THE EFFEC OTF FERTILISATIO ONN TH PERMEE - ABILITY …jeb.biologists.org/content/jexbio/9/1/69.full.pdf · His method was to measure the diameters of individual eggs at definite intervals

69

THE EFFECT OF FERTILISATION ON THE PERME-ABILITY TO WATER AND ON CERTAIN OTHER

PROPERTIES OF THE SURFACE OF THE EGGOF PSAMMECHINUS MILIARIS

BY A. D. HOBSON, M.A.(Lecturer in Experimental Zoology, University of Edinburgh, and RayLankester Investigator at the Marine Biological Laboratory, Plymouth.)

(Received 8th September, 1931.)

(With Twenty Text-figures.)

AMONG the numerous changes in the sea-urchin egg which have been shown byvarious workers to occur at the time of and subsequent to fertilisation none haveattracted more attention than that of increased permeability. R. S. Lillie (1916)showed that fertilisation or artificial activation by means of butyric acid causes theeggs of Arbacia to swell more rapidly when placed in hypotonic sea water. Heconcluded from his experiments that the surface of the sea-urchin egg becomesmore permeable to water as the result of activation.

Assuming that the egg is surrounded by a semi-permeable membrane, Lillieconsidered that the principal factors controlling the rate of swelling in hypotonicsolutions were (1) the difference in osmotic pressure between the cell contents andthe surrounding medium, (2) the frictional resistance of the membrane to thepassage of water, (3) the area of the membrane. He concluded that "the forcesof elasticity, cohesion, and surface tension.. .are undoubtedly negligible in com-parison with osmotic pressure," and that the behaviour of the egg could be describedapproximately in terms of the osmotic gradient alone.

Other workers (McCutcheon and Lucke, 1926; Northrop, 1927; McCutcheon,Lucke and Hartline ,1931; Lucke, Hartline and McCutcheon ,1931) have examined thekinetics of swelling of sea-urchin eggs in more detail, and have introduced variouscorrections into Lillie's original treatment of the problem. They agree with him, how-ever.in leaving out of consideration the mechanical properties of the cell surface. Withthe exception of Vies (1926), no attempt has been made to investigate the elastic proper-ties of the surface of the sea-urchin eggs and their changes subsequent to fertilisation.

The conclusion of R. S. Lillie that fertilisation is followed by an increasedpermeability of the egg surface to water is now generally accepted and has beensupported by the observations of a number of authors (e.g. R. S. Lillie, 19166;Herlant, 1918a; Page, 1929) that fertilisation is followed by an increase in the rateof cytolysis in hypotonic solutions. The time taken for cytolysis to take place under

jo A. D. HOBSON

such conditions has been considered to be a measure of the resistance of the cellsurface to the passage of water. While this may be the factor which is predominantin determining the rate of cytolysis, other conditions, such as the extensibility ofthe cell surface, may be important and should not be neglected.

The evidence for an increase in permeability to dissolved substances is not sosatisfactory. The work of McClendon (1910) and of Gray (1916) indicates a decreasein the electrical resistance of sea-urchin eggs following fertilisation. These authorsconcluded that this shows an increased permeability to electrolytes. Herlant (1918 a)deduced changes in the permeability in sea-urchin eggs by means of the plasmolysismethod. As will be shown later in the present paper, the reaction of fertilised eggsto hypertonic solutions varies in a very striking manner according to the stage ofdevelopment which has been reached. Moreover, the behaviour of the egg dependslargely on the physical properties of its superficial region and not necessarily on itspermeability alone.

The time relations of the permeability changes during the period betweenfertilisation and cleavage are not well known. The sea-urchin egg is especiallysusceptible to cytolysis by hypotonic solutions while the fertilisation membrane isbeing formed immediately after fertilisation (Just, 1922 a; Page, 1929). Lillie (1918)concluded that the maximum permeability to water was reached about 20 min.or longer after fertilisation. Gray (1916) found a fairly steady decrease in theelectrical resistance of eggs following fertilisation. Using the plasmolysis method,Herlant (1918) concluded that the permeability rises to a maximum at 50 min.after fertilisation. He also (1918) found a brief period of high susceptibility tohypotonic sea water just after activation. After this susceptibility (permeability)decreases until the spindle appears, when it increases again.

In the experiments described in the present paper the changes in permeabilityto water following fertilisation have been examined in more detail. The mechanicalproperties of the surface region of the egg as exhibited by its behaviour in hypertonicand hypotonic solutions have also been investigated, and an attempt has been madeto correlate these with what is known of the alterations in permeability.

THE SWELLING OF EGGS IN HYPOTONIC SEA WATER.

R. S. Lillie (1916) has established the fact that fertilised and artificially activatedeggs swell more rapidly than normal unfertilised eggs when placed in diluted seawater. His method was to measure the diameters of individual eggs at definiteintervals by means of a screw eyepiece micrometer. On the assumption that theegg is a sphere, the volume can be calculated from such measurements and a curveshowing the rate of increase of size can be plotted. Since the swelling of the eggis presumably due to increase in its water content, it was concluded that the curveobtained in this way gives an indication of the rate at which water can diffusethrough the cell surface. This conclusion involves the assumption that the cellmembrane is not damaged or fundamentally altered in structure either by themechanical stretching or by the lowering of salt concentration inherent in the experi-

The Effect of Fertilisation on the Surface of the Egg o /P. miliaris 71

ment. That this assumption is probably justified is shown by the fact that in40 per cent, sea water Lillie found that the rate of increase in volume is not appre-ciably altered for the first few minutes.

Lillie's method has since been employed by other workers, notably McCutcheonand Lucke (1926), for the investigation of the osmotic properties of cells.

A serious objection to the use of the screw micrometer or to any other methodof direct measurement at present available is that a very small number of eggs canbe examined simultaneously. Since it is necessary to employ different eggs fordifferent experiments in the same series, relatively gross changes in behaviour

1800 -

1700 -

1800 -

1700

1600

1500

1400

1300

1200

1100

1000

90i 900

4 5 6Minutes

8 9 1& 4 5 6Minutes

10

Fig. 1. Graph showing typical swelling curves of unfertilised eggs and of eggs at certain timesafter fertilisation when placed in 50 per cent, sea water. © unfertilised; ^ 1 min., Q 3 min.,£§ s min., A 15 min., V 20 min. after fertilisation.

can alone be detected. In Psammechinus miliaris there may be a considerablevariation in the rate of swelling in hypotonic solutions of individual eggs obtainedfrom the same female and subjected to conditions as nearly as possible identical.Moreover the speed with which experiments can be performed is important. Itwas found that eggs which have remained for a long time in sea water may swellat a markedly slower rate than when first removed from the female.

For these reasons among others the method of making direct measurementsof individual eggs was abandoned after considerable trial in favour of photography.For this purpose a Leitz microcamera was used. In the latest design of this instru-ment cinematograph film is used. This has a number of advantages. The film

72 A. D. HOBSON

(Perutz Leica Special) is fast and fine grained, enabling short exposures to be usedand giving fine detail. The time and necessary disturbance of the apparatus involvedin changing plates is avoided. At least 36 exposures can be made without changingthe film. Lastly, the cheapness of the film compared with plates is not its smallestadvantage.

600 -

10 30 40 50Minutes after fertilisation

60 70 80

Fig. 2. Graph showing the average volume of water entering an egg before and at various timesafter fertilisation during the first 2 min. after exposure to 50 per cent, sea water.

A Pointolite lamp was usually employed as the source of illumination. ANo. 2 Leitz objective and a x 10 or x 12 ocular was used. After development thefilm was placed in a simple, vertical projection apparatus and the negative imageswere measured on squared paper. The apparatus was calibrated by photographingthe scale of a stage micrometer. The apparatus gave a total magnification of about200 diameters.

The eggs were first photographed lying in a flat-bottomed dish in normal seawater. A small sample of eggs with as little sea water as possible was then trans-

The Effect of Fertilisation on the Surface of the Egg of P. miliaris 73

ferred to another dish containing the diluted sea water. One min. after the trans-ference the first photograph was taken and thereafter exposures were made atintervals of 1 min. usually for a period of 10 min. In this way the behaviour ofany number up to about 40 eggs could be followed simultaneously.

In presenting the results of these experiments I have endeavoured, for thepresent, to avoid entering into the controversy as to the correct evaluation of the"permeability constant" of the egg. I have therefore given in Fig. 1 the swellingcurves in 50 per cent, sea water for unfertilised and for fertilised eggs from thesame female, taken at different intervals after insemination. Fig. 2, which illustratesthe same experiment, shows the increase in volume of the eggs in the first 2 min.after placing in 50 per cent, sea water (sea water diluted with an equal volumeof distilled water). In Fig. 1, in order to economise space, are illustrated only6 out of the total number of 16 swelling curves measured in the course of theexperiment.

It will be seen that the changes in permeability of the surface of the eggs towater after fertilisation are not simple. In the experiment illustrated in Figs. 1 and2 the rate of swelling increased rapidly after fertilisation, reaching a maximum3 min. after insemination. Five min. after insemination the rate of swelling wasmarkedly slower. After this the rate increased fairly steadily, reaching a maximumat 36-40 min. after insemination. After this the rate remained approximatelyuniform until 60-65 min. after fertilisation. The experiment was discontinued after75 min. as cleavage began.

A somewhat unexpected feature of this experiment is the sharp decrease inpermeability preceding cleavage. It is necessary that this should be confirmed byfurther work, as only one of the experiments performed to examine the rate ofswelling of fertilised eggs was continued up to the time of cleavage.

One point illustrated by Fig. 1 may be mentioned. It will be noted that thecurve for eggs 3 min. after fertilisation is not continued for more than 3 min. afterthe eggs were placed in the hypotonic sea water. This is because the eggs are at thisstage very susceptible to the cytolysing action of hypotonic solutions. Out of 38 eggsat the beginning of the experiment 22 had burst by the time the third photographwas taken and only 5 eggs survived 4 min. This matter will be discussed later.

THE EXTENSIBILITY OF THE SURFACE LAYER OF THE EGG.

Page (1929) has noted that the egg of Arbacia during the phase followingfertilisation, in which it is relatively resistant to hypotonic solutions, swells to agreater extent before cytolysing than during a phase of susceptibility. Fig. 1illustrates this point in Psammechinus miliaris. The swelling of eggs placed in thehypotonic solution 3 min. after fertilisation could not be determined for more than3 min., since such a large proportion had undergone cytolysis. The average volumeof the eggs remaining intact at the end of 3 min. was about 14 x io5/x3. This repre-sents approximately the limit of extension which the egg surface can withstand.At no other stage in this experiment were the eggs found to be so susceptible.

74 A. D. HOBSON

In other experiments eggs were placed in a series of dilutions of sea water andexamined after about i hour. The proportion of cytolysis was determined approxi-mately and the eggs were then returned to normal sea water. It was found thatthose eggs whose volume had been increased almost to the bursting point showeda wrinkled surface when returned to normal sea water. The limit of elasticity istherefore somewhat lower than the breaking point. Table I shows the result ofsuch an experiment. There is a slight but constant variation in resistance whichagrees with the results mentioned in the previous paragraph. During the susceptibleperiod immediately following fertilisation the eggs cytolyse as the result of a smallerincrease in volume than when they are more resistant. The change in the mechanicalproperties of the cell surface is also illustrated by the failure to recover from asmaller degree of stretching, even if this does not reach the breaking point. It maybe noted that a marked degree of wrinkling of the surface on return to normal seawater is always followed by cytolysis. Recovery is only possible if shrinkage isaccompanied by a smooth or only very faintly wrinkled surface.

Table I.

Minutesafter

fertilisa-tion

o (un-fertilised)

i

35

IO

IS2O

25

%

90%sea

water

oo

<S<S

<s< 5<S<S

Cytolysis of eggs after i

8o%sea

water

oo

<S<S

<s<s<s<s

70%sea

water

oo

<S<S

<s< 5< 5<S

6o%sea

water

oIO

<S

<s<s<5<S<S

hr. in dilutions of sea water

5o%sea

water

0

851 0

<S< 51 0

1 0 0

<S

40%sea

water

701 0 0

SO755060

1 0 080

30%sea

water

1 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 0

20%sea

water

1 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 0

Maximum con-centration of

sea water whichcaused appear-

ance of wrinklesin eggs returned tonormal sea water

foo46 0 %

40%40%4 0 %5 0 %so%

TYPES OF CYTOLYSIS IN TAP WATER1.

The appearance of the eggs undergoing cytolysis in tap water varies considerably.The behaviour of the unfertilised egg has already been described in a previouspaper. The essential features may be briefly recapitulated. The egg swells uniformlyup to a certain point and then a slight bulge appears on one side. Over this areathe surface is slightly irregular. The cytoplasm becomes clearer in the region nearthe bulge, and the granules become much less numerous (Fig. 3). This changespreads over the whole of the cytoplasm and at the same time the surface of theegg becomes perfectly smooth and spherical (Fig. 4). At no stage is there anyvisible outflow of the contents of the egg.

1 The Plymouth tap water is collected from the granite district of Dartmoor and is almostfree from dissolved salts.


After fertilisation the type of cytolysis changes. Half a minute after inseminationthe behaviour is similar to that of unfertilised eggs. One minute after inseminationcytolysis begins as a bulge occurring on one side only of the egg. It is similar tothat found in the unfertilised egg but more pronounced (Fig. 5). Two minutesafter insemination the bulge is still more marked (Fig. 6). After this an increasingnumber of eggs burst at two or more points on the surface (Fig. 7). After cytolysisis complete the egg does not return to its original spherical form as was the casebefore fertilisation.

The bulges in the fertilised egg appear to be due to a local breakdown of thecell surface. The cytoplasm flows out at these points but gelates rapidly. There isno visible scattering of the cell contents. The surface of the egg is irregular overthe areas of outflow but preserves its original smooth contour elsewhere.

There is thus a striking difference between the behaviour of both unfertilisedand fertilised sea-urchin eggs and the unfertilised eggs of Teredo described in a

Fig. 3. Fig. 4.

Fig. 3. Unfertilised egg cytolysing in tap water. Note the wrinkled surface and the clearer appear-ance of the cytolysed portion of the egg. Stippling diagrammatic and merely represents relativedistribution of granules.Fig. 4. Unfertilised egg completely cytolysed in tap water. Note smooth surface and few granules.Stippling as in Fig. 3.

subsequent paper (Hobson, 1931). In the latter the egg bursts at one point and thecontents flow out and disperse, leaving behind only a crumpled vitelline membrane,which is stout and comparatively inextensible. This probably resembles moreclosely the condition found in the fertilised sea-urchin egg if allowance is madefor the fact that the cytoplasm of the Teredo egg disperses while that of the sea-urchin egg does not. The behaviour of both is what would be expected if the egg weresurrounded by a layer which was capable of only slight extension. In such circum-stances the tendency for the egg to swell must be compensated either by the rapiddiffusion of substances into the surrounding medium or by the surface layerrupturing at one or more points. The behaviour of the egg of Teredo in hypertonicsolutions (Hobson, 1931) shows that the vitelline membrane is permeable tosalts as is the case with the fertilisation membrane in the sea-urchin. It is probablealso that the hyaline plasma layer ("ectoplasm" of Gray) is also readily permeableto salts in spite of Just's assertion (1928) that "the mobile hyaline plasma layer isthe plasma membrane of the egg regulating exchange with the environment."Nevertheless these structures will not permit the rapid diffusion of the more

76 A. D. HOBSON

complex substances which must be present in the egg. Since, therefore, the osmoticpressure of the contents of the egg cannot be lowered, at any rate sufficientlyrapidly, by exosmosis, the surface layer must rupture when it has been stretched toits breaking point.

In the unfertilised sea-urchin egg the type of cytolysis may be explained if it isassumed that the fertilisation membrane is already present on the unfertilised egg.If this is so, the egg may be assumed to be surrounded by a thin membrane whichis both extensible and elastic and which is readily penetrated by salts (Hobson,1927). We may therefore interpret the behaviour of the egg when immersed intap water as follows. The egg swells uniformly up to the point at which the surfaceregion of the cytoplasm beneath the vitelline membrane (fertilisation membrane)is no longer capable of further extension. Whether this surface region of thecytoplasm is the plasma membrane or the cortex does not matter for the present

Fig. 6. Fig. 7.Fig. 5-

Fig. 5. Complete cytolysis of egg placed in tap water 1 min. after fertilisation. A slight bulge withirregular surface marks the point at which cytolysis began. Stippling as in Fig. 3.Fig. 6. Complete cytolysis of egg placed in tap water 2 min. after fertilisation. The bulge at thepoint where cytolysis began is more pronounced. Stippling as in Fig. 3.Fig. 7. Complete cytolysis of egg placed in tap water 5 min. after fertilisation. The egg surfacebroke down at more than one point. Stippling as in Fig. 3.

argument. When the surface of the cytoplasm gives way on one side of the eggthe resistance to the internal pressure is no longer uniform and a slight bulge isformed in the vitelline membrane. Salts diffuse out of the egg and the osmoticpressure of the contents thus becomes lowered. Finally the elasticity of the vitellinemembrane enables the cytolysed egg to regain its spherical form.

THE ACTION OF HYPERTONIC SOLUTIONS.

In the following experiments various hypertonic solutions were employed,including sea water concentrated by evaporation and sea water whose osmoticpressure was raised by addition of varying amounts of z-\M NaCl or i-6M glycerol.All the solutions gave essentially similar results as regards the form adopted bythe plasmolysed egg.

Among the most striking of the differences between fertilised and unfertilisedeggs are those which occur in response to hypertonic solutions. Moreover, thefertilised eggs are found to vary in their behaviour in these circumstances in an


equally remarkable manner at different stages during the period between fertilisationand cleavage. It will be best to describe first of all the types of plasmolysis foundin the unfertilised egg and in the fertilised egg at various times after fertilisation.

The unfertilised egg retains its smooth surface during the first stage of itscontraction. Then fine wrinkles begin to appear all over its surface and these becomemore sharply defined as the egg becomes more strongly plasmolysed (Fig. 8). Theshape remains roughly spherical.

As soon as fertilisation has occurred the behaviour of the egg in hypertonicsolutions changes. The wrinkling of the surface becomes, as a rule, somewhatcoarser and the shape is more irregular than that of the unfertilised egg. Oftenthere is on one side a deep hollow (Fig. 9). If the fertilisation membrane has becomecompletely separated from the egg surface before immersion in the hypertonic

Fig. 9.

Fig. 8. Photograph of plasmolysed unfertilised egg. Hypertonic solution composed of 50 c.c. seawater + 25 c.c. 2'4MNaCl.Fig. 9. Plasmolysis of egg 1 min. after fertilisation. Hypertonic solution composed of 50 c.c. seawater + 25 c.c. 2'4MNaCl.

solution it behaves normally, except that its diameter is less than is found in normalsea water. The reasons for this have been discussed in a previous paper (Hobson,1927). If the process of separation is not complete it may apparently be inhibitedby the hypertonic solution. In this case the surface of the egg may be drawn outto a fine point which remains attached to the fertilisation membrane. Over thisregion the fertilisation membrane is somewhat flattened (Fig. 10).

The description given above applies to eggs which have been fertilised for notmore than about 1 min. (at i7°-i8° C) . Two minutes after fertilisation the plasmo-lysed egg exhibits much coarser wrinkles which may take the form of prominentridges bounding concave areas of the cell surface (Fig. 11). These concave areasare usually small compared with those found in eggs exposed to the hypertonicsolution at a later stage of development. This type of plasmolysis, which is exhibitedin its most characteristic form by eggs which have been fertilised for about 15 min.,corresponds very closely with that which I have already described in the eggs of

i§ A. D. HOBSON

Teredo (1931) and termed "polyhedral." Its appearance in the eggs of Psamm-echinus miliaris is probably determined by physical conditions at the surface insome respects similar to those found in Teredo. Generally the type of plasmolysisfound in the eggs of Psammechinus tested 2 min. after fertilisation is intermediatebetween the "wrinkled" and the "polyhedral."

Fig. 10. Fig. 11.

Fig. 10. Inhibition of membrane separation in egg placed in hypertonic solution half a minuteafter fertilisation. Stippling diagrammatic.Fig. 11. Polyhedral plasmolysis of egg placed in hypertonic solution (50 c.c. sea water + 25 c.c.

M NaCl) 2 min. after fertilisation.

Fig. 12. Fig. 13.

Fig. 12. Photograph of an egg placed in hypertonic solution (50 c.c. sea water + 25 c.c. 2-4MNaCl)4 min. after fertilisation, showing formation of gelatinous layer.Fig. 13. Camera lucida drawing of egg placed in hypertonic solution (50 c.c. sea water + 50 c.c.•2,-^M NaCl) S min. after fertilisation. The thick gelatinous layer has ruptured at one point andcontracted, exposing the surface of the cytoplasm. Stippling diagrammatic.

Three minutes after fertilisation the response of the egg to treatment withhypertonic solutions undergoes an abrupt change. The egg remains perfectlyspherical, except that there may be a slight initial wrinkling of the surface whichpasses off as the egg contracts further. A clear layer now appears over the wholeof the egg surface. The thickness of this layer varies with the concentration of thesolution employed. Sometimes its outer surface is smooth and at others the layerseems as though composed of a number of bubbles on the surface of the egg (Fig. 12).The material of which the clear layer is composed is a stiff jelly enclosing a numberof vesicles of various sizes and a few granules. The consistency is demonstrated by


its behaviour when ruptured. Fig. 13 is a camera lucida sketch of an egg placed instrong hypertonic solution (50 c.c. normal sea water + 50 c.c. sea water evaporatedto about 35 per cent, of its original volume) 5 min. after fertilisation. The clear layeroriginally covered the whole surface of the egg but it ruptured at one point. Itselastic, gelatinous nature is shown by the way in which it has contracted, exposinga considerable area of the surface of the granular protoplasm, and also by theirregular nature of the torn surfaces.

The behaviour of such eggs when they are returned to normal sea water is alsointeresting. If the gelatinous layer is not very thick it ruptures as the egg swells,contracts, and forms a rounded mass at one side of the egg (Fig. 14). If the egghas been exposed to a fairly strong hypertonic sea water (50 c.c. normal sea water4- 25 c.c. 2-4 Af NaCl) exovates may be formed on return to normal sea water.The outer part of the cytoplasm is apparently gelated and can be distinguished

Fig. 14. Fig- 15-Fig. 14. Egg returned to sea water after exposure to hypertonic solution 5 min. subsequent tofertilisation. Two stages in the contraction of the gelatinous layer to form a rounded mass on one sideof the egg. Stippling diagrammatic.Fig. 15. Egg returned to sea water after exposure to hypertonic solution 5 min. subsequent tofertilisation. Note the formation of an exovate and the material of the gelatinous layer at the equator.Stippling diagrammatic and merely represents relative distribution of granules.

from the inner part by its coarser granulation. As the egg swells the clear layerfirst ruptures and contracts. Almost at the same time the gelated outer part of thecytoplasm also bursts and the inner, finely granular part is squeezed out, forming awell-marked exovate (Fig. 15) which may become completely separated; the eggthen bears a close superficial resemblance to a 2-cell stage, the material of the clearlayer usually collects in the groove between the exovate and the rest of the egg.The surface of the exovate is perfectly smooth and naked. That of the corticalpart is covered by a thin, transparent, usually irregular layer beneath which is thegranular cytoplasm whose surface is produced into fine processes.

The origin of the clear gelatinous layer of these eggs is not easy to distinguish.If the process of development is watched under the high power of the microscopethe first stage seems to be that the surface of the granular cytoplasm becomesirregular and withdraws, leaving behind a clear zone with a smooth outer surface.The irregularities of the cytoplasmic surface become more pronounced and develop

So A. D. HOBSON

into somewhat ill defined radial processes. If the hypertonic solution is strong andthe reaction of the egg consequently extreme, these processes can no longer bemade out and the surface of the granular cytoplasm is fairly smooth.

This type of reaction to hypertonic solutions is found in eggs about 3 to 9 min.after fertilisation. As already mentioned, it appears suddenly and its disappearanceis nearly as abrupt. Eggs which have been fertilised about 7-9 min. usually exhibit,when first placed in the hypertonic solution, a transitory irregularity of shape.The egg becomes slightly polyhedral but rapidly becomes spherical and the clearsurface layer described above develops. This transitory polyhedral phase soonbecomes more marked and lasts for a longer time. Ten minutes after fertilisationthe plasmolysis is typically polyhedral (Fig. 16).

Fig. 16. Photograph showing polyhedral plasmolysis of eggs20 min. after fertilisation.

The condition which I have called polyhedral plasmolysis is found most typicallyduring that stage in the development of the egg beginning about 10 min. afterfertilisation and ending shortly before cleavage. As a rule the surface of the eggremains free from the small wrinkles which are so characteristic a feature of theplasmolysis of the unfertilised egg. There are several large depressions in thesurface similar to those formed if a ball of clay is pressed between the finger tips.The hyaline plasma layer, which is now present, follows all the irregularities of theegg surface.

The polyhedral type of plasmolysis just described continues to occur until theeggs have reached the stage of development about 15 min. before cleavage. Afterthis it becomes less and less well marked and finally disappears. Just before cleavagebegins the eggs contract smoothly and remain spherical.


PLASMOLYSIS AS A MEASURE OF PERMEABILITY.

Herlant (1918a, 19186) has made use of hypertonic solutions in an attempt toinvestigate the changes in permeability to salts undergone by sea-urchin eggs afterfertilisation. He found that eggs placed in strongly hypertonic solution (100 partssea water + 40 to 45 parts z\ M NaCl) did not become plasmolysed unless theyhad been fertilised 25—30 min. previously. After this stage of development plasmo-lysis became more and more intense and then decreased and disappeared entirelyat the diaster stage. Plasmolysis did not appear again until just after separationof the blastomeres. Plasmolysed eggs were found to be much more resistant to thecytolytic action of the hypertonic solution than those which were not, thus sup-porting the conclusion that plasmolysis is really an indication of decreased per-meability to salts. Runnstrom (1924) found that the eggs of Paracentrotus lividusbecame most strongly plasmolysed 15-20 min. after fertilisation at the period ofgreatest development of the sperm aster. He obtained similar results with Psamtn-echinus miliaris.

In working with cells such as the eggs of the sea urchin it must be realised thatdeformation produced by osmotic removal of water is not a simple problem.The nature and physical properties of the cell surface and of its investing mem-branes must be important factors in determining the behaviour of the cell as awhole in the hypertonic solution. If the egg is surrounded by a thin elastic mem-brane, as is the case before fertilisation, it may be expected to shrink smoothly untilthe membrane is relaxed. After relaxation is complete further decrease in volumewill result in the membrane, if it is attached tightly to the egg surface, beingthrown into wrinkles. The dimensions of these wrinkles will depend mainly on thethickness of the membrane. In a thin membrane the wrinkles will be small andnumerous, while in a thick membrane they will be fewer and larger. Moreover,if the egg is invested by a thick membrane which is almost inelastic or which,being elastic, is not stretched appreciably, distortion will be induced by a relativelysmall decrease in volume. In considering the action of hypertonic solutions onthe shape of the cell it is, therefore, necessary to distinguish between those effectswhich are due to the rate at which dissolved substances can penetrate the surfaceand those for which the physical properties of the superficial part of the cell areresponsible.

The behaviour of the sea-urchin egg at different stages of development is agood example of the necessity for the above considerations.

The unfertilised egg is enclosed within (a) the true surface membrane of thecell or plasma membrane, (b) the vitelline membrane. Both of these are thin andare elastic and stretched to some extent, as is shown by the spherical form of theegg and its ability to shrink appreciably without becoming wrinkled. As has alreadybeen pointed out, if the identity of the vitelline membrane of the unfertilised eggwith the fertilisation membrane is accepted, it is probable that the former is elastic.The form of the plasmolysed unfertilised egg is therefore in accordance with whatis known of the structure and physical properties of its superficial layers.

JEB'IXl 6

82 A. D. HOBSON

During the first 2 or 3 min. after fertilisation the behaviour of the egg inhypertonic solutions is not so easy to explain. The fertilisation membrane has beenseparated from the surface and, since it is extremely permeable to salts, has noinfluence on the form of the egg. The surface of the egg is passing through a periodof radical change, as is shown, for example, by the results obtained with hypotonicsolutions. It is naked and there is no evidence to show that the superficial layerof the cytoplasm is any more rigid than before fertilisation.The wrinkling of the surface of the plasmolysed eggsupports these conclusions. The circular hollow coincideswith the region from which the fertilisation membranefirst arises. Even in eggs remaining in normal sea watera slight flattening can often be seen in this part of theegg, especially if, for some reason, the separation of themembrane is abnormally slow or incomplete. This pheno-menon has also been noted and figured by Hyman (1923)in the eggs of Strongylocentrotusfranciscanus. Fig. 17 shows Fig. 17. Local contractionan outline sketch of a somewhat extreme case found in $££§£%?£ EfiSan egg fertilised in a small volume of water under a separation of fertilisationcoverslip. It seems probable that the surface layer of m

r ^ * J £ S t ipp l ing dia"the egg is less rigid and capable of resisting distortion

in the region surrounding the reception cone and from which the fertilisationmembrane first separates.

Towards the end of this period of about 3 min. following fertilisation thewrinkling becomes distinctly coarser and approaches more or less closely the poly-hedral type. This may indicate a slight increase in the thickness of the solid surfacelayer, but there is no definite evidence bearing on this point.

The second period after fertilisation is that in which the egg, when placed in ahypertonic solution, shrinks smoothly and becomes covered with a clear gelatinouslayer. The appearance of the gelatinous layer may be preceded by a transientphase of slight deformation of the polyhedral type. This becomes more markedand persists longer as the end of this period of development approaches. Whatevermay be the nature and origin of the material composing the gelatinous layer itis evident that it is distinctly elastic, as is shown by the contraction which occurswhen it is ruptured. This property is sufficient to account for the spherical formof the egg in the hypertonic solution.

The third period after fertilisation is characterised by the presence of thehyaline plasma layer over the surface of the egg and by the markedly polyhedralform of the egg when placed in hypertonic solutions. The hyaline plasma layer atthis stage follows closely all the irregularities of the cell surface. It appears to besolid and relatively inelastic. This structure is probably responsible for the polyhedralform of the plasmolysed egg during this period of development. If eggs arefertilised in normal sea water and transferred a few minutes later to calcium-free sea water they develop normally, except for the absence of the hyalineplasma layer. Such eggs, if placed in hypertonic sea water at the appropriate


stage, do not exhibit polyhedral plasmolysis. They shrink smoothly and remainspherical.

As cleavage approaches, the structure of the hyaline plasma layer changes. Itsinner part becomes fluid while the outer layer remains solid. Concurrently withthis change in structure the behaviour of the egg in hypertonic solutions alters.The irregularity of shape so characteristic of the earlier stage of developmentbecomes less and less marked. The egg shrinks smoothly at first and then becomeswrinkled. The hyaline plasma layer remains spherical, surrounding the egg.

The behaviour of the hyaline plasma layer at this stage in the development ofthe egg is well illustrated by the experiments of Gray (1924). He showed that thedistance between the cytoplasm and the outer surface of the hyaline plasma layeris increased if the egg is placed in hypertonic sea water. He considers the hyalineplasma layer at this stage to be composed of an outer solid membrane enclosingfluid material.

Runnstrom (1924) has also noted the change in behaviour of the hyaline plasmalayer. He says: "Erst am Anfang des Diasterstadiums wird die hyaline Schichtbei der Plasmolyse von der Eioberfiache abgehoben und in der letzteren Halftedes Amphiasterstadiums wird die Abhebung der hyalinen Schicht noch hoher."

Sometimes, when the eggs are presumably slightly abnormal, the hyalineplasma layer remains gelated even during cleavage. In such cases plasmolysis ofthe egg remains polyhedral.

As soon as the egg begins to elongate, just before cleavage, the result of treat-ment with hypertonic solutions is to cause the appearance of a groove round theequator corresponding in position with the cleavage groove which would sub-sequently appear in normal sea water. Water seems to be extracted more readilyfrom those parts of the cytoplasm not included in the asters, with the result thatthe form of the plasmolysed egg at this stage is a somewhat distorted picture of theamphiaster.

The account just given of the different types of behaviour of the sea-urchin eggwhen placed in hypertonic solutions at various stages of development will make itclear that plasmolysis does not provide a suitable method for the estimation ofrelative permeability to dissolved substances in this material The changes in thenature and physical properties of the superficial region of the egg are so profoundthat it is impossible to compare the results obtained at different stages in the develop-ment of the eggs.

THE CYTOLYTIC ACTION OF HYPERTONIC SOLUTIONS.

A number of experiments were performed to determine the rate of cytolysisof unfertilised and of fertilised eggs at different stages of development. It washoped in this way to obtain some evidence of the changes in permeability to salts.This method has already been employed by Herlant (1918 a) who placed eggs in amixture of 100 parts of sea water and 40-45 parts of 2|MNaCl. He found thatplasmolysis of fertilised eggs did not occur until 25-30 min. after fertilisation,

6-2

84 A. D. HOBSON

and disappeared from the middle of the diaster stage until the completion ofcleavage. Plasmolysed eggs were characterised by their resistance to the cytolyticaction of the hypertonic solution.

50

Minutes after fertilisationFig, 18. Graph showing the time taken for 50 per cent, of eggs to cytolyse in 50 c.c. sea

water + SO c.c. 2-^M NaCl. For description see text.

In my experiments an even stronger solution was used composed of equalvolumes of sea water and 2 ^ M NaCl. As an estimate of the cytolytic action of thesolution, the time taken for approximately 50 per cent, of the eggs to cytolyse wasmeasured. The results were not very satisfactory, as the eggs seemed to fall into


two categories with regard to their response to the hypertonic solution. Figs. 18and 19 show the results of experiments performed with the eggs of two differentindividuals. In the experiment shown in Fig. 18 the resistance of the unfertilisedeggs was fairly high. Half a minute after fertilisation the resistance fell markedly.It rose to a maximum at 2 min. after fertilisation, and then fell once more to aminimum at 5-6 min. So far the behaviour strongly recalls that found in responseto hypotonic solutions. After this the cytolysis time increased, and it soon becameimpossible to estimate owing to the change in character of the process. In theunfertilised eggs and in the fertilised eggs up to 8-10 min. after fertilisation cyto-lysis takes place suddenly and is characterised by blackening and swelling of theegg. After this period of development is over the cytolytic process changes in

10 15 20Minutes after fertilisation

Fig. 19. Graph showing time taken for 50 per cent, of eggs to cytolyse in 50 c.c. seawater + 50 c.c. z-$M NaCl. For description see text.

character. The swelling of the egg is less marked and darkening of the protoplasmproceeds gradually from the surface inwards. It becomes, in consequence, almostimpossible to measure the cytolysis time. It is clear, however, that the eggs becomemuch more resistant after the susceptible period already noted as being mostintense at about 5 min. after fertilisation.

The other type of response to the cytolytic action of the hypotonic solution isillustrated in Fig. 19. Here the resistance of the eggs is considerably lower than inthe example described above. The behaviour of the eggs is essentially similar,except that the period of resistance at about 2 min. after fertilisation cannot bedetected.

It should be noted that the phase of least resistance in both categories of eggscoincides with the period during which the clear gelatinous layer, already described,forms at the surface of the plasmolysed egg.

86 A. D. HOBSON

THE CYTOLYTIC ACTION OF HYPOTONIC SOLUTIONS.

Changes in the resistance of fertilised eggs to cytolysis in hypotonic solutionhave been described by several authors (Herlant, 1918 c; R. S. Lillie, 19166; Just,1922a, 1922b, 1928a, 19286; Page, 1929). The method usually employed is tomeasure the time taken for cytolysis to occur in the hypotonic solution. Lillie(19166) and Just (19286) have also adopted the method of returning the eggs tonormal sea water after a certain length of exposure to the hypotonic solution anddetermining the percentage of cleavage.

-aG

30 40Minutes after fertilisation

Fig. 20. Graph showing the time for 100 per cent, cytolysis of unfertilisedand of fertilised eggs in tap water.

In the following experiments the time taken for cytolysis to occur in tap waterwas determined. Previous workers using this method have usually used dilutedsea water as the cytolysing medium, although Just has also used distilled or tapwater. It seems preferable in this type of experiment to employ either tap wateror distilled water. The presence of even a comparatively small concentration ofsalts greatly increases the cytolysis time, and, apart from the desirability of a rapidexperimental method, there may be secondary changes occurring in the egg whichmay obscure those which it is required to investigate. The eggs are in any case


being placed in extremely abnormal conditions, and it is therefore desirable toreduce as far as possible the time during which these conditions act.

The eggs were fertilised in normal sea water and transferred to a relativelylarge volume of tap water, and the time in seconds taken for 100 per cent, cytolysis tooccur measured with a stop watch. Fig. 20 shows the results of such an experi-ment. It will be seen that these are essentially in agreement with those .of previousworkers, although certain differences should be noted. The cytolysis time decreasesprogressively until a minimum is reached at about 1 min. after fertilisation. Thisphase presumably corresponds with that which Just (1928 a) found to correspondwith the period of membrane formation in Arbacia and especially in Echinarachnius.After this the cytolysis time begins to increase until it reaches a maximum at about5 min. after fertilisation. This is in agreement with the resistant phase found byPage (1929) 2-6 min. after insemination in Arbacia. The resistant phase is followedby a decrease in the cytolysis time, which reaches its lowest point generally 12-15min. after fertilisation but sometimes sooner.

Only a few experiments were taken up to the time of cleavage. Generally therewas found to be little change in susceptibility at this stage of development of theegg. If anything there was a slight increase in the cytolysis time when the eggswere beginning to elongate. The experiment illustrated was exceptional in showinga markedly decreased susceptibility before cleavage. These results appear to bein agreement with Just's (1918) observation on Arbacia, but I have failed to detectthe period of susceptibility described by him in this paper at the "streak" stageof the aster. It is possible that observations were not taken at sufficiently shortintervals during this period of development. It is noteworthy that Page's (1929)figures do not show any period of special susceptibility before cleavage. He foundthat "as the time for cleavage approaches the eggs become progressively moresusceptible."

DISCUSSION.

The experiments described in the preceding sections of this paper show thatthe egg of Psammechinus miliaris in its relation to altered osmotic conditions of theenvironment is characterised by clearly marked types of behaviour which corre-spond in time with particular phases of development. It is not, however, alwayseasy to see the reason for this correspondence. These phases are summarised inTable II. It must be remembered that the times given in the table are somewhatarbitrary. There is considerable variation in different batches of eggs, but the se-quence of events is always the same.

There can be no doubt that fertilisation is followed almost immediately by aseries of important changes in the structure and properties of the egg surface.These changes begin almost immediately after fertilisation, so far as can be de-termined, but they are not complete until a considerable space of time has elapsed.The first 10 min. after fertilisation are characterised by a somewhat complexseries of changes, the meaning of which cannot so far be determined.

A. D. HOBSON

Table II.

Condition ofsurface of egg

Type of plas-molysis

Resistance tocytolysis byhypertonicsolutions

Rate of swellingin hypotonicsolutions

Resistance tocytolysis bytap water

Approximatetime scale

Un-fertilised

Vitellinemem-brane only

• present

Wrinkled

High

Slow

High

—

Fertilised,phase I

Fertilisation mem-brane elevatedduring earlypart of thisphase

Wrinkled, but morecoarsely, tendingto polyhedral asend of phaseapproaches

Either (i) steadilydecreasing or (2)lower followed bybrief period ofhigher resistance

Fast

Low

0—4 min. afterfertilisation

Fertilised,phase II

No visiblechange

Roughly spheri-cal with clear,gelatinous layeron surface

Lower than inphase I

Slower than inphase I

Higher than inunfertilised


Fertilised,phase III

Hyaline plasmalayer present ingelatinous con-dition

Polyhedral

Low at first butrapidly increas-ing. Cytolysisbecomes grad-ual instead ofsudden

Increases tomaximumwhich ismaintained

Low

10-50 min. afterfertilisation

Fertilised,phase IV

Hyaline plasmalayer now com-posed of fluidenclosed withinsolid outermembrane

Polyhedral typegradually dis-appears

Decreases (thisphase only in-vestigated in oneexperiment)

Increasesslightly


Cleavage assumed to begin 60 min. after fertilisation.

During the first 3 min. after fertilisation the principal morphological changeis the formation of the fertilisation membrane, separation of which is completein healthy eggs in about 60 sec. The mechanical properties of the cell surface donot appear to be profoundly altered during this period, although it takes a slightlysmaller amount of stretching to cause its breakdown. This is shown by the slight butconstant difference in the dilution of sea water necessary to cause cytolysis. Alsothe limit of elasticity is slightly lower; a smaller degree of stretching (induced byincrease in volume of the egg) is necessary to produce wrinkling of the egg surfacewhen the pressure is released.

The tension in the cell surface is probably slightly lower than in the unfertilisedegg, as is shown by the tendency towards distortion which is particularly markedin overripe eggs, especially during the period of elevation of the fertilisationmembrane from the surface of the egg.

The rate at which water enters the egg from a hypotonic solution is definitelyincreased during this period. In the experiment illustrated in Figs. 1 and 2 themaximum volume of water entering the eggs in 2 min. from 50 per cent, sea waterduring this phase is about 154 per cent, of that entering the unfertilised eggs underthe same conditions. This result is borne out by those obtained by studying thetime taken for cytolysis to occur in tap water. Here there is a brief but clearly


marked phase of susceptibility. This phase has also been noted by other authors,especially Just (1928 a). The susceptible phase can also be seen in some of thefigures illustrating Page's (1929) experiments, although he does not call attentionto it. Just (19286) has associated this phase particularly with separation of thefertilisation membrane, since he found that eggs treated with tap water while thisprocess was taking place burst in that region from which the membrane was actuallylifting. It should be noted, however, that the susceptibility continues to increasefor some time after membrane separation is complete. Evidence has already beenpresented which favours the view that the cell surface is more permeable to wateras well as less resistant to mechanical disturbance during this period, and thesechanges may be, at any rate partly, the direct consequence of the process of mem-brane separation.

In the second phase after fertilisation the most conspicuous feature is thedevelopment of a clear, gelatinous layer at the surface of the plasmolysed egg. Thenature of this material was not determined. Its origin lies in the most superficialregion of the egg in which, so long as it remains in normal sea water, no surfacechange can be detected microscopically. It is probably significant that this phaseimmediately precedes that in which the hyaline plasma layer becomes visible.It may be that the material composing the gelatinous layer represents that whichlater forms the hyaline plasma layer, but in this case it must undergo a considerablechange in its mechanical properties. The gelatinous layer is strikingly elastic, whilethe hyaline plasma layer is almost inelastic.

The permeability of the eggs at this stage, as shown by the rate of swelling,is lower than that at the maximum of the first phase, although still considerablyhigher than that of the unfertilised eggs. This relation is not entirely borne outby the results obtained by measuring the cytolysis time in tap water. The eggsare more resistant to cytolysis than before fertilisation. This was also found byPage (1929) in the eggs of Arbacia. He noted that the resistant eggs swell to alarger size before cytolysis than do relatively susceptible eggs. This point is of someimportance, as it shows that caution must be exercised before accepting the cytolysistime in hypotonic solutions as a relative measure of permeability to water.

This phase of development is characterised by a relatively low degree of re-sistance to the cytolytic action of extremely hypertonic solutions. The resistancedoes not, however, reach a minimum until the end of this phase or the beginningof the next. The minimal value may be maintained for a variable length of time,which did not exceed 10 min. in any of the cases studied.

The third phase of development of the fertilised egg as here defined extendsfrom about 10 min. after fertilisation until cleavage. It is characterised morpho-logically by the presence of the hyaline plasma layer surrounding the egg. Atfirst this layer is gelatinous and closely adherent to the surface of the egg, as isshown by the way in which it follows all the irregularities of the plasmolysed egg.The polyhedral type of plasmolysis typical of this phase is due to the inelasticnature of the hyaline plasma layer. When it is absent, as in calcium-free sea water,the egg shrinks much more smoothly. As cleavage approaches, the polyhedral

90 A. D. HOBSON

form of the plasmolysed egg becomes much less marked and the hyaline plasmalayer tends to remain spherical. It is known (Gray, 1924) that, at the time ofcleavage the hyaline plasma layer consists of a solid outer membrane enclosingfluid material. As Prof. Chambers has pointed out to me, Brownian movementcan be seen in the interior of the hyaline plasma layer about the time of the firstcleavage. It seems, then, that in the hyaline plasma layer we have to do with astructure which is at first of a solid, gelatinous nature, but which later becomesfluid except for the outermost part which remains as a solid film. At first, therefore,the form adopted by the plasmolysed egg is regulated by the presence of thisgelatinous, inelastic layer which is firmly attached to the surface. Later the eggis free to shrink in a manner controlled only by the nature of the protoplasm ofwhich it is composed, since, apart from the fertilisation membrane, it is surroundedby a solid membrane which is freely permeable to salts and is separated from thecell surface by a narrow space filled with fluid. The behaviour of the egg underthese conditions is well illustrated by Gray's (1924) figures. The conclusions herepresented are in accordance with those of Gray as given in his recent book (1931).They do not agree with those put forward in his original paper (1924), in whichthe increase in thickness of the hyaline plasma layer (ectoplasm) in hypertonicsolutions was ascribed to swelling of the material of which this layer is composed.

The cytolytic action of hypertonic solutions during this phase of the develop-ment of the egg has already been described. It is doubtful how far the rate at whichcytolysis takes place may be accepted as a measure of the permeability of the cellsurface to salts. Herlant (1918a) pointed out that cytolysis of fertilised eggs inhypertonic solutions occurs more rapidly in eggs which are not readily plasmolysed.It must be emphasised, however, that plasmolysis is most easily induced in thethird phase of the fertilised egg, and that this is probably due largely to the me-chanical effect of the presence of the inelastic, gelatinous, hyaline plasma layercovering the surface. Moreover, as has already been pointed out, the type ofcytolysis is peculiar in that it progresses slowly even after it has begun, instead ofbeing accomplished with almost explosive suddenness.

During phase II and the earliest part of phase III, the resistance to hypertoniccytolysis is very low compared with that found in the unfertilised egg. This is ingeneral agreement with the results obtained by Gray (1916) in measuring theconductivity of egg suspensions. The more recent work of Cole (1928), however,throws some doubt on the conclusion of Gray (1916) and of McClendon (1910)that the electrical resistance of the egg surface decreases after fertilisation.

From the data presented in this paper it is only safe to conclude that fertilisationdecreases the resistance of the egg surface to the destructive effects of high con-centrations of salts in the surrounding medium. The resistance reached a minimumat 10-15 m m - after fertilisation. There is sometimes, but not constantly, a relativelyresistant period in phase I, the conditions for whose occurrence are not known.After the minimum has been reached the resistance tends to rise but at the sametime the situation becomes complicated by a change in the nature of the cytolyticprocess which renders the estimation of its rate of progress a matter of great


difficulty. Further investigation is needed before the factors underlying this changecan be elucidated.

SUMMARY.

1. A photographic method is described for recording volume changes in sea-urchin eggs.

2. The behaviour of the eggs of Psammechinus miliaris, both before and atvarious intervals after fertilisation, in relation to osmotic changes in the surroundingmedium have been investigated.

3. The rate of entrance of water from hypotonic sea water into the egg increasesimmediately after fertilisation takes place, rises to a first maximum at about3 min. after fertilisation. It then falls to a comparatively low value at about 5 min.after fertilisation. After this the rate increases steadily to a maximum value whichis reached about 35 min. after fertilisation. It remains steady until just beforecleavage when, in the single experiment continued until this stage of development,it decreased very markedly.

4. The action of hypertonic solutions on the egg has been examined. Severaltypes of plasmolysis occur and are characteristic of different stages in the develop-ment of the egg after fertilisation. The type of plasmolysis is determined principallyby the physical properties of the egg surface. The plasmolysis method is of littleuse in this material for the determination of relative permeability to dissolvedsubstances at different stages of development.

5. The rate of cytolysis in tap water has been investigated and its relation topermeability of the egg surface to water is considered. There is a susceptible periodfollowed by one of resistance during the first 5-10 min. after fertilisation. The rateof cytolysis is conditioned, not only by the rate of entrance of water but also bythe degree to which the cell surface will withstand stretching. The latter may be asignificant factor.

6. The rate of cytolysis in extremely hypertonic solutions of sea water + NaClhas been examined. It increases to a maximum at about 5-10 min. after fertilisation.Thereafter it decreases. Cytolysis in the unfertilised egg and just after fertilisationis a sudden process. Later it becomes more and more gradual and progressesslowly from the surface to the interior of the egg. The relation between the rate ofcytolysis and permeability is uncertain.

I wish to express my gratitude to the Trustees of the Ray Lankester Investigator-ship, since it was during the tenure of this appointment that this work was done,and to thank Dr E. J. Allen, F.R.S., and the staff of the Laboratory of the MarineBiological Association at Plymouth for their interest and help. I am indebted tothe Earl of Moray Endowment of the University of Edinburgh for a grant coveringpart of the expenses of this research. I wish also to thank my wife, whose continuedassistance has been of the greatest value.

92 A. D. HOBSON

REFERENCES.

COLE, K. S. (1928). Joum. Gen. Physiol. 12, 37.GRAY, J. (1916). Phil. Trans. Roy. Soc. B, 207, 481.

(1924). Proc. Camb. Philosoph. Soc. Biol. Ser. 1, 166.(1931). A Text-Book of Experimental Cytology. Cambridge University Press.

HERLANT, M. (1918a). C.R. Soc. de Biol. 81, 151.(19186). Arch, de Zool. exp. g&n. 57, 511.

HOBSON, A. D. (1927). Proc. Roy. Soc. Edin. 47, 94.(1931). Joum. Exp. Biol. 9, 93.

HYMAN, L. H. (1923). Biol. Bull. 45, 254.JUST, E. E. (1922a). Amer. Joum. Physiol. 61, 516.

(19226). Amer. Joum. Physiol. 61, 505.(1928a). Physiol. Zool. 1,26.(1928&). Protoplasma, 5, 97.

LILLIE, R. S. (1916a). Amer. Journ. Physiol. 40, 249.(19166). Joum. Exp. Zool. 21, 369.(1918). Amer. Joum. Physiol. 45, 406.

LUCK£, B., HARTLINE, H. K. and MCCUTCHEON, M. (1931). Journ. Gen. Physiol. 14, 405.MCCLENDON, J. F. (1910). Amer. Journ. Physiol. 27, 240.MCCUTCHEON, M. and LUCRE, B. (1926). Journ. Gen. Physiol. 9, 697.MCCUTCHEON, M., LUCRE, B. and HARTLINE, H. K. (1931). Journ. Gen. Physiol. 14, 393.NORTHROP, J. H. (1927). Journ. Gen. Physiol. 11, 43.PAGE, I. H. (1929). Brit. Journ. Exp. Biol. 6, 219.RUNNSTROM, J. (1924). Ada Zool. 5, 345.VLES, F. (1926). Arch, de Physique Biol. 4, 263.

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THE EFFEC OTF FERTILISATIO ONN TH PERMEE - ABILITY …jeb.biologists.org/content/jexbio/9/1/69.full.pdf · His method was to measure the diameters of individual eggs at definite intervals