DEVELOPMENTAL

BIOLOGY

l&,370-380

(1992)

Confocal Microscopy of Fertilization-Induced Dynamics in Sea Urchin Eggs STEPHEN A. STRICKER,*~~VICTORIA

E. CENTONZE,-~STEPHEN

Calcium

W. PADDOCK,~'~ANDGERALDSCHATTEN-~

*Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131; and tlntegrated i%iversity of %‘iscmsin, kfadism, Wisconsin 53706 Accepted September

19,

Microscqp?~ Resource,

1991

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calciumsensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

0 1992 Academic

Press, Inc.

INTRODUCTION

Following fertilization, the egg undergoes a rapid rise in intracellular free calcium ([Ca2+]i). The increase in [Ca”‘], triggers several important events, including cortical granule release and metabolic activation (Longo, 1989; Epel, 1989). In sea urchins, microscopic analyses employing calcium-sensitive probes have shown that the fertilization-induced rise in [Ca”‘], begins at the site of sperm fusion and sweeps through the egg in a wavelike fashion (Eisen et al, 1984; Eisen and Reynolds, 1985; Jaffe, 1985; Hafner et al., 1988; Mohri and Hamaguchi, 1991). Whether fertilization affects calcium levels only in the peripheral ooplasm or throughout the interior of the sea urchin egg, however, is difficult to ascertain by conventional microscopic techniques, because out-of-focus rays tend to obscure subcellular organization in these relatively bulky cells. Recently, confocal microscopy has been introduced as a means of producing thin optical sections that reveal details not normally discernible by conventional light microscopy (Amos, 1988; Fine et al., 1988; Inoue, 1989; Shotton, 1989; Shuman et aZ., 1989). The optical sections obtained with a confocal microscope can also be collected as a vertical “z-series” of sections through the specimen and subsequently reconstructed to yield highly informative three-dimensional (3-D) renderings (Brakenhoff et ah, 1989; Carrington et aZ., 1989; Robert’ To whom corresponds should be addressed. 2 Present address: Howard Hughes Medical Institute, Department of Molecular Biology, University of Wisconsin Madison, Wisconsin 53706. 0012-1606192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in my form reserved.

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Nicoud et aZ., 1989; Shotton and White, 1989; Lechleiter et all, 1991). Previous developmental studies usingconfocal microscopy have typically been performed on fixed embryonic material (White et al, 1987; Cheng and Summers, 1989; Stricker and Schatten, 1989; Wright et aZ., 1989; Fredrikson, 1990). Although such analyses provide valuable information, they are based on disjunct, static stages that are subject to artifacts of fixation and interspecimen variation. Moreover, the use of fixed material precludes precise analyses of dynamic events involving subcellular components such as diffusible ions. In this paper, we show that confocal laser scanning microscopy (CLSM) and time-lapse reconstructions can be used to image free calcium ions during the process of fertilization in living sea urchin eggs labeled with calcium-sensitive fluorescent dyes. Such studies yield several new findings, including the observation that the fertilization-induced calcium wave propagates throughout the interior of the egg rather than just in the cortex and that fertilization also causes the egg nucleus to undergo an apparent transient rise in free calcium. MATERIALS

AND METHODS

Animals and gametes. Ripe adult specimens of the sea urchin Lytechinus pi&us were purchased from Marinus, Inc. (Long Beach, CA), and maintained at 15-18°C in recirculated aquaria containing artificial seawater (“Instant Ocean”). Gametes were obtained by intracoelomic injection of 0.55 M KC1 and were subsequently processed according to standard techniques. Microinjections. To monitor the temporal and spatial

STRICKERETAL.

Co?tfoal Microsc~

distribution of calcium during fertilization, dejellied eggs were gently attached to a protamine-sulfatecoated coverslip that had been glued over a hole in the bottom of a 60-mm petri dish. The eggs were then immersed in 20 ml of artificial seawater (ASW) and microinjected with a stock solution of either 8 mM fluo-3, potassium salt, or 5 mM calcium green, potassium salt. Both dyes were purchased from Molecular Probes, Inc., and were dissolved in an injection buffer consisting of 10 mM Hepes, 100 miM potassium aspartate, pH 7.2. Microinjections were carried out using a Zeiss Axiovert 10 inverted microscope and an Eppendorf 5170/5242 pressure injection system. (Note that microinjections of these dyes were performed because (i) both fluo-3 and calcium green are calcium-sensitive probes that can be excited by the 48%nm line of the confocal microscope’s argon-ion laser and (ii) simple incubation of the eggs in the permeant, acetoxymethylester form of the dyes does not yield a utilizable signal.) Cdocal microsco~. The confocal system used in these studies consisted of either a Bio-Rad MRC-600 laser scanning confocal unit interfaced with a Nikon Optiphot upright microscope or a Noran Odyssey realtime laser scanning system attached to a Nikon Diaphot inverted microscope. To optimize cell viability, the 25 mW argon-ion laser of the Bio-Rad confocal microscope was set at f power, and the beam was further attenuated by means of a 0.1 or 1% transmittance neutral density filter and a fluorescein filter cube (Bio-Rad Corp.) situated in the optical path. Most experiments were run with the confocal aperture set between 4 and 9. The Noran microscope was used with the standard 488~nm emission from the argon-ion laser attenuated to 25% by the acousto-optical deflector system. In most cases, the confocal slit was set to 10 pm. Unless stated otherwise, the figures presented in this paper are of fluo-3-loaded specimens examined with the Bio-Rad CLSM. For all studies, the specimen dish was placed on a thermoelectric cooling stage (KT controller, United Technology, Whitehouse Station, NJ) set at 18°C. In analyses using the Bio-Rad system, observations were made with either a Leitz 25X (0.6 N.A.) or a Zeiss 40X (0.75 N.A.) water immersion lens that was submerged in the seawater medium contained in the dish. Specimens examined with the Noran CLSM were viewed with a Zeiss 40X (0.65 N.A.) or 16X (0.32 N.A.) “dry” objective. Time-lapse calcium imaging. To conduct time-lapse calcium imaging studies, one to several dye-loaded eggs were viewed by CLSM at an optical plane near the eggs’ equators. Directly prior to the addition of sperm, a time-lapse recording set at approximately I-set intervals was initiated for up to 15-20 min following insemination for specimens examined with the Bio-Rad micro-

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scope. Similar time-lapse recordings were conducted with the Noran CLSM, although the faster scan rate of this microscope allowed optical sections to be collected every 2 sec. It should be noted that the exact timing of the fertilization-induced rise in dye fluorescence could not be pinpointed relative to when and where sperm-egg fusion occurred. This is because the dye-loaded specimens were continuously imaged at a single optical plane. Such confocal imaging in turn precluded observations of sperm-egg fusion, unless the sperm happened to fuse in the optical plane that was being monitored. Following each time-lapse run, the eggs under examination were viewed by conventional bright-field optics. Fertilization could thus be verified by observing the fertilization envelope and/or the changes that take place in egg nuclear morphology following fusion with the male pronucleus and the subsequent formation of the first mitotic spindle. To enhance the signal-to-noise ratio of the confocal images, each optical section obtained with the Bio-Rad instrument was accumulated for two 256 X 256 scans at normal scan rates (~1 set/full scan), whereas specimens examined with the Noran CLSM were subjected to a 16-frame jumping average. A modified program for collecting serial z-sections was used such that the stepper motor increment was set to 0 and an optical section was thus collected at nearly the same level within the egg at the maximum frequency of every 4 set (Bio-Rad) or 2 set (Noran), which represented the time required for scanning the specimen, storing the data, and resetting the stepper motor. During the time-lapse runs, the data sets were written onto the hard disk of the confocal microscope’s host computer and subsequently subjected to a standardized histogram shift to boost the overall brightness of each optical section followed by a pseudocolor conversion. In all of the images presented in this paper, blue colors are indicative of relatively low fluorescence intensities and [Ca2’]i levels, whereas reds represent higher intensities and free calcium concentrations. For hard copy output of the pseudocolored images, color prints were obtained from a Sony UP-5000 color video printer or from slides taken of the video monitor using Ektachrome 400 film (Kodak). Fluorescence inter&g measurements and 3-D reconstructions. To compare fluorescence intensities from different regions of the optical sections taken with the BioRad CLSM, relative pixel intensities were measured in rectangular areas of similar size in the following six regions of the optical section: (1) in the cortex near the putative site of sperm entry (i.e., where the calcium wave began), (2) in the adjacent region of the subcortex, (3) in the adjacent region of the egg center, (4) in the subcortex opposite to the origin of the calcium wave, (5)

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in the cortex opposite to the origin of calcium wave, and (6) in the nucleus (if it was present in the optical section). For volumetric reconstructions of confocal data sets, the time-lapse series of single optical sections through fluo-&loaded eggs were transferred to a Silicon Graphics IRIS 4D-70GT workstation, and 3-D renderings were achieved using the Voxel View software of Vital Images, Inc. (Fairfield, IA). Thus, the circular optical sections were stacked together to form a cylinder that represented the optical section over time. RESULTS

Flue-&Loaded Specimens: Fertilixation-Induced Calcium Dynamics In all fluo-3-loaded eggs examined, a wave of increased fluorescence spread across the egg cytoplasm following fertilization (Fig. 1). The increase in fluo-3 fluorescence typically occurred within 20-60 see after sperm addition and corresponded to the fertilization-induced calcium wave, based on the fact that: (i) it was observed only in fertilized eggs, and (ii) it typically traveled at a velocity of 3-10 pm/set, which agreed well with the results of conventional microscopic studies of calcium dynamics in L p&us and other echinoid species (Swann and Whitaker, 1986, Hafner et aL, 1988; Hamaguchi and Hamaguchi, 1990; Mohri and Hamaguchi, 1991). Moreover, treatment of nonfertilized, Auo-3loaded specimens with 0.01% digitonin or a 20 PM solution of the low-fluorescence form of calcium ionophore, 4-bromo-A23187 (Molecular Probes, Inc.), caused a rapid and circumferential burst of fluorescence, rather than the wave-like pattern observed after fertilization (data not shown). Since sperm-egg fusion was not observed during any of the time-lapse runs, it was impossible to determine the patterns of wave propagation relative to the site of sperm entry. Regardless of where the sperm entered, however, the fertilization-induced elevation in fluo-3 fluorescence invariably swept across the entire optical sec-

tion, Thus, it was clear the calcium wave traveled throughout the interior of the egg rather than just in the cortex. In addition to the eytoplasmic wave of increased AUO3 fluorescence that was observed following fertilization, the egg pronucleus in fertilized specimens underwent a transient increase in fluorescence (Figs. 1 and 2). Prior to insemination, the fluorescence of the nucleus was typically lower than that of the surrounding cytoplasm, but following fertilization the nucleus became more intensely fluorescent than did the surrounding cytoplasm (Figs. 2D and 2E). Moreover, the relative rise in fluo-3 fluorescence (i.e., AF/F where AF is the increase in fluorescence following fertilization and F is the resting fluorescence prior to fertilization) was invariably higher in the nucleus than in the cytoplasm. It should be noted that the relative rise in intranuclear fluorescence exceeded that of the cytoplasm regardless of the position of the nucleus within the optical section. Following observation of the fertilization-induced calcium wave, none of the fluo-3-loaded specimens underwent normal development, although neighboring eggs which had not been microinjected with fluo-3 cleaved normally. Fluo-3-loaded cells only proceeded to cleave if they were allowed to develop without being illuminated by the laser. Flue-3-Loaded Specimens: of Calcium Dgnamics

Volumetric

Reconstructions

To analyze calcium waves further, the optical sections in each data set were stacked together to yield a cylinder that represented the confocal image over time (Fig. 3). The transient increase in fluorescence representing the calcium wave formed a narrow, orange-red band around the pseudocolored cylindrical reconstruction following sperm fusion (Fig. 3A). In addition, such reconstructed cylinders suggest that fertilization caused the egg to change its shape and position during the time-lapse run, since a marked deformation in the outline of the cylinder was typically present in fertilized specimens (Fig.

FIG. 1. Time-lapse confocal laser scanning microscopy (CLSM) of an optical section through two sea urchin (L pi&us) eggs that had been microinjected with the fluorescent calcium indicator fluo-3 to monitor the fertilization-induced calcium wave (note that only part of the second egg is shown in the lower left corner of each frame). Fluorescence intensities have been pseudocolored such that blue regions are indicative of relatively low fluorescence intensities and intracellular free calcium [Ca”‘],, whereas redder areas correspond to higher fluorescence intensity and [Caz’]i. (A) Prior to insemination. (B) 52 set following the addition of sperm to the specimen dish; the wave of elevated fluo-3 fluorescence has just begun to sweep through the fertilized egg (arrow). Note that the fluo-3-injected egg in the lower left corner was apparently damaged by the microinjection procedure and thus serves as a control because it did not fertilize or show a change in fluorescence intensity. (C) 56 set postinsemination; fluo-3 fluorescence is elevated throughout half of the optical section. (D) 68 set postinsemination; the entire optical section exhibits an elevation in fluorescence intensity. Note that the nucleus (double arrowheads) is now visible again and has undergone a marked increase in fluo-3 fluorescence. (E) 84 set postinsemination; compared to the center of the egg, the cortical region (orange rim) shows a higher fluorescence intensity. (F) 6.5 min postinsemination; Auo-3 fluorescence has returned toward preinsemination resting levels. The egg has moved relative to the optical axis. Thus, the nucleus is no longer in the optical section, and the section size is smaller. The pseudocolored gray scale indicates relative fluorescence intensity. Black/blue, low intensities; red/orange, high intensities. Scale bar = 50 pm.

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FIG. 2. (A-C) Intensity measurements of nuclear fluorescence in three different fluo-3-injected eggs following fertilization. The relative fluorescence intensities within the nucleus were measured every 4 set in the optical sections obtained by time-lapse CLSM. The approximate time at which sperm was added is marked by the arrowhead. In each case, the nucleus undergoes a rapid and transient rise in flu03 fluorescence following sperm fusion. (D) CLSM optical section of a fluo-&loaded egg showing relatively low fluorescence in the nucleus (double arrowheads) at the onset of the calcium wave. (E) CLSM optical section 22 set after the onset of the calcium wave showing increased nuclear fluorescence. Note that the nucleus invariably showed the highest relative increase in flue-3 fluorescence of any region within the egg, regardless of the position of the nucleus in the optical section. Scale bars for D and E = 50 pm.

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Frc. 3. Calcium dynamics in reconstructions of optical sections obtained by time-lapse CLSM. An optical section was collected near the equator of a fluo-&injected egg every 3-4 sec. The individual optical sections were then stacked together using the Voxel View volume rendering program of Vital Images, Inc., to yield a cylindrical reconstruction that represents the optical section over time. (A) A whole cylinder showing calcium dynamics as viewed from the outside of the egg. Note that the egg appears to change shape and to shift its position following sperm fusion, since the edge of the cylinder is irregular (double arrows); in reconstructions of nonfertilized specimens, there are no such irregularities in the periphery of the cylinder. The calcium wave corresponds to the orange-red band around the edge of the optical section. (B) A longitudinal section of the cylindrical reconstruction, exposing the center of the optical section (the cut cylinder is displayed as a mirror image of the full cylinder’s orientation in A). Note that (i) an elevation in fluo-3 fluorescence occurs across the entire optical section and (ii) the cortical region of this specimen exhibits a higher and more prolonged increase in fluorescence intensity. The horizontal scale bar = 50 pm. The vertical scale bar corresponds to 1 min.

3A, double arrows) but was absent in fluo-3-loaded eggs that failed to fertilize (data not shown). When viewed along a midlongitudinal plane to observe internal regions within the cylinder, elevated fluo-3 fluorescence could be seen across the entire opti-

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cal section. In most specimens, however, the elevation in fluorescence was not uniform throughout the egg, as the cortex often became brighter and remained ao for a longer time than did the more central regions of the egg (Fig. 3B).

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TIME (rain) FOG. 4. Relative fluorescence intensity measurements in different regions of a fluo-&injected egg viewed by time-lapse CLSM. An optical section was collected every 3-4 set near the equator of the egg for up to 20 min following insemination. The relative fluorescence intensity was then measured in rectangles of similar area positioned in the following regions of each optical section: (i) cortex, (ii) nucleus, (iii) subcortex (“Subcor”), and (iv) center of an egg that underwent fertilization, as well as in (v) the cortical cytoplasm of an egg that did not fertilize (“Unfert”). The four peaks in fluorescence intensity represent the free calcium elevation that occurs in each of the four regions measured in the optical section as the fertilization-induced calcium wave passes through the egg. The nucleus, which was located in the subcortical to central region of this egg, shows the greatest postfertilization increase in fluorescence intensity relative to its preinsemination resting level. Note that the unfertilized egg shows no significant change in fluorescence intensity.

STlucKER

Fluc&Loaded L$xci~: Calcium L&ycamti

Intensity

ET AL.

CorlfocalMicroscapg of Calcium Dynamics

Measurements

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Based on measurements of relative fluorescence intensities, the fertilization-induced calcium wave caused the egg cortex to undergo a peak increase of 50.8 + 29.5% (N = 6) over the resting levels observed prior to insemination (Fig. 4). The subcortical and central regions of the egg, on the other hand, showed a 37.4 +- 19% (N = 9) maximum increase during the calcium wave (Fig. 4). This difference in the fluorescence intensity increase was only marginally significant (P < 0.10; Mann-Whitney U test). No difference in the amplitude of fluorescence increase was observed either between the subcortical region and the center of the egg or between cortical regions located at opposite sides of the optical section. Moreover, fluo-&injected eggs that failed to fertilize displayed no detectable increase in fluorescence intensity during the 20 min of data collection following insemination. To determine how long the calcium levels remained elevated, elapsed times were measured for the period between the onset of the fluorescence rise and the point where the fluorescence returned to halfway between the peak intensity and the preinsemination resting value. For the cortical regions, these times averaged 2.63 + 0.62 min (N = 6), whereas the subcortex or center of the egg averaged 2.07 f 0.98 min (N = 9), a difference that was only significant at P < 0.10 (Mann-Whitney U test). Based on intensity measurements made on three time-lapse runs that included the nucleus in the optical plane of section, fertilization caused the nucleus to undergo an average increase of 90.6 -+ 83% in fluorescence intensity over that observed prior to insemination. The relative increase in intranuclear fluo-3 fluorescence (range = 2’7-185%) showed great variability, since a nucleus positioned near the site of wave origin tended to display a much higher rise than one positioned toward the center of the egg. Moreover, on one occasion where the nucleus was centrally positioned within the egg, the absolute peak in fluo-3 fluorescence was slightly lower in the nucleus than it was in the cortical cytoplasm (cf. Fig. 4). The relative rise in intranuclear fluorescence over prefertilization levels, however, was always higher in the nucleus than in the surrounding cytoplasm, regardless of where the nucleus occurred in the optical section. As with the cytoplasmic wave of increased duo-3 fluorescence, the nuclear fluorescence in fertilized eggs remained only transiently elevated before eventually diminishing toward the resting levels observed prior to insemination. In three time-lapse sequences where the

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nucleus was in the optical plane of section, the intensity of the nuclear fluorescence returned to a value that was halfway between the peak intensity and the preinsemination resting level within an average of 2.1 f 0.62 min following the onset of the rise in intranuclear calcium levels. Calcium-Green-L4xwkd Specimens The general patterns of calcium dynamics observed in calcium-green-loaded specimens were similar to those reported for fluo-&loaded eggs, in that the wave of elevated fluorescence swept across the entire optical section and the nucleus underwent a highly noticeable spike in dye fluorescence in all fertilized eggs examined (Fig. 5). Two major differences were evident in the calcium-green-loaded eggs, however. First, unlike fluo-3loaded specimens where the nucleus was typically less fluorescent than the surrounding cytoplasm, the nucleus of eggs microinjected with calcium green was as fluorescent as the cytoplasm. In fact, in some cases, the nuclear fluorescence was higher than that of the cytoplasm. Second, whereas fluo-&loaded eggs did not undergo normal development after being imaged for the calcium wave, calcium-green-loaded specimens developed normally following time-lapse examinations of fertilization-induced calcium dynamics on either the BioRad or the Noran CLSM (Fig. 6). DISCUSSION This study demonstrates that calcium dynamics can be monitored in fertilized sea urchin eggs using timelapse confocal laser scanning microscopy. Although it might seem that laser illumination in a confocal laser scanning microscope is too intense to be used on living cells, the extremely short dwell time of the laser beam at any one point in the specimen (typically l-10 psec for a galvanometer-driven beam scan in an instrument such as the Bio-Rad CLSM (Tsien and Waggoner, 1989)) tends to facilitate observations of living cells, at least in a time-lapse mode where the cells are allowed a recovery period prior to subsequent examination. The fluo-&loaded specimens observed in this study appeared to fertilize normally, but only those receiving infrequent laser illumination proceeded to exhibit normal cleavage (i.e., none of the fluo-&injected eggs irradiated for the entire 20-min period of the time-lapse run cleaved normally). The exact conditions that will allow cleavage of fluo-&loaded specimens in such time-lapse confocal sequences remain to be determined. Calcium-green-loaded eggs, however, undergo normal cleavage following examination of the fertilization-induced calcium wave. Such findings indicate that calcium green as used in this study has less phototoxic effects

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FIG. 5. Fertilization-induced calcium waves in calcium-green-loaded eggs. Note that only part of the second egg is shown in the lower left hand corner. (a) Prior to fertilization. (b-f) A calcium wave spreads across the eggs after fertilization has occurred. Note that the nucleus (n) undergoes a fluorescence spike. Unlike fluo-&loaded specimens where the nucleus is typically less fluorescent than the surrounding cytoplasm prior to fertilization, the nuclear signal of eggs microinjected with calcium green is as fluorescent as the cytoplasm (cf. a, d). Thus, the nuclear spike is not simply due to a dye artifact, in which the calcium indicator is excluded from the nucleus prior to fertilization and subsequently rushes into the nucleus after fertilization occurs. Scale bar = 50 pm.

than does fluo-3. Moreover, the fact that the eggs proceed to cleave supports the view that the calcium clynamics reported here reflect normal events occurring within these cells. In addition to supplying data on calcium dynamics, the 3-D reconstructions of flue-3-loaded specimens indicate that fertilization causes a change in the shape and position of the egg during the time-lapse run, as has been suggested in previous studies using conventional light microscopy (e.g., Schatten, 1981). The changes in cell shape and position noted in the 3-D reconstructions appeared to be related to the process of fertilization, rather than simply some artifact of microscopy, since 3-D reconstructions of nonfertilized, fluo-&injected eggs did not exhibit deformations in their outlines. Such a contraction and/or translocation of the fertilized specimen during the time-lapse run would in turn help to explain the artifactual thin rim observed in some stages (e.g., Figs. lC-lE), which could arise from averaging specimen and background gray values at the edge of the moving cell during acquisition of the signal. Previous conventional microscopic studies of calcium dynamics employing the photoprotein aequorin or the quantitative, ratiometric dye fura- have determined

that the resting level of cytoplasmic free calcium in whole, unfertilized sea urchin eggs is approximately 100 nM, but directly after fertilization [Ca2’]i rises to 1-5 pJ4 (Steinhardt et d, 1977; Eisen and Reynolds, 1985; Tsien and Poenie, 1986). Whether [Ca’+l, typically increases in a uniform fashion throughout the egg or undergoes a patchy elevation in restricted regions of the ooplasm such as the cortex has not been fully ascertained in previous studies, but the confocal microscopic observations presented here indicate that fertilization in sea urchins causes a global elevation in free calcium throughout the whole egg rather than just in the cortex. In the fish calcium wave 01-yziu.s latipes, the fertilization-induced is only propagated at the periphery of the egg (Gilkey et al, 1978), but in this species the interior of the egg is filled with a large membrane-bound yolk compartment, which presumably impedes the transmission of the calcium wave. As noted previously, fluo-3 and calcium green were chosen as the calcium-sensitive probes in this study so that the 488-nm excitation line of the 25-mW argon-ion laser could be utilized. Since neither dye undergoes the spectral shift upon binding calcium as occurs when a ratiometric probe such as fura- is used to detect cal-

STRICKERETAL.

Confocal Microscop?/ of Calcium Dynamics

FIG. 6. Calcium dynamics in a calcium-green-loaded egg examined with a Noran Odyssey laser scanning confocal microscope. Unlike fluo-3-loaded specimens which do not cleave after having been imaged for their fertilization-induced calcium waves, eggs microinjected with calcium green can be examined in a time-lapse mode from fertilization through cleavage. (a) After the fertilization-induced calcium wave has spread across the entire optical section. (b, c) In preparation for first cleavage; note the lower signal in the mitotic apparatus (ma). (d-i) Cleavages as viewed by time-lapse CLSM. Scale bar = 50 pm.

cium, accurate calibrations of absolute free calcium levels are difficult to perform on cells loaded with fluo-3 or calcium green (Bright et ak, 1989; Tsien, 1989). Such calibrations are especially confounded by the spherical nature of cells such as eggs, owing to differences in the path length and the amount of dye loading that may occur across the cell. Nevertheless, methods have been proposed for quantification of the fluo-3 signal (e.g., Kao et al., 1989), and fluo-3 calibrations have been applied to confocal microscopic studies of calcium dynamics (Hernandez-Cruz et ab, 1990; Gehring et al., 1990). As pointed out by Kao et al., (1989), however, the fluo-3 calibration procedure is predicated on the assumption that dye behavior within cells is identical to that observed in vitro. Since it is not clear that the behavior of the calcium indicator in sea urchin eggs is actually the same as what occurs in vitro (see discussion below), we have chosen not to attempt quantitative calibrations of the fluo-3 or calcium green data obtained in this study. Instead, we have relied on fluorescence intensity measurements expressed in arbitrary units rather than in nanomolar concentrations of free calcium to describe the general patterns of calcium dynamics during fertilization. Although such uncalibrated data fail to yield exact measurements of calcium levels within the fertilizing

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eggs, fluo-3 fluorescence intensities can provide valuable information regarding changes in [Ca’+& relative to the resting fluorescence intensities observed prior to the onset of fertilization. As discussed by Cornell-Bell et al. (1990), the rise in fluo-3 fluorescence divided by the resting fluorescence is a sensitive indicator of trends in calcium concentrations that take place when the same region of the cell is compared over time. Accordingly, our studies suggest that the relative rise in calcium tends to be slightly higher and more prolonged in the peripheral ooplasm than in the center of the egg. Such a finding is consistent with the results of a previous investigation that indicated the highest rise in [Ca2+]i occurs in the cortex, near the site of sperm fusion (Swann and Whitaker, 1986). As discussed previously, our data do not allow comparisons to be made between the resting levels of calcium that occur in the cortex vs the center of the egg, since possible differences in dye loading, path length, and/or cellular viscosities can greatly affect the measured intensities. Assuming that these differences remain relatively constant over the period of observation, however, it is likely that comparisons of the relative changes in dye fluorescence are valid to make. It should be noted, however, that the spherical nature of the eggs and thus the highly dissimilar path lengths from the center to the cortical regions of the specimen may confound even such comparisons of relative changes in fluorescence intensities. This conclusion is based on the observation that eggs which adhere tightly to the coverslip and are thus much flatter in shape tend to show a less pronounced difference in relative fluorescence increase between the cortex and the center of the egg (Stricker et ak, unpublished observations), presumably because there is less of a difference in path length when compared to more spherical specimens. Thus, until more quantitative measurements of calcium dynamics are made, preferably by a ratioing procedure, it remains to be determined if the cortex actually experiences a higher rise in free calcium than does the center of the egg following fertilization. In vitro analyses have shown that fluo-3 can increase its fluorescence 35 to 40-fold upon binding calcium (Minta et ak, 1989). In our studies, the maximum observed increase in fluo-3 fluorescence was only about S-fold (185%), and in many cases the rise in fluo-3 fluorescence was much less. Although it is unlikely that the in vitro behavior of the dye would exactly match that observed in. vivo, at least two explanations could account for the seemingly low enhancement of flue-3 fluorescence in living sea urchin eggs. First, large amounts of the injected dye could chelate enough free calcium to dampen the fertilization-induced increase in calcium. Although we suspect that higher elevations in dye fluorescence could be obtained if lower concentrations of

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injected dye were used, we have chosen to load sufficient amounts of the calcium indicator, so that relatively low laser intensities could be used on these living cells. In any case, it is important to note that the free calcium in the eggs was not overly chelated by the dyes, judging from the fact that lifting of the fertilization envelope and normal cleavage could be obtained. A second possible reason for the relatively low rise in fluorescence intensity could be that much of the injected probe becomes compartmentalized and thus cannot respond to increases in free calcium, as has been reported in conventional microscopic analyses of calcium dynamics in other cell types (Malgaroli et al, 1987). Such a compartmentalization is consistent with the observations of scattered patches of persistent fluorescence in dye-loaded cells, following mild treatment with digitonin (Stricker et ab, unpublished observations). Previous light microscopic studies using fura- have noted differences in the resting levels of nuclear versus cytoplasmic free calcium in several types of cells (Williams et ak, 1985, 1987). Moreover, nuclear free calcium levels have been shown to undergo rapid elevations based on: (i) CLSM studies of fluo-3-loaded sympathetic neurons subjected to electrical stimulation (HernandezCruz et ak, 1990), (ii) similar analyses of PDGF-stimulated smooth muscle cells (Dilberto et ab, 1991), and (iii) in vitro investigations of calcium uptake in isolated liver nuclei (Nicotera et al, 1989, 1990). We also believe that the fertilization-induced elevation in intranuclear fluo-3 fluorescence observed in this study reflects a true change in nuclear free calcium levels, based on the following key observation: the egg pronucleus is typically less fluorescent than the cytoplasm prior to fertilization but subsequently undergoes a dramatic and transient increase in fluorescence following sperm fusion. The fact that the nuclear fluo-3 signal changes from low to high and back to low again following fertilization makes it unlikely that the nuclear spike in fluorescence is solely due to an artifact of dye loading in the nucleus. Although the increase in nuclear fluo-3 fluorescence is most easily explained as indicating a fertilization-induced elevation in nuclear free calcium levels, we cannot rule out the possibility that this rise in fluorescence intensity occurs because the nuclear envelope in the unfertilized egg is impermeant to fluo-3 but upon fertilization becomes altered so that the dye enters the nucleus, before somehow being excluded again to the resting levels observed prior to insemination. The dye artifact explanation seems unlikely, however, when the results of studies using calcium green are considered. In eggs loaded with this dye, the nuclear fluorescence is equal to, or slightly greater than, the fluorescence of the surrounding cytoplasm prior to fertilization, but subsequently the nucleus undergoes a

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transient rise in fluorescence following fertilization. Thus, a nuclear signal is evident in unfertilized specimens, and the postfertilization spike in the nucleus of these cells is not simply due to an initial exclusion of the dye from the nucleus followed by a sudden inflow after fertilization. Although it is not completely clear why the nucleus shows a higher fluorescence in specimens loaded with calcium green vs fluo-3, preliminary descriptions of calcium green indicate that this dye is more fluorescent than fluo-3 in low-calcium environments (Molecular Probes, Inc., information publication, No. 13). Thus, it is possible that the calcium indicators used in this study load equally well into the nucleus, but the difference observed in the prefertilization nuclear signal is due to dissimilar spectral properties of the dyes. If in fact nuclear calcium levels are elevated during fertilization, the source for such an increase in calcium has yet to be determined. It is possible that calcium ions released from a general cytoplasmic pool of sequestered calcium (Poenie and Epel, 1987; Oberdorf et al, 1988; Henson et al., 1989) simply accumulate in the nucleus and reach relatively high levels owing to a reduced buffering capacity for calcium in the interphase nucleoplasm. Alternatively, as has been indicated in studies of other cell types (Burgoyne et ah, 1989), the rise in intranuclear free calcium could involve calcium release from specific stores in the nuclear envelope or perinuclear cytoplasm, which may be differentially regulated from the general cytoplasmic pool of sequestered calcium. The biological significance of changes in nuclear calcium levels remains unclear, since there is little direct evidence regarding the regulation and possible functions of intranuclear calcium in cells. Calcium-mediated events have, however, been implicated in a variety of important nuclear activities including modulation of chromatin structure and function, gene expression, nucleocytoplasmic transport, and changes in nuclear architecture (e.g., White, 1985; Yasuda et al, 1987; Dessev et al, 1988; Kalinich and Douglas, 1989; Pate1 et aZ., 1989). Whether or not the nuclear-associated calcium dynamics observed in this study actually play a significant role in early development is currently under investigation. We are grateful to V. Argiro, P. DeVries, R. Summers, and W. Van Zandt for their expert help, and to S. J. Smith for his original guidance in using confocal laser scanning microscopy on living cells. We also thank Noran Instruments, Inc., for technical support, and two anonymous reviewers for their insightful criticisms. This study was supported by NIH Grant HD-12913 to G.S. and a Resource Allocation grant from the University of New Mexico to S.A.S. The Integrated Microscopy Resource for Biomedical Research is an NIH Biomedical Technology Resource (RR570).

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REFERENCES

Amos, W. B. (1988). Results obtained with a sensitive confoeal scanning system designed for epifluorescence. Cell Motil Cytoskel. 10, 54-61.

Brakenhoff, G. J., Van Spronsen, E. A., Van Der Voort, H. T. M., and Nanninga, N. (1989). Three-dimensional confocal fluorescence microscopy. In “Methods in Cell Biology” (D. L. Taylor, and Y. L. Wang, Eds.), Vol. 30, pp. 379-398. Academic Press, San Diego. Bright, G. R., Fisher, G. W., Rogowska, J., and Taylor, D. L. (1989). Fluorescence ratio imaging microscopy. In “Methods in Cell Biology” (D. L. Taylor, and Y. L. Wang, Eds.), Vol. 30, pp. 157-192. Academic Press, San Diego. Burgoyne, R. D., Cheek, T. R., Morgan, A., O’Sullivan, A. J., Moreton, R. B., Berridge, M. J., Mata, A. M., Colyer, J., Lee, A. G., and East, J. M. (1989). Distribution of two distinct Caa+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells. Nature 342,72-74. Carrington, W. A., Fogarty, K. E., Lifschitz, L., and Fay, F. S. (1989). Three-dimensional imaging on confocal and wide-field microscopes. In “The Handbook of Biological Confocal Microscopy” (J. Pawley, Ed.), pp. 137-146. IMR Press, Madison. Cheng, P. C., and Summers, R. G. (1989). Image contrast in confocal light microscopy. In “The Handbook of Biological Confocal Microscopy” (J. Pawley, Ed.), pp. 163-179. IMR Press, Madison. Cornell-Bell, A. H., Finkbeiner, S. M., Cooper, M. S., and Smith, S. J. (1990). Glutamate induces calcium waves in cultured astrocytes: Long-range glial signaling. Science 247,470-473. Dessev, G., Iovcheva, C., Tasheva, B., and Goldman, R. (1988). Protein kinase activity associated with the nuclear lamina. Proc. Nat1 Acad Sci. USA 85,2994-2998.

Dilberto, P. A., Periasammy, A., and Herman, B. (1991). Distinct oscillations in cytosolic and nuclear free calcium in single intact living cells demonstrated by confocal microscopy. In “Proceedings of the 49th Annual Meeting of the Electron Microscopy Society of America” (G. W. Bailey and E. L. Hall, Eds.), pp. 228-229. San Francisco Press, San Francisco. Eisen, A., Kiehart, D. P., Wieland, S. J., and Reynolds, G. T. (1984). Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs. J. Cell BioL 99,1647-1654. Eisen, A. D., and Reynolds, G. T. (1985). Source and sinks for the calcium released during fertilization of single sea urchin eggs. J. Cell BioL

100,1522-1527.

Epel, D. (1989). Arousal of activity in sea urchin eggs at fertilization. In “The Cell Biology of Fertilization” (H. Schatten and G. Schatten, Eds.), pp. 361-385. Academic Press, San Diego. Fine, A., Amos, W. B., Durbin, R. M., and McNaughton, P. A. (1988). Confocal microscopy: Applications in neurobiology. Trends Neuro sci 11,346-351.

Fredrikson, M. (1990). Embryological study of Hwminium monorchis (Orchidaceae) using confocal scanning laser microscopy. Am. J. Bot. 77,123-127. Gehring, C. A., Williams, D. A., Cody, S. H., and Parish, R. W. (1990). Phototropism and geotropism in maize coleoptiles are spatially correlated with increases in cytosolic free calcium. Nature 345,528530. Gilkey, J. C., Jaffe, L. F., Ridgway, E. B., and Reynolds, G. T. (1978). A free calcium wave traverses the activating egg on the medaka, OQ/zias latipes. J. Cell BioL 76,448-466. Hafner, M., Petzelt, C., Nobiling, R., Pawley, J. B., Kramp, D., and Schatten, G. (1988). Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2. Cell MotiL CytoskeL 9,271-277. Hamaguchi, Y., and Hamaguchi, M. S. (1990). Simultaneous investigation of intracellular Ca*+ increase and morphological events upon fertilization in the sand dollar egg. Cell Struct. Fund. 15,159-162.

of Calcium

Dynamics

379

Henson, J. H., Begg, D. A., Beaulieu, S. M., Fishkind, D. J., Bonder, E. M., Terasaki, M., Lebeche, D., and Kaminer, B. (1989). A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: Localization and dynamics in the egg and first cell cycle embryo. J Cell BioL 109,149-161. Hernandez-Cruz, A., Sala, F., and Adams, P. R. (1990). Subcellular calcium transients visualized by confocal microscopy in a voltageclamped vertebrate neuron. Science 247,858-862. Inoue, S. (1989). Foundations of confocal scanned imaging in light microscopy. In “The Handbook of Biological Confocal Microscopy” (J. Pawley, Ed.), pp. 1-13. IMR Press, Madison. Jaffe, L. F. (1985). The role of calcium explosions, waves, and pulses in activating eggs. In “Biology of Fertilization” (C. B. Metz and A. Monroy, Eds.), pp. 127-165. Academic Press, New York. Kalinich, J. F., and Douglas, M. G. (1989). In vitro translocation through the yeast nuclear envelope-signal-dependent transport requires ATP and calcium. J. Biol Chem. 264,17,979-1’7,989. Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989). Photochemitally generated cytosolic calcium pulses and their detection by fluo3. J. BioL Chem 264,8179-8X%. Lechleiter, J., Girard, S., Peralta, E., and Clapham, D. (1991). Spiral calcium wave propagation and annihilation in Xenopus Zoevis oocytes. Science 252,123-126. Longo, F. J. (1989). Egg cortical architecture. In “The Cell Biology of Fertilization” (H. Schatten and G. Schatten, Eds.), pp. 105-138. Academic Press, San Diego. Malgaroli, A., Milani, D., Meldolesi, J., and Pozzan, T. (1987). Furameasurement of cytosolic free Ca*+ in monolayers and suspensions of various types of animal cells. J. Cell. Biol 105,2145-2155. Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989). Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. BioL Chem. 264,8171-8178. Mohri, T., and Hamaguchi, Y. (1991). Propagation of transient Ca*’ increase in sea urchin eggs upon fertilization and its regulation by microinjecting EGTA solution. CeU Struct. Funct. 16,157-165. Nicotera, P., McConkey, D. J., Jones, D. P., and Orrenius, S. (1989). ATP stimulates Ca*+ uptake and increases the free Ca*’ concentration in isolated rat liver nuclei. Proc. NatL Acad. Sci USA 86,453457. Nicotera, P., Orrenius, S., Nilsson, T., and Berggren, P. 0. (1990). An inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in liver nuclei. Proc. Natl.

Acad

Sci. USA 87,6858-6862.

Oberdorf, J. A., Lebeche, D., Head, J. F., and Kaminer, B. (1988). Identification of a calsequestrin-like protein from sea urchin eggs. J. BioL

Chem

263,6806-6809.

Patel, R., Twigg, J., Crossley, I., Golsteyn, R., and Whitaker, M. (1989). Calcium-induced chromatin condensation and cyclin phosphorylation during chromatin condensation cycles in ammonia-activated sea urchin eggs. J. Cell Sti 12, (Suppl.), 129-144. Poenie, M., and Epel, D. (1987). Ultrastructural localization of intracellular calcium stores by a new cytochemical method. J. His&hem. Cytochem.

35,939-956.

Robert-Nicoud, M., Arndt-Jovin, D. J., Sehormann, T., and Jovin, T. M. (1989). 3-D imaging of cells and tissues using confocal laser scanning microscopy and digital processing. Eur. J. Cell BioL 48, (Suppl. 25), 49-52. Schatten, G. (1981). The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus vuriegatus: Time-lapse video microscopy. J. MorphoL 167,231-247. Shotton, D. (1989). Confocal scanning optical microscopy and its applications for biological specimens. J. CelL Sci. 94,175-206. Shotton, D., and White, N. (1989). Confocal scanning microscopy-3Dimensional biological imaging. Trends Biochem 14,435-439. Shuman, H., Murray, J. M., and Dilullo, C. (1989). Confocal microscopy: An overview. BioTechniques 7,154-161.

380

DEVEWPMENTAL

BIOLOGY

Steinhardt, R., Zucker, R., and Schatten, G. (1977). Intracellular calcium release at fertilization in the sea urchin egg. Deu. Biol. 58, 185-196. Stricker, S. A., and Schatten, G. (1989). Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish. Dev. Biol. 135,87-98. Swann, K., and Whitaker, M. (1986). The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs. J. Cell Biol. 103,2333-2342. Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. In “Methods in Cell Biology” (D. L. Taylor and Y. L. Wang, Eds.), Vol. 30, pp. 127-156. Academic Press, San Diego. Tsien, R. Y., and Poenie, M. (1986). Fluorescence ratio imaging: A new window into intracellular ionic signaling. Trends Biochem. 11,450455. Tsien, R. Y., and Waggoner, A. (1989). Fluorophores for confocal microscopy: Photophysics and photochemistry. In “The Handbook of Biological Confocal Microscopy” (J. Pawley, Ed.), pp. 153-161. IMR Press, Madison.

VOLUME

149,1992

White, B. A. (1985). Evidence for a role of calmodulin in the regulation of prolactin gene expression. J. Biol. Chem 260,1213-1217. White, J. G., Amos, W. B., and Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence microscopy. J. Cell Biol. 105,41-48. Williams, D. A., Becker, P. L., and Fay, F. S. (1987). Regional changes in calcium underlying contraction of single smooth muscle cells. Science 235,X44-1648. Williams, D. A., Fogarty, K. E., Tsien, R. Y., and Fay, F. S. (1985). Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature 318,558-561. Wright, S. J., Walker, J. S., Schatten, H., Simerly, C., McCarthy, J. J., and Schatten, G. (1989). Confocal fluorescence microscopy with the TANDEM scanning light microscope. J. CeU Sti. 94,617-624. Yasuda, H., Mueller, R. D., and Bradbury, E. M. (1987). Chromatin structure and histone modifications through mitosis in plasmodia of Physarum polycephalum. In “Cell Biology: A series of monographs, ” “Molecular Regulation of Nuclear Events in Mitosis and Meiosis” (R. A. Schlegel, M. S. Halleck, and P. N. Rao, Eds.), pp. 319-361. Academic Press, Orlando.