oscillations, also revealed additional [Ca2+]j rises atthe pronuclear stage (Poenie et al., 1985; Steinhardt, 1990;Gillot and Whitaker, 1994; Ciapa et al, 1994; Strieker, 1995).Similar to the present observations, the spatial propagation of

these Ca2+ signals differed from the fertilization-induced Ca2+

response in sea urchin (Gillot and Whitaker, 1994) and starfishzygotes (Strieker, 1995). This change in the spatial propagationof Ca2+ waves may be related to the migration of the Ca2+

stores with the highest sensitivity to CICR, which serve as thesource of a local detonating Ca2+ discharge triggering theglobal CICR in human oocytes (Tesarik et al, 1995), fromthe oocyte periphery to the perinuclear region of pronuclear

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Table I. Developmental changes in the characteristics of the Ca2+ signalling system of human preimplantation embryos

Period Features of [Ca2+], rises PKCaccumulation

ERaccumulation

Temporalpattern

Spatialpropagation

Ryanodineeffect

Pre-pronuclearPronuclearCleavage

SpikingSpikingSinusoidal plus spiking

Periphery-to-centreCentre-to-peripheryCentre-to-periphery

InhibitionInhibitionStimulation

Cell peripheryPerinuclearPerinuclear

Cell peripheryPerinuclearPerinuclear

PKC = protein kinase C; ER = endoplasmic reticulum.

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Figure 7. Ca2+ signals recorded in a representative blastomere of a4-cell embryo that has developed from the 2-cell embryo whoserecords are shown in Figure 5. Fluo-3 fluorescence was recorded atintervals of 2 s. The other blastomeres showed a similar record.

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Figure 8. Ca2+ signals recorded in a representative blastomere of a4-cell embryo in which no cell division occurred either during, orclose to, the recording period (representative of data obtained withtwo 2-cell embryos and six 4-cell embryos). Fluo-3 fluorescencewas recorded at intervals of 2 s. The other blastomeres showed asimilar record.

zygotes. The observed reorganization of ER and redistributionof PKC coinciding with this reversal of the spatial dynamicsof Ca2+ signals may reflect this underlying mechanism. Earlierelectron microscopic observations on human pronuclearzygotes also showed the presence of abundant ER structuresin the perinuclear region (Soupart and Strong, 1974; Sundstrom

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Figure 9. Effect of the addition of 4 mM ryanodine (Ry) on Ca2+

signals recorded in the embryo whose previous Ca2+ records areshown in Figure 7 (representative of data obtained with one 4-cellembryo, two 4-cell embryos and one 6-cell embryo). (A) Record ofthe whole embryo. (B) Record of a representative blastomere. Fluo-3 fluorescence was recorded at intervals of 2 s.

et al, 1981; Sathananthan and Trounson, 1985), contrastingwith the accumulation of this organelle in the periphery ofunfertilized human oocytes (Zamboni et al., 1972; Sundstromet al, 1985; SundstrOm and Nilsson, 1988).

It is possible that at least part of the Fim-1 fluorescencesignal detected in the perinuclear region of human pronuclearzygotes in this study corresponded to protein kinase M ratherthan PKC. This is corroborated by recent observations onhamster ionophore-activated oocytes probed with Rim-1(Gallicano et al, 1995), a PKC reporter dye analogous to Fim-1 used in the present study. These authors have demonstrated

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Figure 10. Spatial characteristics of Ca2+ waves, protein kinase C (PKC) distribution and endoplasmic reticulum (ER) organization in thecleavage period. (A) Sequential confocal images of a 2-cell embryo showing a small [Ca2+]j increase in blastomere 1 (bl) (marked with an arrowin the corresponding record shown in Figure SB) and of a large [Ca2+]j increase in blastomere 2 (b2) (marked with an arrow in the correspondingrecord shown in Fig. SC). (B) Sequential confocal images of a 2-cell embryo showing a small [Ca2+]j increase in blastomere 1 (bl) (marked witha double arrow in die corresponding record shown in Figure SB) and an intermediate-intensity [Ca2+jj increase in blastomere 2 (b2) (markedwith a double arrow in the corresponding record shown in Figure SC). (C) Sequential confocal images of a 4-cell embryo showing a small[Ca2+jj increase in blastomere 1 (bl) (marked with an arrow in the corresponding record shown in Figure 6B) and an intermediate-intensity[Ca2+]i increase in blastomere 4 (M) (marked with an arrow in the corresponding record shown in Figure 6E). Fluo-3 fluorescence emitted fromthe equatorial plane of the embryos shown in panels A-C was recorded at intervals of 2 s. The numbers displayed in these panels indicate thetime (s) after the recording of the first image of each series presented. (D) Confocal imaging of PKC in a 4-cell embryo (representative of dataobtained with one 2-cell and two 4-cell embryos). (E) Confocal imaging of ER in a 4-cell embryo (representative of data obtained with one 2-cellembryo and two 4-cell embryos). The dyes used and the mode of image presentation are those described in Figure 2.

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M.Sousa, A.Barros and J.Tesarik

a shift in the Rim-1 fluorescence intensity maximum from theoocyte periphery, where the dye labelled PKC, towards thecentral region of the oocyte, where the dye mainly labelledprotein kinase M. This latter represents the isolated catalyticsubunit of PKC devoid of the membrane-binding domain,probably the result of proteolytic down-regulation of PKC(Gallicano et al., 1995).

Cleavage periodTwo types of Ca2+ signals were detected in this study: abackground sinusoidal rhythm, observed at any phase of thecell cycle, and superimposed clusters of higher Ca2+ spikesoccurring shortly before cell division. Although the temporalpattern of this Ca2+ signal was quite different when comparedwith the first cell cycle after fertilization, the spatial propagationof Ca2+ waves was similar to the pronuclear period. ER andPKC also showed a perinuclear accumulation in blastomeres ofcleaving embryos, similar to pronuclear zygotes. Surprisingly,ryanodine exhibited a quite opposite effect on these Ca2+

signals when compared with the previous stages. The earlyfertilization-induced series of Ca2+ oscillations has been sug-gested to rely on cyclic exchanges of Ca2+ ions between theInsP3-sensitive and the ryanodine-sensitive Ca2+ stores (Sousaet al., 1996a; Tesarik and Sousa, 1996), and a similar mechan-ism may underlie the Ca2+ oscillations of pronuclear zygoteswhich show a similar sensitivity to ryanodine. The absence ofryanodine inhibition of the [Ca2+]j rises occurring in cleavinghuman embryos may be related to a change in the Ca2+

oscillation mechanism involving a modification of the role ofthe ryanodine-sensitive Ca2+ stores. Alternatively, the respons-iveness of the ryanodine receptors may have changed, eitheras a consequence of the synthesis of new classes of the receptoror owing to conformational changes of the structure of theexisting receptors. Further research is needed to elucidate thesequestions. Experiments carried out in the sea urchin also havesuggested that the mechanism of the cell-cycle-related Ca2+

transients in embryonic cells is different from that of thefertilization-induced Ca2+ signals of freshly fertilized oocytes(Ciapa et al., 1994).

Similar to our observations, a unique type of Ca2+ signaltypically preceded mitosis in cleaving starfish embryos(Strieker, 1995). In spontaneously cleavage-arrested embryosobserved in this study, fluctuations of [Ca2+]j disappearedprogressively. This finding suggests that the changes in [Ca2+],reported in this study represent developmentally regulatedsignals required for cleavage of human embryos. The depend-ence of cleavage on InsP3-driven Ca2+ signals has beendemonstrated experimentally in the sea urchin (Ciapa et al,1994) and starfish (Strieker, 1995). These Ca2+ signals arebelieved to be governed by a 'cytoplasmic clock' that isindependent of cytokinesis because they still continue inembryos whose cleavage has been arrested artificially bycolchicine or protein synthesis inhibition (Kubota et al., 1993;Ciapa etal, 1994; Keating etal, 1994; Strieker, 1995). Hence,these Ca2+ signals appear to be important for, but independentof, cell division in early embryos.

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Previous studies have focused on the fertilization-inducedCa2+ signals in human zygotes (Taylor et al., 1993; Tesarikand Sousa, 1994; Tesarik and Testart, 1994; Tesarik et al.,1994, 1995; Sousa et al., 1996a,b) and on the possibleconsequences of artificial modifications of these signals inspecial conditions associated with different techniques ofmicromanipulation-assisted fertilization (Tesarik and Sousa,1994; Tesarik, 1995, 1996). This study is the first to show thatdevelopmentally regulated changes in [Ca2+]j, presumablyplaying a signalling role, continue at later stages of the firstcell cycle as well as during cleavage of human embryos. Thesedata should stimulate further study of the relationship betweenthese Ca2+ signals, on the one hand, and embryo qualityon the other, including participation in the early matemo-embryonic crosstalk necessary for embryo implantation(Edwards, 1995). The action of many of the factors known tobe implicated in this crosstalk, such as platelet-activating factoror components of the interleukin-1 system, involve Ca2+ as asecond messenger. The evaluation of the eventual role ofembryonic Ca2+ signals in the synthesis of, or response to,implantation-preparing molecules is an intriguing challengefor future research.

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Received on My 1, 1996; accepted on October 30, 1996

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