J. Embryol. exp. Morph. Vol. 64, pp. 259-274, 1981
Printed in Great Britain © Company of Biologists Limited 1981
259
Cytoplasmic regulation of the duration of
cleavage in amphibian eggs
By CHRISTIAN AIMAR1, MICHEL DELARUE
AND CLAUDE VILAIN
From the Laboratoire cTImmunologie Comparee, Universite
P. et M. Curie, Paris
SUMMARY
Relations between a cytoplasmic species specificity and the duration of cleavage cycles were
investigated by reciprocal injections of egg cytoplasm. Xenogenic cytoplasm induces an
early or delayed cleavage of the recipient egg depending on the chronological specificity of the
injected cell cytoplasm. Activity of the so-called cleavage timing system (CTS) was first
detected in the cytoplasm of maturing oocytes at the stage of germinal vesicle breakdown
(GVBD). This specific cytoplasmic property was not dependent on the maturation promoting
factor (MPF). Relations between the CTS and other cytoplasmic components which are
known to induce cleavage are discussed.
INTRODUCTION
Cleavage of the amphibian egg has been investigated recently as a model for
the study of the regulation of cell division.
The cleavage pattern has been extensively studied in Ranapipiens (Chulitskaia,
1970) and Ambystomamexicanum (Signoret & Lefresne, 1971, 1973;Hara, 1977).
It was shown that the cleavage stage consists of two successive phases: synchronous and asynchronous cell divisions. These are connected by a transitional
period which is characterized by a progressive desynchronization of cell division.
Some of the various factors which can be expected to be implicated in the
onset, maintenance and peculiarities of these different cleavage phases have been
investigated. It was shown that the duration of the synchronous period is
dependent on cytoplasmic factors in Ambystoma and Rana (Signoret & Lefresne,
1973; Chulitskaia, 1970) and more precisely in Xenopus on the amount of
deoxyribonucleoside triphosphates present in the egg (Landstrom, Lovtrup
Rein & L0vtrup, 1975). The time of occurrence of the transitional period could
be related to cytoplasmic and nuclear factors. The end of the cleavage stage
appeared to be under the control of cytoplasmic components. (Signoret &
Lefresne, 1973; Landstrom et ah 1975).
1
Author's address: Laboratoire d'Immunologie Comparee, Universite P. et M. Curie,
75230 Paris, Cedex 05, France.
260
C. AIMAR, M. DELARUE AND C. VILAIN
The factors involved in the rhythm of cell division during the different
cleavage phases are still imperfectly identified. It is evident that the chronology
of cleavage, during the synchronous period, is variable and species specific. It
was observed that the rhythm of cytokinesis is related to previous characteristics
of the native cytoplasm (Signoret & Lefresne, 1973) and that it is maintained in
experimentally enucleated eggs (Aimar & Delarue, 1976). Hara, Tydeman &
Kirschner (1980) have shown that the cell cycle persists in non-nucleated
fragments of normally fertilized eggs. These different reports show that the
factors involved in the endogenous cleavage rhythms can be present in the egg
cytoplasm.
The objective of the present work was to study the relationships between the
duration of the cell cycle and the species specific properties of the cytoplasm. For
this purpose exchanges of cytoplasm between eggs of amphibian species with
various cleavage timings were performed. Under these conditions we have shown
that the cleavage rhythm can be experimentally modulated. These results emphasize that the endogenous cleavage rhythm is correlated with specific characteristics of the cytoplasm. We have determined that this cytoplasmic characteristic
appears, first, in the cytoplasm of progesterone-stimulated oocytes, i.e. at the
same time as the Maturation Promoting Factor (MPF). This cytoplasmic factor
is known to induce the break-down of the germinal vesicle (GVBD) and the
reinitiation of meiosis (Masui & Markert, 1971). Later a cyclic appearance of an
MPF activity was found in the cytoplasm of the cleaving embryo (Wasserman &
Smith, 1978). Consequently the relationships between MPF and a cytoplasmic
factor which would control the specific chronological cleavage rhythm were
investigated.
MATERIALS AND METHODS
Four amphibian species were selected. P. waltlii and X. laevis were raised in
the laboratory. B. bufo and B. calamita (B. cal.) were collected from fields.
The first cleavage furrow started respectively about 6 h after fertilization
(18 °C) in P. waltlii eggs and 2\ h for eggs of the three other species. However,
the delay of the first cleavage in P. waltlii is widely variable.
Following cleavages occurred synchronously about 90 min apart in P. waltlii,
30 min in X. laevis and 60 min in B. bufo and B. cal. eggs. Unfertilized enucleated
eggs which were experimentally activated exhibited an abortive cleavage called
'fragmentation'. This is characterized by irregular furrows on the surface of the
animal pole (Fig. 1A and B). In the virgin eggs the fragmentation occurred with
the same delay as the first cleavage of fertilized eggs (Aimar & Delarue, 1976).
As suggested by recent observations (Hara et al. 1980), fragmentation could be
considered as an event with a biological significance equivalent to that of the
first cleavage of the fertilized egg. Thus, virgin or fertilized eggs were both used
to study the role of the cytoplasm in the establishment of the endogenous
rhythm of cleavage.
Cytoplasmic control of cleavage
261
Oocytes selection
Oocytes were aseptically removed from adult females anaesthetized with MS
222 (Sandoz) 1 g/1. They were then carefully isolated with watchmaker's forceps. Oocyte follicular layers were enzymatically disrupted with collagenase
(1 mg/ml) for 6 h at 28-30 °C (X. laevis) or 4 h at 26 °C (other species) in
medium A: 88 mM-NaCl; 1 mM-KCl; 0-33 mM-Ca (NO3)2; 0-41 mM-CaC12;
0-82 mM-MgSO4; 2-4 mM-NaHCO3; 2-0 mM-Tris-HCl; 10 mg/1 streptomycin
pH 7-4. Oocytes were washed, sorted according to their developmental stages
and stored at 18 °C in the medium A for a period which did not exceed 48 h.
Maturation of oocytes
The maturation of full-grown oocytes was induced by progesterone (10~6 M)
in medium A. The appearance of a depigmented area at the animal pole of the
oocyte is indicative of GVBD stage (first meiotic metaphase). For X. laevis and
P. wait Hi, this stage occurred respectively within about 3-4 h and 10 h of
progesterone stimulation.
Preparation of virgin eggs
Ovulation of virgin females was induced by intracoelomic injection of gonadotropin. P. waltlii received pituitary gonadotropin (10 Fevold units) and X.
laevis chorionic gonadotropin (500 i.u.). For B. bufo and B. cat., hormonal
stimulation was obtained by injection of a soluble extract of crushed bovine
pituitary gland (2 ml/animal). P. waltlii eggs were dejellied with watchmaker's
forceps 3 to 15 min after laying, and eggs of other species were dejellied with
dithiothreitol DTT (5 10~3 M) in medium OR2: 82-5 mM-NaCl; 2-5 mM-KCl;
1 m.M-CaCl2; 1 mM-MgCl2; 1 mM-Na2PO4; 3-8 mM-NaOH; 5-0 mM-Hepes-HCl,
pH 7-8). Female pronuclei at second meiotic metaphase were either removed or
destroyed by means of a 90 sec ultraviolet irradiation. Virgin eggs were activated
by an electrical discharge or by pricking them with a sharp glass needle. Activation of eggs was complete within about 75 min for P. waltlii and 20-30 min for
other species.
Preparation of a MPF active cytoplasmic extract from X. laevis oocytes
The extraction procedure was derived from the Drury technique (1978).
Maturing X. laevis oocytes, at the GVBD stage, were collected and stored at
0-5 °C in medium OR2. They were rinsed in modified Masui solution (MMS) and
centrifugated at 12000 g for 40 min at 2 °C in medium B: 250 mM-Sucrose; 25
mM-Na-Glycerol-PO4; 2-5 mM-MgSO4; 4 mM-EGTA; 35 mM-NaF; pH 6-5.
MPF activity of the clear soluble supernatant portion obtained was judged
suitable if 80 % of oocytes were able to mature after injection of 100 ml of this
fraction. The extract obtained under these conditions was active for at least 60
days if stored at - 7 0 ° C .
262
C. AIMAR, M. DELARUE AND C. VILAIN
Cytoplasmic transfers
These were performed in Petri dishes lined with agar (2 %) and half-filled with
sterile Steinberg solution at 18 °C. Operated germs were cultured under the same
conditions. Injections or sampling of total or fractionated cytoplasm were
performed with micropipettes of appropriate internal diameters ranging from 30
to 40 [im. A Leitz micromanipulation apparatus was used.
Cytological study
Germs were fixed with Zenker solution, embedded in paraffin and serially
sectioned (10 fim) for light microscopy. Nuclei were stained by the FeulgenRossenbeck method and cytoplasmic structures with light green dye.
RESULTS
(1) Reciprocal injections of xenogenous cytoplasm into virgin eggs
Reciprocal cytoplasm transfers were carried out in P. waltlii and X. laevis
virgin eggs. Eggs were previously activated at the same time as control groups of
the same layings.
The cytoplasm from the animal hemisphere of the just activated egg was
removed then immediately replaced by an equal volume of xenogenous cytoplasm originating from an equivalent localization (Fig. 1C). The amounts of
injected cytoplasm, 900 and 800 nl respectively for P. waltlii and X. laevis, were
equal to 40 and 50 % of the egg volume.
Development of operated eggs and of control groups in the nine experimental
series was observed up to the first cleavage furrowing. On an average, more than
85% of the control and 60% of the operated eggs cleaved (Table 1). Later a
necrotic process was observed in all germs, within 3-4 h of laying for X. laevis
eggs and within 6-10 h of laying for P. waltlii eggs. Germs at different developmental stages were fixed for cytological studies. At first, their nuclei formed
atypical mitotic figures, in particular metaphases were associated with monopolar spindles. Later on, all female pronuclei were involved in pycnotic degeneration.
The experimental results showed that the cleavage rhythm of the eggs was
specifically modified by the heterochronous cytoplasm injected. The duration of
the first cycle of division was 154 ± 14 min for normal X. laevis eggs and 382 ±
68 min for normal P. waltlii eggs. In comparison with these control values, for
Xenopus eggs injected with Pleurodeles cytoplasm this duration was delayed by
46 % (P < 0-001). In reciprocal experiments, the duration of the cell cycle of
P. waltlii eggs was on the average 20 % shorter (P < 0-01) than normal. For the
most extreme variations observed, the duration of cleavage was delayed by 100 %
or shortened by 33 % respectively for X. laevis and P. waltlii eggs (exp. 5 and 3).
r
20-20
25-21
103-86
30-18
21-17
24-24
25-23
25-21t
Normal
aI
30-25
——
22-15
28-26t
exp
X. laevis
—
20-20
20-19
20-10
18-16
18-10
18-18
40-27
20-18
Normal
al
20- 8
37-28
48-38
exp
P. waltlii
15- 6
14-11
26-16
22-15
14-10
47-35
27-24
14- 3
cytfP.w.j
X.I.
——
15 -4
28-13
28-21
36-26
31- 9
50-36
9- 7
19-10
cyt (X.I.)
P.w.
Recipient eggs
150
150
150
150
180
150
165
165
125
195
—
210
210
270
255
240
285
150
X.I. cyt(P.w.)
Controls X.I.
+ 30
—
+ 40
+ 40
+ 50
+ 70
+ 45
+ 72
+ 20
%
X
360
450
345
480
465
350
360
270
- 2
(360)
—
300
300
300
300
375
240
260
265
cyt(X.l.) P.w.
P.w. Control
-17
-33
-13
-37
-20
-31
-28
/o
Recipient eggs
Duration of the first cleavage (mean values, in min)
-22
292
154
227
382
273-230 80-66 134-129 95-74 179-120 216-126
+ 46
100-84% 100-82% 100-96% 100-77% 100-67% 100-58%
(±14) (±44)
(±41) (±68)
X.I.: Xenopus laevis eggs; P.w.: Pleurodeles waltlii eggs; cyt(P.w.) or cyt (X.].): Species of injected cytoplasm.
* Controls-normal: normal virgin eggs which were electrically activated. Controls-experimental: virgin eggs in which removal then injection
of isologous cytoplasm (900 nl. and 800 nl respectively for P.w. and X.I. eggs) were performed just after activation procedure.
t First column, number of operated eggs; second column: number of cleaving eggs.
% Percentage in ieference to control groups.
Total/
average
3
4
5
6
7
8
9
2
1
Experiment
Controls*
Number of eggs
Table 1. Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs
ON
3
Total/average
Fertilized eggs
4
5
6
X. laevis
B. bufo
B. bufo
B. bufo
B. cal
B.cal
1
2
3
Virgin eggs
Total/average
Donor
species
Experiment
no.
Nature of
donor eggs
Bufo
150
150
150
150
150
150
150
150
7-4
32-17
13- 5
52-26
15-12
40-19
19- 7
59-26
Control
Number of
recipient
eggs
240
360
300
300
360
360
360
360
180 (-25%)
270 (-25%)
150 (-50%)
200 (-33%)
300 (-17%)
270 (-25%)
205 (-40%)
237 (-33%)
Control
P. waltlii
(cyt. Bufo)
P. waltlii
Duration of the first cleavage (in min)
Table 2. Injection of the cytoplasm from various amphibian species into P. watllii virgin eggs
>
p
<
o
d
tn
r
en
o
50
p
4
ON
Cytoplasmic control of cleavage
265
In order to demonstrate that the injection procedure by itself did not provoke
the alteration of cleavage duration, six experimental series (Table 1) consisting of
removal then injection of isologous cytoplasm in P. waltlii and X. laevis virgin
eggs (cyt. P.w
> P.w eggs; cyt. X.I
> X.I eggs) was performed. For each
experimental series, operated eggs (controls-experimental) and electrically
activated eggs (controls-normal), used as control, cleaved at the same time,
statistical't test' of Student showing no significant difference at P 0-01.
Delay of cleavage of X. laevis recipient eggs was related to the chronological
type of the P. waltlii donor eggs. It was more marked if the P. waltlii donor eggs
divided slowly. Thus, a long delay of cleavage in X. laevis eggs, as in experiment
6, corresponded to the injection of cytoplasm of P. waltlii eggs for which the first
cleavage occurred 465 min after fertilization. It was also found that delay of
cleavage of X. laevis recipient egg was prolonged if these eggs belong to a
'slow-cleaving' type (compare series 8 and 9). Consequently, the timing of
cleavage of an operated egg appeared to be the result of an interacting system
between qualitative cytoplasmic factors specific to the injected cytoplasm and to
the recipient egg.
(2) Effects of egg cytoplasm of various species on the cleavage of?, waltlii virgin
eggs
With the procedure previously used, P. waltlii eggs were injected with cytoplasm from one of three amphibian species: X. laevis, B. bufo and B. calamita.
The duration of the first cleavage was normally the same for each species. For
three experiments (52 operated eggs) B. bufo and B. cal. eggs were virgin but
activated, for the two other experiments (59 eggs) they were at the 2- or 4-cell
stage.
An average of half of P. waltlii eggs injected with B. bufo or B. cal. cytoplasm
cleaved (Table 2).
Whatever the species of injected cytoplasm was, X. laevis or B. bufo, the
duration of the first cycle of P. waltlii recipient eggs was shortened, on the average by 25 %. Virgin or fertilized Bufo eggs induced identical effects on the duration of the cleavage of the P. waltlii recipient eggs.
Our data supports the hypothesis that the duration of cell cycles during the
cleavage phase in various amphibian species is controlled by the same kinds
of biochemical events. Presumably, closely related cytoplasmic components
would occur in species whose cleavages chronologies are the same.
(3) Effects of X. laevis cytoplasm on the development of fertilized P. waltlii eggs
At the apical pole of one ot the blastomeres of P. waltlii eggs at the 2-or 4-cell
stage, 250 or 125 nl of X. laevis cytoplasm were injected, corresponding to one
quarter of the cell volume respectively. Just before these injections, an equivalent
volume of cytoplasm had been removed from the recipient cells.
Three types of development were observed in the 63 operated eggs (Table 3).
266
C. AIMAR, M. DELARUE AND C. VILAIN
Table 3. Effects ofX. laevis cytoplasm on cleavage of
P. waltlii eggs
Cleavage duration
j
0
+
Experiments
no.
1
2
3
4
5
6
7
8
Number
of eggs
9
11
7
12
5
10
5
4
(
3
1
5
3
1
5
«
)
5
6
—
9
4
4
(b)
1
4
—
—
—
—
—
—
2
—
—
1
—
4
1
4
Total
63
26
28
5
4
(100%) (41-5%) (44-5%)
(8%)
(6%)
In reference to control groups: + , rapid or accelerated cleavage; - , development arrest
(a) no permanent arrest (b) arrest and cytolysis; 0, no effect on cleavage.
In some cases (6 %) the injected cytoplasm did not change the natural cleavage
rhythm of the eggs. In 52-5% of operated eggs an arrest of development was
observed.
It was irreversible for a few eggs which became rapidly necrotic and temporary for the others which were blocked for one hour at their initial stage
before cleaving.
X. laevis cytoplasm induced a variation of the cleavage rhythm of all other
P. waltlii eggs (41-5%) which can be classified into three groups of equal size.
In the first group, the initial cleavage of the tested cells was shortened
on the average by 30 min, as compared to other cells of the same embryos
and the duration of subsequent cell divisions was progressively lengthened.
Consequently at the 3rd or 4th cycle of the cleavage phase all blastomeres,
whether injected or not, cleaved synchronously. It is worth pointing out that the
recipient cells had not effected more divisions than the other cells of the eggs.
Figure 1 (AandB). Cleavage (fragmentation) of a P. waltlii egg which was enucleated
then activated (6 h old); external view (A) serial section (B) of 'cleavage furrows'
in theanimal hemisphere of the egg. (C). Animal hemisphere of P. waltlii egg injected
with cytoplasm of B. bufo egg (dark area). (D, E and F). Development of a P. waltlii
egg injected with cytoplasm of X. laevis egg, at the 4-cell stage. (D). Two earlier
cleavages in the site of injection (arrow); recipient egg in the course of 8-cell stage.
(E). New division (arrow) of a'rapid cleaving blastomere'; recipient egg in thecourse
of 16-cell stage. (F). The same egg at morula stage (20 h development). Note the
reduced size of the cells, (limited by arrows) in the area of injection. Magnifications:
A, D, E a n d F : G x 3 6 ; B and C: G x 8 5 .
Cytoplasmic control of cleavage
267
t
•
*
>
K
v.
^sSW'^'
PV«
\
t
w
-
*
*
-
.
268
C. AIMAR, M. DELARUE AND C. VILAIN
Table 4. Chronology of the cell cycles of a P. waltlii egg
injected with X. laevis cytoplasm
Chronology of cleavage (in min)
Cycles no.
4
5
6
7
8
9
10
Control eggs
Experimental eggs
64-8
(+61-1%)*
/ 2 5 2^.
( + 61-1%)
136-8
(+31-6%)
/
93-6 ^ \
(+31.6%)
198
(+25-5%)147-6f
169-2 (+14-5%)
255-6
( + 22-5%) 198
216 (+15-5%)
309-6
(+15-1%) 262-8
284-4 ( + 8-1%)
367-2
( + 5-9%) 345-6
345-6 (+5-9%)
424-8
(+2-5%) 414
414 ( + 2-5%)
* % of variation of the duration of cell cycle by reference to controls.
t Lineage of small cells.
908070607
50-
\
4030-
20- 8
/\
/\
/
/
/
/
/
/
/
/
\
\
\
\
\
\
\
X
/\
/\
/\
/\
/\
/\
/\
/\
/\
10
Cleavage cycles
Figure 2. Duration of cell cycles of a P. waltlii egg injected with X. laevis cytoplasm.
(1) Control group eggs. (2, 3 and 4) Operated egg, (3) lineage of small cells, (4) lineage
of normal size cells.
In a second group of eggs, the recipient blastomeres cleaved rapidly and the
cells subsequently divided early either once or twice (Fig. 1D, E and F). Blastomeres resulting from these cell divisions had the size of animal hemisphere cells
of an egg at the theoretical 16-, 32- or 64-cell stage. At the same time other
cells in the same embryos were not cleaving or exhibited a single cycle of division
as the control did. Thereafter all cells of the embryos re-entered the synchronous
phase of cleavage. As a consequence of injections of cytoplasm, the animal
hemispheres of the embryos were comprised of two populations with different
cell size. (Fig. 1F) Further development proceeded with a rhythm identical to
that of control groups. However, losses or necrosis of external cells were observed
Cytoplasmic control of cleavage
269
at the tail-bud stage. This cellular damage did not alter the development of
embryos which had been observed up to the larval stages.
The third group was composed of individuals combining both developmental
characteristics: early cleavage and the formation of two cell populations.
Chronological events from the 3rd to the 10th cleavage cycles of an egg of this
group were observed by microcinematography and are shown in Table 4 and in
Fig. 2. The 4th cell cycle was 40 min earlier than for control groups. Early cleavage was later offset by a gradual delay in the duration of the cell cycle. At the
5th cycle two kinds of cells segregated - cellular sizes and the duration of the
cell cycles were different. By the 9th cycle, those two kinds of blastomeres
divided synchronously. From these observations, it may be seen that injection of
xenogenic cytoplasm induced a desynchronization of cleavages, and a characteristic shortening of the cell cycle for the injected blastomeres. As cleavage
proceeded the effect of the xenogenic cytoplasm decreased progressively suggesting that a quantitative cytoplasmic factor is required for the control of the
timing of cleavage.
(4) Comparison of the effects ofX. laevis karyoplasm and cytoplasm injected into
P. waltlii eggs
Karyoplasm (200 nl) and cytoplasm (800 nl) from X. laevis oocytes and from
virgin eggs was injected in P. watltii eggs. Just before these injections an equivalent volume of cytoplasm had been removed from the recipient eggs. The nuclei
used came from full-grown immature oocytes or full-grown oocytes which were
incubated for 2 h with progesterone (10~6 M) and still exhibited an intact germinal
vesicle. Injected cytoplasm had been removed respectively from full-grown
oocytes, from oocytes at three different stages of maturation: progesterone
stimulation (2 h of hormonal incubation), rupture of germinal vesicle stage
(GVBD) and post-GVBD stage, and from virgin eggs.
The karyoplasm or the cytoplasm of full-grown oocytes or oocytes newly
stimulated with progesterone failed to change the chronology of recipient eggs
(Table 5). Most of them (80%) cleaved at the same time as control groups. On
the other hand, the cleavage duration was shortened when the injected cytoplasm
was taken from eggs at subsequent development stages. The number of reacting
eggs was different depending on the oocyte stage at which the cytoplasm was
sampled. After injection of cytoplasm at GVBD and post-GVBD stages and in
the virgin eggs stage, the ratio of eggs cleaving faster than the control group was
82, 37 and 71 % respectively.
On the average 20 % of operated eggs did not cleave. For the two series of
eggs injected with cytoplasm of maturing oocyte, a large proportion (71 and
48 %) of these uncleaved eggs were blocked at the metaphase II stage of meiosis.
The 'cytostatic factor' (Masui & Markert, 1971; Meyerhof & Masui, 1979)
present in the cytoplasm of maturing oocytes may be involved in the blockage of
these eggs.
Injections
+
id.
control
Timing of cleavage
-
no
cleavage
Fast cleavage
effect
Oocyte (stage 5)
G.V.
32
0
24
0
3
5
0
89%*
0%
11%*
16%t
cyt.
47
13
20
0
80
0
78%
22%
25%
0%
Maturing oocyte
7
G.V.
0
30
23
0
0
before
23%
77%
0%
0
GVBD
28
6
0
12
10
cyto.
55%
45%
0%
21%
Maturing
57
7
1
14 (10)
35
cyto.
oocyte GVBD stage
16%
2%
24%
+
82%
Maturing
7
10
2
42
cyto.
23(11)
+/oocyte post-GVBD
37%
10%
53%
54%
Virgin egg
cyto.
4
0
0
14
10
+
0%
29%
71%
* Percentage on number of cleaving eggs ''col. 4, 5 and 6).
t Percentage on total number of experiment eggs (col. 3), in braces, number of eggs blocked at metaphase II stage.
+ , Fast cleavage; —, Delayed cleavage.
Stage of
donor cells
Number
of
eggs
Table 5. Injection ofX. laevis karyoplasm and cytoplasm into P. waltlii virgin eggs
p
d
w
AIMAR, M
p
Z
d
LARUE.
ILA
Cytoplasmic control of cleavage
271
In a control group, P. waltlii eggs were injected with nucleic and cytoplasmic
material, 200 nl and 800 nl respectively, of the same species and taken from
oocytes and eggs at the same development stages as in the above experiment. As
many as 75 % of recipient eggs cleaved at the same time as unoperated eggs; for
the others cleavage was delayed. These results indicate that the timing of cell
cycle does not depend on a direct effect of components confined in the oocyte
nucleus. Tt seems to be related to a specific, qualitative but not quantitative, feature of the oocyte cytoplasm expressed as soon as the germinal vesicle ruptures.
(5) Effects of the X. laevis Maturation Promoting Factor (MPF) on cleavage of
P. waltlii and X. laevis eggs
The effects on the cell cycle of cytoplasmic extracts showing MPF activity have
been compared to the effects of the injection of a similar quantity of total cytoplasm from virgin eggs.
100 nl of MPF cytoplasmic fraction extracted from X. laevis oocytes were
injected into virgin eggs of X. laevis and P. waltlii species.
For six experimental groups (52 eggs), MPF was injected in eggs of the X.
laevis species. As many as 74 % of developing eggs cleaved at the same time as
control groups, i.e. 150 min after activation. The remainder of the eggs (26%)
cleaved about 15 min earlier than control groups.
Eighteen groups of 15 recipient eggs of P. waltlii species, on the average, were
tested. 164 of these eggs, i.e. 62% cleaved. For five series, eggs presented a
delayed cleavage of about 30 min in comparison to control groups which cleaved
in 375 ± 15 min after activation. For all other series, the cell cycle period was the
same as for control groups. Under these experimental conditions, MPF extracted from Xenopus oocytes did not seem to induce any significant reduction of
the cleavage duration of P. waltlii eggs, contrary to the results with transfers of
cytoplasm of X. laevis egg.
DISCUSSION AND CONCLUSION
A controlled alteration of the cell cycle has been experimentally induced in
amphibian eggs. Reciprocal cytoplasm transfers between virgin P. waltlii and
X. laevis eggs induced a fast or delayed cleavage of the recipient eggs related to
the specific type of the injected cytoplasm. Fertilized eggs subjected to this
experimental procedure gave rise to two cell populations with different cycles of
division. Our data demonstrates that the cycle duration is related to qualitative
properties of the egg cytoplasm. This result agrees with the recent work of Hara,
Tydeman & Kirshner (1980) who showed that a cytoplasmic rhythm is maintained in enucleated egg fragments. In addition, our experiments suggest that
delay of the cell cycle is, for an operated egg, dependent on a quantitative effect
of the injected xenogenic cytoplasm.
Various cellular components are able to induce cleavage of an egg. Some of
272
C. AIMAR, M. DELARUE AND C. VILAIN
them are cellular bodies, such as basal bodies or centrioles, involved in the
mitotic apparatus. These are able to induce eggs furrows in amphibian or
echinodea eggs (Heideman & Kirshner, 1975; Mailer et ah 1976; Hirano &
Ishikawa, 1979). Other fractions, as in our experiments, originate from amphibian
oocyte cytoplasm. Sawai (1976) has demonstrated that subcortical cytoplasm of
cleaving eggs induces the formation of early and irregular furrows on newt eggs.
However, 'this furrow-inducing cytoplasmic component' (FIC) is only present
in the subcortical area of a cleaving egg but not in a newly activated egg such as
we have used.
Ca 2+ present in the egg cytoplasm, seems to play a major role in cell division.
Intracellular variations of calcium concentration obtained by microinjections of
calcium ions or ionophore 123.187 induce cortical constriction in Ilyanassa eggs
(Conrad & Davis, 1977) and in Rana pipiens eggs (Hollinger & Schuetz, 1976).
However, the effects obtained after injection of Ca 2+ in an egg seem to be different from those observed in our experiments. Ca 2+ induced constrictions lacked
any denned orientation and started much earlier (2 to 5 min) than those induced
by injection of total cytoplasm. Moreover, the consequences of cytoplasm injections on the development of the recipient egg were observed for several cell
cycles as opposed to the short-time action of injected Ca 2+ ions.
A Ca2+-dependent factor involved in the meiotic process was recently investigated. This' Maturation Promoting Factor' (MPF) was first detected in the
cytoplasm of maturing oocytes (Masui & Markert, 1971) then in the cleaving
egg (Wasserman & Smith, 1978). Furthermore, it was found that extracts of
cultivated mammalian cells exhibit a mitogenic effect identical to that obtained
with MPF (Sunkara, Wright & Rao, 1979; Nelkin, Nichols & Vogelstein, 1980).
In our experiments, injection into eggs of a cytoplasmic extract showing MPF
activity does not alter the cleavage timing. This factor does not seem to control
the duration of cell cycle.
Our data showed that the determination of the cleavage delay is established
during the course of oogenesis. As demonstrated, an endogenous rhythm is
peculiar to the cytoplasmic fraction of the oocyte at the GVBD stage. It is not
related to the cytoplasm or to the nucleoplasm of the full-grown oocytes or to the
cytoplasm of those newly stimulated with progesterone.
At the GVBD stage, the mixture of the nuclear and cytoplasmic components
allows molecular interactions between various components previously stored in
the vitellous cytoplasm or confined in the oocyte nucleus. As a consequence,
modifications in the physicobiochemical characteristics of the oocyte insure new
cellular properties (Smith, 1975). Some of these are related to the cleavage
process. Activation of the cortical contractile system (Hollinger & Schuetz,
1976) or the ability of the egg to elaborate asters and to undergo normal cleavage
(Heideman & Kirschner, 1978) are relevant to these events. Besides, the ability
of an egg to be activated appears to be related to germinal vesicle breakdown
(Bellanger & Schuetz, 1975; Hollinger & Schuetz, 1976). As for these different
Cytoplasmic control of cleavage
273
systems involved in the process of cleavage, a cleavage timing system (CTS)
acting as a cytoplasmic clock comparable to that observed by Hara et al.
(1980) seems to become established or to become activated at this developmental stage just preceeding cleavage of the egg.
We are most grateful to M. Jean Desrosiers, photographer for his technical assistance.
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{Received 8 December 1980, revised 23 March 1981)