69
THE EFFECT OF FERTILISATION ON THE PERMEABILITY TO WATER AND ON CERTAIN OTHER
PROPERTIES OF THE SURFACE OF THE EGG
OF PSAMMECHINUS MILIARIS
BY A. D. HOBSON, M.A.
(Lecturer in Experimental Zoology, University of Edinburgh, and Ray
Lankester Investigator at the Marine Biological Laboratory, Plymouth.)
(Received 8th September, 1931.)
(With Twenty Text-figures.)
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 Lucke, 1926; Northrop, 1927; McCutcheon,
Lucke and Hartline ,1931; Lucke, 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 Vies (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, 19166;
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
AMONG
jo
A. D. HOBSON
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 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 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 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 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 behaviour
1800 -
1800 -
1700 -
1700
1600
1500
1400
1300
1200
1100
1000
90i
900
4
5
Minutes
6
8
9
1&
4
5
6
10
Minutes
Fig. 1. Graph showing typical swelling curves of unfertilised eggs and of eggs at certain times
after 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 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
72
A. D. HOBSON
(Perutz Leica Special) is fast andfinegrained, 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.
600 -
10
60
30
40
50
Minutes after fertilisation
70
80
Fig. 2. Graph showing the average volume of water entering an egg before and at various times
after 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. A
No. 2 Leitz objective and a x 10 or x 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 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 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 x io5/x3. 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.
A. D. HOBSON
74
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.
Table I.
Minutes
after
fertilisation
o (unfertilised)
i
3
5
IO
IS
2O
25
%
sea
water
8o%
sea
water
sea
water
o
o
<S
<S
o
o
<S
<S
o
o
<S
<S
<s
<5
<s
<s
<s
<s
90%
<S
<S
Maximum concentration of
sea water which
caused appearance of wrinkles
20%
sea in eggs returned to
water normal sea water
Cytolysis of eggs after i hr. in dilutions of sea water
70%
6o%
sea
water
5o%
sea
water
40%
sea
water
o
0
70
IO
85
100
<S
10
SO
<S
<5
75
<s
<5
<s
<s
<5
<5
<S
<S
<S
50
60
10
100
100
<S
80
30%
sea
water
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
f6o0o4
%
40%
40%
40%
50%
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 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.
1
The Plymouth tap water is collected from the granite district of Dartmoor and is almost
free from dissolved salts.
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
75
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
Fig. 3.
Fig. 4.
Fig. 3. Unfertilised egg cytolysing in tap water. Note the wrinkled surface and the clearer appearance of the cytolysed portion of the egg. Stippling diagrammatic and merely represents relative
distribution 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 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
76
A. D. HOBSON
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
Fig. 6.
Fig. 7.
Fig. 5Fig. 5. Complete cytolysis of egg placed in tap water 1 min. after fertilisation. A slight bulge with
irregular 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 the
point 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 surface
broke 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 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 z-\M NaCl or i-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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
77
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
Fig. 9.
Fig. 8. Photograph of plasmolysed unfertilised egg. Hypertonic solution composed of 50 c.c. sea
water + 25 c.c. 2'4MNaCl.
Fig. 9. Plasmolysis of egg 1 min. after fertilisation. Hypertonic solution composed of 50 c.c. sea
water + 25 c.c. 2'4MNaCl.
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
i§
A. D. HOBSON
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."
Fig. 10.
Fig. 11.
Fig. 10. Inhibition of membrane separation in egg placed in hypertonic solution half a minute
after 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 and
contracted, exposing the surface of the cytoplasm. Stippling diagrammatic.
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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
79
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
4- 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- 15Fig. 14. Egg returned to sea water after exposure to hypertonic solution 5 min. subsequent to
fertilisation. Two stages in the contraction of the gelatinous layer to form a rounded mass on one side
of the egg. Stippling diagrammatic.
Fig. 15. Egg returned to sea water after exposure to hypertonic solution 5 min. subsequent to
fertilisation. 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 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
So
A. D. HOBSON
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).
Fig. 16. Photograph showing polyhedral plasmolysis of eggs
20 min. after fertilisation.
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 andfinallydisappears. Just before cleavage
begins the eggs contract smoothly and remain spherical.
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
81
PLASMOLYSIS AS A MEASURE OF PERMEABILITY.
Herlant (1918a, 19186) 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 z\ 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. Runnstrom (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 Psamtnechinus 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.
JEB'IXl
6
82
A. D. HOBSON
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 Strongylocentrotusfranciscanus. Fig. 17 shows Fig. 17. Local contraction
an outline sketch of a somewhat extreme case found in $££§£%?£
EfiS
an egg fertilised in a small volume of water under a separation of fertilisation
coverslip. It seems probable that the surface layer of m r ^ * J £ S t i p p l i n g dia"
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 calciumfree 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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
83
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.
Runnstrom (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 Eioberfiache abgehoben und in der letzteren Halfte
des Amphiasterstadiums wird die Abhebung der hyalinen Schicht noch hoher."
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 2|MNaCl. He found that
plasmolysis 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 of
cleavage. Plasmolysed eggs were characterised by their resistance to the cytolytic
action of the hypertonic solution.
50
Minutes after fertilisation
Fig, 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 equal
volumes of sea water and 2 ^ 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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
85
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
10
15
20
Minutes after fertilisation
Fig. 19. Graph showing time taken for 50 per cent, of eggs to cytolyse in 50 c.c. sea
water + 50 c.c. z-$M NaCl. For description see text.
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.
86
A. D. HOBSON
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 c; R. S. Lillie, 19166; Just,
1922a, 1922b, 1928a, 19286; Page, 1929). The method usually employed is to
measure 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 to
normal sea water after a certain length of exposure to the hypotonic solution and
determining the percentage of cleavage.
-a
G
30
40
Minutes after fertilisation
Fig. 20. Graph showing the time for 100 per cent, cytolysis of unfertilised
and of fertilised eggs in tap water.
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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
87
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."
DISCUSSION.
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.
A. D. HOBSON
Table II.
Fertilised,
phase I
Un-
fertilised
Fertilised,
phase II
Fertilised,
phase III
Fertilised,
phase IV
Hyaline plasma Hyaline plasma
layer present in layer now comgelatinous con- posed of fluid
dition
enclosed within
solid outer
membrane
Polyhedral type
Polyhedral
gradually disappears
Condition of
surface of egg
Vitellinemem- Fertilisation membrane only
brane elevated
• present
during early
part of this
phase
No visible
change
Type of plasmolysis
Wrinkled
Roughly spherical with clear,
gelatinous layer
on surface
Resistance to
cytolysis by
hypertonic
solutions
High
Rate of swelling
in hypotonic
solutions
Slow
Fast
Slower than in
phase I
Resistance to
cytolysis by
tap water
Approximate
time scale
High
Low
Higher than in
unfertilised
Low at first but
rapidly increasing. Cytolysis
becomes gradual instead of
sudden
Decreases (this
Increases to
maximum
phase only investigated in one
which is
maintained
experiment)
Increases
Low
slightly
0—4 min. after
fertilisation
4—9 min. after
fertilisation
10-50 min. after 50—60 min. after
fertilisation
fertilisation
—
Wrinkled, but more
coarsely, tending
to polyhedral as
end of phase
approaches
Either (i) steadily
decreasing or (2)
lower followed by
brief period of
higher resistance
Lower than in
phase I
Cleavage assumed to begin 60 min. after fertilisation.
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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
89
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 (19286) 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
90
A. D. HOBSON
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 m m - 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
The Effect of Fertilisation on the Surface of the Egg of P. miliaris
91
difficulty. Further investigation is needed before the factors underlying this change
can be elucidated.
SUMMARY.
1. A photographic method is described for recording volume changes in seaurchin eggs.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 Investigatorship, 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.
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.