J. Cell Sci. 55, 327-339 (1982) 327Printed in Great Britain © Company of Biologists Limited 1982

STRUCTURAL CHANGES IN DIVIDING

SEA-URCHIN EGGS INDUCED BY THE

VOLATILE ANAESTHETIC HALOTHANE

ROBERT E. HINKLEY* AND EDWARD L. CHAMBERSDepartments of Anatomy and Anaesthesiology, and Physiology and Biophysics,The University of Miami School of Medicine, Miami, Florida 33101, U.S.A.

SUMMARYFertilized Lytechinus eggs exposed to the volatile anaesthetic halothane before metaphase do

not undergo cytoplasmic cleavage. This effect has been correlated with the failure of thecontractile ring to assemble. Comparative studies on mitotic apparatuses isolated from controland halothane-treated cells show that halothane significantly impairs both spindle and astergrowth as early as metaphase. When transferred to control solutions, halothane-treated cellsinitiate furrowing activity in association with either the first or second mitotic division, depend-ing on the duration of the exposure to anaesthetic and the concentration employed. In contrastto these effects, halothane has no effect on any aspect of the cleavage process if applied laterthan metaphase. In this case, furrows develop even in the presence of halothane, deepenprogressively, and complete cell division. These observations confirm and extend previousstudies on echinoderm eggs exposed to volatile anaesthetics, and support the view that anaes-thetic agents indirectly prevent cell cleavage by inhibiting the growth of the mitotic apparatus.

INTRODUCTIONVolatile anaesthetics have been reported to inhibit the growth and division of

many cell types including plant cells (Nunn, Lovis & Kimball, 1971), protozoa(Nunn, Dixon & Moore, 1968), echinoderm eggs (Wilson, 1901; Lillie, 1914), anda variety of cells in tissue culture (Nunn, Sturrock & Ho well, 1976; Jackson, 1972;Telser & Hinkley, 1977). Despite these observations few attempts have been made toexamine the effects of volatile anaesthetics on the morphology of dividing cells. Thereis evidence, however, that volatile anaesthetic agents can reversibly inhibit theassembly of the mitotic apparatus and prevent cell cleavage. In an early report, forexample, Wilson (1901) noted that ether caused a reversible 'fading' of the mitoticapparatus of dividing sea-urchin eggs and subsequently delayed or prevented cellcleavage. These observations have been confirmed in a more recent study (Swann,1954), and Rappaport (1971) has obtained experimental evidence indicating that theabsence of cleavage in etherized sea-urchin eggs is causally related to the impaireddevelopment of the mitotic apparatus.

When reconsidered at the ultrastructural level, these observations suggest thatvolatile anaesthetics inhibit cell division by reversibly impairing the assembly ofmitotic microtubules and inhibiting the formation of the contractile ring, the organelle

• Author for correspondence at Department of Anatomy.

328 R. E. Hinkley and E. L. Chambers

responsible for cell cleavage (Schroeder, 1972, 1975). Although light-microscopicstudies support this view, no direct attempt has been made to examine the effects ofvolatile anaesthetic agents on the cytoskeletal organelles responsible for cell division.In the studies reported here, halothane, a widely used halogenated hydrocarbonanaesthetic agent, has been shown to inhibit the division of sea-urchin eggs in afashion identical to that reported for ether. Halothane-induced reductions in thegrowth of the mitotic apparatus have been demonstrated by comparing mitoticapparatuses isolated from control and halothane-treated cells, while the absence ofcleavage has been correlated ultrastructurally with the failure of the contractile ringto assemble.

MATERIALS AND METHODS

Sea urchins (Lytechittus variegatus) were induced to spawn by injecting 0-5 M-KC1 into thebody cavity. Eggs were collected and washed in natural sea water (SW) (pH 8'3) to remove thejelly layer, while sperm were collected in concentrated form and diluted just prior to fertiliza-tion. Eggs were fertilized in 60 mm plastic tissue-culture dishes containing sea water with20 mM-TAPS buffer (2-hydroxy-i, i-bis(hydroxy-methyl ethyl)-amino-i-propane sulphonicacid), pH 8-3 (SW-TAPS), and allowed to develop to prophase. In several experimentsfertilization membranes were removed with 1 M-urea (pH 76) immediately after insemination.

For exposure to anaesthetic, eggs were transferred to gas-tight vials containing SW-TAPSequilibrated with 0-5-2-5 mM-halothane (2-bromo, 2-chloro-i: 1 :i-trifluroethane; AyeretLaboratories, New York). Control eggs were incubated in vials containing only SW-TAPS.The duration of anaesthetic exposure varied from 3 to 30 min. All anaesthetic-induced effectswere tested for reversibility by reincubating halothane-treated eggs in fresh SW-TAPS.

To determine the effects of halothane on furrowing activity and the structure of preassembledcontractile rings, eggs in early to mid-cleavage were transferred individually to vials containingSW-TAPS equilibrated with 2-5 mM-halothane. After 2-10 min the SW-TAPS-halothanesolution was rapidly decanted and replaced with glutaraldehyde fixative.

All eggs were fixed for electron microscopy at room temperature in either: (1) 2%glutaraldehyde/83 % SW (v/v, with distilled water) followed by 0-5 % osmium tetroxide/75 %SW (Schroeder, 1972); or (2) 1 % glutaraldehyde/phosphate buffer followed by 4 mM-osmiumtetroxide (Maupin-Szamier & Pollard, 1978). Thereafter, all cells were dehydrated in ethanolthrough hydroxypropyl methacrylate and flat-embedded in Epon 812 (Brinkley, Murphy &Richardson, 1967). Equatorial and meridonal sections of control and halothane-exposed cellswere stained in methanolic uranyl acetate and lead citrate and examined in an Hitachi 11 C ora Philips 300 electron microscope. The presumptive furrowing regions of halothane-exposedcells were located by thick-sectioning whole eggs to determine the polar axis of the mitoticapparatus. Eggs in which the plane of section passed through both poles were then thin-sectioned and the cortex midway between the poles was photographed in the electron micro-scope.

Mitotic apparatuses (MAs) were isolated from control and halothane-treated cells by com-bining the protocols of Sakai, Shimoda & Hiramoto (1977) with those of Forer & Zimmerman(1974). In these experiments, a single batch of eggs was fertilized, treated with 1 M-urea(pH 7-6) to remove fertilization membranes, and then washed three times with 20 vol. ofcalcium-free SW (Goldstein, 1953) containing 20 mM-TAPS buffer (CFSW-TAPS), pH 8-3.After the third wash, the cells were resuspended in CFSW-TAPS and half of the suspensionplaced in each of two plastic tissue-culture dishes. When the eggs reached late prophase, thecells in one dish were transferred to a gas-tight vial containing CFSW-TAPS pre-equilibratedwith 1-5 mM-halothane, while those in the other dish were transferred to an identical vialcontaining only CFSW-TAPS. When the control cells reached metaphase, the cells in bothvials were collected, washed in 1 M-dextrose, and resuspended in 20 vol. of an MA isolationbuffer (Sakai et al. 1977) containing o-i % Triton X-100. The suspensions were allowed to

Halothane and cell division 329

stand for 1 min and were then shaken vigorously to liberate the MAs. The MA suspensionswere immediately layered over an equal volume of stabilization buffer (Forer & Zimmerman,1974) consisting of 50% glycerol, 10% dimethyl sulphoxide, 5 mM-MgSO4 and 1 mM-EGTAin 6 mM-pho8phate buffer (pH 6-8) and centrifuged at 1500 g for 15 min. The loosely pelleted MAswere resuspended in 0-5 ml fresh stabilization buffer and photographed at a uniform magnificationusing Nomarski illumination. Spindle length and width, as well as maximum astral fibrelength, were determined for 65 metaphase MAs isolated from each group.

RESULTS

The first cleavage furrow of dividing Lytechinus eggs contains a contractile ring(CR) resembling that described in other echinoderm cells (Schroeder, 1975). At lowmagnification the CR appears as an electron-dense layer approximately 0-05-0-1 /mithick immediately below the plasma membrane, which smoothly conforms to thefurrow contour (Figs. 1-3). Individual microfilaments comprising the CR are orientedcircumferentially with respect to the cell equator and have diameters in the 30-80 Arange (Fig. 3).

Halothane has a differential effect on cell cleavage depending on the time at whichexposure to anaesthetic is started. When applied at metaphase or before, halothaneconcentrations as low as 0-5 mM inhibit the growth of the mitotic apparatus andcompletely abolish all furrowing activity associated with the first cleavage process(Figs. 4-6). If applied later than metaphase, halothane causes reversible decreases inthe size of the mitotic apparatus but has little discernible effect on the developmentor progression of furrows or on the completion of cell cleavage, even at concentrationsup to 2-5 nriM. Moreover, cells that had entered cleavage prior to exposure to anaes-thetic continued to divide when transferred to SW-TAPS equilibrated with 2-5 mM-halothane, with no evidence of furrow arrest or reversal.

Ultrastructural studies show that halothane prevents CR formation when appliedbefore metaphase. The presumptive furrowing regions of halothane-treated cellsremain convex in outline, possess lengthy microvilli at the cell surface, and havea cytoplasmic organization indistinguishable from that of pre-cleavage cortices(Figs. 5-6). Although furrowing activity and CR formation were completely abolishedat the lowest concentration of anaesthetic tested (0-5 HIM), anaesthetic concentrationsin the 0-5-1-0 mM range seemed to have little effect on the progress of mitosis. Asa consequence, prolonged exposure to relatively low concentrations of halothaneresulted in the formation of binucleated cells (Figs. 7-9) at about the same time thatcontrol eggs reached the two-cell stage.

Cells exposed to halothane before metaphase resume furrowing activity whentransferred to fresh SW-TAPS. Whether cleavage resumes in association with thefirst or second mitotic division, however, clearly depends on the duration of exposureto anaesthetic and the concentration employed. For example, eggs exposed to lowconcentrations of halothane (0-5-1-0 mM for 5-15 min) rapidly initiate furrowing inconjunction with the first mitotic division when returned to control conditions.Although the predominant cleavage pattern exhibited by these cells is symmetrical(annular) in type, resembling that of control cells, many eggs developed incompleteor 'unilateral' furrows such as those shown in Figs. 11-12. Unilateral furrows extend

33° R. E. Hinkley and E. L. Chambers

Halothane and cell division 331

around only part of the cell circumference and always appear in a plane perpendicularto the axis of the mitotic apparatus. These furrows, like their symmetrical counter-parts, form near the end of the first mitotic process and ultimately regress, therebyresulting in the formation of binucleated cells. Eggs exposed to higher concentrationsof anaesthetic (or for longer durations) require longer periods of recovery in SW-TAPS before initiating furrowing activity. Such cells resume cleavage in associationwith the second mitotic division by the process illustrated in Figs. 13-18. In thisinstance, two unilateral furrows usually form at right angles to one another, eithersimultaneously or in rapid succession, and partition the egg into four blastomeres.The cells produced by this furrowing process have a distinctive 'lobed' appearanceand are typically unequal in size.

Table 1. Comparison of metaphase mitotic apparatuses isolated fromcontrol and halothane-treated eggs

Maximum astral Spindle length Spindle widthfibre length (/tm) (j"m) (/im)

Control 18-5 (9-8-25-4) 191 (15-5-24-6) 98 (7-4-14-7)

1-5 mM-halothane 12-0 (8-9-16-4) 13-1 (9-8-16-4) 8-1 (5-7-11-5)

N = 65 for each group. See Materials and Methods for conditions of exposure to halothaneand isolation protocol. Maximum astral fibre length and spindle length (pole-to-pole distance)are decreased by 35-4 % and 31-4 %, respectively. Spindle width was approximated by measur-ing the diameter of the metaphase plate. Data presented are from one of six experiments. Ineach case similar results were obtained.

MAs isolated from halothane-treated cells are consistently smaller at all stages ofmitosis than those isolated from control cells. This observation was expected, sincestudies on intact eggs had shown that halothane inhibits MA growth in vivo. MAsisolated at metaphase from control cells and cells exposed to 15 mM-halothane areshown in Figs. 19-23 and are compared in Table 1. These studies clearly show thathalothane inhibits both spindle and aster growth and that these effects are detectableas early as metaphase.

Fig. 1. Nomarski micrograph of control cells in early cleavage, x 295.Fig. 2. Meridional section through the cleavage furrow of a control cell such asthose shown in Fig. 1. The contractile ring (er) appears as a continuous dense layer,005-0-1 fim thick, immediately below the cell membrane, fm, fertilization membrane.X I I 600.

Fig. 3. Para-meridional section through the cleavage furrow of a control cell fixed in1 % glutaraldehyde followed by 4 mM-osmium tetroxide. Microfilaments comprisingthe contractile ring measure 30-80 A in diameter, x 66000.

332 R. E. Hinkley and E. L. Chambers

Halothane and cell division 333

DISCUSSION

Cell cleavage is accomplished by a circumferentially oriented band of actin filamentscalled the 'contractile ring', which appears transiently at the cell equator shortly afterthe onset of telophase (Schroeder, 1972). Owing to the actin composition of thefilaments comprising the contractile ring (Schroeder, 1973), as well as the presence ofmyosin (Fujiwara & Pollard, 1978) and tropomyosin (Ishimoda-Takagi, 1979) in thecleavage furrow, the contractile ring is thought to generate the annular force forcleavage through an actomyosin-like interaction (Schroeder, 1973; Mabuchi & Okuno,1977; Meeusen, Bennett & Cande, 1980). In the experiments reported here, we haveshown that fertilized sea-urchin eggs fail to cleave when exposed to the volatileanaesthetic halothane. This effect occurs only when exposure to halothane is startedat or before metaphase and has been correlated ultrastructurally with the failure ofthe contractile ring to appear. In striking contrast, however, cells exposed to halothanelater than metaphase undergo completely normal cleavage. In this instance, furrowseven in the presence of anaesthetic, progressively deepen, and successfully completecleavage. As expected, the cleavage furrows of these cells contained contractile ringsindistinguishable from those of control cells (data not shown). These observationsindicate that halothane has no direct effect on the structure of CR microfilaments oron the actomyosin-like interaction that generates the contractile force for cleavage.Instead, the prevention of cleavage by halothane appears to be limited to an inhibitionof the process that initiates contractile ring assembly.

Although considerable information has accumulated regarding the structure andfunction of the contractile ring (see Schroeder, 1975 for a review), comparativelylittle is known about cellular conditions and factors that initiate contractile ringformation in vivo. In echinoderm eggs, however, there is evidence that cleavage (and,by implication, contractile ring formation) is both temporally and spatially linked tomitosis through the establishment of rather precise geometrical relationships betweena pair of asters and the cell surface (Hamaguchi, 1975; Hiramoto, 1971; Rappaport,1968, 1969). These relationships are thought to occur sometime during early-midanaphase as the asters increase dramatically in size (Hamaguchi, 1975; Rappaport,1969). Although the mechanism by which aster growth influences the timing of cleav-age remains to be identified (see Rappaport, 1975), astral growth appears to be an

Fig. 4. Eggs that were transferred to 1 mM-halothane for 15 min beginning at prometa-phase. At the time of photography eggs in parallel control cultures were in mid-cleavage. Compare with Fig. 1. X295.

Fig. 5. Meridional section through the presumptive furrowing region (see text formethods) of a halothane-treated cell similar to those shown in Fig. 4. In this case,however, the fertilization membrane was removed with urea immediately followingfertilization. The cell surface remains convex, possesses numerous microvilli, andcontains no evidence of contractile ring formation. Control (urea-treated) eggs were inmid-cleavage by this time. Compare with Fig. 2. x 13200.

Fig. 6. The presumptive furrowing region of a halothane-treated cell at highermagnification. The cortex generally resembles that of pre-clcavage cells, x 38250.

334 R. E. Hinkley and E. L. Chambers

16 18

Hahthane and cell division 335

important structural prerequisite for cleavage initiation. In this context, it has longbeen recognized that eggs that fail to cleave when exposed to volatile anaestheticsconsistently contain a smaller mitotic apparatus than their control counterparts(Wilson, 1901; Lillie, 1914; Swann, 1954; Rappaport, 1971). A similar effect has nowbeen demonstrated in echinoderm eggs exposed to halothane. On the basis of theseobservations, Rappaport (1971) has suggested that volatile anaesthetics indirectlyprevent cleavage by inhibiting aster growth. Such an effect would, in turn, precludethe establishment of the necessary aster-cell surface relationships required to initiatecontractile-ring assembly and induce cleavage. Experimental support for this hypothe-sis has been obtained by Rappaport (1971) in studies showing that cortical furrowingcould be induced in etherized sea-urchin eggs simply by mechanically narrowing the'gap' between the diminutive mitotic apparatus and the cell surface. Consistent withthis view, comparative studies of isolated mitotic apparatuses show that halothanemarkedly inhibits aster growth at concentrations that prevent cleavage. Moreover,reductions in aster growth are clearly evident as early as metaphase, well in advanceof the time during anaphase at which the cleavage mechanism is normally established.

Recovery experiments show that the prevention of cleavage by halothane is rever-sible, although extensive modification of the second cleavage process invariablyoccurs. The simultaneous formation of two furrows during the second mitoticdivision is entirely predictable if the role that a pair of asters plays in determining theplane of cleavage in a normally dividing cell (see Rappaport, 1969) is extended to thespecial case in which four asters are present in a single egg. Only the most commoncleavage pattern observed following exposure to halothane is illustrated in Figs. 13-18.This pattern occurs only when the two mitotic apparatuses are parallel. We havenoted numerous variations in this pattern, however, which seem to be related to thepositioning of the four asters relative to one another. For example, when the axes ofthe mitotic apparatuses are not parallel, furrows form but not at right angles. In these

Fig. 7. Egg exposed to 1 mM-halothane for 15 min beginning at prometaphase.Although cleavage is prevented under these conditions, mitosis continues and resultsin the formation of binucleated eggs, x 260.Figs. 8-9. Binucleated cells induced by halothane (1 mM, 30 min). Control eggs hadreached the two-cell stage when these cells were photographed, x 260.Fig. 10. Halothane-treated cell containing eccentrically located mitotic apparatus.Such cells commonly appear in halothane-treated cultures and subsequently undergounilateral cleavage, x 260.Figs. 11-12. Halothane-treated cell (0-5 mM, 5 min) undergoing unilateral cleavagein fresh SW-TAPS. These furrows invariably develop near the end of the firstmitotic division and are transient, x 260.Figs. 13-14. Cells undergoing the second mitotic division in the presence of i-o mM-halothane. Note the presence of two mitotic apparatuses, x 260.Figs. 15-18. Halothane-exposed cells similar to those shown in Figs. 13-14 butreincubated in fresh SW-TAPS to permit cleavage initiation. Predictability, these cellsdevelop two furrows at right angles to one another, which partition the egg into fourblastomeres. x 260.

336 R. E. Hinkley and E. L. Chambers

Figs. 19-23. Representative metaphase MAs isolated from control (Figs. 19-20) andhalothane-treated (Figs. 21-23) cells at the same time after fertilization. See text forconditions of exposure to halothane and method of isolation, x 650.

cases the resulting blastomeres may be unusually different in size and occasionallythree-celled embryos appear, consisting of two blastomeres each having a singlenucleus and one large binucleated blastomere. These observations indicate clearlythat relatively brief exposures to halothane may profoundly affect embryogenesis.Moreover, in studies to be reported elsewhere, we have shown that all of the effectsof halothane described here will occur at clinically useful concentrations, thus raisingthe possibility that the lethality and purported teratogenicity of volatile anaestheticsobserved in avian (Anderson, 1968) and mammalian embryos (Basford & Fink, 1968)may be related in some way to early alterations in the division process.

The methods used here to isolate MAs preserve little besides the chromosomes andmicrotubule framework of the mitotic apparatus (personal observation). Although thelength of individual microtubules is difficult to determine in thin sections, differencesin the size of MAs isolated from control and halothane-treated cells are readilyapparent at the ultrastructural level. Accordingly, transient reductions in the growth

Halothane and cell division 337

of the mitotic apparatus during exposure to halothane almost certainly reflect areversible inhibition of microtubule assembly. A similar conclusion was reached byNunn & Allison (1972) in birefringence studies of dividing sea-urchin eggs exposed tohalothane. While the ability of volatile anaesthetics, particularly halothane, to inhibitthe assembly of microtubules has been demonstrated in a number of systems, bothin vivo (Allison et al. 1970; Livingston & Vergara, 1979; Telser, 1977) and in vitro(Okuda & Ogli, 1976; Hinkley & Telser, unpublished observations), the mechanismby which volatile anaesthetics inhibit microtubule assembly remains to be determined.It has been proposed, however, that volatile anaesthetics may inhibit microtubuleassembly (or cause the depolymerization of preassembled tubules) in one of two ways:either (1) through a direct molecular interaction between the anaesthetic moleculeand tubulin dimer, which transiently precludes dimer-dimer interactions; or (2) byaltering the microtubule environment (Allison et al. 1970; Hinkley, 1978). Theability of volatile anaesthetics to bind to globular proteins and induce reversibleconformational changes has been demonstrated in model physico-chemical studies(Balasubramanian & Wetlaufer, 1966; DiPaolo & Sandorfy, 1974; see Woodbury,D'Arrigo & Eyring, 1975 for a review). It has yet to be shown, however, whethervolatile anaesthetic agents can induce analogous conformational changes in eithertubulin or protein factors such as microtubule-associated proteins (MAPs) thatregulate the assembly—disassembly of microtubules (Sakai, 1980). Alternatively, itis entirely possible that halothane inhibits microtubule assembly by altering environ-mental parameters, particularly ionic conditions, which are known to affect micro-tubule assembly in vitro and which may regulate the growth and function of themitotic apparatus in vivo (Sakai, 1980; Kiehart, 1981). Although aster microtubulesthemselves appear to play no direct role in the initiation of cleavage (Asnes &Schroeder, 1979), future studies with isolated sea-urchin MAs and cortices mayclarify the mechanism by which halothane and other volatile anaesthetics inhibit thegrowth of the mitotic apparatus and ultimately prevent cleavage.

This research was supported by NIH Research Career Development award 1 KO4 GM-00173 (R.E.H.) and a Travel Grant from the Burroughs Wellcome Fund. We wish to thankDr A. Telser for reviewing the manuscript.

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{Received 19 August 1981)