J. Cell Set. 54, 35-46 (1982) 35Printed in Great Britain © Company of Biologists Limited 1982

SURFACE CONTRACTION WAVES INAMPHIBIAN EGGS

MITSUKI YONEDA*, YOSHITAKA KOBAYAKAWA,HIROSHI Y. KUBOTA AND MASAO SAKAIDepartment of Zoology, Faculty of Science, Kyoto University,Kyoto 6o6, Japan

SUMMARY

Circular waves of change in brightness, known as 'surface contraction waves' (SCW-i andSCW-2), propagate over the animal surface of amphibian eggs at each cycle of cleavage.Movement of carbon particles attached to the egg surface indicated that SCW-i involvesexpansion of the egg surface, whereas SCW-2 accompanies surface contraction. Stiffness ofthe cortex as measured by applying negative pressure through a micropipette increasedconcomitantly with the passage of SCW-2. Measurement of stiffness at two loci on the eggsurface with two sets of pipettes confirmed the spatio-temporal coincidence of the wave ofstiffness and SCW-2. The stiffness showed either no change or even a slight decrease on passageof SCW-i. Thus SCW-2 is a genuine wave of 'contraction', but SCW-i can more properlybe called a ' surface relaxation wave'.

INTRODUCTION

By time-lapse cinemicrography, Hara (1971) observed wave-like changes in bright-ness of the surface of axolotl eggs, propagating as circular zones over the animalhemisphere towards the vegetal region. This change in brightness, termed a 'surfacecontraction wave' (SCW) by Hara, was composed of two closely spaced waves, thefirst (SCW-i) appearing 30 min before cleavage and the second (SCW-2) immediatelyin advance of the cleavage furrow. Hara noticed that both the waves start at the(uture initiation point of the cleavage furrow, no matter where the furrow firstappeared on the animal hemisphere. Similar waves were also reported in eggs ofXenopus (Hara, Tydeman & Hengst, 1977). Fig. 1 illustrates the pattern of SCWsin Xenopus eggs.

Temporal and spatial correlations between SCWs and cleavage suggest that theformer reflect a certain cellular event, which prepares for the formation of the cleavagefurrow. Another feature of interest is that the waves appear periodically even innon-nucleate egg-fragments, in the absence of cleavage (Hara, Tydeman & Kirschner,1980; Kirschner, Gerhart, Hara & Ubbels, 1980; Sakai & Kubota, 1981). Thus thewaves represent a cyclic activity of cytoplasm that proceeds by itself independentlyof the nuclear division cycle.

The changes in brightness are, however, so vague and slow that we can hardlyidentify them in real time. Hence time-lapse cinemicrography has been the sole

• Author for correspondence.

M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

Fig. i. Diagrammatic representation of the surface contraction waves (SCW-iand 2) in the egg of Xenopus. SCW-i is a bright circular area {B -> C), which appearsprior to cleavage and expands over the egg surface. SCW-2 is a circular dark zone(C -»• E), which propagates immediately ahead of the advance of the cleavagefurrow.

means of easily visualizing them. This could be the reason why such a simplephenomenon has passed unnoticed in the long history of amphibian embryology.

In this paper a detailed account of SCWs is presented. First, the spatio-temporalpattern of SCWs was analysed by a photo-kymographic device. Second, some physicalchanges of the egg surface during passage of SCWs were investigated, with a viewto relating SCWs to the 'wave of stiffness', which is also known to propagate overthe surface of the newt egg (Sawai & Yoneda, 1974). The results obtained show thatSCW-2 is a genuine wave of contraction of the egg surface, while SCW-i may bea wave of relaxation.

MATERIALS AND METHODS

Preparation of materials

Fertilized eggs of the newt, Cynops pyrrhogaster, and the frog, Xenopus laevis, were denudedof their fertilization envelopes in full-strength modified Steinberg's solution composed of:0-34% NaCl, 0005 %KC1, 0-008% Ca(NO,),.4H,O, 0-0205 % MgSO4.7H,O and 00001 %phenol red, buffered to pH 7-0 with 0-05 % HEPES and NaOH. Normally fertilized eggswere not fully appropriate for observation of SCWs, since the cleavage involved a considerablechange in shape of the denuded eggs, which complicated the pattern of SCWs. For observationof SCWs in the absence of cleavage, eggs were either injected with 100 nl of modifiedSteinberg's saline containing i mg/ml colchicine (Hara et al. 1980) or divided into halvesby placing a short glass rod onto each egg so as to obtain non-dividing egg-fragments (Sakai &Kubota, 1981; Kobayakawa & Kubota, 1981).

Time-lapse recording

Eggs or egg-fragments were allowed to rest in agar-coated vessels with their animal regionsfacing upward in modified Steinberg's saline. The materials were illuminated aslant fromtwo directions and imaged from above with either a time-lapse cine camera (Bolex equippedwith Nikon CFMA) or a time-lapse video system (National NV-8030).

Carbon marking

To detect any local stretching and contraction of the surface, eggs were loaded with min-ute carbon particles (Norit, 'extra') on their animal hemisphere. Movement of theparticles was analysed either by frame-by-frame tracing or with a photo-kymograph. As wasshown by Sakai & Kubota (1981), rounding-up and relaxing movements of the whole bodyof eggs are invariable concomitants of SCWs in non-dividing as well as in normal eggs (cf.Figs. 9, 11, 13). These movements will cause an apparent shift of the position of particlesmeasured from above. To minimize such an effect only those particles that were lying withinthe central two-thirds of the largest diameter of the egg were analysed.

Surface waves in amphibian eggs

P2 i . . . . i Pi

37

Fig. 2. Observation chamber. Side and front views. Drawn to scale except for theegg. AG, agar bed; Pi and Pa, micropipettes; VM and HM, objectives of verticaland horizontal microscopes; H, height of the bulge. D, inner diameter of thepipette.

Analysis by photp-kymograph

Cinematographic films were projected through a narrow slit onto a sheet of photographicpaper loaded on a rotating drum in a dark box. The dark box was accurately positionedbeforehand so that the image of a thin rectangular portion of the egg surface, including theinitiation centre of SCWs, lay along the slit. Since the photographic paper was moving ata constant rate perpendicular to the slit, we could visualize, on a still picture, the change inbrightness of the egg surface in a radial direction as a function of time.

Stiffness of the egg surface

Stiffness was determined with a 'cell elastimeter' (Mitchison & Swann, 1954; Selman &Waddington, 1955; Sawai & Yoneda, 1974), in which a bulge was sucked out of the eggsurface by a definite amount of negative pressure applied through a micropipette of knowninner diameter (D). The 'stiffness' of the egg surface was defined as the negative pressure(P) divided by the height of the bulge (H). The micropipette was connected to a cylindricalreservoir of saline into which a movable glass rod was dipped vertically. A definite amountof negative pressure within the pipette was applied by raising the rod a given amount whichwas monitored by a dial gauge. Raising it by 1 cm yielded a negative pressure of 853 Nm"'.Before each experiment the level of saline in the reservoir was adjusted for hydrodynamicequilibrium, by monitoring oil droplets of milk suspended in the saline until the dropletsbecame stationary in the pipette. For eggs of Cynops, the negative pressure was applied for2 min and the final form of the bulge was recorded using a horizontal microscope. Negativepressure was then removed and the pipette was withdrawn to allow the egg to resume itssmooth contour. Since the time taken for the bulge to relax back depended on the appliedpressure, a negative pressure was chosen so that the stiffness could be measured at intervalsof either 5 or 10 min. For eggs of Xenopiu, the stiffness was measured every 3 min, of which1 min was for suction and remaining 2 min for intermission. To observe SCWs and thebulge simultaneously two microscopes, one horizontal and the other vertical, were assembledtogether. With the aid of a special image-processer (National WJ-545), two images of theegg taken from above and from the side were recorded in a single video tape, by means ofwhich perfectly synchronous replay of both images was possible (cf. Fig. 10). To conform tosuch an optical system, a T-shaped observation chamber was made (Fig. 2). Its widened topgave a sufficient area of flat surface of saline, thereby assuring an undistorted image of thematerial viewed through the vertical microscope, while the lower narrow portion allowedclose access to the 10 x objective of the horizontal microscope. Attempts were also made tomeasure the stiffness at two loci on the egg surface by using two pipettes.

M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

1 mm Time (h)Fig. 3. Photo-kymogram of SCWs in a colchicine-injected egg of Cynops at 22 CC.A cinematographic film was projected through a narrow slit onto photographic paperon a rotating drum, by which a change in the brightness of the narrow rectangularportion of the egg surface was visualized on a still picture.

RESULTS

Propagation of SCWs and movement of the cell surface

Two surface contraction waves, SCW-i and SCW-2, propagating at each cleavageare so different from each other that it is easy to distinguish them; SCW-i is a circularexpansion of a bright area with its ' wave-front' well-demarcated, although in someeggs this wave was too faint to be detected. The second one (SCW-2) propagatesas a circular dark zone with vague boundaries. Strictly speaking, however, SCW-iand SCW-2 do not both start from a point on the egg surface, but originate as anarea of changed brightness. These characteristics of the two waves are common toeggs of Cynops and Xenopus. In the following description, the term 'centre ofinitiation of SCWs' will be used to denote simply the geometrical centre of theoriginating area.

A photo-kymogram taken of a colchicine-injected egg of Cynops (Fig. 3) visualizescentrifugal propagation of SCW-i (bright zone) and SCW-2 (dark zone) in succession,both of which repeat with a fairly constant interval, although SCW-i progressivelydecayed. The duration of the passage of SCW-i and 2 in each region was about20 and 15 min, respectively, and the speed of propagation was about 50 /im/min at22 °C. Figs. 4 and 5 are photo-kymograms at a faster time-scale for normal andnon-dividing eggs. A very bright area immediately after SCW-2 in the normal egg(Fig. 4) represents the pigment-free pale surface expanding on both sides of thecleavage furrow, but the overall pattern of the SCWs was similar to that in thenon-dividing egg (Fig. 5). Dark wavy lines in Figs. 4 and 5 represent the movementof carbon particles. It is apparent at a glance that, before the passage of SCW-2, theparticles tend to move in a centripetal direction towards the initiation centre ofSCWs, and after SCW-2 they move centrifugally away from the centre.

Surface waves in amphibian eggs

150

Time (min)Fig. 4. A photo-kymogram of SCWs at the first cleavage of the normal egg ofCynops, at 17 CC.Fig. 5. A photo-kymogram on expanded time-scale of the first round of SCWsshown in Fig. 3.

These movements of individual carbon particles were analysed on original recordsby frame-by-frame tracing. The position of each particle was expressed in polarcoordinates (r, 6) by taking the initiation centre of SCWs as the origin. Usually theparticles moved in an almost radial direction with respect to the initiation centresof SCWs on the surface of non-dividing eggs, so that the polar angle (6) of eachparticle remained practically unchanged. Fig. 6 shows the change in the distance (r)from the centre of six particles lying in a nearly straight line (6 = 300). As wasindicated in the photo-kymograms, and here too, successive movements of particlesin centripetal and centrifugal directions were evident. This pattern of movementswas initiated by the innermost particle and followed by outer particles sequentially

M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

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Fig. 6. Movement of carbon particles expressed as the distance from the initiationcentre of SCWs, at 21 °C. Numerals (at right) indicate the polar angle (0) of eachparticle in the polar coordinate. Those particles that were lying almost in a singleradius (8 = 300) are shown.

Fig. 7. Movement of carbon particles at various polar angles (indicated by numeralsat right), at 21 °C.

in step with the SCWs. Particles lying at various polar angles (Fig. 7) still exhibitedthe same pattern of movement as those measured for particles aligned in one direction(Fig. 6). The similarity in the patterns of Figs. 6 and 7, therefore, indicates a uniformand concentric propagation of the wave of relaxation-contraction along the eggsurface. To determine the extent of local contraction and relaxation of the surface,distances between adjacent particles aligned along a single radius were plotted asrelative values in Fig. 8. Each region first expanded by some 10-20% in lineardimension, after which it contracted down to about 70-80 % of the most expandedstate. Maximum relaxation and maximum contraction were observed mostly duringthe passage of SCW-i and 2, respectively.

Stiffness of the egg surface

The correlation between SCWs and the stiffness of the egg surface was studiedmainly in non-nucleated egg-fragments of Cynops. Egg-fragments separated as latein the one-cell stage as possible were selected for this purpose, since if separated atearlier stages both fragments often cleaved, possibly due to the activity of spermnuclei incorporated by natural polyspermy. In non-dividing eggs (Figs. 9, 10), SCW-ipassed in advance of the stiffness. SCW-2 corresponded with the maximum stiffening(in 2 egg-fragments out of 6) or came shortly behind it (in 4 out of 6, cf. Fig. 13).A similar temporal relation was observed in non-nucleated fragments derived from

Surface waves in amphibian eggs 41

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Fig. 8. Distances between adjacent particles aligned in a single radius, showinglocal stretching and contraction of the surface of a colchicine-injected egg of Cynops.Distances are expressed as relative values with reference to the initial distance.Thick broken and solid lines refer to the passage of SCW-i and SCW-2, respectively;21 °C.

i

1 h

Fig. 9. Stiffness (upper curve) of a non-dividing fragment of Cynops. Bars I and 2under the curve indicate the passage of SCW-i and 2, respectively, across the pointunder the pipette. D = 252 fim; P = 4-26 Nnv2; 21 °C. The lower curve showsthe change in the height of the fragment, indicating its cyclic rounding-up.

M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

^^^L B ^ ^ ^ C ^^^ ° ^ ^ ^ J

Fig. 10. One of the rare cases in which SCW-2 could be identified in still pictures.Non-dividing egg-fragment of Xenopus; 21 °C. The top view (above) and side view(middle) of the fragment were simultaneously recorded on a single video tape.In the top view, the tip of the pipette is seen to the left as a bright spot. In the sideview, right and left are reversed. Line drawings (below) show SCW-2. Bar representsO'S mm. A. SCW-2 originates as dark area. B. Ten-min after A; SCW-2 is passingthrough the region under the pipette. The side view indicates the increased stiffness(decreased height of the bulge), c. Five min after B; SCW-2 is leaving the pointunder the pipette. The fragment is maximally rounded up. D. Five min after c;SCW-2 reaches the equator; stiffness is decreasing.

1 h

Fig.non

11. Change in stiffness (upper curve) and rounding-up (lower curve) of a-nucleate fragment of Xenopus. D = 174/Jin. P = 4-26 Nm"1. A, 20 °C; B, 19 °C.

eggs of Xenopus (Fig. n ) . In both Cynops and Xenopus the rise in stiffness wassometimes preceded by a brief fall. This period, when present, corresponded withthe passage of SCW-i.

Fig. 12 represents the results from experiments on normally dividing eggs ofCynops. Here, too, there was a temporal correlation between SCW-2 and stiffening,

Surface waves in amphibian eggs 43

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Fig. 12. Stiffness at a point on the surface (marked by circles in drawings above)of normal eggs of Cynopt. Bars i and 2 are SCW-i and 2; D = 252 fim. A, P = 170Nm"', 21 °C. B, P = 2-56 Nm"', 22 CC. Short bars in drawings represent the initialcleavage furrow, which appeared at the time marked by arrows in the Figure.Arrowheads indicate the time of appearance of the second cleavage furrow.

though with some fluctuation, probably due to difficulty in measuring the sameportion of the surface of the dividing egg, which changes its shape as the cleavagefurrow advances.

To explore the spatial as well as temporal coordination of SCWs and the waveof stiffness, a glass capillary with a thinned central portion was cut into halves soas to give a pair of pipettes with the same inner diameter. Both pipettes were connectedto a common reservoir of saline and used to measure the stiffness at two loci on theegg surface simultaneously. Fig. 13 illustrates the result with a non-dividing fragmentof Cynops. The first SCW-2 was initiated from a point nearer to the left pipette andreached there about 12 min before reaching the right pipette. This compares withthe lag in the increase in stiffness measured between the left and right pipettes, whichwas 10-15 m m - The initiation centre of the second SCW-2 moved away from thefirst one to such an extent that the dark zone of SCW-2 reached the two loci atnearly the same time. Correspondingly, the time-course of the second stiffening atboth loci was almost coincident. The time relation between the loci was reversed inthe third round owing to a further shift in the initiation centre, but still the consistenttemporal correlation between SCW-2 and the wave of stiffness persisted; bothreached the site of the left pipette some 5 minutes after reaching the right pipette.These observations indicate that the wave of stiffness and SCW-2 share a commoncentre of initiation and a common speed of propagation. Most probably both wavesare due to a common change in the structure of the cortical layer that propagates asa circular wave over the egg surface.

44 M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

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200

100

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Fig. 13. Simultaneous measurement of the stiffness at two loci (L, R) on the surfaceof a non-dividing egg-fragment of Cynops. The centre of initiation of SCW-2 ismarked by crosses in the drawings above. SCW-i was not conspicuous in thisfragment. Bars L and R indicate passages of SCW-2 to loci L and R; D = 145 fim.P = 8-53 Nnr1; 22 CC. The apparent high value of calculated stiffness is due tothe small diameter (D) of the pipette used, rather than to real variation amongbatches; Mitchison & Swann's model experiment using rubber balloon (their fig. 15)indicates that the numerical values of stiffness (= P/H) may vary in proportion to2)-i » T n e i o w e r c u r v e shows cycles of rounding-up of the fragment.

DISCUSSION

The term 'surface contraction wave' coined by Hara (1971) has involved a tacitassumption that the observed waves are due to the contraction of some structureat or near the egg surface. Stiffness and contraction of the egg surface during thepassage of SCW-2 as revealed in the present study substantiate the notion thatSCW-2 is essentially a wave of 'contraction'. In the case of SCW-i, however, there isno evidence of contraction. Conversely, the expansion of the egg surface and thedecrease in the stiffness sometimes observed indicate that SCW-i is actually a waveof relaxation, or a 'surface relaxation wave'.

A tendency to round-up at mitosis is a feature common to most animal cells, andits possible relevance to the formation of the cleavage furrow has been stressed bySchroeder (1981). In amphibian eggs, undoubtedly, SCW-2 is exclusively responsiblefor the rounding-up of the whole body of the egg occurring periodically in non-dividing as well as normal eggs. What appears characteristic of amphibians is thatthe increase in rigidity of the cell surface to effect rounding-up is not synchronousover the entire surface, but travels as a wave with a speed of around 1 jim/s. Thespatio-temporal correlation between SCW-2 and the advance of the cleavage furrowmay thus indicate that the former is a preparatory step by the cortex in furrow

Surface waves in amphibian eggs 45

formation, by acquiring reactivity to the so-called 'furrow-inducing cytoplasm'found by Sawai (1972).

By using time-lapse cinematography we are certain of the fairly circular andconcentric propagation of SCWs. Such a regularity in the spatial pattern and occasionalshift of the initiation centre of SCWs, as exemplified in Fig. 13, may suggest thatSCW-2 are self-propagating waves evoked by some sort of chain reaction involvingeither biochemical or mechanical interaction among adjacent regions of the eggcortex.

The changes in the brightness of the egg surface, manifest as SCWs, could bedue to accumulation or dispersion of pigment granules by cortical contraction orrelaxation, but any changes in surface architecture, for example, in shape anddistribution of microvilli, may affect the brightness of the egg surface as well. Howcontraction and relaxation relate to the change in brightness has yet to be clarified.

The cyclic appearance of SCWs and the wave of stiffness in non-nucleate egg-fragments of amphibians (Sawai, 1979; Hara et al. 1980; Kirschner et al. 1980;Sakai & Kubota, 1981) are manifestation of an autonomous cyclic activity of the cyto-plasm, as also detected in egg-fragments of sea-urchins (Yoneda, Ikeda & Washitani,1978) and annelids (Shimizu, 1981). An understanding of the molecular and structuralbases of SCWs should, therefore, afford some clue as to the nature of the cytoplasmiccycle.

This work was partly supported by a grant of the Ministry of Education, Japan, no. 484009to M.Y. The authors' thanks are due to Mr Yuzo Kadokawa, now in Nagoya University, who,in our laboratory in 1979, found the efficacy of colchicine injection in observing SCWs. Weexpress our gratitude to Dr Thomas E. Schroeder, who gave us an opportunity to read hispaper in advance of publication.

REFERENCES

HARA, K. (1971). Cinematographic observation of 'surface contraction waves' (SCW) duringthe early cleavage of axolotl eggs. Wilhelm Roux Arch. EntwMech. Org. 167, 183-186.

HARA, K., TYDEMAN, P. & HENGST, R. T. M. (1977). Cinematographic observation of 'post-fertilization waves' (PFW) on the zygote of Xenopus laevis. Wilhelm Roux Arch. EntwMech.Org. 181, 189-192.

HARA, K., TYDEMAN, P. & KIRSCHNER, M. (1980). A cytoplasmic clock with the same periodas the division cycle in Xenopus eggs. Proc. natn. Acad. Set. U.S.A. 77, 462-466.

KIRSCHNER, M., GERHART, J. C, HARA, K. & UBBELS, G. A. (1980). Initiation of the cellcycle and establishment of bilateral symmetry in Xenopus eggs. In The Cell surface: Mediatorof developmental processes (ed. S. Subtelny & N. K. Wessels), pp. 187-215. New York,London: Academic Press.

KOBAYAKAWA, Y. & KUBOTA, H. Y. (1981). Temporal pattern of cleavage and the onset ofgastrulation in amphibian embryos developed from eggs with the reduced cytoplasm.J. Embryol. exp. Morph. 62, 83-94.

MITCHISON, J. M. & SWANN, M. M. (1954). The mechanical properties of the cell surface.I. The cell elastimeter. J. exp. Biol. 31, 443-460.

SAKAI, M. & KUBOTA, H. Y. (1981). Cyclic surface changes in the non-nucleate egg fragmentof Xenopus laevis. Develop., Growth Differ. 23, 41-49.

SAWAI, T. (1972). Roles of cortical and subcortical components in cleavage furrow formationin amphibia. X Cell Sci. 11, 543-556.

SAWAI, T. (1979). Cyclic changes in the cortical layer of non-nucleated fragments of thenewt's egg. J. Embryol. exp. Morph. 51, 183-193.

46 M. Yoneda, Y. Kobayakawa, H. Y. Kubota and M. Sakai

SAWAI, T. & YONEDA, M. (1974). Wave of stiffness propagating along the surface of thenewt egg during cleavage. J. Cell Biol. 60, 1-7.

SCHROEDER, T. E. (1981). The origin of cleavage forces in dividing eggs. A mechanism intwo steps. Expl Cell Res. (In Press.)

SELMAN, G. G. & WADDINGTON, C. H. (1955). The mechanism of cell division in the cleavageof the newt's egg. J. exp. Biol. 32, 700-733.

SHIMIZU, T. (1981). Cyclic changes in shape of a non-nucleate egg fragment of Tubifex(Annelida, Oligochaeta). Develop., Growth Differ. 33, 101-109.

YONEDA, M., IKEDA, M. & WASHITANI, S. (1978). Periodic change in the tension at the surfaceof activated non-nucleate fragments of sea-urchin eggs. Develop., Growth Differ. 20, 329—336.

(Received 7 August 1981)