/ . Embryol. exp. Morph., Vol. 17, 2, pp. 331-340, April 1967
Printed in Great Britain
331
A regional change
in the rigidity of the cortex of the egg of
Rana nigromaculata following extrusion of
the second polar body
By T. KUBOTA 1
From the Biological Laboratory, Liberal Arts College,
Kagoshima University, Japan
Measurements of the consistency of the protoplasm by following the movement of cellular granules under centrifugal force have been made in various
materials (e.g. for cytoplasm, Heilbrunn, 1928; Heilbrunn & Wilson, 1948;
Allen, 1960; for cortex, Brown, 1934; Wilson, 1951; Zimmerman, Landau &
Marsland, 1957). In amphibian eggs such measurements were performed by
Konopacka (1908) and Odquist (1922) on the cytoplasm and by Selman &
Waddington (1955) on the cortex. However, since the observations of the latter
authors were restricted to dividing eggs, a report on the cortical consistency
of uncleaved amphibian eggs determined by the centrifugal method is not
available.
In the present work, by applying a weak centrifugal force to uncleaved eggs of
Rana nigromaculata, it was found that a rigidity change took place regionally
on the cortex of fertilized eggs and of certain activated eggs after extrusion of
the second polar body.
MATERIALS AND METHODS
The eggs used were obtained from pituitary-stimulated females of Rana
nigromaculata. For insemination, a suspension of macerated testes was added
to eggs placed in a dry Petri dish. For activation, eggs were pricked with a glass
needle in the presence or absence of the frog's blood, following which spring
water was added. Experiments were performed at room temperatures (17-23 °C).
In order to determine the relative rigidity of the egg cortex at various stages,
eggs of the same batch were subjected to the same centrifugal force at regular
intervals, and the degree of displacement of pigment granules from the cortex was
compared among these eggs. A force of 250g (a radius of 13 cm c. 1300 rev./min)
was applied for a definite period (50-60 sec for different series). Since the
1
Author's address: The Biological Laboratory, Liberal Arts College, Kagoshima University,
Kamoike-cho, Kagoshima, Japan.
332
T. KUBOTA
frog egg orients with the animal pole on the centripetal side during centrifugation, the granules of the animal side are dislodged from the surface toward the
vegetal side, resulting in a fading of the tint of the pigment cap. The degree of
fading indicates ease of dislocation of the pigment granules, and consequently
the relative fluidity (inverse of rigidity) of the cortex.
For cytological observations, eggs were fixed in Zenker's fluid and Smith's
fluid with or without calcium chloride, and stained with bromphenol blue
(Mazia, Brower & Alfert, 1953) or triply stained with bromphenol blue, fast
green and acid fuchsin.
EXPERIMENTAL RESULTS
The rigidity changes in fertilized eggs
Rigidity change around the time of the extrusion of the second polar body. In
eggs which had just finished rotation, the rigidity of the cortex was so high that
no change in the pigment cap was seen even after centrifuging for 120 sec. But
30-45
Fig. 1. The regional change in the cortical rigidity as revealed by centrifuging fertilized eggs at successive intervals after second polar body extrusion. The black part in
each figure indicates a region where the rigidity has reached a higher level, i.e. the
region where the pigment remains on the surface. The dotted part indicates a region
where the rigidity is low, i.e. where the pigment is dislocated.
when eggs were taken 10-15 min later at the stage of the extrusion of the second
polar body, the pigment cap faded slightly after centrifugation for 60 sec,
indicating a decrease in the rigidity of the cortex. The rigidity further dropped
and reached the lowest level for the uncleaved stage several minutes later. In
other words, the dark brown tint of the pigment cap became yellowish brown
after a standard centrifugation of 250g for 50-60 sec.
Regional rigidity change after the polar body extrusion. About 15 min after polar
body extrusion (c. 5 min after the minimum noted above) a local increase in
rigidity was noted and the affected region continued to enlarge for 30-45 min
in 17 series (17-23 °C). The sequence is shown in Fig. 1, which was reconstructed
from observations made at brief intervals. The initial stage just described
corresponds to Fig. 1, b. In the figure the region where the rigidity reached a
Regional change in cortex rigidity
333
higher level, i.e. the region which retained a pigment patch after centrifugation,
is indicated in black. As illustrated in the figure, an initially small patch expanded
to cover eventually the entire animal hemisphere.
These growing patches possess the following two characteristics. First, the
patches may first appear in any part of the hemisphere and their shapes at early
stages may differ according to the places of their appearance (Fig. 1, b, c, b', c').
However, at some stage the patches lying near the animal pole (br, c') spread
more quickly toward the nearest equator (d') so that, after reaching the equator,
the shape of such a patch became more or less the same as that of a patch
lying near the equator from the beginning (e,f). Secondly, the spreading speed
of the pigment patch increased as the patch enlarged. In general, it took about
half as much time to spread over the second half of the hemisphere as the first
half.
Relation between the region of the rigidity change and sperm entrance point. In
some eggs, a black spot indicating the point of sperm entry still remained on the
centrifuged egg surface after fixation. In these cases the black mark was always
found on the median line of the patch. A recognition of the sperm pronucleus
was also possible in eggs centrifuged somewhat longer at a stage corresponding
to Fig. 1, c or d. In these eggs, a small white spot frequently appeared on the
patch due to a localized displacement of pigment granules. The white spot is
also on the median line of the patch, and it was ascertained from sections that
the sperm pronucleus lies below the white spot. The above findings indicate that
the regional change starts from the point of sperm entry.
The rigidity change in activated eggs
On pricking with a glass needle, activation occurred in 90-95 % of the eggs of
R. nigromaculata.
The cortical rigidity of activated eggs decreased at first. A subsequent rise
sometimes occurred regionally as in fertilized eggs, but it sometimes occurred
in an overall fashion.
Regional rigidity change. This type of change rather frequently occurred in
eggs activated with blood, but very rarely in eggs activated in the absence of
blood. The patches of activated eggs appeared near the pricked point in the
same way as the patches of fertilized eggs appeared around the point of sperm
entry. The size of the patches varied from one individual to another.
Overall rigidity change. In activated eggs showing an overall change, the
cortical rigidity remained at the minimum level for longer than in normal
fertilization (Fig. 2, a-e), and then the rigidity rose uniformly over the entire
hemisphere (Fig. 2,/). The rise was so rapid as to be complete in less than 10 min.
Once the change is complete (Fig. 2, g) there is no way of knowing which of
the two types of changes the eggs have undergone.
The rigidity changes and cleavage in activated eggs. Since the regional rigidity
334
T. KUBOTA
change was found to occur more frequently after pricking with blood, the
correlation between rigidity change and cleavage was investigated.
The eggs activated with blood were separated into those showing the regional
change and those showing the overall change, and then the number of cleaved
eggs in each group was counted. In the former group, the furrow was distinctly
seen in 27 of 30 eggs 1 | h after activation and the furrow remained in 22 eggs
after 4 | h, whereas in the latter group such a furrow was found at the respective
times in only 1 and 2 of 27 eggs.
It is clear that those activated eggs displaying the regional rigidity change have
a greater capacity for cleavage. According to Bataillon (1911; cf. also Bataillon
& Tchou Su, 1929) the blood cells introduced into the cytoplasm upon pricking
act as the 'second factor' promoting cytaster formation, thus increasing the
frequency of cleavage. Therefore, the occurrence of the regional rigidity change
in activated eggs in the presence of blood may be related also to cytaster
formation. To test this hypothesis sections were examined.
30-45 min
1 min,
Fig. 2. The overall change in cortical rigidity observed in activated eggs. A rapid rise
in rigidity occurs uniformly over the entire animal hemisphere.
Observations of sections
The first batch of eggs was inseminated. Parts of it were centrifuged at regular
intervals and fixed together with uncentrifuged control eggs. The second batch
was activated rather than inseminated. The eggs were sectioned parallel to a
plane including the median line of the pigment patch or the point of sperm
entry.
Sperm aster of uncentrifuged eggs. In normal, uncentrifuged eggs two concentric layers were recognized surrounding the sperm nucleus, the outer ring
being compact and the inner one coarse in texture. The outer ring stained light
blue with bromphenol blue and was in contact with the egg surface on one side.
Such a configuration staining light blue was already seen around the sperm head
at the stage of the second polar body extrusion (i.e. c. 15 min before the pigment
patch would appear in centrifuged eggs of the same batch; Fig. 3a). Fifteen
minutes later, at the stage corresponding to Fig. 1, b, the outer ring developed
to such an extent that its periphery came in contact with half the animal
cortex (Fig. 3 b). A little later corresponding to Fig. 1, c, the contact area expanded to two-thirds of the animal hemisphere (Fig. 3 c). In other words, the
Regional change in cortex rigidity
335
contact between the light blue ring and the egg cortex preceded the spreading of
pigment patch detectable by centrifugation.
The concentric figures are judged to be the sperm aster, because the figures
accord well in shape, size and position with (1) sperm asters of Rana pipiens
photographed by Subtelny & Bradt (1963) and are similar in appearance to (2)
the aster of the first cleavage in the present material.
Sperm aster of centrifuged eggs. In centrifuged eggs the concentric figures
were also seen around the sperm nucleus, resembling the uncentrifuged control
eggs at the same stage except that they stained less well with bromphenol blue.
In most cases, the contact between the two pronuclei was observed in eggs fixed
after pigment patches covered two-thirds of the animal hemisphere (Fig. l,f-h).
Cytaster of centrifuged, activated eggs. In activated eggs showing the regional
pigment pattern, well-developed concentric figures were found under the pigment patch. Being in activated eggs, the figures cannot be other than cytasters.
On the other hand, activated eggs showing the overall rigidity change lacked
such cytoplasmic figures.
Fig. 3. Axial sections of fertilized, uncentrifuged eggs, showing appearance and
development of a light-blue figure (densely dotted part) and its contact with the egg
cortex, (a) At a stage of the second polar body extrusion. The light-blue configuration surrounds the oval sperm head. In the present material, a pillar of large yolk
granules (filled circles) extends to the animal pole, (b) 15 min later at a stage corresponding to Fig. 1, b. Two migrating pronuclei (open circles) are found, one inside
and one on the periphery of the light-blue ring, (c) A little later, corresponding to
Fig. 1, c.
All the observations of the sections further strengthen the previously expressed
possibility that the regionally high rigidity is always underlain by the sperm
aster or cytaster.
Relation between rigidity change and determination of bilateral symmetry
Under natural conditions the median plane of many anuran embryos is
known to be determined at the uncleaved stage by the point of sperm entry.
Since the regional change in rigidity is also initiated around the point of sperm
entry, the two phenomena may be closely related. For this reason, the critical
time when the median plane is established in the egg of R. nigromaculata was
ascertained and was compared with that of the rigidity change.
At each of eight stages between egg rotation and completion of the regional
rigidity change, ten eggs were placed in a moist chamber with the egg axis
inclined 90° and 15 min later the chamber was flooded. Although the eggs
reoriented themselves, the return was not complete.
336
T. KUBOTA
If inclined before the stage corresponding to Fig. 1, / , the eggs formed a single
dorsal lip of the blastopore on the side where the border of the inclined pigment
cap was the highest. But, when inclined at stages later than Fig. 1,/, some eggs
formed two groves, one on the highest side and the other on any other side,
which eventually became continuous to make a large blastopore.
Table 1. The effects of inclining uncleaved eggs 90° at various stages on the subsequent axial differentiation, expressed in the ratios of anomalous embryos to the
total
Insemination
A
Rotation
t t t t t t t
Time of inclination
Appearance of two
initial blastopores
B
Period of rigidity change
_0_
9
0
10
0
10
0
10
0
9
0
10
3
10
t
t t
rr-
2
30
13
30
11
28
0
29
5
29
1
Irregularity or doubling of neural fold
29
14
27
Abnormality in late tail-bud embryo
2
29
9
28
8
28
17
27
Time of inclination
I
Appearance of two initial blastopores
i
This may indicate that the position of the dorsal lip on the highest side must
be determined anew by rotation, while the other groove must have been partially
determined before the egg was inclined. As long as one adopts this criterion, the
critical stage at which the bilateral symmetry is established in the eggs of R.
nigromaculata must be around that shown in Fig. l,/(see Table 1, A).
To know in more detail the status of the determination of bilateral symmetry,
observations were repeated on the position of the dorsal lip of the blastopore,
the position and shape of the neural fold and the shape of the embryo at the late
tail-bud stage. The results are summarized in Table 1, B. As shown in the table,
eggs inclined after late period of the regional change often showed various
abnormalities at gastrula, neural and tail-bud stages, indicating that the
determination of bilateral symmetry had occurred before inclination. However,
determination at this time is not yet rigid, because in embryos forming two
neural folds, one fold on the highest side predominated over the other fold,
suppressing the latter from differentiating into organs. It is reasonable to
Regional change in cortex rigidity
337
conclude that axial determination in eggs of R. nigromaculata begins at the late
stage of the regional change in cortical rigidity.
DISCUSSION
The rigidity change in other kinds of eggs. Zimmerman et al. (1957) working
with Arbacia eggs reported that the rise of the cortical 'gel strength' following
insemination is closely related in time to the development of the sperm aster.
They suggested the possibility that a gelation in the cortex occurs first, which
quickly spreads to the deeper cytoplasm, where it leads to the development of the
aster. This view may be compatible with observations that the formation of the
sperm aster in Chaetopterus eggs occurs following the rigidity rise in the cortex
(Heilbrunn & Wilson, 1948; Wilson, 1951). The application of the concept to the
frog egg, however, poses a difficulty, for the cortical rigidity does not rise until
the sperm aster had developed to a. considerable extent.
In Rana fusca, Konopacka (1908) centrifuged the eggs l | - 2 h (contact
between pronuclei) after insemination at 230g for 5-20 min and Odquist (1922)
after 1, 2 and 3 h at 700g for 3 min. Neither of these workers described any
regional change in pigmentation. In the present experiments, a force of 250g
was applied to eggs of R. nigromaculata for only 1 min. In the present material,
the pigment patches were much obscured by altering the centrifugal period
even by 10 sec. It is of the utmost importance to use a mild centrifugation and
the procedures employed by the above two investigators might have been too
drastic. This interpretation is supported by the fact that the same regional
pattern was observed in Rana limnocharis as long as mild centrifugation
procedures were applied.
The rigidity change and the determination of bilateral symmetry. In eggs of
several anurans, the grey (or clear) crescent is said to appear on the side opposite
the point of sperm entry (reviews: Holtfreter & Hamburger, 1955; Clavert, 1962;
Pasteels, 1964; Lovtrup, 1965). In eggs of R. fusca, Ancel & Vintemberger (1948)
pointed out that the future site of the crescent is indicated by direction of an
inclination of the pigment cap at the stage of the contact of pronuclei. The
border of the pigment on the side of the sperm entry is lower, the crescent
appearing later on the higher side. In activated eggs the pigment cap is also
inclined, but neither the direction of the inclination nor the position of the
crescent (Brachet, 1911) has a relation to the pricked point. Ancel & Vintemberger (1948) noticed that sections of fertilized egg show a concentration of
cortical pigment granules around the point of sperm entry, whereas activated
eggs show no such accumulation. From these observations, they speculated that
the inclination of the pigment cap toward the side of the sperm entry might be
brought about by a local increase in load in the cortex owing to a concentration
of the cortical material (pp. 122-3).
In the eggs of R. nigromaculata, as mentioned already, the rise in the cortical
338
T. KUBOTA
rigidity starts from the sperm entry point. If an increase in rigidity of the cortical
gel signifies a synaeresis of the gel, the specific gravity of the cortex around the
sperm pronucleus will surpass that of the rest of the cortex and will act as a load.
Furthermore, around the time when the regional change approaches completion
at the stage of syngamy, the determination of the median plane of the embryo
occurs. In other words, the development of the regional pattern of pigmentation
as revealed by a mild centrifugation of the eggs of R. nigromaculata and R.
limnocharis seems to offer excellent visual evidence for the concept proposed by
Ancel & Vintemberger (1948).
SUMMARY
1. By applying a weak centrifugal force to the eggs of Rana nigromaculata,
regional differences in the ease or difficulty of dislocating the pigment from the
cortex are revealed as pale or dark areas of the cortex.
2. In fertilized eggs, a region of a high rigidity (black patch) appears around
the point of sperm entry after polar body formation. It spreads to cover the
entire animal half roughly by the time of syngamy.
3. In some cases of activated eggs, especially among those pricked in frog's
blood, a similar regional pattern is seen. In the majority of them, and particularly
in those pricked in the absence of blood, an overall change in rigidity occurs
with no regionally.
4. The end of the regional change of the cortex and the onset of bilateral
symmetry coincide in time.
5. Studies of sections show that a regional pattern is always underlain by a
spermaster or a cytaster, while in eggs showing the overall change no aster was
seen.
6. Among activated eggs those undergoing the regional change are more
likely to enter cleavage.
RESUME
Modification regionale de la rigidite du cortex de Vceuf de Rana nigromaculata a
la suite de Vextrusion du deuxieme globule polaire
1. Par l'application d'une faible force centrifuge aux oeufs de Rana nigromaculata, des differences regionales dans la facilite ou la difficulte de disloquer
le pigment du cortex sont mises en evidence sous la forme de zones pales ou
sombres dans le cortex.
2. Dans les oeufs fecondes, une region a rigidite elevee (tache noire) apparait
autour du point d'entree du spermatozoide apres formation du globule polaire.
Elle s'etend et recouvre la moitie animale entiere, approximativement au
moment de ramphimixie.
3. Dans quelques oeufs actives, en particulier parmi ceux qui ont ete piques
dans du sang de grenouille, on observe une structure regionale semblable. Mais
dans la majorite d'entre eux, et en particulier chez ceux qui ont ete piques en
Regional change in cortex rigidity
339
l'absence de sang, une modification generate de la rigidite intervient sans
regionalisation.
4. La fin de la modification regionale du cortex coincide avec le debut de la
symetrie bilaterale.
5. Les recherches sur coupes ont montre qu'une structure regionale se trouve
toujours en rapport avec un spermaster ou un cytaster, alors qu'on n'a pas
observe d'aster dans les ceufs presentant la modification generate.
6. Parmi les oeufs actives, ceux qui subissent la modification regionale entreront
plus vraisemblablement en segmentation.
The author would like to thank Professor K. Dan for his valuable advice and kind help
in preparing the manuscript.
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{Manuscript received 29 September 1966)