Among the numerous changes in the sea-urchin egg which have been shown by various workers to occur at the time of and subsequent to fertilisation none have attracted more attention than that of increased permeability. R. S. Lillie (1916) showed that fertilisation or artificial activation by means of butyric acid causes the eggs of Arbacia to swell more rapidly when placed in hypotonic sea water. He concluded from his experiments that the surface of the sea-urchin egg becomes more permeable to water as the result of activation.
Assuming that the egg is surrounded by a semi-permeable membrane, Lillie considered that the principal factors controlling the rate of swelling in hypotonic solutions were (1) the difference in osmotic pressure between the cell contents and the surrounding medium, (2) the frictional resistance of the membrane to the passage of water, (3) the area of the membrane. He concluded that “the forces of elasticity, cohesion, and surface tension …are undoubtedly negligible in comparison with osmotic pressure,” and that the behaviour of the egg could be described approximately in terms of the osmotic gradient alone.
Other workers (McCutcheon and Lucké, 1926; Northrop, 1927; McCutcheon, Luck é and Hartline, 1931; Lucké, Hartline and McCutcheon, 1931) have examined the kinetics of swelling of sea-urchin eggs in more detail, and have introduced various corrections into Lillie’s original treatment of the problem. They agree with him, however, in leaving out of consideration the mechanical properties of the cell surface. With the exception of Viés (1926), no attempt has been made to investigate the elastic properties 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 increased permeability of the egg surface to water is now generally accepted and has been supported by the observations of a number of authors (e.g. R. S. Lillie, 1916 b; Herlant, 1918a; Page, 1929) that fertilisation is followed by an increase in the rate of cytolysis in hypotonic solutions. The time taken for cytolysis to take place under such conditions has been considered to be a measure of the resistance of the cell surface to the passage of water. While this may be the factor which is predominant in determining the rate of cytolysis, other conditions, such as the extensibility of the cell surface, may be important and should not be neglected.
The evidence for an increase in permeability to dissolved substances is not so satisfactory. The work of McClendon (1910) and of Gray (1916) indicates a decrease in the electrical resistance of sea-urchin eggs following fertilisation. These authors concluded that this shows an increased permeability to electrolytes. Herlant (1918 a) deduced changes in the permeability in sea-urchin eggs by means of the plasmolysis method. As will be shown later in the present paper, the reaction of fertilised eggs to hypertonic solutions varies in a very striking manner according to the stage of development which has been reached. Moreover, the behaviour of the egg depends largely on the physical properties of its superficial region and not necessarily on its permeability alone.
The time relations of the permeability changes during the period between fertilisation and cleavage are not well known. The sea-urchin egg is especially susceptible to cytolysis by hypotonic solutions while the fertilisation membrane is being 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 the electrical 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 to hypotonic 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 permeability to water following fertilisation have been examined in more detail. The mechanical properties of the surface region of the egg as exhibited by its behaviour in hypertonic and hypotonic solutions have also been investigated, and an attempt has been made to 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 activated eggs swell more rapidly than normal unfertilised eggs when placed in diluted sea water. His method was to measure the diameters of individual eggs at definite intervals by means of a screw eyepiece micrometer. On the assumption that the egg is a sphere, the volume can be calculated from such measurements and a curve showing the rate of increase of size can be plotted. Since the swelling of the egg is presumably due to increase in its water content, it was concluded that the curve obtained in this way gives an indication of the rate at which water can diffuse through the cell surface. This conclusion involves the assumption that the cell membrane is not damaged or fundamentally altered in structure either by the mechanical stretching or by the lowering of salt concentration inherent in the experiment. That this assumption is probably justified is shown by the fact that in 40 per cent, sea water Lillie found that the rate of increase in volume is not appreciably altered for the first few minutes.
Lillie’s method has since been employed by other workers, notably McCutcheon and Lucké (1926), for the investigation of the osmotic properties of cells.
A serious objection to the use of the screw micrometer or to any other method of direct measurement at present available is that a very small number of eggs can be examined simultaneously. Since it is necessary to employ different eggs for different experiments in the same series, relatively gross changes in behavior can alone be detected. In Psammechinus miliaris there may be a considerable variation in the rate of swelling in hypotonic solutions of individual eggs obtained from the same female and subjected to conditions as nearly as possible identical. Moreover the speed with which experiments can be performed is important. It was found that eggs which have remained for a long time in sea water may swell at a markedly slower rate than when first removed from the female.
For these reasons among others the method of making direct measurements of 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 instrument cinematograph film is used. This has a number of advantages. The film (Perutz Leica Special) is fast and fine grained, enabling short exposures to be used and giving fine detail. The time and necessary disturbance of the apparatus involved in changing plates is avoided. At least 36 exposures can be made without changing the film. Lastly, the cheapness of the film compared with plates is not its smallest advantage.
A Pointolite lamp was usually employed as the source of illumination. A No. 2 Leitz objective and a × 10 or × 12 ocular was used. After development the film was placed in a simple, vertical projection apparatus and the negative images were measured on squared paper. The apparatus was calibrated by photographing the scale of a stage micrometer. The apparatus gave a total magnification of about 200 diameters.
The eggs were first photographed lying in a flat-bottomed dish in normal sea water. A small sample of eggs with as little sea water as possible was then transferred to another dish containing the diluted sea water. One min. after the transference the first photograph was taken and thereafter exposures were made at intervals of 1 min. usually for a period of 10 min. In this way the behaviour of any number up to about 40 eggs could be followed simultaneously.
In presenting the results of these experiments I have endeavoured, for the present, 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 swelling curves in 50 per cent, sea water for unfertilised and for fertilised eggs from the same female, taken at different intervals after insemination. Fig. 2, which illustrates the 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 volume of distilled water). In Fig. 1, in order to economise space, are illustrated only 6 out of the total number of 16 swelling curves measured in the course of the experiment.
It will be seen that the changes in permeability of the surface of the eggs to water after fertilisation are not simple. In the experiment illustrated in Figs. 1 and 2 the rate of swelling increased rapidly after fertilisation, reaching a maximum 3 min. after insemination. Five min. after insemination the rate of swelling was markedly slower. After this the rate increased fairly steadily, reaching a maximum at 36-40 min. after insemination. After this the rate remained approximately uniform until 60-65 min. after fertilisation. The experiment was discontinued after 75 min. as cleavage began.
A somewhat unexpected feature of this experiment is the sharp decrease in permeability preceding cleavage. It is necessary that this should be confirmed by further work, as only one of the experiments performed to examine the rate of swelling 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 the curve for eggs 3 min. after fertilisation is not continued for more than 3 min. after the eggs were placed in the hypotonic sea water. This is because the eggs are at this stage very susceptible to the cytolysing action of hypotonic solutions. Out of 38 eggs at the beginning of the experiment 22 had burst by the time the third photograph was 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 following fertilisation, in which it is relatively resistant to hypotonic solutions, swells to a greater extent before cytolysing than during a phase of susceptibility. Fig. 1 illustrates this point in Psammechinus miliaris. The swelling of eggs placed in the hypotonic solution 3 min. after fertilisation could not be determined for more than 3 min., since such a large proportion had undergone cytolysis. The average volume of the eggs remaining intact at the end of 3 min. was about 14 × 105μ3. This represents 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.
In other experiments eggs were placed in a series of dilutions of sea water and examined after about i hour. The proportion of cytolysis was determined approximately and the eggs were then returned to normal sea water. It was found that those eggs whose volume had been increased almost to the bursting point showed a wrinkled surface when returned to normal sea water. The limit of elasticity is therefore somewhat lower than the breaking point. Table I shows the result of such an experiment. There is a slight but constant variation in resistance which agrees with the results mentioned in the previous paragraph. During the susceptible period immediately following fertilisation the eggs cytolyse as the result of a smaller increase in volume than when they are more resistant. The change in the mechanical properties of the cell surface is also illustrated by the failure to recover from a smaller degree of stretching, even if this does not reach the breaking point. It may be noted that a marked degree of wrinkling of the surface on return to normal sea water is always followed by cytolysis. Recovery is only possible if shrinkage is accompanied by a smooth or only very faintly wrinkled surface.
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 previous paper. The essential features may be briefly recapitulated. The egg swells uniformly up to a certain point and then a slight bulge appears on one side. Over this area the surface is slightly irregular. The cytoplasm becomes clearer in the region near the bulge, and the granules become much less numerous (Fig. 3). This change spreads over the whole of the cytoplasm and at the same time the surface of the egg becomes perfectly smooth and spherical (Fig. 4). At no stage is there any visible outflow of the contents of the egg.
After fertilisation the type of cytolysis changes. Half a minute after insemination the behaviour is similar to that of unfertilised eggs. One minute after insemination cytolysis begins as a bulge occurring on one side only of the egg. It is similar to that found in the unfertilised egg but more pronounced (Fig. 5). Two minutes after insemination the bulge is still more marked (Fig. 6). After this an increasing number of eggs burst at two or more points on the surface (Fig. 7). After cytolysis is complete the egg does not return to its original spherical form as was the case before fertilisation.
The bulges in the fertilised egg appear to be due to a local breakdown of the cell surface. The cytoplasm flows out at these points but gelates rapidly. There is no visible scattering of the cell contents. The surface of the egg is irregular over the areas of outflow but preserves its original smooth contour elsewhere.
There is thus a striking difference between the behaviour of both unfertilised and fertilised sea-urchin eggs and the unfertilised eggs of Teredo described in a subsequent paper (Hobson, 1931). In the latter the egg bursts at one point and the contents flow out and disperse, leaving behind only a crumpled vitelline membrane, which is stout and comparatively inextensible. This probably resembles more closely the condition found in the fertilised sea-urchin egg if allowance is made for the fact that the cytoplasm of the Teredo egg disperses while that of the seaurchin egg does not. The behaviour of both is what would be expected if the egg were surrounded by a layer which was capable of only slight extension. In such circumstances the tendency for the egg to swell must be compensated either by the rapid diffusion of substances into the surrounding medium or by the surface layer rupturing at one or more points. The behaviour of the egg of Teredo in hypertonic solutions (Hobson, 1931) shows that the vitelline membrane is permeable to salts as is the case with the fertilisation membrane in the sea-urchin. It is probable also that the hyaline plasma layer (“ectoplasm” of Gray) is also readily permeable to salts in spite of Just’s assertion (1928) that “the mobile hyaline plasma layer is the plasma membrane of the egg regulating exchange with the environment.” Nevertheless these structures will not permit the rapid diffusion of the more complex substances which must be present in the egg. Since, therefore, the osmotic pressure of the contents of the egg cannot be lowered, at any rate sufficiently rapidly, by exosmosis, the surface layer must rupture when it has been stretched to its breaking point.
In the unfertilised sea-urchin egg the type of cytolysis may be explained if it is assumed 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 which is both extensible and elastic and which is readily penetrated by salts (Hobson, 1927). We may therefore interpret the behaviour of the egg when immersed in tap water as follows. The egg swells uniformly up to the point at which the surface region of the cytoplasm beneath the vitelline membrane (fertilisation membrane) is no longer capable of further extension. Whether this surface region of the cytoplasm is the plasma membrane or the cortex does not matter for the present argument. When the surface of the cytoplasm gives way on one side of the egg the resistance to the internal pressure is no longer uniform and a slight bulge is formed in the vitelline membrane. Salts diffuse out of the egg and the osmotic pressure of the contents thus becomes lowered. Finally the elasticity of the vitelline membrane 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 osmotic pressure was raised by addition of varying amounts of 2·4 M NaCl or 1·6M glycerol. All the solutions gave essentially similar results as regards the form adopted by the plasmolysed egg.
Among the most striking of the differences between fertilised and unfertilised eggs are those which occur in response to hypertonic solutions. Moreover, the fertilised eggs are found to vary in their behaviour in these circumstances in an equally remarkable manner at different stages during the period between fertilisation and cleavage. It will be best to describe first of all the types of plasmolysis found in 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 its contraction. Then fine wrinkles begin to appear all over its surface and these become more sharply defined as the egg becomes more strongly plasmolysed (Fig. 8). The shape remains roughly spherical.
As soon as fertilisation has occurred the behaviour of the egg in hypertonic solutions changes. The wrinkling of the surface becomes, as a rule, somewhat coarser and the shape is more irregular than that of the unfertilised egg. Often there is on one side a deep hollow (Fig, 9). If the fertilisation membrane has become completely separated from the egg surface before immersion in the hypertonic solution it behaves normally, except that its diameter is less than is found in normal sea 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 inhibited by the hypertonic solution. In this case the surface of the egg may be drawn out to a fine point which remains attached to the fertilisation membrane. Over this region the fertilisation membrane is somewhat flattened (Fig. 10).
The description given above applies to eggs which have been fertilised for not more than about 1 min. (at I7°-i8° C.). Two minutes after fertilisation the plasmolysed egg exhibits much coarser wrinkles which may take the form of prominent ridges bounding concave areas of the cell surface (Fig. 11). These concave areas are usually small compared with those found in eggs exposed to the hypertonic solution at a later stage of development. This type of plasmolysis, which is exhibited in 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 Teredo (1931) and termed “polyhedral.” Its appearance in the eggs of Psammechinus miliaris is probably determined by physical conditions at the surface in some respects similar to those found in Teredo. Generally the type of plasmolysis found in the eggs of Psammechinus tested 2 min. after fertilisation is intermediate between the “wrinkled” and the “polyhedral.”
Three minutes after fertilisation the response of the egg to treatment with hypertonic solutions undergoes an abrupt change. The egg remains perfectly spherical, except that there may be a slight initial wrinkling of the surface which passes off as the egg contracts further. A clear layer now appears over the whole of the egg surface. The thickness of this layer varies with the concentration of the solution employed. Sometimes its outer surface is smooth and at others the layer seems 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 number of 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 in strong hypertonic solution (50 c.c. normal sea water + 50 c.c. sea water evaporated to about 35 per cent, of its original volume) 5 min. after fertilisation. The clear layer originally covered the whole surface of the egg but it ruptured at one point. Its elastic, gelatinous nature is shown by the way in which it has contracted, exposing a considerable area of the surface of the granular protoplasm, and also by the irregular nature of the torn surfaces.
The behaviour of such eggs when they are returned to normal sea water is also interesting. 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 egg has been exposed to a fairly strong hypertonic sea water (50 c.c. normal sea water + 25 c.c. 24M NaCl) exovates may be formed on return to normal sea water. The outer part of the cytoplasm is apparently gelated and can be distinguished from the inner part by its coarser granulation. As the egg swells the clear layer first ruptures and contracts. Almost at the same time the gelated outer part of the cytoplasm also bursts and the inner, finely granular part is squeezed out, forming a well-marked exovate (Fig. 15) which may become completely separated; the egg then bears a close superficial resemblance to a 2-cell stage, the material of the clear layer 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 cortical part is covered by a thin, transparent, usually irregular layer beneath which is the granular 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 microscope the first stage seems to be that the surface of the granular cytoplasm becomes irregular and withdraws, leaving behind a clear zone with a smooth outer surface. The irregularities of the cytoplasmic surface become more pronounced and develop into somewhat ill defined radial processes. If the hypertonic solution is strong and the reaction of the egg consequently extreme, these processes can no longer be made 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 disappearance is 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 clear surface layer described above develops. This transitory polyhedral phase soon becomes more marked and lasts for a longer time. Ten minutes after fertilisation the plasmolysis is typically polyhedral (Fig. 16).
The condition which I have called polyhedral plasmolysis is found most typically during that stage in the development of the egg beginning about 10 min. after fertilisation and ending shortly before cleavage. As a rule the surface of the egg remains free from the small wrinkles which are so characteristic a feature of the plasmolysis of the unfertilised egg. There are several large depressions in the surface 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 the egg surface.
The polyhedral type of plasmolysis just described continues to occur until the eggs have reached the stage of development about 15 min. before cleavage. After this it becomes less and less well marked and finally disappears. Just before cleavage begins the eggs contract smoothly and remain spherical.
PLASMOLYSIS AS A MEASURE OF PERMEABILITY
Herlant (1918a, 1918 b) has made use of hypertonic solutions in an attempt to investigate the changes in permeability to salts undergone by sea-urchin eggs after fertilisation. He found that eggs placed in strongly hypertonic solution (100 parts sea water + 40 to 45 parts M NaCl) did not become plasmolysed unless they had been fertilised 25-30 min. previously. After this stage of development plasmolysis became more and more intense and then decreased and disappeared entirely at the diaster stage. Plasmolysis did not appear again until just after separation of the blastomeres. Plasmolysed eggs were found to be much more resistant to the cytolytic action of the hypertonic solution than those which were not, thus supporting the conclusion that plasmolysis is really an indication of decreased permeability to salts. Runnström (1924) found that the eggs of Paracentrotus lividus became most strongly plasmolysed 15-20 min. after fertilisation at the period of greatest development of the sperm aster. He obtained similar results with Psammechinus miliaris.
In working with cells such as the eggs of the sea urchin it must be realised that deformation produced by osmotic removal of water is not a simple problem. The nature and physical properties of the cell surface and of its investing membranes must be important factors in determining the behaviour of the cell as a whole in the hypertonic solution. If the egg is surrounded by a thin elastic membrane, as is the case before fertilisation, it may be expected to shrink smoothly until the membrane is relaxed. After relaxation is complete further decrease in volume will result in the membrane, if it is attached tightly to the egg surface, being thrown into wrinkles. The dimensions of these wrinkles will depend mainly on the thickness of the membrane. In a thin membrane the wrinkles will be small and numerous, 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 relatively small decrease in volume. In considering the action of hypertonic solutions on the shape of the cell it is, therefore, necessary to distinguish between those effects which are due to the rate at which dissolved substances can penetrate the surface and those for which the physical properties of the superficial part of the cell are responsible.
The behaviour of the sea-urchin egg at different stages of development is a good example of the necessity for the above considerations.
The unfertilised egg is enclosed within (a) the true surface membrane of the cell or plasma membrane, (b) the vitelline membrane. Both of these are thin and are elastic and stretched to some extent, as is shown by the spherical form of the egg and its ability to shrink appreciably without becoming wrinkled. As has already been pointed out, if the identity of the vitelline membrane of the unfertilised egg with 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 what is known of the structure and physical properties of its superficial layers.
During the first 2 or 3 min. after fertilisation the behaviour of the egg in hypertonic solutions is not so easy to explain. The fertilisation membrane has been separated from the surface and, since it is extremely permeable to salts, has no influence on the form of the egg. The surface of the egg is passing through a period of radical change, as is shown, for example, by the results obtained with hypotonic solutions. It is naked and there is no evidence to show that the superficial layer of the cytoplasm is any more rigid than before fertilisation. The wrinkling of the surface of the plasmolysed egg supports these conclusions. The circular hollow coincides with the region from which the fertilisation membrane first arises. Even in eggs remaining in normal sea water a slight flattening can often be seen in this part of the egg, especially if, for some reason, the separation of the membrane is abnormally slow or incomplete. This phenomenon has also been noted and figured by Hyman (1923) in the eggs of Strongylocentrotus franciscanus. Fig. 17 shows an outline sketch of a somewhat extreme case found in an egg fertilised in a small volume of water under a coverslip. It seems probable that the surface layer of the egg is less rigid and capable of resisting distortion in the region surrounding the reception cone and from which the fertilisation membrane first separates.
Towards the end of this period of about 3 min. following fertilisation the wrinkling becomes distinctly coarser and approaches more or less closely the polyhedral type. This may indicate a slight increase in the thickness of the solid surface layer, but there is no definite evidence bearing on this point.
The second period after fertilisation is that in which the egg, when placed in a hypertonic solution, shrinks smoothly and becomes covered with a clear gelatinous layer. The appearance of the gelatinous layer may be preceded by a transient phase of slight deformation of the polyhedral type. This becomes more marked and persists longer as the end of this period of development approaches. Whatever may be the nature and origin of the material composing the gelatinous layer it is evident that it is distinctly elastic, as is shown by the contraction which occurs when it is ruptured. This property is sufficient to account for the spherical form of the egg in the hypertonic solution.
The third period after fertilisation is characterised by the presence of the hyaline plasma layer over the surface of the egg and by the markedly polyhedral form of the egg when placed in hypertonic solutions. The hyaline plasma layer at this stage follows closely all the irregularities of the cell surface. It appears to be solid and relatively inelastic. This structure is probably responsible for the polyhedral form of the plasmolysed egg during this period of development. If eggs are fertilised in normal sea water and transferred a few minutes later to calcium-free sea water they develop normally, except for the absence of the hyaline plasma layer. Such eggs, if placed in hypertonic sea water at the appropriate stage, do not exhibit polyhedral plasmolysis. They shrink smoothly and remain spherical.
As cleavage approaches, the structure of the hyaline plasma layer changes. Its inner part becomes fluid while the outer layer remains solid. Concurrently with this change in structure the behaviour of the egg in hypertonic solutions alters. The irregularity of shape so characteristic of the earlier stage of development becomes less and less marked. The egg shrinks smoothly at first and then becomes wrinkled. The hyaline plasma layer remains spherical, surrounding the egg.
The behaviour of the hyaline plasma layer at this stage in the development of the egg is well illustrated by the experiments of Gray (1924). He showed that the distance between the cytoplasm and the outer surface of the hyaline plasma layer is increased if the egg is placed in hypertonic sea water. He considers the hyaline plasma layer at this stage to be composed of an outer solid membrane enclosing fluid material.
Runnström (1924) has also noted the change in behaviour of the hyaline plasma layer. He says: “Erst am Anfang des Diasterstadiums wird die hyaline Schicht bei der Plasmolyse von der Eioberfläche abgehoben und in der letzteren Hälfte des Amphiasterstadiums wird die Abhebung der hyalinen Schicht noch höher.”
Sometimes, when the eggs are presumably slightly abnormal, the hyaline plasma layer remains gelated even during cleavage. In such cases plasmolysis of the egg remains polyhedral.
As soon as the egg begins to elongate, just before cleavage, the result of treatment with hypertonic solutions is to cause the appearance of a groove round the equator corresponding in position with the cleavage groove which would subsequently appear in normal sea water. Water seems to be extracted more readily from those parts of the cytoplasm not included in the asters, with the result that the form of the plasmolysed egg at this stage is a somewhat distorted picture of the amphiaster.
The account just given of the different types of behaviour of the sea-urchin egg when placed in hypertonic solutions at various stages of development will make it clear that plasmolysis does not provide a suitable method for the estimation of relative permeability to dissolved substances in this material, The changes in the nature and physical properties of the superficial region of the egg are so profound that it is impossible to compare the results obtained at different stages in the development of the eggs.
THE CYTOLYTIC ACTION OF HYPERTONIC SOLUTIONS
A number of experiments were performed to determine the rate of cytolysis of unfertilised and of fertilised eggs at different stages of development. It was hoped 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 a mixture of 100 parts of sea water and 40-45 parts of M NaCl. He found that plasmolysis of fertilised eggs did not occur until 25-30 min. after fertilisation, and disappeared from the middle of the diaster stage until the completion of cleavage. Plasmolysed eggs were characterised by their resistance to the cytolytic action of the hypertonic solution.
In my experiments an even stronger solution was used composed of equal volumes of sea water and 2.4 M NaCl. As an estimate of the cytolytic action of the solution, the time taken for approximately 50 per cent, of the eggs to cytolyse was measured. 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. 18 and 19 show the results of experiments performed with the eggs of two different individuals. In the experiment shown in Fig. 18 the resistance of the unfertilised eggs 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 a minimum at 5-6 min. So far the behaviour strongly recalls that found in response to hypotonic solutions. After this the cytolysis time increased, and it soon became impossible to estimate owing to the change in character of the process. In the unfertilised eggs and in the fertilised eggs up to 8-10 min. after fertilisation cytolysis takes place suddenly and is characterised by blackening and swelling of the egg. After this period of development is over the cytolytic process changes in character. The swelling of the egg is less marked and darkening of the protoplasm proceeds gradually from the surface inwards. It becomes, in consequence, almost impossible to measure the cytolysis time. It is clear, however, that the eggs become much more resistant after the susceptible period already noted as being most intense at about 5 min. after fertilisation.
The other type of response to the cytolytic action of the hypotonic solution is illustrated in Fig. 19. Here the resistance of the eggs is considerably lower than in the example described above. The behaviour of the eggs is essentially similar, except that the period of resistance at about 2 min. after fertilisation cannot be detected.
It should be noted that the phase of least resistance in both categories of eggs coincides with the period during which the clear gelatinous layer, already described, forms at the surface of the plasmolysed egg.
THE CYTOLYTIC ACTION OF HYPOTONIC SOLUTIONS
Changes in the resistance of fertilised eggs to cytolysis in hypotonic solution have been described by several authors (Herlant, 1918 a; R. S. Lillie, 1916 b;,Just, 1922a, 1922b, 1928a, 1928b; Page, 1929). The method usually employed is to measure the time taken for cytolysis to occur in the hypotonic solution. Lillie (1916 b) and Just (1928 b) have also adopted the method of returning the eggs to normal sea water after a certain length of exposure to the hypotonic solution and determining the percentage of cleavage.
In the following experiments the time taken for cytolysis to occur in tap water was determined. Previous workers using this method have usually used diluted sea water as the cytolysing medium, although Just has also used distilled or tap water. It seems preferable in this type of experiment to employ either tap water or distilled water. The presence of even a comparatively small concentration of salts greatly increases the cytolysis time, and, apart from the desirability of a rapid experimental method, there may be secondary changes occurring in the egg which may 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 to reduce as far as possible the time during which these conditions act.
The eggs were fertilised in normal sea water and transferred to a relatively large volume of tap water, and the time in seconds taken for 100 per cent, cytolysis to occur measured with a stop watch. Fig. 20 shows the results of such an experiment. It will be seen that these are essentially in agreement with those of previous workers, although certain differences should be noted. The cytolysis time decreases progressively until a minimum is reached at about 1 min. after fertilisation. This phase presumably corresponds with that which Just (1928 a) found to correspond with 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 about 5 min. after fertilisation. This is in agreement with the resistant phase found by Page (1929) 2-6 min. after insemination in Arbacia. The resistant phase is followed by a decrease in the cytolysis time, which reaches its lowest point generally 12-15 min. after fertilisation but sometimes sooner.
Only a few experiments were taken up to the time of cleavage. Generally there was found to be little change in susceptibility at this stage of development of the egg. If anything there was a slight increase in the cytolysis time when the eggs were beginning to elongate. The experiment illustrated was exceptional in showing a markedly decreased susceptibility before cleavage. These results appear to be in agreement with Just’s (1918) observation on Arbacia, but I have failed to detect the period of susceptibility described by him in this paper at the “streak” stage of the aster. It is possible that observations were not taken at sufficiently short intervals during this period of development. It is noteworthy that Page’s (1929) figures do not show any period of special susceptibility before cleavage. He found that “as the time for cleavage approaches the eggs become progressively more susceptible.”
The experiments described in the preceding sections of this paper show that the egg of Psammechinus miliaris in its relation to altered osmotic conditions of the environment is characterised by clearly marked types of behaviour which correspond in time with particular phases of development. It is not, however, always easy to see the reason for this correspondence. These phases are summarised in Table II. It must be remembered that the times given in the table are somewhat arbitrary. There is considerable variation in different batches of eggs, but the sequence of events is always the same.
There can be no doubt that fertilisation is followed almost immediately by a series of important changes in the structure and properties of the egg surface. These changes begin almost immediately after fertilisation, so far as can be determined, but they are not complete until a considerable space of time has elapsed. The first 10 min. after fertilisation are characterised by a somewhat complex series of changes, the meaning of which cannot so far be determined.
During the first 3 min. after fertilisation the principal morphological change is the formation of the fertilisation membrane, separation of which is complete in healthy eggs in about 60 sec. The mechanical properties of the cell surface do not appear to be profoundly altered during this period, although it takes a slightly smaller amount of stretching to cause its breakdown. This is shown by the slight but constant difference in the dilution of sea water necessary to cause cytolysis. Also the limit of elasticity is slightly lower ; a smaller degree of stretching (induced by increase in volume of the egg) is necessary to produce wrinkling of the egg surface when the pressure is released.
The tension in the cell surface is probably slightly lower than in the unfertilised egg, as is shown by the tendency towards distortion which is particularly marked in overripe eggs, especially during the period of elevation of the fertilisation membrane from the surface of the egg.
The rate at which water enters the egg from a hypotonic solution is definitely increased during this period. In the experiment illustrated in Figs. 1 and 2 the maximum volume of water entering the eggs in 2 min. from 50 per cent, sea water during this phase is about 154 per cent, of that entering the unfertilised eggs under the same conditions. This result is borne out by those obtained by studying the time 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 the figures illustrating Page’s (1929) experiments, although he does not call attention to it. Just (1928 b) has associated this phase particularly with separation of the fertilisation membrane, since he found that eggs treated with tap water while this process was taking place burst in that region from which the membrane was actually lifting. It should be noted, however, that the susceptibility continues to increase for some time after membrane separation is complete. Evidence has already been presented which favours the view that the cell surface is more permeable to water as well as less resistant to mechanical disturbance during this period, and these changes may be, at any rate partly, the direct consequence of the process of membrane separation.
In the second phase after fertilisation the most conspicuous feature is the development of a clear, gelatinous layer at the surface of the plasmolysed egg. The nature of this material was not determined. Its origin lies in the most superficial region of the egg in which, so long as it remains in normal sea water, no surface change can be detected microscopically. It is probably significant that this phase immediately precedes that in which the hyaline plasma layer becomes visible. It may be that the material composing the gelatinous layer represents that which later forms the hyaline plasma layer, but in this case it must undergo a considerable change in its mechanical properties. The gelatinous layer is strikingly elastic, while the 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 considerably higher than that of the unfertilised eggs. This relation is not entirely borne out by the results obtained by measuring the cytolysis time in tap water. The eggs are more resistant to cytolysis than before fertilisation. This was also found by Page (1929) in the eggs of Arbacia. He noted that the resistant eggs swell to a larger size before cytolysis than do relatively susceptible eggs. This point is of some importance, as it shows that caution must be exercised before accepting the cytolysis time in hypotonic solutions as a relative measure of permeability to water.
This phase of development is characterised by a relatively low degree of resistance to the cytolytic action of extremely hypertonic solutions. The resistance does not, however, reach a minimum until the end of this phase or the beginning of 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 extends from about 10 min. after fertilisation until cleavage. It is characterised morphologically by the presence of the hyaline plasma layer surrounding the egg. At first this layer is gelatinous and closely adherent to the surface of the egg, as is shown 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 inelastic nature 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 form of the plasmolysed egg becomes much less marked and the hyaline plasma layer tends to remain spherical. It is known (Gray, 1924) that, at the time of cleavage the hyaline plasma layer consists of a solid outer membrane enclosing fluid material. As Prof. Chambers has pointed out to me, Brownian movement can be seen in the interior of the hyaline plasma layer about the time of the first cleavage. It seems, then, that in the hyaline plasma layer we have to do with a structure which is at first of a solid, gelatinous nature, but which later becomes fluid 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 this gelatinous, inelastic layer which is firmly attached to the surface. Later the egg is free to shrink in a manner controlled only by the nature of the protoplasm of which it is composed, since, apart from the fertilisation membrane, it is surrounded by a solid membrane which is freely permeable to salts and is separated from the cell surface by a narrow space filled with fluid. The behaviour of the egg under these conditions is well illustrated by Gray’s (1924) figures. The conclusions here presented 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 which the increase in thickness of the hyaline plasma layer (ectoplasm) in hypertonic solutions was ascribed to swelling of the material of which this layer is composed.
The cytolytic action of hypertonic solutions during this phase of the development of the egg has already been described. It is doubtful how far the rate at which cytolysis takes place may be accepted as a measure of the permeability of the cell surface to salts. Herlant (1918a) pointed out that cytolysis of fertilised eggs in hypertonic solutions occurs more rapidly in eggs which are not readily plasmolysed. It must be emphasised, however, that plasmolysis is most easily induced in the third phase of the fertilised egg, and that this is probably due largely to the mechanical effect of the presence of the inelastic, gelatinous, hyaline plasma layer covering the surface. Moreover, as has already been pointed out, the type of cytolysis is peculiar in that it progresses slowly even after it has begun, instead of being accomplished with almost explosive suddenness.
During phase II and the earliest part of phase III, the resistance to hypertonic cytolysis is very low compared with that found in the unfertilised egg. This is in general agreement with the results obtained by Gray (1916) in measuring the conductivity 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 fertilisation decreases the resistance of the egg surface to the destructive effects of high concentrations of salts in the surrounding medium. The resistance reached a minimum at 10-15 min. after fertilisation. There is sometimes, but not constantly, a relatively resistant 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 same time the situation becomes complicated by a change in the nature of the cytolytic process which renders the estimation of its rate of progress a matter of great difficulty. Further investigation is needed before the factors underlying this change can be elucidated.
A photographic method is described for recording volume changes in sea-urchin eggs.
The behaviour of the eggs of Psammechinus miliaris, both before and at various intervals after fertilisation, in relation to osmotic changes in the surrounding medium have been investigated.
The rate of entrance of water from hypotonic sea water into the egg increases immediately after fertilisation takes place, rises to a first maximum at about 3 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 which is reached about 35 min. after fertilisation. It remains steady until just before cleavage when, in the single experiment continued until this stage of development, it decreased very markedly.
The action of hypertonic solutions on the egg has been examined. Several types of plasmolysis occur and are characteristic of different stages in the development of the egg after fertilisation. The type of plasmolysis is determined principally by the physical properties of the egg surface. The plasmolysis method is of little use in this material for the determination of relative permeability to dissolved substances at different stages of development.
The rate of cytolysis in tap water has been investigated and its relation to permeability of the egg surface to water is considered. There is a susceptible period followed by one of resistance during the first 5-10 min. after fertilisation. The rate of cytolysis is conditioned, not only by the rate of entrance of water but also by the degree to which the cell surface will withstand stretching. The latter may be a significant factor.
The rate of cytolysis in extremely hypertonic solutions of sea water + NaCl has 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 fertilisation is a sudden process. Later it becomes more and more gradual and progresses slowly from the surface to the interior of the egg. The relation between the rate of cytolysis 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 Marine Biological Association at Plymouth for their interest and help. I am indebted to the Earl of Moray Endowment of the University of Edinburgh for a grant covering part of the expenses of this research. I wish also to thank my wife, whose continued assistance has been of the greatest value.
The Plymouth tap water is collected from the granite district of Dartmoor and is almost free from dissolved salts.