/. Embryol. exp. Morph. 85, 33-46 (1985)
33
Printed in Great Britain © The Company of Biologists Limited 1985
Localization of the factors producing the periodic
activities responsible for synchronous cleavage in
Xenopus embryos
ATSUNORI SHINAGAWA
Department of Biology, Faculty of Science, Yamagata University, Yamagata 990,
Japan
SUMMARY
This paper investigates the localization within the Xenopus egg of the factors responsible for
the periodic activities such as the cyclic rounding-up and flattening, related to the cleavage cycle.
Denuded eggs were bisected along the boundary line between the animal and the vegetal
hemispheres immediately after being rotated through 90° off the vertical axis (Early Bisection).
The resulting animal halves, though prevented from cell division by colchicine, showed typical
periodic rounding-up as previously observed in enucleated egg fragments, whereas the vegetal
halves did not. This result indicates that the factors inducing the periodic rounding-up are not
distributed uniformly throughout the egg but localized mostly in the animal hemisphere.
Furthermore, the distribution of these factors between the cortex and endoplasm of the animal
hemisphere was investigated. Eggs were separated into animal and vegetal halves following
incubation for 30min after the 90°-off axis rotation (Late Bisection). During this incubation the
endoplasmic components become relocated in the rotated egg under the force of gravity. After
the rotation, the Late-Bisected vegetal halves showed typical cyclic rounding-up in contrast to
those formed by Early Bisection. These results suggest that the factors inducing the periodic
rounding-up (and probably also many other cyclic activities, closely linked with the rounding-up
movement) are localized in endoplasmic components which can be displaced by gravity from the
animal to the vegetal hemisphere of the Xenopus egg.
INTRODUCTION
Early cleavage of animal embryos can be distinguished from the usual cell
division in somatic cells not only because each cell cycle in this stage consists
mostly of S-, and M-phases but also because during this period embryos show no
synthesis of mRNA, and no increase in cell mass (Flickinger, Lauth & Stambrook,
1970). Moreover, during this period each blastomere cleaves synchronously at a
regular interval characteristic of the species, 30min at 21 °C in Xenopus embryos
for example (Boterenbrood, Narraway & Hara, 1983). It has, therefore, been
considered that early embryos might have a particular mechanism which regulates
Key words: Xenopus laevis, cleavage, cytoplasmic cycle.
34
A. SHINAGAWA
the cycles of early cleavage. Study of the specific timing mechanisms in early
cleavage is thus important for an understanding of early development.
Recently it has been reported that anucleate egg fragments formed by bisecting
uncleaved eggs show cyclic changes similar to those seen in normally cleaving
embryos. These include cyclic change in stiffness (Yoneda, Ikeda & Washitani,
1978), cyclic rounding-up and flattening, and the cyclic appearance of surface
contraction waves (SCWs; Sawai, 1979; Hara, Tydeman & Kirschner, 1980;
Sakai & Kubota, 1981; Sakai & Shinagawa, 1983; Shinagawa, 1983). Furthermore,
it has also been reported that those eggs in which the nucleus is prevented from
mitosis by antimitotic agents persist likewise in the cyclic changes. The periodicity
of the cyclic changes in either enucleated, or colchicine-injected eggs is comparable to that of the cleavage cycle in normal embryos. These results
consequently suggest that the cyclic divisions during early development might be
regulated by certain factors present in the cytoplasm and/or the cortex, independent of the nucleus.
Kirschner, Gerhart, Hara & Ubbels (1980) consider the factors to constitute a
master oscillator of early cleavage, which could regulate the cyclic change of
various activities such as DNA synthesis, duplication of centrioles, mitosis, and
surface contraction waves. Even nuclei inserted into the cytoplasm of another egg
are entrained by the surrounding cytoplasm so as to be in harmony with the phase
of the cytoplasmic cycle quite independently of the original phase of the nuclei
(Graham, Arms & Gurdon, 1966; Sakai & Shinagawa, 1983). This may suggest a
relationship between the cytoplasmic factors mentioned above and the maturation
promoting factor or the cytostatic factor, known to be likewise in the cytoplasm
and to control the morphology of the nucleus (Wasserman & Smith, 1978; Lye,
Newport & Kirschner, 1983).
The localization within the egg of the factors producing these various
periodicities is, however, not known. The present study was undertaken in an
attempt to answer this question. Most of the phenomena apparent on the surface
of amphibian eggs originate at the animal pole, and propagate toward the vegetal
hemisphere; for example the SCWs (Yoneda, Kobayakawa, Kubota & Sakai,
1982), the waves of stiffness in the surface (Sawai & Yoneda, 1974; Yoneda et al.
1982), and the waves of activity of furrow-inducing components (Sawai, 1972).
This raises the possibility that the factors inducing the periodic activities of the egg
are not located uniformly throughout the egg but are restricted to the animal
hemisphere.
In order to test this possibility, the cyclic changes in animal and vegetal halves,
formed by bisecting uncleaved Xenopus eggs, were compared with each other. In
addition, to ascertain whether the cytoplasmic factors are localized in the
endoplasm or in the cortex of an egg, the same comparison was carried out
between those halves after the constitution of the endoplasmic components, but
not of the cortical components, had been altered.
Localization of periodic activity
35
MATERIALS AND METHODS
Egg preparation
Eggs of Xenopus laevis were obtained by natural mating following injection with 200i.u. and
300i.u. of human gonadotrophic hormone (Gonatropin, Teikoku-zoki Co. Ltd, Tokyo) into a
male and a female respectively. Jelly was removed by carefully stirring eggs in 10 % modified
Steinberg's saline containing 1 % sodium thioglycolate (Wako Pure Chemical Industries Ltd,
Osaka, Japan) at pH9-10 for 2min. Dejellied eggs were treated with 0-5 % pronase in modified
Steinberg's saline (Sakai & Kubota, 1981; 58-2mM-NaCl, 0-67 mM-KCl, 0-34mM-Ca(NO3)2,
0-83mM-MgSO4, 0-30mM-HEPES) at pH6-8 for lmin, and the fertilization envelope was
removed with forceps.
Bisection of fertilized eggs
Bisection of fertilized Xenopus eggs was carried out by two procedures:
(1) Early Bisection. Denuded eggs were placed in a Petri dish covered with 2 % agar and
rotated 90° off the vertical axis; consequently the animal/vegetal axis of the eggs was oriented
horizontally. A fine glass rod was gently put on the egg along the boundary line between the
animal and the vegetal hemispheres immediately after the egg was rotated 90° off axis. The region
of the egg under the glass rod was gradually squeezed until the egg was separated into halves
(Fig. 1). This bisection was always initiated within 30 min after fertilization because at later times
the eggs become too rigid to be bisected. The zygote nucleus was usually included in the
resulting animal half fragment, which went through the cycles of cleavage (Fig. IF). After the
separation was completed, each half fragment was injected with 25 nl of colchicine dissolved in
modified Steinberg's saline (1 mg/ml) so as to arrest cleavages. For precise comparison of the
periodicity in an animal half with that in a vegetal half, both should be inhibited from cleavage
by injection with colchicine. This avoids the alteration of periodicity caused by formation of a
mitotic apparatus in one of the fragments (Sluder, 1979; Shinagawa, 1983). This bisection is
called 'Early Bisection' for convenience in the present study.
(2) Late Bisection. Denuded eggs were placed in a Petri dish and rotated 90° off axis in the
same manner as above. They were injected with colchicine soon after the rotation. At 30min
after rotation, afineglass rod was placed on the boundary line of the hemispheres, to accomplish
bisection. This delayed bisection was usually initiated about 50 min after fertilization, when eggs
would have normally become too rigid for the operation. However, it was possible to succeed in
the bisection because the cytoplasm of the egg had been liquified by colchicine. Thus, in this
procedure colchicine was used for liquifying the egg cytoplasm as well as for inhibiting the
fragments from cell division. This bisection is called 'Late Bisection'. The timing of the bisection
is the main difference between the two procedures.
Observation of cyclic activity in half fragments
Cyclic activity in animal and vegetal halves was recorded as a cyclic change in cell diameter,
which reflects the cyclic and sequential rounding-up and flattening of the cell. The periodicity of
the cyclic rounding-up is known to represent also that of other cyclic activities including SCWs
and cyclic change in stiffness (Sakai & Kubota, 1981; Yoneda et al. 1982). Each half fragment
was observed from above, and its diameter was measured every 3 min at room temperature
(21 °C to 23 °C). A minimum diameter measured from above corresponds to a maximum
rounding-up as viewed from the side. For minimizing the effects of unexpected temperature
variation, both halves were placed side by side in a Petri dish. Comparison of the periodicity was
always carried out with a pair of fragments derived from a single egg.
Histology
After the periodic activity in the half fragments was observed, each wasfixedfor about half a
day in Herry's fixative followed by alcoholic dehydration, and embedding in paraffin wax (m.p.
36
A. SHINAGAWA
52-54°C, Nakarai Chemicals, Ltd, Kyoto, Japan). Serial sections of the fragments were cut at
8jUm. After deparaffination, sections were stained for 20-30 min in a solution containing both
0-4 % orange G and 0-2 % aniline blue (Azan's staining).
RESULTS
Periodic activity in the animal and vegetal half fragments formed by Early Bisection
Fig. 2 illustrates the cyclic change in diameter of the animal and vegetal halves
formed by bisection of the eggs immediately after 90°-off axis rotation (Early
Bisection). Since Fig. 2 shows the diameter of fragments observed from above, the
minima of the plot correspond to the times of maximum rounding-up of the
fragment, viewed from the side. Though animal halves were prevented from cell
B
i
6
F
•
25
i
30
t
10 G
l^
|
^[
15 _H
70
Fig. 1. Bisection of the fertilized Xenopus egg immediately after the egg has been
rotated through 90° off the vertical axis. (A) Three minutes after placing a glass rod on
the egg (about 15 min after fertilization). Times (min) after placing the glass rod are
indicated on the right. The egg is completely separated into two halves at about 30 min
after placing the glass rod (G). The animal half fragment alone goes through the cycles
of cleavage because of including the zygote nucleus (H). Scale bar equals lmm.
Localization of periodic activity
37
division by injection with colchicine, a significant cyclic change in diameter
nonetheless occurs. The period of the cyclic change in diameter of the animal
halves is definitely longer than that of cleavage cycle (Fig. 2A). The maximum
change of diameter for the animal half is 8-15 %. Thus, the cyclic change in the
animal halves can be equated with that previously observed in anucleate eggs
(Sakai & Kubota, 1981; Shinagawa, 1983). On the other hand, vegetal halves do
not show as large cyclic changes in diameter (2-5 %) as do animal halves, although
in many cases, cyclic changes can be detected also in vegetal halves (Fig. 2C).
Moreover, some vegetal halves failed to display detectable rounding-up (Fig.
2A,B).
In order to ascertain the difference in periodicity between the two halves, the
data of Fig. 2 and of similar experiments are plotted in Fig. 3. The time of the 2nd
(circles), 3rd (closed triangles), 4th (squares) and 5th (open triangles) rounding-up
in the vegetal halves is plotted against that of the corresponding rounding-up in
their animal-half partners. The points of the plot stay above a line with a slope of
1-8
\
•
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1-6 - o o o oo o o o o o o o o o o o o
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met er of egg f ragments (
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°oo° O O O O O O oO
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Time after first rounding-up (min)
•
120
Fig. 2. Changes in diameter of the animal (•), and vegetal (O) halves formed by
bisecting the eggs rotated through 90° off the vertical axis shortly before the bisection
(Early Bisection). To facilitate the comparison, the time scale is shifted so as to align
the first rounding-up of each half at time 0 in the graph. In reality, the first rounding-up
in the vegetal half lags behind that in the animal half by 10 to 40 min (C). Arrows in (A)
indicate the cleavage time of the control egg derived from the same batch.
38
A. SHINAGAWA
45 °, clearly revealing that the period of cyclic rounding-up in the vegetal halves is
longer than that in their animal-half partners. The ratio of the former to the latter
is actually 1-26 (±0-14 S.D.) on average. These results conclusively indicate that
the typical periodic activity is not distributed uniformly throughout an egg but is
localized mainly in the animal hemisphere.
Periodic activity in animal and vegetal halves formed by Late Bisection
Fig. 4 illustrates the cyclic change in diameter of the animal and vegetal halves
formed by bisecting the eggs at 30min after the 90°-off axis rotation (Late
Bisection). The diameter of the animal halves changes as extensively and as
quickly as that of the animal halves formed by Early Bisection. However, vegetal
halves formed by Late Bisection change their diameter much more than those
formed by Early Bisection. As shown in Fig. 4A,B the period and extent of cyclic
rounding-up in the Late-Bisected vegetal halves is very similar to that in their
animal-half partners, although in some experiments the former remain somewhat
1
180
A
A
_•
A
150
•
A
•
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y
• •
120
A
A
/
90
•
60
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f
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'
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60
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90
120
Rounding-up time of animal halves (min)
Fig. 3. The time of 2nd ( • ) , 3rd (A), 4th ( • ) , 5th (A) rounding-up in 17 vegetal
halves obtained by Early Bisection plotted against the timing of the corresponding
rounding-up in their cleavage-arrested animal half partners. The time of 1st roundingup in each half is taken as 0.
Localization of periodic activity
39
slower than the latter (Fig. 2C). However, this difference in periodicity between
the two halves is much smaller than in those pairs obtained by Early Bisection.
Fig. 5 further documents the similarity in period of the cyclic rounding-up in
both halves of Late-Bisected eggs. In contrast to Fig. 3, each point is located close
to a line with a slope of 45 °, revealing that the period of cyclic rounding-up in the
vegetal halves formed by this procedure has become equalized to that in their
animal-half partners. The ratio of the former to the latter is 1-07 (±0-07 S.D.) on
average.
Distribution of cytoplasm in the animal and vegetal halves formed by Early or Late
Bisection
As seen in Fig. 6A, there is a polarity not only of the cortex but also of the
endoplasm of the egg. Externally, the cortex of the animal hemisphere can easily
be distinguished from that of the vegetal one because the former is thick and well
pigmented, while the latter is thin and poorly pigmented. The animal portion of
A
o
1-6
~
o o oo o
oo
ooo
°oooQ
ooo oo ° o
o
1-8
O Oo O
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1-6
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1-4
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CO
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1-8
ooooo° °° o
o
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O o
ooooo
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1-6
1-4
30
60
Time after first rounding-up
90
120
Fig. 4. Changes in diameter in the animal halves (•), prevented from mitosis, and the
vegetal halves (O), formed by bisecting the eggs rotated through 90° off the vertical
axis at 30min prior to the bisection (Late Bisection). To facilitate the comparison, the
time scale is shifted so as to align the first rounding-up in each half at time 0: In reality,
1st rounding-up in the vegetal half sometimes lags behind that in the animal half by up
to 15min.
40
A. SHINAGAWA
the surface appears as a clear-cut margin, while the vegetal portion of the surface
seems to lack such a marginal structure. Internally, as described by Nieuwkoop
(1977), the animal hemisphere of the egg contains a relatively large amount of
small to medium-sized yolk platelets embedded in rather abundant cytoplasm,
while the vegetal hemisphere is composed of a large quantity of mainly large yolk
platelets, the amount of inteFstitial cytoplasm being scanty.
The distribution of cytoplasmic components in animal and vegetal halves
formed by Early Bisection is shown in Fig. 6B and C respectively. The animal half
fragment is packed mainly with small to medium-sized yolk platelets embedded in
a large amount of yolk-free cytoplasm, while the vegetal counterpart is occupied
predominantly by large-sized yolk platelets, apparently lacking yolk-free
cytoplasm. The surface of the animal half (Fig. 6B) appears as a clear-cut margin
while that of the vegetal half (Fig. 6C) lacks such a marginal structure. Thus, the
two halves resemble the animal and the vegetal hemisphere of the intact egg
respectively.
180
0
30
60
90
120
Rounding-up time of animal halves (min)
Fig. 5. The time of 2nd ( • ) , 3rd (A), 4th ( • ) , 5th (A) rounding-up in 12 vegetal
halves obtained by Late Bisection plotted against the timing of the corresponding
rounding-up in their cleavage-arrested animal half partners.
Localization of periodic activity
41
Fig. 7B,C shows the distribution of endoplasmic components in the animal and
vegetal half fragments formed by Late Bisection respectively. As pointed out by
Elinson (1983), the endoplasm of fertilized Xenopus eggs becomes fluid about
15min after fertilization so that the endoplasmic components can easily be
relocated during the incubation of the eggs for 30 min following the 90 °-off axis
; .**'. •
V
B
Fig. 6. Distribution of the endoplasmic components in an intact egg (A), and in the
animal and vegetal halves (B and C respectively) formed by bisecting the egg rotated
through 90° off the vertical axis shortly before the bisection. The broken line in (A)
indicates the boundary between the animal (a), and vegetal (v) hemispheres, situated
upper and lower on the plate respectively. Scale bar equals 500 jum.
42
A. SHINAGAWA
rotation. Consequently, heavy yolk granules are translocated toward the lower
portion of the rotated egg, and simultaneously, yolk-free cytoplasm is translocated
toward the upper portion of the rotated egg, across the pigmentation boundary
line between the hemispheres (Fig. 7A).
V
7A
B
Fig. 7. Distribution of the endoplasmic components in the intact egg rotated through
90° off the vertical axis at 30min before the bisection (A), and in the animal and
vegetal halves (B and C respectively) formed by bisecting the egg rotated through 90°
off the vertical axis at 30 min prior to the bisection (Late Bisection). The broken line in
(A) indicates the boundary between the animal (a), and the vegetal (v) hemispheres,
situated left and right on the plate respectively. Scale bar equals 500 ^m.
Localization of periodic activity
43
The resulting animal half still contains a large amount of yolk-free cytoplasm
and fine yolk platelets, and has gained a small amount of large yolk platelets,
probably derived from the endoplasm of the vegetal hemisphere (Fig. 7B). It
should be noted particularly that the vegetal counterpart now contains a rather
large amount of yolk-free cytoplasm in its upper portion, in an amount similar to
the animal half, in contrast to the vegetal half formed by Early Bisection (compare
Fig. 7C with Fig. 6C). Figs 7B,C thus clearly show that both halves obtained by
Late Bisection contain the same kinds of endoplasmic components including yolkfree cytoplasm, even though the cortex in the two halves remains different in
respect to thickness and pigmentation.
On the basis of the above observation of the periodicity and cytoplasmic
constitution of the fragments, it could be concluded that the displaceable
endoplasmic components lying in the animal hemisphere, probably in the yolkfree cytoplasm, contain the factors inducing the cyclic rounding-up and perhaps
other cyclic activities.
DISCUSSION
The amphibian egg clearly has various kinds of polarity along the
animal/vegetal axis, such as the polarity of distribution of cytoplasmic
components (Nieuwkoop, 1977; Herkovits & Ubbels, 1979), that of the
distribution of RNA (Capco, 1982), that of developmental competence (Grunz,
1977), and the structural and functional polarity in the cortex (Goldenberg &
Elinson, 1980). It seems plausible that there may also be a polarity in the
distribution of the factors producing the periodic activities of the Xenopus egg.
The first experiment involving Early Bisection revealed that the typical cyclic
rounding-up can be seen only in the animal halves, indicating that the factors
inducing it are localized mainly in the animal hemisphere. The second experiment
involving Late Bisection further suggested that the factors are present not in the
cortex but in the displaceable endoplasmic components, since a vegetal half gains
the capacity to show typical rounding-up once it acquires the endoplasmic
components displaced from the animal hemisphere. This is in good agreement
with the study on the cytoplasmic regulation of the duration of cleavage in
amphibian eggs (Aimar, Delarue & Vilain, 1981). The acquisition of the typical
cyclic rounding-up in these vegetal halves should exclude the possibility that the
absence of shape changes in the vegetal halves formed by Early Bisection is caused
merely by the inability of the vegetal half to express the periodic activities, for
example, because of shortage of the contractile system. It seems more likely that
the vegetal half of a normal egg fails to originate the cyclic changes as vigorously as
does the animal half. This lesser activity of the vegetal half is perhaps due to lesser
amount of the endoplasmic components containing the factors. The rather typical
44
A. SHINAGAWA
cyclic rounding-up in some vegetal halves might be due to a greater amount of the
factors, unintentionally introduced during bisection.
Though there are other endoplasmic components in the animal hemisphere such
as fine yolk granules, yolk-free cytoplasm would be the most probable candidate
for including the factors since it contains abundant organelles and enzyme systems
(Herkovits & Ubbels, 1979) lacking in the yolk platelets. The factors present in the
endoplasmic components may comprise the 'Master Oscillator', proposed by
Kirschner etal. (1980) as controlling the progress of the cell cycle. Although in the
present study the factors producing the periodicity were assayed only by recording
the cyclic rounding-up of the egg, it seems very likely that other cyclic activities,
including SCWs and cyclic change in stiffness, are regulated likewise by the same
factors because they seem to be closely linked with the rounding-up movement
either temporally or mechanically (Sakai & Kubota, 1981; Yoneda etal. 1982).
If appearance of SCWs is triggered by the factors present in the mass of yolkfree cytoplasm underlying the animal pole, this would explain why SCWs always
appear initially at the animal pole of eggs, and propagate toward the vegetal
hemisphere. This assumption is consistent with studies demonstrating that the
endoplasm plays a much more important role than the cortex in determining the
furrow-appearance site and axes of developing embryos (Neff, Malacinski,
Wakahara & Jurand, 1983; Ubbels, Hara, Koster & Kirschner, 1983).
For characterization of those factors it may be important to note the relation
between them and the maturation promoting factor, thought to be a proteinous
substance (Wasserman & Smith, 1978; Lye et al. 1983). The previous, and the
present studies reveal that there are many similarities between both factors: (1)
each is present in endoplasm, and is translocatable or transplantable (Lye et al.
1983); (2) each causes changes with a periodicity corresponding to the cleavage
cycle (Wasserman & Smith, 1978); (3) each is capable of affecting the morphology
of a nucleus (Wasserman & Smith, 1978; Sakai & Shinagawa, 1983); (4) each might
be derived at least partly from germinal vesicle materials (Herkovits & Ubbels,
1979).
Though the nature of the factors is unrevealed, it might be related to a complex
of calcium and the calcium-sequestering system (Harris, 1978; Kiehart, 1981). The
free calcium, reportedly affecting the contractile systems, cytoskeletons, and
mitotic apparatus, might participate in inducing the autonomous oscillation.
Further study is required to characterize, at subcellular and molecular levels, the
cytoplasmic factor producing the autonomous oscillation.
The author thanks Dr T. Sawai for his support in carrying out the present study, and also
thanks Professor M. Yoneda for his valuable advice.
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{Accepted 9 October 1984)