Cortical Changes in Acipenserid Eggs during
Fertilization and Artificial Activation
by T. A. DETTLAFF 1
From the Institute of Animal Morphology of the Academy of Sciences
of the U.S.S.R., Moscow
WITH FOUR PLATES
I N the complex chain of interrelated processes which make up the early changes
in the egg during activation (see Tyler, 1941, 1955; Runnstrdm, 1952; Rothschild, 1956; T. Yamamoto, 1956; Allen, 1958) many phenomena are still unclear : for example, the problem of the fertilization impulse, the rate of its spread,
and its relationship with the other changes in the egg; the problem of the origin
of the perivitelline space colloid and of its relation to the material of the cortical
granules (Runnstrom, 1952; Rothschild, 1956; Allen, 1958). The main objects
of investigation have hitherto been the eggs of sea urchins and, to a lesser
extent, of teleost fishes, so that a danger exists that the sequence of phenomena
characteristic of these eggs may be supposed to hold generally. Acipenserid
eggs, because of certain peculiarities of their structure and development, are a
suitable object for studies of this kind.
The work presented aims at describing cortical structures and their changes on
fertilization and artificial activation in Acipenserid eggs; and at elucidating the
relationship between fertilization impulse, cortical granule expulsion, perivitelline
space formation, change in the properties of the egg membranes, and the stimulation of the nucleus. Special attention was given to the study of acid mucopolysaccharides and their alteration in the activation process, as well as to the effect
of treatment of living eggs with Na-periodate.
Some of the data presented in this work have already been published elsewhere (Dettlaff & Ginsburg, 1954; Dettlaff, 1957).
MATERIAL AND METHODS
Investigations were carried out on the eggs of sturgeon—Acipenser giildenstaclti cokhicus V. Marti, of sevruga—A. stellatus Pall., and of white sturgeon—
Huso huso L. The material was collected during 1955-9 in Rogozhkino sturgeon
hatchery (Lower Don). The eggs were procured from females which had been
given a hypophyseal injection. Living observations on the changes in fertilized
1
Author's address: Institute of Animal Morphology of the Academy of Sciences of the U.S.S.R.,
Lenin Avenue 33, Moscow V-71, U.S.S.R.
[J. Etnbryol. exp. Morph. Vol. 10, Part 1, pp. 1-26, March 1962]
B2
2
T. A. DETTLAFF
eggs and the study of fixed eggs in section (in 6 lots of sturgeon eggs, 2 of sevruga
eggs, and one of white sturgeon) were made in parallel; comparison of the
dynamics of the changes in fertilized eggs and in eggs activated by pricking
was carried out on the eggs of two sturgeon and one white sturgeon female. The
eggs were pricked with a glass needle at the border of the animal region, or in
the vegetative pole. Activation by pricking allows precise recording of the time
of stimulation (which coincides with the moment of pricking) and hence precise
study of the dynamics of the egg-changes (see also Ginsburg, 1961).
Living observations were carried out under a binocular dissecting microscope
with incident light, and in a vertical chamber (Ancel & Vintemberger, 1948) in
transmitted light with the aid of a compound microscope. To study the eggs in
sections, precisely timed fixations were made: during the first 10 seconds after
insemination (or pricking), every 1-2 seconds; later, up to 30 seconds, every
5 seconds; during the next 4 minutes, at intervals of 15 and 30 seconds; up to
the onset of the first division at intervals of 5 and 15 minutes; and then at the
stages of 2, 4, 8, and many blastomeres. The eggs were fixed in Sanfelice and
Bouin fluids, and in 4 per cent, formalin, and embedded in paraffin; 5-10 eggs
were cut at each stage. Sections 7 /u, thick were stained by the azan and Scruff's
iodine methods, with Heidenhain's iron haematoxylin, and by Feulgen counterstained with light green. In order to study acid polysaccharides of the cortical
layer and their changes during activation, sturgeon eggs were fixed at successive
stages with Shabadash (1949), Bouin-Allen, and Carnoy's fluids, and with lead
acetate and formalin by the Sylven method. We employed Schiff's iodine method,
p retreating sections with (a) diastase prepared from germinated barley seeds by
Ivanov's technique (1946), (b) crystalline ribonuclease, (c) hyaluronidase from
bovine testis (put at my disposal by L. G. Smirnova), and (d) an unpurified
aqueous extract of an acetonized homogenate of sturgeon testis1 possessing
hyaluronidase activity; staining was carried out with an old aqueous solution
of pyronin and 0-5 per cent, aqueous solution of toluidine blue. In order to detect
proteins, the eggs were stained with sublimate-phenol-blue (according to
Mazia, Brower, & Alfert, 1953).
Particular methods employed in individual experiments are described in the
text.
RESULTS
Unfertilized egg
Acipenserid eggs are covered with an external jelly membrane and with two
layers of vitelline membrane (zona radiata interna and externa). The membrane
layers can be distinguished only under high magnifications. The membranes
closely adjoin the egg surface. The inner border of the zona radiata interna in
the living egg is invisible. Over the region of the polar spot where the spindle
1
Extract preparation was carried out under the guidance and with the active participation of
Dr. R. I. Tatarskaya.
CORTICAL CHANGES IN ACIPENSERID EGGS
3
of the 2nd maturation division lies, micropylar canals are located (Plate 1,
fig. 1).
The cortical layer of the unfertilized egg is formed by a layer of cytoplasm free
of yolk and pigment granules1 and by a deeper-lying layer of cortical granules
(Dettlaff, 1957) which merges into a layer of pigment granules (Plate 1, figs. 6a,
la). The cortical layer possesses this structure even in the oocyte, at the stage
before nuclear membrane dissolution (Plate 1, fig. 2).
The animal portion of the egg is rich in cytoplasm; it contains small-granular
yolk and yolk-free lacunae, some substances of which are involved in the formation of the perivitelline space colloid (Dettlaff & Ginsburg, 1954).
Cortical layer and egg-membranes
Cortical granules are oval or spherical and form a continuous layer over the
whole egg surface, including the funnel of maturation. The largest cortical
granules in sturgeon and sevruga reach 2-2x3-8 /x. Egg membranes are stained
similarly with aniline blue and light green. A positive Schiff-iodine reaction of
granules and egg membranes was preserved after 3- and 20-hour incubation of
sections at 37-38° C. in 1 percent, solutions of diastase and testicular hyaluronidases, while in control preparations of liver and umbilical cord the staining
specific to glycogen and hyaluronic acid completely disappeared during this
time; glycogen present in large amount in the animal portion of the egg also
ceased to be detected.
After staining sections with an old pyronin solution for 2-5 days, the cortical
granules were equally intensively pink-coloured, whether or not the sections
were pretreated with ribonuclease solution (1 mg. per 1 ml. borate buffer at
pH 7-5 during 3-20 hours at 37° C ) . On keeping the preparations over 24 hours
in 0-5 per cent, aqueous toluidine blue, the granules showed y-metachromasia.
The latter, however, as in the case of cortical granules of sea urchins (Monne &
Harde, 1951#), was not always distinctly revealed.
The Schiff-positive reaction, and the metachromatic staining with toluidine
blue and pyronin, point to the presence of acid polysaccharides in the cortical
granules. Since their specific staining is preserved after the action of hyaluronidases (including the species-specific one) which split both hyaluronic and
chondroitinsulfuric acids (Pearse, 1953) they can be assumed to contain acid
mucopolysaccharides of heparin type. (Employing both the histochemical and
the clot methods, we failed to find the species-specificity in the action of testicular
hyaluronidases noticed by Takashima, Mori, and Kawano (1955), when comparing hyaluronidase activity of the extracts from bovine and sturgeon testes.)
Unlike cortical granules, egg membranes contain neutral mucopolysaccharides: they did not show a clear metachromasia with toluidine blue, and the
1
Allen (1958) suggested that this layer in sea urchins should be called the luminous hyaline layer,
in contrast to the ectoplasm hyaline layer formed after fertilization.
4
T. A. DETTLAFF
zona radiata was not at all stained with pyronin, while the jelly membrane was
stained light violet.
Polysaccharides in granules, as well as in membranes, appear to be firmly
linked to proteins, since they were preserved when fixed with non-specific
fixatives and when sections were heated. A small amount of protein was revealed
in them on treatment of sections with sublimate-bromine-phenol-blue. Trypsin
diminished the firmness of the membranes (G. M. Ignatieva, unpublished),
and this also suggests the presence of proteins in them.
No lipid membrane could be detected in the cortical granules of sturgeons.
It can be seen in preparations made by Ginsburg (1956), who studied lipids in
sturgeon oocytes and eggs (fixation with formol-calcium, embedding in gelatine,
cutting with freezing microtome and staining with Sudan black B), that the
cortical layer of the cytoplasm contains diffusely dispersed lipids, while cortical
granules lack these. Because of this, the granules are clearly seen. Under maximal
magnifications of the light microscope no lipid concentration can be seen on
their surface.
Diffuse acid mucopolysaccharides cannot be revealed in the cortical layer of
sturgeon eggs by means of the above methods, while the Hale (1946) method
employed for this purpose in sea urchins (Monne & Slautterback, 1950;
Immers, 1956; Runnstrom & Immers, 1956) gave in my experiments a nonspecific coloration; mucopolysaccharides of the cortical granules are not revealed at all by this method.
Treatment of living sturgeon and sevruga eggs with 0-01 N solution of Na
periodate for 1-5-15 minutes brought about a depolymerization of the membrane polysaccharides, but it did not affect the polysaccharides of the cortical
granules.
In sections through such eggs treated with Schiff's solution without preliminary immersion of the sections in NaIO 4 solution, cortical granules remained
unstained at all time intervals (Plate 1, fig. 6b), while the coloration intensity of
the membranes progressively increased. In control sections of the same eggs
pretreated with NaIO 4 solution before Schiff's reagent, the granules showed a
typical PAS-positive reaction (Plate 1, fig. 6a). Diminishing the concentration
of NaIO 4 solution with which control sections were treated gave preparations
in which the coloration intensity of the membranes hardly differed from that in
the experimental sections, but cortical granules, nevertheless, were stained with
the same intensity as the membranes. Since a 15-minute exposure to the action
of 0-01 N Na periodate solution was close to an injurious one, the conclusion
must be drawn that the cortical layer in the living sturgeon egg appears to be
impermeable to Na periodate. In this connexion it is of interest that Na periodate
does not activate sturgeon eggs (HO- 3 and l-10~4 M solutions neither damage
the eggs nor activate them even with a 6-hour exposure; 1-10-1 and l-10~2 M
solutions damage the eggs after a 30-minute exposure without activating them,
while with a 5-minute exposure they neither damage nor activate the eggs).
CORTICAL CHANGES IN ACIPENSERID EGGS
5
Lacunae of karyoplasm in the cytoplasm of the animal egg region
A network of lacunae of varying size was described in the cytoplasm of the
animal region of the egg of A. ruthenus by Salensky (1881). This network arises
during the maturation period as a result of the dissolution of the nuclear membrane and the consequent passage of the karyoplasm into the cytoplasm
(Plate 1, figs. 2, 3). Like the oocyte nucleus, these lacunae are stained with
aniline blue. The staining is more successful after formalin fixation of the eggs.
After fixation with Sanfelice fluid, round globules of a hydrophilic colloid may
be seen in these karyoplasmic areas of the animal region (Plate 1,fig.4). In nonactivated eggs kept for some time in water, these globules, after fixation and
passing through the alcohols, shrink more strongly than the rest of the cytoplasm, so that they often appear to lie in larger vacuoles (Plate 1, fig 5). These
globules will be called 'the globules of the hydrophylic colloid' until their
composition has been finally elucidated.
Cortical changes in the egg during fertilization and
artificial activation by pricking
Living observations
After insemination or pricking the egg surface rapidly undergoes a change:
the coloration alters in shade, and the membranes become transparent. The
membrane layers can now be easily distinguished, and the inner border of the
zona radiata interna appears. In fertilized eggs these changes start in the micropylar region and spread in a wave-like fashion over the egg surface, reaching
the opposite pole in between 2 and 4 minutes, depending on the temperature.
In the eggs activated by pricking, the changes start from the pricked area and
spread towards the animal pole at a higher velocity than towards the vegetative
pole (cf. also Ginsburg, 1960, 1961). The spread of these changes can be clearly
followed only in certain darker-pigmented lots of eggs.
The brightening of the membranes is followed by their rapid swelling, connected with their active water uptake from the external medium (Zotin, 1955;
Dettlaff & Ginsburg, 1954) (Text-fig. 3). The jelly-membrane acquires an alveolar structure and becomes sticky.
After the appearance of stickiness the eggs begin to turn within the membranes
with the animal pole upwards. In some lots the rotation of the eggs is completed
by 20-25 minutes, in others somewhat later.
When examining the eggs laterally under the microscope in a vertical chamber,
the inner border of zona radiata interna can be seen, but the space between it
and the egg surface is still lacking. Only at the termination of the egg rotation,
15-20 minutes after insemination (17-8—180° C), the animal region of the egg
begins to flatten and between this region and the internal layer of the zona
radiata a narrow perivitelline space becomes visible for the first time and
rapidly widens during the subsequent 20-25 minutes (see also Dettlaff &
Ginsburg, 1954).
T. A. DETTLAFF
Observation of a living egg under a dissecting binocular shows that the
turbid, somewhat opalescent colloid is secreted from the animal portion of the
egg into the perivitelline space. In sturgeons the secreted substance is slightly
violet and can be clearly seen.
Whether eggs are fertilized or activated by pricking, the perivitelline space
is formed simultaneously and in the same manner. Independently of the site
of pricking (in the animal or vegetative region of the egg), it arises over the
animal region.
Concomitantly with the rotation of the egg and with the formation of the
perivitelline space, a hardening of the egg membranes takes place. By the onset
of the first division their toughness reaches its maximum (Zotin, 1953).
Microscopic investigation
Expulsion of cortical granules and formation of the fertilization membrane'. The
first changes in the cortical layer of sturgeon eggs were observed 3 seconds after
insemination (17-8-18-0° C ) ; small-sized, optiWO
cally empty vacuoles appeared near the micro/
pylar canals, in the cortical layer, among the
granules; the contours of the granules were no
longer clearly seen. During some seconds subsequently the size of the vacuoles somewhat
increased and, at the same time, the process of
vacuolization spread laterally (Plate. 1,fig.7). The
percentage
of eggs in which the early stages of the
50
cortical changes could be detected rapidly increased
during the first 8 seconds after insemination and
by 11-15 seconds reached its maximum (Text-
1
I
//
/
7
•
fig. 1); i.e. it underwent the same changes as the
fertilization percentage in experiments on the insemination rate (Ginsburg, 1957), or in experiments on chelating Ca-ions with versene solution
i
i
i
4 8 12 16 at different intervals after the addition of sperm
Time after
(Dettlaff, 1958). In the eggs showing the first signs
insemination (sec.) of activation, spermatozoa were found at the
entrance of the terminal canal where, according
TEXT-FIG. 1. The percentage of
eggs showing in section the
to Ginsburg's (1957) data, an effective contact of
signs of cortical reaction, at difgametes is achieved. Thus, the cytoplasm of the
ferent times after insemination.
cortical layer responds to the encounter with a
spermatozoon by the formation of vacuoles during the first 1-2 seconds.
1
The cytoplasmic areas with their granule material, enclosed between vacuoles,
acquire a columnar form. At this time the whole material of the columns is
stained by aniline blue and shows a PAS-positive reaction. In those places where
individual granules are preserved within the columns, the latter can be clearly
CORTICAL CHANGES IN ACIPENSERID EGGS
7
seen to consist not only of the granule material but of the cytoplasm as well. With
the later enlargement of the vacuoles the columns stretch, get thinner, and
separate from the egg (Plate 1, fig le). In the eggs of some females, apparently
after over-ripening of the eggs, as well as in non-fertilized eggs which have
remained in water for a long time, small vacuoles can also be found sometimes in the cytoplasm of the cortical layer, but they are more often located
under the granular layer and not among the granules. The appearance of
vacuoles in this case does not lead to a rapid spreading of the cortical reaction
and to the detachment of granules. It seems that this requires some other changes
in the properties of the surface layer.
The bulk of the columnar substance with the protoplasmic membrane uniting
them passes then to the internal surface of the zona radiata interna (Plate 1,
fig. le). Fragments of cytoplasmic filaments are left on the egg surface, and
individual unchanged granules can be found. In some eggs the separation of the
columnar material could be observed in the
centre of the animal region 5 seconds after insemination or pricking. Whether the detachment of the columns described above is a
result of their separation, or whether the latter
is accelerated in the process of histological
treatment, can hardly be decided.
Having once started in the animal portion of
the egg, the changes described spread in all
directions and gradually embrace the whole
egg surface. After 60 seconds the contact between the egg and the membranes in the animal
region is preserved only in the area of the
TEXT-FIG. 2. Velocity of the spread of
micropylar canals (Ginsburg, 1957; Dettlaff,
cortical changes in sturgeon eggs.
1958). In sections of eggs fixed 20-40 seconds
Levels of cortical reaction reached at
different times after insemination
after insemination all intermediate stages from
{t. = 17-8-18° C).
separated to completely unchanged granules
can be seen (Plate 1, figs. la-e).
The velocity of spread of the cortical changes gradually decreases from the
animal towards the vegetative pole (Text-fig. 2). When eggs are activated by
pricking, the cortical layer undergoes the same changes as after fertilization but
they start from the site of pricking. The time of termination of the separation
of cortical granules is similar to that after fertilization. Comparison of the
spread of the appreciable colour change in the living egg during the first 2-3
minutes after insemination or pricking with the changes in the layer of cortical
granules as seen in sections shows that they agree.
It is interesting to note that one of the first manifestations of the inadequacy
of eggs is distortion of the cortical reaction. After a prolonged exposure of
eggs to the body fluid, or at an unfavourable temperature, the uniformity in the
8
T. A. DETTLAFF
spread of the cortical reaction is lost. The velocity of cortical changes concomitantly decreases, or, in parts of the egg surface, they do not proceed at all.
This latter abnormality occurs particularly often in white sturgeon eggs at unfavourable temperatures. In that part of the egg where no extrusion of cortical
granules has taken place, the membranes are not elevated over the egg surface
and do not undergo changes. Those parts of the egg which have not undergone
cortical changes do not subsequently undergo cleavage, and eggs with a partial
discoidal cleavage thus arise.
After the termination of the extrusion of cortical granules, the external surface
of the egg has a relatively even outer contour (Plate 3, fig la) and sometimes
contains individual unexpelled cortical granules.
Cytoplasmic columns containing the material of the cortical granules, which
were separated from the egg in the process of the cortical reaction, adjoin from
within the zona radiata interna and form on its inner surface a kind of fringe.
More or less isolated columns fuse with each other. From this moment the
vitelline membrane with the underlying layer containing the substances of the
cortical granules corresponds to the so-called fertilization membrane of sea
urchins (Motomura, 1941; Runnstrom, 1948; Endo, 1952).
Mucopolysaccharides of the cortical granules do not undergo chemical
change in the process of egg activation: the fringe on the inner surface of the
vitelline membrane, which contains mucopolysaccharides, shows the samehistochemical reactions as the cortical granules: the positive Schiff iodine reaction
is preserved after the treatment of sections with diastase and hyaluronidascs
(Plate 2, figs. 3a, b).
The extrusion of cortical granules and the formation of the fertilization membrane completes the first phase of the cortical changes. Because of them the
egg is released from close contact with the membranes and begins to rotate
within them with its animal pole upward. The egg membranes swell and become
sticky.
Formation of the perivitelline space. The first isolation of the egg from the egg
membranes takes place as a result of the extrusion of cortical granules and of
the formation of a slightly swollen granular layer (internal portion of the fertilization membrane) on the inner surface of the vitelline membrane. The formation of the extended perivitelline space over the animal region of the egg takes
place later and is connected with the secretion of a hydrophilic colloid from the
eggGlobules of hydrophilic colloid which were described in an unfertilized egg
and which separate within the lacunae of the karyoplasm do not fuse with the
rest of the cytoplasm and, after activation, gradually rise to the surface cytoplasmic layer of the animal region. Because of this the surface layer of the animal
region becomes enriched with a light hydrophilic colloid (Plate 2, figs, la-e;
Plate 3, figs. \a-d). At first globules form isolated islets in the surface layer,
while later on a continuous light layer appears in the animal portion of the egg
CORTICAL CHANGES IN ACIPENSERID EGGS
9
(Plate 2, fig. le, Plate 3, figs. b-d). At the egg surface the shape of the globules
changes (cf. Plate 2, fig. 2 a, b) while their content acquires a somewhat increased water content and undergoes vacuolization. The swelling of the colloid
is particularly clearly seen in formol-fixed eggs (Plate 3, fig. 2a). This effect
may, however, be increased in the case of formol fixation by post-mortem
swelling of the colloid.
The colloid appears in the perivitelline space (after formol fixation) 10-15
minutes after activation; during the subsequent 30-40 minutes its volume
rapidly increases on account both of extrusion of new colloid from the egg
—
•
r»•—
m
—J
/•
-o_,
;•—*O
i
•T
On
O<
00
fcor
• o
30
60
Time after
90
insemination
120
(min.)
150
TEXT-FIG. 3. The entrance of water into the sevruga egg at different times after insemination. 1, into the egg covered by all its membranes; 2, into the egg covered
only by the inner vitelline membrane (taken from Zotin, 1955).
(Plate 3, fig. 2b) and of water uptake from the external medium (Zotin, 1955)
(Text-fig. 3). At this time the animal region flattens considerably. In sections
the pigment turns out to be dispersed at these stages in the thicker cytoplasm
layer; its individual granules are carried away with the colloid to the perivitelline space. The colloid extruded is sometimes very vacuolated at the egg surface
(Plate 3, fig. 2d). In some cases the light layer at the egg surface and the colloid
of the perivitelline space pass imperceptibly one into the other (Plate 3,fig.2e).
In other cases (Plate 3, fig. 2c) they are distinguishable, but there exists a direct
connexion between the light surface layer and the colloid of the perivitelline
space. In this picture it is difficult to determine the border between the egg
surface and the colloid of the perivitelline space. By 45-50 minutes (18° C.)
10
T. A. DETTLAFF
the volume of the perivitelline space in the sturgeon reaches almost its maximal
size; by this time the extrusion of the colloid from the egg ceases as well.
Simultaneously with the rise of hydrophilic colloid globules to the surface
layer, the endoplasm of the animal region acquires a more uniform structure
(Plate 2, figs. Id, e); karyoplasmic lacunae which pierce it disappear. By the
stages of the first and second cleavage divisions the hydrophilic colloid also
disappears from the surface layer of the egg (Plate 2, fig. 1/; Plate 3, fig. le).
The egg surface acquires a smooth appearance. In the process of cleavage hydrophilic substances that were secreted at previous stages into the perivitelline space
are seen in the crevices between the blastomeres and, later on, in the blastocoel
(see also Zotin, 1961). The volume of the perivitelline space over the animal
portion of the embryo diminishes at this time, as can be seen both in section
and by vital observation in the vertical chamber. Some of the aniline blue
stained substance does not, however, pass to the perivitelline space, and instead submerges together with the pronuclei from the egg surface inside the
animal part of the egg.
During the period of cleavage a small amount of substance stained by aniline
blue is also found in the pathways of the presumptive cleavage furrows. Prior
to the appearance of a furrow on the egg surface its pathway is prepared in the
cytoplasm of the animal region. This pathway consists of a chain of optically
empty vacuoles surrounded by single pigment granules (Plate 4, fig. 1) and terminates in a colloid aggregation stained by aniline blue (Plate 4, fig. 2). Later on.
the surface egg layer with pigment granules deepens in the pathway prepared
by the vacuoles and forms adjacent surfaces. The vacuoles in the pathways of
the presumptive cleavage furrow in sturgeon eggs were described for the first
time by Peltzam (1886) and then by Ginsburg (1959).
The hydrophilic colloid secreted from the egg into the perivitelline space
differs from the material of the cortical granules not only in its origin but also
in its chemical composition. Unlike the cortical granules, it does not contain
mucopolysaccharides: the PAS-positive reaction of the light layer (Plate 2,
fig. 3d) is due to the presence of glycogen granules and can be eliminated by
a preliminary diastase treatment of the section (Plate 2, fig. 3b). Thanks to this,
it can be distinguished from the substances of discharged cortical granules.
Apart from glycogen, traces of RNA and protein can be revealed in the hydrophilic colloid. Elective staining with aniline blue facilitates their detection but it
does not reveal their chemical nature, though there are some indications that
aminosaccharides can be revealed by this method (Monne & Harde, 195lo;
Kusa, 1956). When stained with aniline blue, the hydrophilic colloid of the light
layer shows a different shade of coloration from the cortical granule material,
which also facilitates their distinction when outside the egg (Plate 2, fig. 3c).
Despite the differences described in cortical granules and the hydrophilic
colloid, their secretion from the egg represents consequent and interdependent
processes: the cortical reaction, accompanied by the extrusion of cortical
CORTICAL CHANGES IN ACIPENSERID EGGS
11
granules, stimulates the rise of hydrophilic colloid globules from the endoplasm
to the egg surface. If the cortical reaction is defective and is not accompanied by
the extrusion of granules, the hydrophilic colloid cannot pass to the perivitelline
space, though it accumulates in the surface layer of the egg under the layer of
cortical granules (Plate 1, fig. 8). (A small amount of the colloid under the membrane, as well as the separation of the membranes from the egg surface in this
case (Plate 1, fig. 8), seem to be artefacts.) It can be clearly seen in this figure
that the cortical granule material and hydrophilic colloid of the light surface
layer exist simultaneously and independently of each other.
Comparison of the results of microscopic investigation with the data of vital
observation in a vertical chamber shows that, while the rise of hydrophilic
colloid globules to the egg surface starts in the first few minutes after activation
and proceeds for 30-40 minutes, the formation of the perivitelline space visible
under the microscope can be observed only after 15-20 minutes; thereafter
during the subsequent 20-25 minutes it rapidly extends. Water entry into the
egg (leaving apart the water taken up by the membranes), according to Zotin's
data (1955), starts also some minutes after activation (in the experiment shown
in Text-fig. 3 after 12 minutes, i.e. during the period of onset of the formation
of a widened perivitelline space or somewhat earlier). The hydrophilic colloid
accumulating in the surface egg layer seems to increase somewhat in water
content and to pass to the perivitelline space, where it swells considerably.
Relationship between the change in egg-membrane properties
and the cortical reaction
The change in egg-membrane properties starting soon after the expulsion of
cortical granules seems, however, not to be causally related to the latter.
In experiments carried out in collaboration with A. I. Zotin (see also Zotin,
1961), sevruga eggs were placed in water with the sperm for 10, 60, and 120
seconds and thereafter kept in 0-1 N NaCl. (Some of the eggs were kept in water
during the whole experiment up to the 4-cell stage, while others were placed in
water after varying periods of time.)
The membranes of the eggs kept in water with the sperm for 60 seconds and
more swelled normally in NaCl solution, became sticky, and hardened. Those
of the eggs placed in 0-1 N NaCl solution 10 seconds after insemination did not
change, but, after replacement in water, they swelled, became sticky, and
hardened even at the stages of 2 and 4 blastomeres. Thus, 0-1 N NaCl solution
does not prevent changes in the membranes themselves, but it blocks some
process in the eggs which proceeds over the period from 10 to 60 seconds after
insemination, and is necessary for the membrane stickiness and hardening. In
order to elucidate the relation of this action of NaCl solution to the extrusion of
cortical granules, the eggs were fixed at the moment of placing them in NaCl
solution and later, when they were taken from NaCl solution, with both
12
T. A. DETTLAFF
unchanged and normally hardened membranes. It turned out that in the eggs
placed into NaCl solution 10 seconds after their insemination in water, the
membranes of which did not become sticky and did not harden, the cortical
reaction proceeded normally, i.e. cortical granules were extruded, the fringe on
the inner surface of the membrane was formed, and the hydrophilic colloid secreted. Since the eggs placed in 0-1 N NaCl solution 60 seconds after insemination
hardened under the same conditions, it can be assumed that the stickiness and
hardness of membranes in sturgeons was related to substances other than
cortical granules and hydrophilic colloid, the expulsion of which from the egg
followed that of cortical granules and could be reversibly blocked by 0-1 N
NaCl solution.
Fertilization impulse, cortical reaction, and stimulation of
nuclear division
The transition of the nucleus to the active state follows the extrusion of
cortical granules. Judging by the analogy with sea urchins and teleost fishes, it
can be expected that the latter is, in its turn, preceded by invisible cortical
changes, the 'fertilization wave' or 'fertilization impulse' (Yamamoto, 1944,
1956; Sugiyama, 1956; Runnstrom, 1956; Rothschild, 1956; Allen, 1958).
Since egg activation is possible without an extrusion of cortical granules
(Motomura, 1934, 1941; Thomopoulos, 1953a; Kusa, 1953a; Ishikawa, 1954;
and many others—see also Rothschild, 1958; Allen, 1958) there are grounds for
believing that stimulation of the nucleus is due to the action of the fertilization
impulse and, therefore, that the transition of the nucleus to the active state can
be used as a criterion for a study of the fertilization impulse.
On fertilization in Acipenserid fishes a spermatozoon enters the egg through
the micropylar canal in the direct vicinity of the female nucleus (Plate 1, fig. 1)
and it is just here that the first visible changes in the layer of cortical granules
arise in the first seconds after the fertilization impulse. In order to judge the
velocity of spread of the fertilization impulse over the egg surface and its relation to the velocity of spread of visible cortical changes (extrusion of cortical
granules), experimental conditions must be created in which the fertilization
impulse arises at a different distance from the nucleus. With this aim the eggs
of two sturgeons and one white sturgeon were activated by pricking with a fine
glass needle: some of them in the border of the animal region, others in the
vegetative pole. Since the expulsion of cortical granules, having started from
the site of pricking, spreads over the cortical egg layer during some minutes, on
pricking in the vegetative pole the process of cortical granule discharge reaches
the region of the nucleus later than on pricking in the animal region. As to
the fertilization impulse, if it spreads in sturgeon eggs with a greater velocity
than the cortical granule discharge (as is suggested by Rothschild & Swann,
1949, for sea urchins), the difference in the pricking site would not bring about a
CORTICAL CHANGES IN ACIPENSERID EGGS
13
considerable difference in the time when the impulse reaches the zone of location
of the nucleus.
The eggs of two sturgeons and of one white sturgeon pricked in the animal
or the vegetative regions were kept at an even temperature (sturgeon eggs at
14-9-15-20 and 16-3-16-50 C , white sturgeon eggs at 21-5-21-6° C ) . In order
>
J/J.
'
Sturgeon
15
/
/
/
>/
/
r
/I 1
>
/
10
5
/
/
n
HI-
/
/
/<
i
/ >A
/
/ /
:/ I
mmmt
10
15
y
•u J
<— — —x a 1
\2
//I A
\\
)\
1
A
V
\
V
\
/
*
\
•
s
\
\
\
V
20
25
30
Time after pricking (min.)
TEXT-FIGS. 4 and 5. Changes in the distance between sister chromosomes (curves 1) and increase
in the spindle length (curves 2) during the second maturation division of eggs, pricked at the
border of the animal zone (a) and in the vegetative pole (v). Text-fig. 4, sturgeon (t. = 14-9—
15-2° C), Text-fig. 5, white sturgeon (t. = 21-5° C).
to follow the changes in the nuclei in detail, the eggs of one sturgeon were fixed
each minute for 10 minutes and then after 15 minutes; the eggs of another
sturgeon were fixed each minute during the period from 8 to 30 minutes after
pricking; the eggs of the white sturgeon were fixed each minute for 20 minutes.
Ten or 4 sturgeon eggs and 5 white sturgeon eggs were pricked at each time.
Sections were stained by iron haematoxylin. Two-hundred and sixty nuclear
figures were investigated. Mitotic patterns were drawn under the microscope by
T. A. DETTLAFF
14
means of a camera lucida; distances between sister chromosomes and the length
of spindles were measured in the drawings.
Apart from this, additional fixations were carried out to find the time required for the cortical reaction to reach the site of the nucleus after pricking in
various areas. Sections were stained by the Heidenhain azan method.
4"
* - -
/
/
A
White sturgeon
/
i
20
j
1
1
\
1
1
I
1A
\
• •
15
— x
x a1
— •
•vJ
— x—— —x a 1
•
•
10
<
:•
••
/
/
/
\
\
\
/
\
i
/
/;
I 1)
1
/
/
I
\
/
/ /I1
1
\
\
1
X
11
f
;=£T<
T
\i
\
/
5
>
,'j
/
0
•
•
10
15
1
?,
Time after pricking (min.)
FIG.
5
The curves presented in Text-figs. 4 and 5 show changes in the distance
between sister chromosomes (1) and in the spindle length of the second maturation division (2) in sturgeon (Text-fig. 4) and white sturgeon (Text-fig. 5) eggs
pricked at the site of the animal (a) and vegetative (v) pole during 30 (sturgeon)
and 20 (white sturgeon) minutes after pricking. Each point represents an
average for 3-4 eggs.
CORTICAL CHANGES IN ACIPENSERID EGGS
15
The dynamics of the process of nuclear activation and of the completion of
the second maturation division is the same on pricking the eggs either in the
animal or in the vegetative region. The prolonged period is occupied by a very
slow separation of sister chromosomes, then follows a period during which
the chromosomes rapidly diverge to the spindle poles and, simultaneously, the
spindle itself lengthens. After reaching its maximal length the spindle shortens,
and then begins the separation of the polar body.
When comparing the stages of nuclear division in white sturgeon and sturgeon
eggs pricked at different places and fixed at the same time interval after pricking,
it turns out that at any time interval, the eggs pricked in the vegetative pole
lag behind the eggs pricked in the animal region (Plate 4, fig 3, A, V). The
differences are most demonstrative in the period of rapid chromosome divergence, when the spindle considerably lengthens during a minute, while the
chromosomes cover a great distance (Text-fig. 4, Plate 4, fig. 3). The time of
retardation of nuclei in the eggs activated by pricking in the vegetative region
is close to the time required for the cortical reaction to spread from the vegetative egg pole to the border of the animal region. In sturgeon it took 3 - 3 |
minutes, in white sturgeon 1^-2 minutes. In some white sturgeon eggs the delay
in nuclear division on pricking in the vegetative region was much greater,1 but
it corresponded to a slow spread of the cortical reaction in some eggs. No
considerable differences could be found in the stages of nuclear development
in white sturgeon eggs at the same time after pricking in the animal region.
A retardation in the stage of nuclear division on pricking in the vegetative
region was also found in the experiment on the second sturgeon, but here it was
less demonstrative, the eggs being fixed early.
DISCUSSION
According to Allen's (1958) classification Acipenserid fishes have to be included in the group of animals with a labile cortex. The first phase of visible
cortical change in Acipenserid eggs is related to cortical granule expulsion and
has many features in common with cortical changes occurring in the eggs of
sea urchins, Saccoglossus, teleost fishes, amphibians, and others. The cortical
granules of Acipenserid fishes contain acid mucopolysaccharides, as do the
cortical granules of sea urchins (Runnstrom, Monne, & Wicklund, 1946; Monne
& Slautterback, 1950, Monne & Harde, 195k; Nakano & Ohashi, 1954),
lamprey (Kusa, 1957ft), the cortical alveoli of many teleost species (Kusa, 1953 a,
ft, 1954, 1956, 1958ft; Thomopoulos, 1953 a, ft; Devillers, Thomopoulos, &
Colas, 1953; Aketa, 1954; K. Yamamoto, 1956; Nakano, 1956; Sakun, 1960;
1
These eggs were not taken into consideration when plotting the curves presented in Text-fig. 4
since some of the fertilized eggs of the same female underwent discoidal cleavage, and the vegetative
region did not participate in the development. It is, however, of interest that in these evidently pathologically developing eggs the time of nuclear stimulation was also correlated with the velocity of the
cortical reaction spread in the same manner as in normally developing eggs.
5084.10
C
16
T. A. DETTLAFF
also see Rothschild, 1958), and the granules of amphibians (Wartenberg, 1956;
Rosenbaum, 1958). As in sea urchins (Vasseur, 1948; Vasseur & Immers, 1949;
Monn6 & Harde, 1951a; Nakano & Ohashi, 1954), they are, in their chemical
composition, similar to egg membranes. No morphologically manifested lipid
membrane of cortical granules can be revealed in Acipenserids by histological
methods; such a membrane is described in sea urchins (Runnstrom, 1956). In
most teleost species a lipid membrane is not found (see Kusa, 1956), though their
alveoli undergo destruction under the influence of lipoid solvents (T. Yamamoto,
1951; Kusa, 19586).
Unlike the case of sea urchins (Allen & Griffin, 1958), changes in the layer of
cortical granules in Acipenserids begin in the first seconds after the establishment of the contact between the spermatozoon and the egg cytoplasm (or after
pricking), that is, after a very short latent period (cf. also Ginsburg, 1961).
The mode of extrusion of the cortical granular material in Acipenserids
differs from the mode of alveolar breakdown in teleosts (Kusa, 1953a, 1956,
19586) and from that of cortical granule discharge in sea urchins described by
Endo (1952). It can be suggested, however, that changes in the teleost alveoli
and in the ectoplasm of the cortical granule layer of Acipenserids are based on
common processes. In this connexion it is interesting to note that narcotics
inhibit cortical changes in the eggs of sea urchins (Hagstrom & Allen, 1956;
Sugiyama, 1956), teleosts (T. Yamamoto, 1956), and Acipenserid fishes (Ginsburg, 1961) in the same way.
After extrusion, the material of the cortical granules adheres to the inner
yolk membrane as in sea urchins (Motomura, 1941; Runnstrom, 1948; Endo,
1952), Saccoglossus (Colwin & Colwin, 1954), and amphibians (Motomura,
1952; Kemp, 1956). As in other animals, the egg of the Acipenserids, owing to
the cortical granule discharge, is freed within the membranes and turns with
its animal pole upwards under the action of gravity (Dettlaff & Ginsburg, 1954).
Changes in sturgeon eggs following the extrusion of cortical granules essentially differ from those described in echinoderm and teleost eggs. While in teleost
fishes and sea urchins the destruction of cortical alveoli and granules is followed
by the formation of the quickly widening perivitelline space (T. Yamamoto,
1939; Motomura, 1941; Runnstrom, 1948, 1952; Devillers, Thomopoulos, &
Colas, 1953; Kanoh, 1953; Kusa, 1953; and others—see also Runnstrom, 1952;
Allen, 1958; Kusa, 1956,1958; Rothschild, 1958), in Acipenserids the formation
of the latter starts considerably later and is related to the secretion of the
hydrophilic colloid globules not described in teleosts and sea urchins. The
movement of these globules towards the egg surface seems, however, to represent
a result of the same activating effect of the cortical reaction on the endoplasm
as does the pigment migration in sea-urchin eggs (Allen & Rowe, 1958; cf.
Allen, 1958). It is of interest that in echinoderms (Runnstrom, 1928; Monne &
Harde, 19516), as well as in Acipenserids homogeneity of the cytoplasm increases in the process of egg activation. In the cytoplasm of unripe sea-urchin
CORTICAL CHANGES IN ACIPENSERID EGGS
17
eggs vacuoles arising in the process of maturation have been described; they
stain with basic dyes. These vacuoles disappear in the course of fertilization as
a result of changes in the physiological state of the cytoplasm (Monne &
Harde, 19516); their expulsion into the perivitelline space was not observed.
There is no general agreement in the literature about the problem of the origin
of the colloid involved in the formation of the perivitelline space (cf. Rothschild, 1956; Allen, 1958). It seems that in different animals it appears in a different manner. In teleost fishes it is secreted after the destruction of cortical
alveoli (T. Yamamoto, 1939); most of the workers believe this colloid to be
contained in the alveoli themselves, which show a high osmotic activity upon
isolation (Kusa, 1957«, 1958a). As to sea urchins, some authors (Runnstrom,
Monne, & Wicklund, 1946; Runnstrom, 1952; Moore, 1951; Endo, 1952)
think that the granules cannot form an osmotically active colloid, while others
(Parpart & Laris, 1955; Afzelius, 1956; Parpart & Cagle, 1957) assume that a
portion of the extruded material of the granules, while polymerizing, forms the
hyaline layer and the colloid of the perivitelline space. Their opinion is shared
by Allen (1958).
At the same time data are available in the literature on the extrusion from the
egg of other substances, along with cortical granules, during the process of
activation: in sea urchins a luminous substance (Moore, 1951) and a transparent
surface layer (Hiramoto, 1954); in Saccoglossus transparent granules and a
jelly-like mass (Colwin & Colwin, 19541); in the brook lamprey fine particles
(Kusa & Ootake, 1959). There are some old (O. Hertwig, 1877; Bialaszewicz,
1912) and recent (Wintrebert, 1933) data on the excretion of a colloid from the
animal portion of amphibian eggs. In amphibians, as well as in Acipenserids,
the release of the egg and its rotation within the membranes precedes the formation of the perivitelline space (Ancel & Vintemberger, 1948). According to unpublished data of the author, in Rana temporaria and Ambystoma mexicanum
the rotation of the egg is connected with the extrusion of cortical granules (described in amphibians by Motomura (1952) and Kemp (1956)), while the formation of the widened perivitelline space, as in Acipenserids, is related to the
colloid expulsion from the animal egg region.
Formations very similar to the globules of the hydrophilic colloid in sturgeon
eggs were described in the eggs of Parascaris equorum (van Beneden, 1883;
Faure-Fremiet, Ebel, & Colas, 1954) under the name 'spheres hyalines'. Like
the hydrophilic globules, they are electively stained with aniline blue, arise
during oocyte maturation in the perinuclear zone, and migrate to the egg surface
after fertilization. Here they fuse and form a continuous layer which widens
and disappears in the perivitelline space thus formed. Hyaline spheres contain
proteins and lack polysaccharides; they are in a peculiar physical state, do not
mix with the cytoplasm, and are soluble in water. The data presented thus show
1
These authors are not sure whether the origin of these substances is not connected with the
material of cortical granules (personal communication).
18
T. A. DETTLAFF
that the extrusion of the globules of hydrophilic colloid in egg activation is not
confined to sturgeons.
The synthesis of the hydrophilic colloid (whose chemical nature is not clear)
appears to proceed in sturgeons in the oocyte nucleus. Since this nucleus
shows considerable hydrophily, though it is likely to proceed in the lacunae of the
karyoplasm after it has passed into the cytoplasm (this problem requires further
investigation). In the process of activation the hydrophilic colloid moves towards the membranes and takes part in the formation of the perivitelline space,
coming to lie between blastomeres at cleavage stages and, it seems, participating
in the formation of the blastocoel (cf. also Zotin, 1961) as well as in that of other
cavities of the embryo, since, according to the data of Zotin & Krumin (1959),
the colloid from the blastocoel moves during gastrulation to the gastrocoel
and, later, into the lumen of the gut.
The passage of some of the substances staining like the karyoplasm with
aniline blue along the pathways of the presumptive cleavage furrow, and the
presence of these substances at the termination of a chain of vacuoles preparing
the plane of blastomere separation, suggests that the hydrophilic colloid may
play the same separating role in the process of egg cleavage as it plays in the
formation of the perivitelline space and the cavities.
Apart from cortical granules and the hydrophilic colloid, there is a substance
(or substances) extruded from the eggs following the cortical granule discharge.
They bring about swelling, appearance of stickiness, and hardening of the egg
membranes (cf. also Zotin, 1961) and differ both from the substance of granules
and from the hydrophilic colloid. It seems that they correspond to the hardening factor in sea-urchin eggs (Motomura, 1950, 1954, Runnstrom, 1952) and
to the hardening enzyme in the eggs of Salmonid fishes (Zotin, 1958).
As to the data on the stimulation of the female nucleus and the velocity of
the spread of the fertilization impulse inducing this stimulation in sturgeon eggs,
there are no similar data for other animals in the literature. The fact that the
delay of nuclear division of eggs pricked in the vegetative egg region, as compared
with eggs pricked in the animal region, is close to the time required for the
spread of the cortical reaction from one site of pricking to the other, speaks
against the suggestion of Rothschild & Swann (1949) that there are considerable
differences in the velocity of the spread of the fertilization impulse and the
extrusion of cortical granules. Ginsburg (1960, 1961) failed to find in sturgeon
eggs a rapidly spreading fertilization impulse even when judging by the sign of
its hypothetical action (Rothschild & Swann, 1949, 1952) on the block to
polyspermy. It is reasonable to conclude that the impulse bringing about the
transition of the nucleus to the active state spreads in the cortical egg layer
at a rate similar to the velocity of the spread of changes in cortical granules.
The action of cortical changes on the egg-cleavage process is not exhausted
by the stimulation of the resting female nucleus to develop; in sturgeons, as in
sea urchins (Allen, 1954, Allen & Hagstrom, 1955), the cortical reaction causes
CORTICAL CHANGES IN ACIPENSERID EGGS
19
an effect upon the endoplasm without which normal movement of the cleavage
nuclei is impossible: a portion of the egg in which cortical changes are lacking
does not undergo cleavage either in the sturgeon or in the sea urchin.1
The last question to be considered deals with the behaviour of acid mucopolysaccharides of the cortical structures in sturgeon eggs in the process of
activation. This problem is of some interest in connexion with the widely known
theory of Runnstrom (1949, 1952) on the inhibitory function of acid mucopolysaccharides in unfertilized sea-urchin eggs. If the facts underlying this
theory could be demonstrated on another object, it would be of great interest.
^ ^ - "
butyric
acid
. FERTILIZATION
s'
IMPULSE
ACROSOME
REACTION
'
/
^
|
> CORTICAL GRANULE ^ ^ ^
^--"•^
BREAKDOWN
^ ^ ^ ^
^ *
<
\
i :
( Latent; ? ;Period)
^
"RAPID BLOCK"
AGAINST POLYSPERMY (?)
^
\
ENDOPLASMIC
ACTIVATION V ^ ^ ^
MEMBRANE
DEVELOPMENT
COLLOIDS FOR
PERIVITELLINE SPACE
HYALINE LAYER
niRFCTFn
STREAMING
" ^ • ^ - - ^ PIGMENT
.
MIGRATION
^ * * NUCLEAR
MOVEMENTS
TEXT-FIG. 6. Scheme comparing changes upon fertilization and artificial activation of echinoderm
eggs (taken from Allen, 1958).
The PAS-iodine method does not reveal the presence of diffusely scattered
acid mucopolysaccharides in the cortical layer of ripe unfertilized eggs of
sturgeons. After activation, during the extrusion of cortical granules, a diffuse
staining of the cytoplasmic columns appears, as in sea urchins (Monne &
Slautterback, 1950; Immers, 1956; Runnstrom & Immers, 1956), but in sturgeons it is due first of all to the disruption of cortical granules inside the cortical
layer. The protoplasmic mass containing these polysaccharides passes later to
the membranes. The surface light layer, which arises in sturgeon eggs after extrusion of cortical granules, either does not contain acid mucopolysaccharides
at all, or contains but a very small amount of them. Thus, the localization and
changes in acid mucopolysaccharides of the cortical layer revealed by the PASiodine method in sea-urchin and sturgeon eggs are not identical. The results
of vital Na-periodate treatment of the Acipenserid are also different from those
in sea urchins (Runnstrom & Kriszat, 1950): it does not activate sturgeon eggs,
causing only a depolymerization of the membranes, and it seems not to penetrate into the egg itself. Differences in the permeability of sea-urchin and sturgeon eggs are revealed by the action of other substances too. (For example
I [A urea solution, though it does activate sturgeon eggs, does not dissolve the
1
Recent experiments have shown that the 'non-fertilized' part of the cortex can neither deepen nor
participate in the formation of cleavage furrows: when running into an area of the 'non-fertilized'
cortex, in sevruga eggs with the mosaic cortical reaction, the cleavage furrow is stopped in its distribution and does not appear in the 'fertilized' surface below this 'non-fertilized' area.
20
T. A. DETTLAFF
material of cortical granules; neutral ATP solution in river-water does not accelerate cortical reaction, and heparin solution does not affect mitosis.) Thus
the data obtained on sturgeons do not provide additional convincing facts in
favour of this theory, though they do not contradict it.
To compare changes upon fertilization and artificial activation of sturgeon
eggs with those in sea-urchin eggs, the scheme put forward by Allen (1958) for
echinoderms (Text-fig. 6) and the same scheme modified for sturgeons (Textfig. 7) are presented here. Along with some similar features, essential differences
can be seen in them which have been discussed above. Further investigations
must show which of these differences are essential and which are a result of the
incompleteness of our present knowledge.
Block against
olyspermy
Fertilization
impulse
Formation of the
• granular part of
membrane
After a short latent
period expulsion of
cortical granules
k
*Acrosome reaction
(or artificial stimulation)
Release of the
factor for membrane
hardening and stickiness
Endoplasm
activation
"Shift of the globules of
the hydrophilic colloid
to the egg surface
Stimulation of the
completion of the
second maturation
division^
Nuclear
movements
Origin of the
perivitelline
space colloid
I
Movement of the
colloid to the crevices
between the blastomeres
According to unpublished data of Ginsburg &_ Dettlaff the spermatozoa of sturgeon
and sevruga form an acrosomal filament under the influence of the egg water.
TEXT-FIG. 7. Allen's (1958) scheme for echinoderms (see Text-fig. 6) modified for sturgeons.
SUMMARY
1. The paper describes the cortical structures of the Acipenserid egg and their
changes during fertilization and artificial activation by pricking, and the relationship between fertilization impulse, cortical granule expulsion, formation
of perivitelline space, membrane changes, and nuclear stimulation. The eggs of
Acipenser guldenstddti colchicus V. Marti, A. stellatus Pall., and Huso huso L.
were studied.
2. The fertilization impulse bringing about nuclear stimulation and the disintegration and discharge of cortical granules spreads in the cortical layer at
CORTICAL CHANGES IN ACIPENSERID EGGS
21
a velocity close to that of the spread of visible changes in the cortical granule
layer.
3. Changes in the layer of cortical granules begin 1-2 seconds after the origin
of the fertilization impulse and spread in a wave-like fashion over the egg
surface, from the place of pricking (or, in the case of fertilization, from the
micropylar canal region) to the opposite egg pole (in sturgeon this process takes
some 3 minutes at 18° C).
4. The extrusion of cortical granules determines the first separation and rotation of the egg within its membranes and makes hydrophilic colloid globules
rise to the egg surface and become extruded; at the time of extrusion of the
cortical granules a substance (or substances?) is released which affects the swelling, stickiness, and hardening of the egg membranes.
5. Formation of the widened perivitelline space in sturgeons, unlike teleosts
and sea urchins, does not directly follow the extrusion of cortical granules, and
is related to the swelling of the colloid excreted from the animal region of the
egg which differs from the cortical granule material. This colloid, unlike the
cortical granules, does not contain sulphated acid mucopolysaccharides, being
related in its origin to the lacunae of the karyoplasm arising in the oocyte cytoplasm during the maturation period. At cleavage stages this colloid enters the
spaces between the blastomeres and participates in blastocoel formation.
6. The localization and fate of sulphated acid polysaccharides of the egg cortical
layer during the process of activation in sturgeons differs from that in sea
urchins. The problem is discussed in relation to Runnstrom's theory of the inhibiting action of acid mucopolysaccharides in unfertilized eggs.
7. The dynamics of the process of completion of the second maturation
division is established: a prolonged period is occupied by very slow separation
of sister chromosomes, then follows a period of the same duration in which
the chromosomes rapidly diverge to the spindle poles and, simultaneously, the
spindle itself lengthens. After reaching its maximal length, the spindle shortens
and then the separation of the polar body begins.
BBIBO^bl
B pa6oTe orracaHbi KopTHKaABHtie CTpyKTypti nviu, oceTpOBbix pw6 H HX H3MeHCHHH npH OIIAO4OTBOpeHHH H aKTHBaiJHH yKOAOM, a TaKHCe B3aHM00TH0meHHfl
MCHC4y HMiryAbCOM OnAO^OTBOpeHHH, Bbl^eAeHHeM KOpTHKaAbHblX rpaHVA, B03HHK-
a^pa. H3yieHbi nftija oceTpa (Acipenser giildenstadti colchicus V.
Marti), ceBpiora (A. stellatus Pall.) H 6eAyrn [Huso huso L.).
i. HMiryAbC onAO^OTBopeHHH, Bbi3bmaioujHH CTHMVAHI!,HK) flApa, a TaioKe p a c na,4 H Bbi^eAeHHe KopTHKaAbHbix rpaHVA, pacnpocTpaHaeTCH B KopTHKaAbHOM CAOe flHIja CO CKOpOCTbK), 6AH3KOH K CKOpOCTH pacnpOCTpaHeHHH BH^HMblX
B CAoe KopTHKaAbHbix rpaHVA.
22
T. A. D E T T L A F F
2. H3MeHeHHH B CAoe KopTHKaAtHbix rpaHyA Ha^HHaioTCfl qepe3 1-2
nocAe BO3HHKHOBeHHH HMnyAtca onAo^OTBopeHHH H BOAHoo6pa3HO pacnpocTpaHHIOTCH n o noBepxHOCTH a n y a OT Mecra yKOAa (HAH B CAyqae onAO^OTBopeHiin,
OT o6AacTH KaHaAbu,eB MHKponHAe) K npoTHBonoAOHCHOMy noAiocy HHiia (y
oceTpa 9TOT npoijecc 3aHHMaeT n p n i 8 ° C . OKOAO 3 MHHVT).
3. Bbi^eAeHne KopTHKaAbHbix rpaHyA o6ycAOBAHBaeT nepBoe o6oco6AeHiie H
noBopoT fliiua BHyTpn o6oAOiieK. O H O CTHMyAHpyeT n o ^ t e M K noBepxHOCTH
HHija rH4po(j)HABHoro KOAAOH^a H ^eAaeT BO3MOJKHWM e r o BbmeAemie 110,4
o6OAO*IKH. O^HOBpeMeHHO C BbI4eAeHHeM KOpTHKaALHBIX rpaHyA H3
ocBo6o>K4aeTCfl BeujecTBO (HAH BeuiecTBa ?) BAHmoujee Ha Ha6yxaHiie,
KAeflKocTH H 3aTBep^eBaHHe nfiijeBbix O6OAO^CK.
4. O6pa30BaHHe pacninpeHHoro nepHBHTeAAHHOBoro npocTpaHCTBa y oceTpoBbix, B OTAH^iHe OT MopcKHx e>KevL H KOCTHCTBIX pbi6, He CAe^yeT Henocpe^CTBeHHO 3a Bbi^eAeHHeM KopTHKaAbHbix rpaHyA H CBH3aHO c Ha6yxaHHeM KO AAOH^a,
Bti^eAflioujerocH H3 aHHMaABHoft ^acTH a a ^ a H He H^eHTHHHoro MaTepHaAy
KopTHKaAbHbix rpaHyA. 9 T O T KOAAOH^, B OTAH^ne OT KopTHKaAbHbix rpaHyA, He
CO^ep^CHT CyAb^)HpOBaHHbIX MVKOnOAHCaxapH^OB H CBH3aH B CBOeM
4CHHH C AaKyHaMH KapHOnAa3MbI, BO3HHKaK)IIJHMH B IXHTOnAa3Me
npoHecce co3peBaHHH. H a CTa^nax 4po6AeHHa KOAAOH^ 3axo4HT B IIJCAH
6AacTOMepaMH H npnHHMaeT yqacTne B o6pa3OBaHHH 6AacTOiieAH.
5. AoKaAH3aHHH H cy^b6a cyAb^npoBaHHbix KHCAWX
KopTHKaAbHoro CAOH HHiia B npoijecce aKTHBayHH y oceTpoBbix
OT TaKOBbix y MopcKHx eaten. Bonpoc o6cyHC4aeTCH B CBH3H C Teopnew
PyHHCTpeMa 06 HHrn6HpyioujeM ^CHCTBHH KHCAWX MyKonoAHcaxapn^oB B
HeOnAO^OTBOpeHHOM HHHC
6. H s y ^ e H a ^HHaMHKa 3aBepineHHH BToporo ^eAeHHH co3peBaHHH. BHa^aAe
AeHHoe o6oco6AeHHe cecTpaHCKHx xpoMOCOM. 3aTeM
KOPOTKHH n e p n o ^ , B Te^eHne KOToporo xpoMocoMbi 6wcTpo
K noAiocaM BepeTeHa H , o^HOBpeMeHHo, y^AHHaeTCH caMO BepeTeHO.
BepeTeHO yKopa^HBaeTCH H Ha^HHaeTcn o6oco6AeHne
ACKNOWLEDGEMENTS
The author is greatly indebted to Drs. A. S. Ginsburg and A. I. Zotin, whose
close contact was a great help in the study of various problems of Acipenserid
fertilization. Grateful acknowledgement is made to Dr. G. M. Ignatieva and
Prof. G. V. Lopashov for critical discussion of the manuscript and to Dr. R. I.
Tatarskaya for consultation about and participation in hyaluronidase preparation, as well as to Miss S. E. Golossovskaya, Mrs. L. A. Filatova, and
Miss R. V. Pagnaeva for their technical assistance.
CORTICAL CHANGES IN A C I P E N S E R I D EGGS
23
REFERENCES
AFZELIUS, B. A. (1956). The ultrastructure of the cortical granules and their products in the sea
urchin egg as studied with the electron microscope. Exp. Cell Res. 10, 257-85.
AKETA, K. (1954). The chemical nature and origin of the cortical alveoli in the egg of the Medaka,
Oryzias latipes. Embryologia, 2, 63-66.
ALLEN, R. D. (1954). Fertilization and activation of sea-urchin eggs in glass capillaries. Exp. Cell
Res. 6, 403-24.
(1958). The initiation of development. A Symposium on the Chemical Basis of Development,
pp. 17-72. Baltimore: Johns Hopkins University Press.
& HAGSTROM, B. E. (1955). Some interrelationships among cortical reaction phenomena in the sea
urchin egg. Exp. Cell Res. (Suppl.) 3, 1-15.
& GRIFFIN, J. L. (1958). The time sequence of early events in the fertilization of sea urchin eggs.
Exp. Cell Res. 15, 163-73.
& ROWE, E. C. (1958). The dependence of pigment granule migration on the cortical reaction
in the egg of Arbacia punctulata. Biol. Bull. Wood's Hole, 114, 113-17.
ANCEL, P., & VINTEMBERGER, P. (1948). Recherches sur le determinisme de la symetrie bilaterale dans
l'oeuf des amphibiens. Bull. biol. Fr. Belg., 31, 1-182.
BENEDEN, E. VAN (1883). Recherches sur la maturation de l'oeuf et la fecondation. Arch. Biol. 4,
265-640.
BIALASZEWICZ, K. (1912). Uber das Verhalten des osmotischen Druckes wahrend der Entwicklung
der Wirbeltierembryonen. Roux' Arch. EntwMech. Organ. 34, 489-540.
COLWIN, L. H., & COL WIN, A. L. (1954). Fertilization changes in the membranes and cortical granular
layer of the egg of Saccoglossus Kowalevskii (Enteropneusta). /. Morph. 95, 1-45.
DETTLAFF, T. A. (1957). Cortical granules and substances secreted from the animal portion of the
egg during the period of activation in Acipenseridae. Doklady Acad. Nauk, SSSR, 116, 341-44
(Russian).
(1958). The role of calcium ions for the stimulation of eggs and spread of cortical reaction in
Acipenseridae. Doklady Acad. Nauk, SSSR, 121, 944-7 (Russian).
& GINSBURG, A. S. (1954). The Embryonic Development of the Sturgeons (Acipenser stellatus, A.
giildenstadti colchicus and Huso huso) in Connection with the Problem of Breeding. Moscow: USSR
Acad. Sci. (Russian).
DEVILLERS, CH., THOMOPOULOS, A., & COLAS, J. (1953). Differentiation bipolaire et formation de
l'espace perivitellin dans l'oeuf de Salmo irideus. Bull. Soc. zool. Fr. 78, 462-70.
ENDO, J. (1952). The role of the cortical granules in the formation of the fertilization membrane in
eggs from Japanese sea urchins. Exp. Cell Res. 3, 406-18.
FAURE-FREMIET, E., EBEL, J. P., & COLAS, J. (1954). Les inclusions proteiques de l'oocyte de Parascaris equorum. Exp. Cell Res. 7, 153-68.
GrNSBURG, A. S. (1956). Lipids in the oocytes and in the egg of Acipenser stellatus Pall. Doklady
Acad. Nauk, SSSR, 111, 236-40 (Russian).
(1957). The time of establishment of contact between the egg and spermatozoon in sturgeons.
Doklady Acad. Nauk, SSSR, 115, 845-8 (Russian).
(1959). Fertilization in sturgeon. I. The fusion of gametes. Cytologia, 1, 510-26 (Russian).
(I960). On the mechanism underlying the elimination of polyspermy in Acipenseridae. Doklady
Acad. Nauk, SSSR, 130, 473-6.
(1961). Block to polyspermy in sturgeon and trout with special reference to the role of cortical
granules (alveoli). /. Embryol. exp. Morph. 9, 173-90.
HAGSTROM, B. E., & ALLEN, R. D. (1956). The mechanism of nicotine induced polyspermy. Exp. Cell
Res. 10, 14-23.
HALE, C. W. (1946). Histochemical demonstration of acid polysaccharides in animal tissues. Nature,
Lond., 157, 802.
HERTWIG, O. (1877). Beitrage zur Kenntniss der Bildung, Befruchtung und Theilung des thierischen
Eies. Morph. Jb. 3,1-86.
HIRAMOTO, I. (1954). Nature of the perivitelline space in sea urchin eggs. Jap. J. Zool. 11, 227-43.
IMMERS, J. (1956). Changes in acid mucopolysaccharides attending the fertilization and development
of the sea urchin. Ark. Zool. 9, 367-75.
ISHIKAWA, M. (1954). Relation between the breakdown of the cortical granules and permeability to
water in the sea-urchin egg. Embryologia, 2, 57-62.
IVANOV, N. N. (1946). Methods of Plant Physiology and Biochemistry. Moscow (Russian).
KANOH, J. (1953). Uber den japanischen Hering (Clupea pallasii Cuvier et Valenc). II. Veranderung
im Ei bei der Befruchtung oder Aktivierung. Cytologia, Tokyo, 18, 67-79.
& ITO, S. (1953). The effect of outer medium upon the cortical change, bipolar differentiation and
24
T. A. D E T T L A F F
karyokinesis at the time of activation in the egg of Japanese dace (Tribolodon hakuensis). Bull.
Jap. Soc. Sci. Fish. 18, 462-7.
KEMP, N. (1956). Electron microscopy of growing oocytes of Rana pipiens. J. biophys. biochem.
Cytol. 2, 281-91.
KUSA, M. (1953a). Significance of the cortical change in the initiation of development of the Salmon
egg (physiological analysis of fertilization in the egg of salmon, Oncorhynchus keta). Annot. zool.
Jap. 26, 73-77.
(19536). On some properties of the cortical alveoli in the egg of the Stickleback. Annot. Zool.
Jap. 26, 138^4.
(1954). The cortical alveoli of salmon egg. Annot. Zool. Jap. 27, 1-6.
(1956). Studies on cortical alveoli in some teleostean eggs. Embryologia, 3, 105-29.
(1957a). Osmotic behaviour of the isolated cortical alveoli of Stickleback eggs. Annot. zool. Jap.
30, 67-70.
(19576). Occurrence of a neutral mucopolysaccharide in the cortical alveoli of Lamprey eggs.
/. Fac. Sci. Hokkaido Univ. (Zoology), 13, 455-7.
(1958a). Explosion of isolated cortical alveoli of the Stickleback egg. Annot. zool. Jap. 31,
212-15.
(19586). Cortical alveoli in some teleostean eggs. Symp. Cell Chent. 8, 101-18.
& OOTAKE, S. (1959). Notes on the particles occurring in the perivitelline space in the egg of
the brook lamprey following insemination. Bull. Yamagata Univ. nat. Sci. 4, 459-68.
MAZIA, D., BROWER, Ph., & ALFERT, M. (1953). The cytochemical staining and measurement of pro-
tein with mercuric-bromphenol blue. Biol. Bull. Wood's Hole, 104, 5-7.
MONNE, L., & SLAUTTERBACK, D. B. (1950). Differential staining of various polysaccharides in sea
urchin eggs. Exp. Cell Res. 1, 477-91.
MONNE, L., & HARDE, S. (1951a). On the cortical granules of the sea urchin egg. Ark. Zool. 1,487-98.
(19516). Changes in the protoplasmic properties occurring upon stimulation and inhibition
of the cellular activities. Ark. Zool. 3, 289-318.
MOORE, A. R. (1951). On the origin and nature of the membranes which result from fertilization in
the eggs of echinoderms. Scientia, 86, 195-9.
MOTOMURA, I. (1934). On the mechanism of fertilization and development without membrane formation in the sea urchin egg, with notes on a new method of artificial parthenogenesis. Sci. Rep.
Tdhoku Imp. Univ. 9, 33-45.
(1941). Materials of the fertilization membrane in the egg of Echinoderms. 5c/. Rep. Tdhoku
Imp. Univ. 16, 345-63.
(1950). On a new factor for the toughening of the fertilization membrane in the eggs of echinoderms. Sci. Rep. Tdhoku Imp. Univ. 18, 561-70. (After Motomura, 1954.)
(1952). Cortical granules in the egg of the frog. Annot. zool. Jap. 25,238-41. (After Kusa, 1956.)
(1954). Inhibition and acceleration of the toughening of the fertilization membrane in the sea
urchin eggs. Sci. Rep. Tdhoku Univ. 20, 158-62.
NAKANO, E. (1956). Changes in the egg membrane of the fish egg during fertilization. Embryologia, 3,
89-103.
& OHASHI, S. (1954). On the carbohydrate component of the jelly coat and related substances
of eggs from Japanese sea urchins. Embryologia, 2, 81-85.
PARPART, A. K., & LARIS, P. S. (1955). A theory for the lifting of the fertilization membrane of the
egg of Arbacia punctulata. Biol. Bull. Wood's Hole, 109, 350-1.
& CAGLE, J. (1957). Contractility of the hyaline layer of Arbacia punctulata. Biol. Bull. Wood's
Hole, 113, 331.
PEARSE, A. G. (1951). Histochemistry, Theoretical and Applied. London: J. Churchill.
PELTZAM, E. D. (1886). Observations on egg segmentation in Acipenser ruthenus. Izvestia obshchestva
lubit. estestvoznania, antropol., etnogr. M. 50, Prot. zool. otdel. 1, 1-206 (Russian).
ROSENBAUM, R. (1958). Histochemical observation on the cortical region of the oocytes of Rana
pipiens. Quart. J. micr. Sci. 99, 159-69.
ROTHSCHILD, LORD (1956). Fertilization. London: Methuen.
(1958). Fertilization in fish and lampreys. Biol. Rev. 33, 372-92.
& SWANN, M. M. (1949). The fertilization reaction in the sea-urchin egg. A propagated response
to sperm attachment. /. exp. Biol. 26, 164-76.
(1952). The fertilization reaction in the sea-urchin. The block to polyspermy. / . exp. Biol.
29, 469-83.
RUNNSTROM, J. (1928). Uber die Veranderung der Plasmakolloide bei der Entwicklungserregung des
Seeigeleies. Protoplasma, 5, 201-310.
(1948). Further studies on the formation of the fertilization membrane in the sea-urchin egg.
Ark. Zool. 40, 1-19.
CORTICAL CHANGES IN A C I P E N S E R I D EGGS
25
RUNNSTROM, J. (1949). The mechanism of fertilization in metazoa. Advanc. Enzymol. 9, 241-327.
(1952). The cell surface in relation to fertilization. Symp. Soc. exp. Biol. 6, 39-88.
(1956). Some aspects of the initiating processes in the fertilization of the sea-urchin egg. {Zool.
Anz. 156,91-101.
— , MONNE, L., & WICKLUND, E. (1946). Studies on the surface layers and the formation of the
fertilization membrane in sea-urchin eggs. </. Colloid Sci. 1, 421-51.
& KRISZAT, G. (1950). Action of periodate on the surface reactions attending the activation of
the egg of the sea urchin, Psammechinus miliaris. Exp. Cell Res. 1, 355-70.
& IMMERS, J. (1956). The role of mucopolysaccharides in the fertilization of the sea-urchin egg.
Exp. Cell Res. 10, 354-63.
SAKUN, O. F. (1960). Chemical nature and significance of inclusion in oocytes of osseous fishes.
Arkh. anat., gist., embr. 38, 38-42 (Russian).
SALENSKY, W. (1881). Recherches sur le developpement du sterlet (Acipenser ruthenus). Arch. Biol. 2,
233-341.
SHABADASH, A. L. (1949). Glycogen histochemistry in the Normal Nervous System. Moscow: Medgiz.
(Russian).
SUGIYAMA, M. (1956). Physiological analysis of the cortical response of the sea urchin egg. Exp. Cell
Res. 10, 364-76.
TAKASHIMA, R., MORI, S., & KAWANO, M. (1955). The role of hyaluronidase in fertilization. Bull.
exp. Biol. 5, 75-81.
THOMOPOULOS, A. (1953a). Sur l'oeuf de PercafluviatilisL. Bull. Soc. zool. Fr. 78, 106-14.
(1953&). Sur l'ceuf de l'epinoche {Gasterosteus aculeatus L.). Bull. Soc. zool. Fr. 78, 142-9.
TYLER, A. (1941). Artificial parthenogenesis. Biol. Rev. 16, 291-336.
(1955). Gametogenesis, fertilization and parthenogenesis. In Analysis of Development, pp. 170212, ed. B. H. Willier, P. Weiss, & V. Hamburger. London; Saunders Co.
VASSEUR, E. (1948). Chemical studies on the jelly coat of the sea-urchin egg. Acta chem. scand. 2,
900-13.
& IMMERS, J. (1949). Genus specificity of the carbohydrate component in the sea-urchin egg
jelly coat as revealed by paper chromatography. Ark. Kemi, 1, 39-41.
WARTENBERG, H. (1956). Topochemische Untersuchungen an den Ovarialeiern von Xenopus laevis
und Rana fusca. Acta Histochem. 3, 25-71.
WINTREBERT, P. (1933). La Mecanique du developpement chez Discoglossuspictus Otth. de l'ovogenese
a la segmentation. Arch. Zool. exp. gen. 75, 501-39.
YAMAMOTO, K. (1956). Studies on the formation of Fish eggs. VIII. The fate of the yolk vesicle in the
oocyte of the smelt, Hypomesus japonicus, during vitellogenesis. Embryologia, 3, 131-8.
YAMAMOTO, T. (1939). Mechanism of membrane elevation in the egg of Oryzias latipes at the time of
fertilization. Proc. imp. Acad. Japan, 15, 272-4.
(1944). Physiological studies on fertilization and activation of fish eggs. I. Response of the cortical layer of the egg of Oryzias latipes to insemination and artificial stimulation. II. The conduction
of the 'fertilization wave' in the egg of Oryzias latipes. Annot. zool. Jap. 22, 74-82, 109-36.
(1951). Action of lipoid solvents on the unfertilized eggs of medaka {Oryzias latipes). Annot.
zool. Jap. 1A, 74-82.
(1956). The physiology of fertilization in the medaka {Oryzias latipes). Exp. Cell Res. 10,
387-93.
ZOTIN, A. I. (1953). Change in mechanical resistance of egg membranes of Acipenseridae embryos
during development. Doklady Akad. Nauk, SSSR, 92, 443-6 (Russian).
(1955). Water uptake by developing eggs of Salmonid and Acipenserid fishes from the surrounding medium. Voprosy ichthyologii, 4, 82-104 (Russian).
(1958). The mechanism of hardening of the Salmonid egg membrane after fertilization or
spontaneous activation. / . Embryol. exp. Morph. 6, 546-68.
(1961). Physiology of Water Exchange in the Embryos of Fishes and Cyclostomata. USSR Acad.
Sci. (Russian).
& KRUMIN, A. I. (1959). Formation of the primary gut cavity in Acipenserid embryos. Zhurn.
obshchei biol. 20, 313-21 (Russian).
EXPLANATION OF PLATES
KEY: C.G., cortical granules; F.M., funnel of maturation; G.H.C, globules of hydrophilic colloid;
G.M., granular material extruded from the egg (inner portion of the 'fertilization membrane'); J.M.,
jelly membrane; L.C. lacunae of caryoplasm; L.L., light layer; M.c.,micropylar canal; N, female nucleus;
p.c, protoplasmic columns; P.G., pigment granules; P.S.C, perivitelline space colloid; v, vacuoles in
the cortical layer; Z.R.I., zona radiata interna; Z.R.E. zona radiata externa.
26
T. A. DETTLAFF
PLATE I
FIG. 1. Polar spot region of the ripe sevruga egg. Fixation with Sanfelice fluid, staining with
Heidenhain's azan.
FIG. 2. Sturgeon oocyte at the stage close to the onset of nuclear membrane dissolution. Fixation
with Sanfelice fluid, staining with Heidenhain's iron haematoxylin.
FIG. 3. Sturgeon oocyte at the stage following nuclear membrane dissolution; the passage of
karyoplasm into the cytoplasm can be clearly seen. (Fixation and staining as in fig. 2.)
FIG. 4. Lacunae of karyoplasm containing a globule of hydrophilic colloid in the animal part of
ripe sturgeon egg. (Fixation and staining as infig.2.)
FIG. 5. Region of the animal part of the ripe sturgeon egg containing the globules of hydrophilic
colloid. Fixation with 4 per cent, formol, staining with Heidenhain's azan.
FIG. 6. Sections through a sturgeon egg treated alive with 001 M NaIO4 solution and then
in sections: (a) treated with 001 M NaIO4 solution and thereafter with Sch iff reagent; (b) with Schiff
reagent only. Fixation with Sanfelice fluid.
FIG. 7. Successive stages of cortical changes in sturgeon egg (from the side to the top of the animal
region of the egg): (a) unchanged granules; (b) vacuoles and protoplasmic columns containing
cortical granule material; (c) and (d) vacuoles enlarged, columns lengthened; (e) expelled granule
material adhering to the inner vitelline membrane. Fixation with Sanfelice fluid, staining with
Heidenhain's azan.
FIG. 8. The cortical part of a sturgeon egg activated without the expulsion of cortical granules.
Cortical granules partly disintegrated; simultaneously, below the cortical granule layer, are seen in
section the substances of the hydrophilic colloid globules which had migrated to the egg surface.
Fixation with Sanfelice fluid, staining with Heidenhain's azan.
PLATE 2
FIG. 1. a-f. Successive stages of changes in the animal region of sturgeon eggs, fixed at different
times after pricking or insemination: a, non-activated ripe egg; b, 1 minute after pricking \ C, 8 minutes;
d, 30 minutes; e, 50 minutes;/, stage of the first cell-division. Fixation with Sanfelice fluid, staining
with Heidenhain's iron haematoxylin.
FIG. 2. The globules of hydrophilic colloid: a, in the endoplasm; b, at the egg surface (1 minute
after pricking). Fixation with Sanfelice fluid, staining with Heidenhain's iron haematoxylin.
FIG. 3. Distribution of acid mucopolysaccharides in the surface layers of the sturgeon egg 30 minutes
after fertilization. Fixation with lead-formol. Sections treated by Schiff-iodine method modified by
Shabadash. a, without hyaluronidase pretreatment; b, after pretreatment of sections with testicular
hyaluronidase; c, control staining with Heidenhain's azan.
PLATE 3
Later stages of changes in the sturgeon egg surface and the origin of the perivitelline space colloid.
Fig. 1,fixationwith Sanfelice fluid, at different times after pricking the egg in the animal region, staining with Heidenhain's iron haematoxylin, / = 14-9-15° C. Fig. 2, fixation with 4 per cent, formol,
at different times after insemination, staining with Heidenhein's azan. / = 17-8-18" C.
FIG. 1. a, egg surface after the discharge of cortical granules (30 seconds); b, globules of hydrophilic
colloid shift to the egg surface (5 minutes); c, the surface layer contains more hydrophilic colloid
substances (after 12 minutes); d, the egg surface layer after 50 minutes; e, egg surface of the 2-cell
division stage.
FIG. 2. a, swelling and vacuolization of the colloidal globules inside the egg (20 minutes); b, part of
the colloid remains in the egg, and part is already in the perivitelline space; c, connexion between
the egg surface and the perivitelline space colloid (30 minutes); d, vacuoles at the egg surface in the
perivitelline space colloid (75 minutes); e, the boundary between the light layer and the perivitelline
space colloid is indistinguishable (78 minutes);/, the expulsion of the perivitelline space colloid is
finished. The egg surface seems smooth (stage of the first cell-division).
PLATE 4
FIG. 1. Vacuoles in the pathway of the presumptive cleavage furrow (V.C.F.).
Fig. 2. The termination of the chain of vacuoles. (c.Ag, colloid aggregation stained by aniline
blue.
FIG. 3. Divergence of sturgeon sister chromosomes and spindle length at different time intervals
after egg activation by pricking (A) into the border of the animal region and (v) into the vegetative
egg pole: a 12, b 14, c 16, d 18 minutes after pricking.
Vol. 10, Part 1
/. Embryol. exp. Morph.
T. A. DETTLAFF
Plate 1
/. Embryol. exp. Morph.
Vol. 10, Part 1
T. A. DETTLAFF
Plate 2
Vol. 10, Part 1
/. Ernbryol. exp. Morph.
O
20
4O
O
6O 8O 100ji
T. A. DETTLAFF
Plate 3
20 40 60 80 100 ji
J. Embryol. exp. Morph.
Vol. 10, Part 1
50>t
T. A. DETTLAFF
Plate 4