/ . Embryol. exp. Morph. Vol. pp. 183-193, 1979
Printed in Great Britain © Company of Biologists Limited 1979
183
Cyclic changes in the cortical layer of non-nucleated
fragments of the newt's egg
ByTSUYOSHI SAWAI
From the Biological Laboratory, Faculty of General Education,
Yamagata University
SUMMARY
Various changes in the cortical layer of the amphibian egg have been observed during cleavage; for example, rounding up in the animal hemisphere, increase in stiffness of the surface and reactivity of the cortex to furrow-inducing cytoplasmic components. The three kinds
of change occur synchronously with the cleavage cycle.
The present experiments were aimed at determining whether the above changes in the
cortical layer are caused exclusively by the nucleus or autonomously by the cytoplasm.
Uncleaved fertilized eggs of the newt, Cynopspyrrhogaster, were divided into two parts, one
nucleated and one non-nucleated, by cutting with a fine glass needle. Special attention was
paid to the behaviour of the non-nucleated fragments, where the above-mentioned changes
were also observed. The cycle of these changes did not necessarily start synchronously
with the cleavage cycle of the nucleated partner, but, once started, the rhythm of both cycles
had almost the same timing. These results suggest that some change in the cortex necessary
for cleavage furrow formation in the amphibian egg is brought about by cyclic change in the
cytoplasm, independently of the nucleus.
INTRODUCTION
A leading hypothesis of amphibian cytokinesis explains the entire process of
cleavage in such a way that, in the initial phase of cytokinesis, the cleavage
furrow is formed by contraction of a bundle of microfilaments underlying the
bottom of the furrow (Bluemink, 1970; Selman & Perry, 1970; Bluemink, 1971;
Kalt, 1971), and in the late phase, new surface is supplied by fusion of membrane-bound vesicles with the existing furrow surface (Bluemink, 1971; Kalt,
1971; Bluemink & de Laat, 1973; Sanders & Singal, 1975).
On the other hand, various changes in the cortical layer of the amphibian
egg have been observed during cleavage. Selman & Waddington (1955) and
Selman, Jacob & Perry (1976) measured changes in the shape of the newt egg
during the cleavage, and Selman & Waddington (1955) and Sawai & Yoneda
(1974) reported that stiffness of the surface of the newt egg increased during the
cleavage. Contraction waves have been observed in the surface of the axolotl
egg (Hara, 1971) and the frog egg (Satoh, 1977). Sawai (1972) found that the
1
Author's address: The Biological Laboratory, Faculty of General Education, Yamagata
University, Yamagata 990, Japan.
184
T. SAWAI
cortex in the newt egg acquired the ability to form a cleavage furrow in response
to furrow-inducing cytoplasmic components. These cortical changes were
localized in the animal pole region when cleavage had just started, and propagated medially as a belt along the surface towards the vegetal pole with
advance of the furrow tip.
The present experiments were designed to investigate whether the changes in
the cortical layer are caused exclusively by activity of the syncaryon or autonomously by activity of the cytoplasm. Uncleaved fertilized eggs of the newt were
divided into two parts, one nucleated and one non-nucleated. Changes in
shape, stiffness of the surface and reactivity of the cortex in the non-nucleated
fragment were investigated with particular care.
MATERIALS AND METHODS
Eggs of the newt, Cynops pyrrhogaster, were used. Spawning of the eggs
was stimulated by injecting about 100 i.u. of pituitary hormone (Gonatropine,
Teikoku Zoki Co. Ltd) into the abdomen of the females every other day.
Experiments were carried out in Holtfreter's saline at room temperature
(20-25 °C).
Cutting operation
Uncleaved fertilized eggs were divided into two parts - nucleated fragments
and non-nucleated ones-in the following way. After removal of the jelly
capsule with scissors, the eggs were put on agar on the bottom of the dish. A
small slit was made in the vitelline membrane with a pair of watchmaker's
forceps, and from the narrow slit, about half of the egg was pushed out with a
hair loop. Thus half the egg was outside and the other half remained within
the vitelline membrane (Fig. 1A). After about 20 min, the constricted part of the
egg, was cut by pressing down gently with a fine glass needle. By these operations,
an egg could be separated into two parts without losing cytoplasm (Fig. 1 B).
The vitelline membrane remaining round a fragment was removed later.
Roughly speaking, the egg was separated into the animal hemisphere including
the nucleus and an anucleate vegetal hemisphere.
Measurement of shape changes
Changes in shape of whole eggs and egg fragments were measured as changes
in height in side view. For the measurement the eggs and fragments were put
in a shallow depression on agar gel and photographed every 10 min in side
view by use of a prism. Heights were directly measured on prints made to a
standard enlargement. All values for the measurements were indicated as
percentages of the initial values (100%).
Non-nucleated fragments of newt's egg
185
Fig. 1. (A) Configuration of the uncleaved egg just before cutting; the right half is
outside and the left half within the vitelline membrane, which is invisible. (B) Nucleated (right) and non-nucleated fragment (left) just after cutting.
Stiffness measurement
Stiffness of the surface of the non-nucleated fragments was measured by the
principle of the cell elastimeter of Mitchson & Swann (1954). The tip of a
small pipette (210 /.im in inner diameter) was gently pressed against the surface
of the egg fragment. A bulge was sucked out from the surface with negative
pressure through the pipette. By plotting values of deformation of the surface
against corresponding values of negative pressure, a straight line was obtained.
The slope of the pressure-deformation curve was termed the stiffness (dyne/
cm 2 /mm deformation).
Reactivity determination
Transplantation of furrow-inducing cytoplasmic components (FIC) was
achieved with a glass micropipette about 50 jam in diameter, using mouth,
suction via a rubber tube (Sawai, 1972). The tip of capillary, inserted into the
cleaving egg, was brought close to the bottom of the cleavage furrow where
FIC was localized. FIC was sucked into the capillary at the site. The loaded
capillary was brought under the cortex of a non-nucleated egg fragment
from within, where FIC was deposited. The volume of cytoplasm injected
was about 50 nl (cf. Sawai, 1976).
186
T. SAWAI
D
Fig. 2. Shape changes in the whole egg during cleavage, in side view. (A) 60 min
before; (B) just at start; (C) 20 min after; (D) 60 min after start of cleavage.
RESULTS
Shape changes in whole eggs, nucleated and non-nucleated fragments
Shape changes in the cleaving amphibian egg deprived of both the jelly
capsule and the vitelline membrane were previously studied in Triturus alpestris
(Selman & Waddington, 1955; Selman et al. 1976). The present experiments
firstly measured the shape changes in the naked whole egg and in nucleated and
non-nucleated fragments of Cynops {Triturus) pyrrhogaster.
Results (Figs. 2, 3) from the whole egg were approximately similar to those
reported previously in the above species (Selman & Waddington, 1955; Selman
et al. 1976). About 90 min before the first cleavage, a slight change occurred
in two of four cases (Fig. 3). The animal half began to round up rapidly at about
20 min before start of the first cleavage. The height of the egg increased 5-13 %
when cleavage had just begun. The initial sign of the cleavage furrow appeared
always at the highest position. After the start of cleavage, both sides of the
furrow increased in height further in the initial phase of cleavage and relaxed
in the late phase. Similar changes also occurred in the second cleavage. The
nucleated egg fragments behaved in the same way as whole eggs during cleavage
(Fig. 4).
Results from non-nucleated fragment are shown in Figs. 5, 6. Before the
start of the first main change, a slight increase in height, with gentle slope,
was observed in two out of four cases (Fig. 6 A). Sudden rounding up was
encountered, in the cases presented in Fig. 6 A, about 60 min after the onset of
the first cleavage in the nucleated partner. As shown in Fig. 7, the time of
occurrence of the first rapid increase in height did not necessarily coincide with
the time of the first cleavage of the sister nucleated fragment. In many cases,
187
Non-nucleated fragments of newt's egg
2nd cleavage
start
130
©O •
120
110
100
90
80 r
—1
0
Time (h)
Fig. 3. Graph showing height changes in four examples of the whole egg. Measurement before cleavage was made at the highest point of contour. Measurement after
cleavage was made at the highest point at the side of the furrow. Ordinate: relative
values of height as percentage of the initial value (100 %). Abscissa: time. —, meaning before; 0, just at start; +, after the start of the first cleavage. Arrows indicate
time of the second cleavage in individual cases.
2nd cleavage
start
130
r>
3rd cl.
start
i
120
no
100
- F^Jv*
80
i
-
i
1
0
i
1
i
2
i
I
i
1
3
4
5
6
Time (h)
Fig. 4. Graphical representation of height changes in nucleated fragments. Points
to be measured, ordinate and abscissa were the same as in Fig. 3.
the first cleavage in the nucleated fragment preceded the increase in height
in the non-nucleated fragment. Once the first rounding up occurred, however,
the succeeding changes tended to proceed with practically the same interval as
the cleavage cycle of nucleated partner. In Fig. 6B, the cycle of the height
change synchronized approximately with the cleavage cycle of the nucleated
half (|) in three cases (O, O, 3 ) , but in the remaining one ( • ) was delayed
about 90 min. The maximum value of the height corresponded to a relative
increase of 10-20 %, approximately as in whole eggs or nucleated fragments.
188
T. SAWAI
Fig. 5. Shape changes of the non-nucleated half in side view. (A) Just before
rising. (B) Maximum rise. (C) After relaxation. Intervals: 30 min in each.
Stiffness changes in surface of non-nucleated halves
Stiffness of the surface of the non-nucleated fragments increased when the
height change was occurring (Fig. 8). The stiffness began to rise about 20 min
before the start of the increase in height, and reached a maximum value two to
four times the initial value after about 30 min, when the height of the fragment
had not yet reached a maximum. After passing a maximum the stiffness decreased, returning roughly to the initial level. A similar change also followed
in the second increase in height. This pattern of stiffness change is similar to
that found in the whole egg (Selman & Waddington, 1955; Sawai & Yoneda,
1974).
Reactivity in cortex of non-nucleated fragments
In the whole egg (Sawai, 1972, 1976), the ability of the cortex to form a
cleavage furrow in response to the furrow-inducing cytoplasmic components
(FlC) was detected only in the cleaving egg, never in the interphase egg. In the
Non-nucleated fragments ofnewfs egg
130
189
(A)
120
110
100
-4
-3
-1
0
Time (h)
2nd cleavage
start
-0-5
0
3
4
Time (h)
Fig. 6. Graph showing height changes in the non-nucleated halves. (A) The changes
in four examples up to the first conspicuous rounding up. Ordinate: the same
as in Fig. 3. Abscissa: time. —, before; 0, just at; +, after the start of the first
cleavage of the nucleated partner. (B) Cyclic height changes in four other examples. Arrows show time of the second to the fourth cleavage in each nucleated
partner.
present experiment, the ability of the cortex of the non-nucleated fragments
to acquire competence to form the furrow, in answer to FIC, was investigated.
FIC was injected under the cortex of the non-nucleated fragments at the stage of
rounding up or of relaxation. At the former stage, furrows were formed at the
site of injection in many cases (Fig. 9). Results were positive in any stage of the
first, the second and the third increase of height (Table 1). At the stage of the
relaxation, results were negative in almost all cases.
DISCUSSION
The present paper shows that the cortical layer of non-nucleated fragments
of the newt egg behaves in a similar way to the cortex of cleaving whole eggs
EMB 51
190
T. SAWAI
Time (h)
Fig. 7. Diagram showing the time of start of the first cleavage in the nucleated
halves (vertical line at time 0), the time of the greatest increase in height in the
non-nucleated sisters (open-circles) and the time of cutting in each case (filled
circles).
1400
i
1050
700
350
-1
1
Time (h)
Fig. 8. Stiffness changes in non-nucleated halves. Ordinate: stiffness (dyne/cm2/mm
deformation). Abscissa: time. —, before; 0, just at; +, after beginning of the first
rounding up. Arrows show time of the second increase in height.
in terms of the shape changes, the increase in stiffness and the reactivity of the
cortex to FIC. Although the above three changes occurred approximately at the
same time among themselves in non-nucleated halves, in many cases the occurrence of the changes was delayed in comparison with those of the sister nucleated
halves. Once the first change happened, however, the rhythm of the succeeding
changes had a timing which tended to approximate that in the cleavage cycle
of the nucleated half.
Non-nucleated fragments ofnewfs egg
191
Fig. 9. Furrows (arrow) induced on the surface of the non-nucleated fragment
by injection of FIC. (A) Beginning of furrow induction, 20 min after FIC injection.
(B-E) Showing development of the induced furrow. Interval: 10 min in each.
The delay in occurrence of the changes may be attributable to differences in
recuperative power from drastic injury, between the two fragments. Another
reason may be the following. Since the various cortical changes shifted from
the animal towards the vegetal pole in the whole egg, the non-nucleated halves,
which were mainly derived from the vegetal hemisphere, may maintain the
same delay when separated by the cut.
In sea-urchin eggs, when enucleated fragments derived from unfertilized
eggs were activated artificially, the thickness of the hyaline layer (Kojima,
1960) and the stiffness of the surface (Yoneda, Ikeda & Washitani, 1978)
changed cyclically with a similar rhythm to that in normal eggs.
The present experiments, and the results obtained from sea-urchin eggs,
mentioned above, may indicate that no activity of the egg nucleus is directly
involved in the changes in the cortical layer observed during the cleavage, at
least in the early developmental stages; i.e. that some alteration in the cortex
necessary for cleavage furrow formation is brought about by cyclic autonomous
activity of the cytoplasm.
In the present experiments, occasionally partial furrows, or, rarely, complete
furrows, were formed in the non-nucleated fragments after prolonged observation (no such cases are included in the present data). They rarely developed
13-2
Total no.
16
1*
2
13
22
20
1
1
35
2(2*)
1
30
1
1
15
13
During 2nd
increase of height
+ , positive; ±, weak reaction; —, negative.
* Cases reacted after rounding up.
After relaxation
During 1st increase
of height
Before 1st increase
of height
Time of FIC transplantation
14
1*
0
13
After relaxation
10
9
0
1
During 3rd
increase of height
Table 1. Furrow formation in non-nucleated fragments by injection of furrow-inducing cytoplasmic components (FIC)
on
H
Non-nucleated fragments ofnewfs egg
193
up to the morula or to the blastula stage (cf. Fankhauser, 1934; Briggs, Green
& King, 1951). There is a possibility that the sperm nuclei or centrioles introduced by polyspermy were activated after separation of the non-nucleated
half from the nucleus in these casss.
The author wishes to express his sincere thanks to Professor K. Dan for his valuable advice
and help in preparing the manuscripts.
This work was supported by grant no. 154241 from the Ministry of Education, Japan.
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BLUEMINK,
(Received 25 October 1978)