J. Cell Set. 32, 137-151 (1978) 13-7Printed in Great Britain © Company of Biologists Limited igyS

THE INDUCTION OF THE ACROSOME

REACTION IN GUINEA-PIG SPERM BY

THE DIVALENT METAL CATION

IONOPHORE A23187

D. P. L. GREENPhysiological Laboratory, Cambridge CB2 3EG, England

SUMMARY

The divalent metal cation ionophore A23187 induces an acrosome reaction in guinea-pigsperm which is dependent on external calcium. Examination of this acrosome reaction byelectron microscopy shows that it is morphologically normal. The known properties of A23187and the morphological similarity between the acrosome reaction and the secretory dischargesof other secretory cells suggests that the immediate cause of the acrosome reaction is an increasein the cytoplasmic free calcium concentration.

INTRODUCTION

The mammalian egg at fertilization is surrounded by two concentric investments:the innermost is the amorphous mass of the zona pellucida; outside this lie the residualfollicle cells of the corona radiata. Sperm are unable to penetrate the zona and sub-sequently fuse with the egg without first undergoing an acrosome reaction.

The acrosome is an intracellular membrane-bound organelle which forms a capover the anterior two-thirds of the nucleus. The acrosome reaction is the fusion of theouter acrosomal membrane to the overlying plasma membrane: part of the outer andthe inner acrosomal membrane are incorporated into the plasma membrane andbecome part of it; the remainder is lost as vesicles of plasma and acrosomal membrane(Barros, Bedford, Franklin & Austin, 1967; Bedford, 1970).

The natural stimulus for the acrosome reaction is unknown. It can, however, beinduced by a number of artificial stimuli (Yanagimachi, 1969; Barros, Arrau &Herrera, 1972; Wooding, 1975; Yanagimachi, 1975) which include follicularfluid, serum and detergents and, in the case of guinea-pig sperm, extended incubationin simple medium (Barros, Berrios & Herrera, 1973; Yanagimachi & Usui, 1974).This spontaneous reaction is dependent on external calcium (Yanagimachi & Usui,1974). An acrosome reaction can also be induced in guinea-pig sperm by the divalentmetal cation ionophore A23187 (Green, 1976; Summers et al. 1976; Talbot et al.1976).

This paper examines the ultrastructure of this A23i87-induced reaction.

138 D. P. L. Green

METHODS

Twenty-five per cent glutaraldehyde was purchased from Koch-Light, ethylene glycoltetra-acetic acid (EGTA) from Sigma, and Ficoll 400 from Pharmacia. All other reagents wereA.R. grade. A23187 was a generous gift from Dr R. L. Hamill of Eli Lilly Laboratories,Indianapolis. Siliconized glassware was used throughout.

Two media were used throughout: calcium medium contained 140 mM NaCl, 4 mM KC1,4 mM HEPES, 10 mM glucose and 2 mM CaClj, pH 7-4; magnesium medium was the same ascalcium medium except that 2 mM MgCla and 100 fiM EGTA were substituted for the 2 mMCaClj. A23187 was used from a stock solution in dimethyl sulphoxide (DMSO) (2 mg/ml)kept at room temperature in the dark.

Epididymal sperm were obtained from adult guinea pigs (0-5-1 kg) killed by dislocation ofthe neck. The vas deferens and cauda epididymis were removed in one piece and sperm flushedout with magnesium medium containing 5% Ficoll 400 (w/v) and spun at 1500 g^ for 5 min.The sperm pellet was washed once in magnesium medium and centrifuged at 1500 g.v fora further 10 min. Sperm were then suspended in either calcium or magnesium medium ata concentration of about 2 x 10' ml.

A preliminary time course for the loss of the acrosome induced by A23187 was estimatedfrom inspection by light microscopy of sperm fixed at suitable intervals after addition ofA23187. The acrosome reaction was induced by addition of 50 fi[ of the stock solution ofA23187 in DMSO to 5 ml of sperm suspension at 37 °C. Samples (0-5 ml) were pipetted intoKarnovsky's fixative in which magnesium and 100 fiM EGTA were substituted for calcium.500 sperm were examined for the loss of the acrosome for each time point.

Sperm in either magnesium or calcium medium were fixed for electron microscopy byaddition of an equal volume of 0-15 M sodium cacodylate buffer, pH 7-2, containing 2%glutaraldehyde. After 1 h at room temperature the fixed suspensions were pelleted in Eppendorftubes at 14000 g,r for 10 min. Pellets were detached from the bottom of the tube with the aidof a needle and given three 10-min washes in o-i M sodium cacodylate buffer, pH 7-4, post-fixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature and given afurther three 10-min washes in c i M sodium, acetate buffer, pH 5-1. The pellets were thenblock-stained with 1% uranyl acetate in o-i M sodium acetate buffer, pH 51 , overnight atroom temperature before dehydration in graded ethanols and propylene oxide and embeddingin Spurr (Spurr, 1969). Silver sections were cut on a Reichert microtome, post-stained withReynold's lead citrate and examined in a Philips 300 electron microscope.

Fig. 1. A stack of guinea-pig sperm heads in head-to-tail section fixed 30 s afteraddition of A23187 to sperm in calcium medium (A, acrosome; N, nucleus). In thecentre are 2 sperm which have not yet undergone an acrosome reaction: b is anapproximately elliptical region containing denser, round material within it, c is thedemarcation line between 2 distinct areas of the acrosome (seen more clearly once theacrosome reaction has taken place) and d and d' are 2 lighter areas close to the sites ofinitial membrane fusion. Immediately above and below the intact sperm are 2 whichhave undergone an acrosome reaction. The areas b and d have become devoid ofmaterial and occupied the area now occupied by b', membrane fusion has occurred ata and a', close to the areas d and d' in the intact acrosome, the demarcation line c hasbecome much clearer, and approximately half-way between this line and the concaveedge of the acrosome has appeared a large cavity, e. For the material lying between cand the concave edge, the cavity represents an approximately 85 % increase in total areafor the loss of no more than 10% of the material (assessed by measuring the areas ofelectron-dense material before and after the acrosome reaction), x 30400.

Induction of acrosome reaction by A23187

\

140 D. P. L. Green

RESULTS

The results in this paper are designed to provide a foundation for the three paperswhich follow (Green, 1978a, b; Green & Hockaday, 1978) by, firstly, showing thatthe acrosome reaction induced in guinea-pig sperm by A23187 in calcium mediumis morphologically normal and, secondly, by establishing the changes which occur inthe morphology of the acrosomal contents during the acrosome reaction.

Guinea-pig sperm agglutinate spontaneously to form well ordered stacks by head-to-head association. Fig. 1 shows a head-to-tail section through such a stack fixed30 s after addition of A23187. The 2 sperm in the centre still have intact acrosomesbut they are flanked above and below by 2 sperm which have undergone the earlystages of the acrosome reaction, including cavitation of the acrosomal matrix.

The acrosomal contents of the intact acrosome have a very characteristic mor-phology: there is the elliptical area b, often containing circular bodies; there is the line c,which marks the division of the acrosomal contents into 2 further regions, and there arethe 2 areas d and d', identified by Friend, Orci, Perrelet & Yanagimachi (1977) asareas of initial membrane fusion during the acrosome reaction.

The sperm which have undergone the acrosome reaction show the 2 areas a and a'of initial membrane fusion, corresponding to the 2 light areas d and d' in the intactsperm; the total disappearance of the area b and its conversion into the swollen spaceV; the increased clarity of the boundary c; and the appearance of a cavity e within thearea delimited by the boundary at c and the concave surface of the acrosome.

The sequence of events which lead from the intact acrosome to the early stage ofthe acrosome shown in Fig. 1 is outlined diagrammatically in Fig. 7 (p. 148). Mem-brane fusion occurs initially at the tip, and possibly on the convex surface of theacrosome; this leads to the immediate loss of the area b; at this stage cavitation beginsin the acrosomal matrix delimited by c and the concave surface of the acrosome togive the cavity e, and the appearance of sperm immediately after the early stages ofthe acrosome reaction shown in Fig. 1.

Some of the evidence for this mechanism is shown in Figs. 2 and 3; the remainderis dealt with in Green (19786). Fig. 2 shows a sperm after membrane fusion and theloss of the area b, but before cavitation has begun: Fig. 3 shows a sperm at the nextstage; cavitation has just begun and the matrix has the appearance of being tornapart. Cavitation in this matrix causes an approximately 85% increase in area forless than 10% loss of material as assessed by area.

Within a few minutes of addition of A23187, all sperm have the appearance shownin Fig. 4. Vestigial remnants of acrosomal material remain attached at the sperm tip,

Fig. 2. Two sperm heads in approximately the same section as Fig. 1. The upper is stillintact but the lower shows an early stage of the acrosome reaction immediately aftermembrane fusion at the tip of the acrosome (arrow). The material in area b of Fig. 1has already disappeared but cavitation has not begun, x 34 800.Fig. 3. The stage immediately after that shown in Fig. 2. Membrane fusion hasoccurred at the tip of the acrosome (arrow), the area b of Fig. 1 has more completelydisappeared and cavitation has begun, x 39000.

Induction of acrosome reaction by A23187 141

CEL 32

142 D. P. L. Green

Fig. 4. The completed A23i87-induced acrosome reaction. The bulk of the acrosomalmaterial has detached but residual vesicles of plasma and outer acrosomal membraneremain attached to the inner acrosomal membrane, x 40 500.

Induction of acrosome reaction by A23187

Fig. 5. Close-up of the area of membrane fusion between plasma and outer acrosomalmembrane of the centre sperm in Fig. 4. x 191 200.Fig. 6. The area immediately posterior to the base of the acrosome showing theadditional bilaminar material extending under the membrane of the equatorial seg-ment and running from it at a distance of 10-20 nm. It appears to bifurcate when itmeets the acrosomal membrane, become amorphous and then rapidly stop, x 138 800.

10-3

144 D- p- L- Green

and fusion of acrosomal and plasma membrane to form vesicles and cisternae hasextended over the sperm surface to give the characteristic double hairpin profile,shown in greater detail in Fig. 5, where the rump of the outer acrosomal membranefuses with the plasma membrane.

Block-staining with uranyl acetate also reveals very clearly the material which liescaudal to the posterior tip of the acrosome (Fig. 6). The precise form of this materialhas been the subject of some controversy (Fawcett, 1965; Fawcett & Ito, 1965;Gordon, 1972). In the guinea pig, it appears as a bilaminar structure, marginallywider than the plasma membrane, from which it runs at a distance of 10-20 nm. Itappears to bifurcate at the tip of the acrosome before becoming amorphous andrapidly petering out. There is no periodic structure holding it to the plasma membraneor the nuclear membrane.

The criteria by which an acrosome reaction can be judged to be morphologicallynormal are very limited: they are basically the occurrence of proper vesiculation andthe formation of the double hairpin structure at the base of the acrosome (Bedford,1968, 1970; Stefanini, Oura & Zamboni, 1969; Yanagimachi & Noda, 1970). By thesecriteria, the acrosome reaction induced in guinea-pig sperm by A23187 is normal.

The time course for the acrosome reaction is dealt with in the following paper(Green, 1978a). However, a few general points will be made here. External calciumis essential; A23187 in magnesium medium has no effect. Neither does DMSO incalcium medium without A23187. A23187 in calcium medium, in addition to inducingan acrosome reaction, also produces immotility; in magnesium medium, A23187 hasno effect on motility.

The acrosome reaction induced in guinea-pig sperm after extended incubation insimple media described by Yanagimachi & Usui (1974) was examined using the mediadescribed in Methods. Sperm incubated in calcium medium for 16 h at 37 °C did notundergo an acrosome reaction but subsequent addition of A23187 caused one withina few minutes. Incubation in magnesium medium for 16 h followed by resuspensionin calcium medium also produced no acrosome reaction. The origin of the differencebetween these results and those of Yanagimachi & Usui (1974) is unknown but ontheir part it may be due to the fluctuations in the pH of the incubation media: bothmedia use bicarbonate but it is titrated off as CO2 by acid neutralization before theincubation begins.

DISCUSSION

There is now increasing evidence that in a wide variety of secretory cells, secretionis a consequence of calcium-mediated stimulus-secretion coupling (Douglas, 1968;

. Rubin, 1970; Baker, 1974). It is the conclusion of this paper that the acrosome reactionis caused by an increase in cytoplasmic free calcium and that this, taken with itsmorphology, strongly suggests that it, too, is an example of stimulus-secretioncoupling.

The concept of stimulus-secretion coupling rests on the following propositions:that secretion is the result of the exocytosis of the contents of intracellular secretory

Induction of acrosome reaction by A23187 145

granules, that exocytosis is caused by fusion of the secretory granule membrane withthe plasma membrane, that cytoplasmic free calcium is normally low, that it rises onthe receipt by the cell of the stimulus to secretion, at least in the region between thesecretory granules and the plasma membrane, and that it is the increase in cytoplasmicfree calcium which causes the fusion of the secretory granule membrane to the plasmamembrane with exocytosis of the granule contents.

The evidence that the acrosome reaction is an example of stimulus-secretioncoupling can be divided into 2 parts: there is the evidence that the acrosome isa secretory organelle whose fusion to the plasma membrane results in exocytosis ofthe acrosomal contents, and secondly, there is the evidence that this fusion is theconsequence of an increase in cytoplasmic free calcium.

The origins and contents of the acrosome are very similar to those of the zymogengranules of the exocrine pancreas (Burgos & Fawcett, 1955; Palade, Siekevitz & Caro,1962; Jamieson & Palade, 1971^,6); both contain zymogens (Meizel & Mukerji,1976), both are formed by budding off of the rough endoplasmic reticulum, bothundergo condensation in conjunction with the Golgi apparatus (the proacrosomalgranules also fuse at this stage to form a single granule) and both move into appositionto the plasma membrane. (In the case of the acinar cells of the pancreas, the geometryof the cell remains fixed and the granules move to the plasma membrane; in thedeveloping sperm, the whole geometry of the cell is changing.) There is one importantdifference between the pancreatic exocrine cell and sperm and that is that sperm areterminal cells: the rough endoplasmic reticulum and Golgi apparatus are lost earlyin sperm development and with them, the ability to synthesize the acrosome. Theacrosome reaction, therefore, commits the cell irreversibly.

The morphology of the acrosome reaction is well established for a number of specieswhich have either undergone an acrosome reaction in the course of fertilization(Bedford, 1968,1970; Stefanini et al. 1969; Yanagimachi & Noda, 1970) or in responseto one of a number of artificial stimuli (Yanagimachi & Usui, 1974; Wooding, 1975;Yanagimachi, 1975). The principle morphological features of the acrosome reactionare the inclusion of the inner acrosomal membrane and a small part of the outer intothe plasma membrane of the head and the fusion of the remainder of the outeracrosomal membrane with the overlying plasma membrane with the formation ofvesicles (Barros et al. 1967) which are subsequently lost during penetration of thezona (Bedford, 1968). The acrosome reaction induced by A23187 is morphologicallynormal when judged by these criteria (Figs. 1, 4, 5). With the exception of theincubation technique of Yanagimachi & Usui (1974), which was found to be irrepro-ducible, the use of A23187 is the only means available for inducing an acrosomereaction rapidly in a whole population of sperm. Using the A23i87-induced reaction,it has been possible to show that proacrosin is activated and that acrosin, the putativezona lysin, is secreted as a consequence of the acrosome reaction (Green, 1978 a);there is separate evidence that the 2 of them are constituents of the acrosome (Green& Hockaday, 1978).

The ability of A23187 to induce an acrosome reaction in calcium medium suggeststhat the reaction is a consequence of an increase in intracellular calcium, and if the

146 D. P. L. Green

acrosome reaction is to be an example of stimulus-secretion coupling, a consequenceof an increase in cytoplasmic free calcium. In those cells where it has been measured,cytoplasmic free calcium is low, possibly no more than ioonM (Harrison & Long,1968; Ashley & Ridgway, 1970; Baker, 1972; Ridgvvay, Gilkey & Jaffe, 1977;Steinhardt, Zucker & Schatten, 1977). Extracellular free calcium, on the other hand,is often about 1 mM, i.e. 4 orders of magnitude greater. There is, therefore, a steepelectrochemical gradient for inward calcium movement at membrane potentialswhich are either negative or moderately positive: in guinea-pig sperm it is about+ iomV(Rink, 1977).

The level of cytoplasmic free calcium which normally exists in cells is the result of3 fluxes: an influx due to calcium leaking through the plasma membrane down itselectrochemical gradient, an efflux due either to calcium pumping or coupled exchangewith another moving into the cell down its own electrochemical gradient, e.g. sodium,or both, and calcium uptake into intracellular organelles. The stimulus to secretionnormally increases cytoplasmic free calcium by increasing the calcium permeabilityof the plasma membrane, thereby allowing external calcium to move into the celldown its electrochemical gradient (Douglas, 1968; Rubin, 1970; Baker, 1974) althoughin some cases, calcium is subsequently released from intracellular stores (Steinhardtet al. 1977).

The divalent metal cation ionophore A23187 can increase cytoplasmic free calciumeither by producing an increase in the inward calcium leak (Babcock, First & Lardy,1976; Desmedt & Hainaut, 1976) or by releasing calcium from intracellular stores(Steinhardt et al. 1977). A23187 causes electroneutral exchange of a number of divalentmetal cations and protons across membranes (Reed & Lardy, 1972; Babcock et al.1976); for practical purposes, the only 2 metal cations which need be considered aremagnesium and calcium. Because ion movement is tightly coupled (Kafka & Holz,1976) the exchange which A23187 induces is not equivalent to the introduction ofa calcium permeability into the membrane: net calcium movement across a membranein one direction occurs only as long as net movement of magnesium and protonsoccurs in the opposite direction. It stops when the activities of the 3 ions collectivelymove into equilibrium on each side of the membrane and this, of course, need notbe when the calcium activities on either side are themselves in electrochemical equilib-rium. Although the effects of A23187 are theoretically so complicated, it is knownempirically that it increases cytoplasmic free calcium (Babcock et al. 1976; Desmedt& Hainaut, 1976; Steinhardt et al. 1977) as well as stimulating secretion in a widevariety of secretory cells (Foreman, Mongar & Gomperts, 1973; Prince, Rasmussen& Berridge, 1973; Chambers, Pressman & Rose, 1974; Cochrane & Douglas, 1974;Plattner, 1974; Steinhardt & Epel, 1974; Steinhardt, Epel, Carroll & Yanagimachi,1974) for which there is evidence of secretion as a concomitant of increased cytoplasmicfree calcium (Kanno, Cochrane & Douglas, 1973; Ridgway et al. 1977; Steinhardtet al. 1977) or calcium uptake (Foreman et al. 1973).

The natural stimulus for the acrosome reaction is unknown but whatever it is, itwill be assumed that its effect is to increase cytoplasmic free calcium, i.e. that A23187is a surrogate stimulus but the increase in calcium is not. The evidence suggests that

Induction of acrosome reaction by A23187 147

any natural stimulus must increase the calcium permeability of the plasma membrane,since A23187 does not induce an acrosome reaction in the absence of external calcium(cf. the sea-urchin egg (Steinhardt et al. 1974)), and that calcium moves into thecytoplasm from the external medium down its electrochemical gradient to causemembrane fusion and exocytosis of the acrosomal contents. There is no need topostulate metabolically dependent calcium uptake into the cell, i.e. inward calciumpumping, as suggested by Gordon (1973): all the evidence suggests quite the contrary,that inward calcium movement is down the electrochemical gradient for calcium andthat calcium pumping is outwards (Babcock et al. 1976). Rink (1977) has effectivelyeliminated a change in membrane potential as the stimulus fcr calcium entry in theacrosome reaction. This leaves a ligand as the most likely stimulus, acting on receptorsin the plasma membrane of the head. Provided that they are restricted to the plasmamembrane overlying the acrosome, they are lost as the result of the vesiculation whichfollows their occupancy: continued occupation would not, therefore, lead to problemsof calcium regulation for the rest of the cell, assuming that desensitization did notrapidly curtail calcium influx immediately after ligand binding.

A23187 is not alone in inducing an acrosome reaction artificially; it can also beinduced by serum, follicular fluid, detergents, etc. (Yanagimachi, 1969; Yanagimachi& Usui, 1974; Wooding, 1975; Yanagimachi, 1975). The incubation media invariablycontain calcium, inter alia, and since the evidence suggests that a rise in intracellularcalcium is the cause of the acrosome reaction, it is likely that these agents all act bysomehow increasing the calcium permeability of the plasma membrane, therebyallowing calcium in and initiating the acrosome reaction. Cells which, for whateverreason, have an increased calcium permeability will, in the presence of externalcalcium, expend increasing quantities of metabolic energy, in the form of ATP, inmaintaining a low cytoplasmic free-calcium level: reducing the level of intracellularATP will impair motility, since flagellar movement employs a magnesium-dependentATPase: at the same time, the increasing levels of cytoplasmic free calcium willinduce the acrosome reaction. This may well explain why the substantial fraction(25-30%) of epididymal sperm which have undergone an acrosome reaction by thetime of isolation (Green, 1978 a) are also immotile since, in these circumstances, bothimmotility and the occurrence of the acrosome reaction represent a collapse in theability of the cell to maintain low cytoplasmic free calcium. Depletion of intracellularATP due to pumping would also explain why A23187 at high concentrations causesimmotility as well as inducing an acrosome reaction, although it may be due todepletion of intracellular free magnesium. The effect of A23187 in causing immotilityis, however, one of dosage and at lower concentrations an acrosome reaction can beinduced, albeit much more slowly, without impairing motility (Talbot et al. 1976).

The mechanism by which an increase in cytoplasmic free calcium causes fusion ofplasma and acrosomal membranes is unknown. Yanagimachi & Usui (1974) suggestedthat calcium entering the cell causes the acrosome to swell and that this swelling, bypushing the acrosomal membrane against the plasma membrane, causes fusion. Themechanism proposed for the origin of the swelling has been examined elsewhere(Green, 19786); however, the evidence suggests that swelling (or more strictly,

148 D. P. L. Green

,t

•l

Fig. 7. A schematic representation of the stages in the acrosome reaction of guinea-pigsperm: A, normal acrosome before reaction; B, reaction starts through initial mem-brane fusion at the leading edge of the acrosome; C, elliptical area (6 in Fig. 1) dis-appears ; D, cavitation begins in area adjacent to concave edge of acrosome; E, cavitationcomplete; and F, acrosomal contents have detached except for vesicles attached to inneracrosomal membrane.

Induction of acrosome reaction by K2 3187 149

cavitation of the acrosomal contents) is the consequence, not the cause, of membranefusion and this is confirmed by electron micrographs (Figs. 2, 3) which show mem-brane fusion preceding cavitation. The sequence of events suggested by the evidenceis shown diagrammatically in Fig. 7. One further argument against a mechanism whichrelies on acrosomal swelling as the cause of membrane fusion is that it would bea mechanism peculiar to sperm, because only in sperm is the geometry of the secretorygranule such that swelling would bring it automatically against the plasma membrane.Membrane fusion does not depend on proacrosin activation or acrosin activity (thelatter a suggestion of Gordon (1973)) since the acrosome reaction takes place in thepresence of a membrane-permeable acrosin inhibitor (Green, 1978 a).

Finally, there is the question of what prevents a premature acrosome reaction. Oneanswer is, of course, the absence of the stimulus. But the evidence suggests that some-thing more is involved. Sperm are incapable of undergoing an acrosome reaction untilthey have been capacitated (Bedford, 1970). One explanation of the need for capaci-tation is that it represents the removal of an inhibitor from the receptor for thestimulus ligand or the calcium channel which it opens. The possibility that theacrosome reaction can be prevented during sperm storage in the epididymis and vasdeferens because of the absence of external free calcium can, at least in the case ofthe latter, be ruled out: measurement of the free calcium in the guinea-pig vasdeferens using a calcium electrode shows it to be about 300/iM (T. J. Rink & R. Y.Tsien, unpublished observations). It must fall at the time of ejaculation because ofthe large injection of citrate, which is more than sufficient to chelate it, from theseminal vesicles (Mann, 1964) but it almost certainly rises in the female reproductivetract as sperm progressively free themselves from the seminal plasma: the free calciumlevel in the tract is unknown but total calcium is about 2-4 mM (Olds & VanDemark,1957; Hamner & Williams, 1965; Holmdahl & Mastroianni, 1965; Restell & Wales,1966). The evidence suggests, therefore, that a low cytoplasmic free calcium ismaintained in sperm by active calcium efflux and a low resting permeability to calciumand that this, possibly together with a high threshold to calcium activation of mem-brane fusion, prevents a premature acrosome reaction.

For the work reported here, and in the subsequent three papers, I wish to acknowledgegratefully the helpful criticism of Professors C. R. Austin and P. F. Baker, Dr R. G. Edwards,Professor Sir Alan Hodgkin, Mr T. J. Rink and Dr R. Y. Tsien and the invaluable technicalassistance of Mr A. R. Hockaday, Also, I wish to acknowledge gratefully the generous gift ofa Zeiss Standard microscope from the Royal Society.

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(Received 13 January 1978)