J. Embryol. exp. Morph. Vol. 23, 2, pp. 385-94, 1970
Printed in Great Britain
385
Cleavage delay and abnormal
morphogenesis in Lymnaea eggs after pulse
treatment with azide of successive
stages in a cleavage cycle
By TOSHIE CAMEY 1 AND W. L. M. GEILENKIRCHEN 2
From the Zoological Laboratory, University of Utrecht
and 'René Rachou' Research Centre, National Institute
of Rural Diseases, Brazil
Heat treatments of short duration of successive stages in a cleavage cycle of
Lymnaea eggs differentially affect the division cycle and morphogenesis. Effects
on the division cycle are most pronounced after a treatment during actual
cleavage, at the end of interphase and during prometaphase. Abnormal morphogenesis is only found when a heat shock is applied during metaphase-anaphase
stages; after treatment development proceeds up to the blastula stage. Development after the blastula stage, however, may be disturbed, leading to death,
exogastrulation or malformations of the embryo.
The results of these experiments indicate that successive division cycles
represent well-defined steps of differing significance for later development
(Geilenkirchen, 1966). Comparable results were obtained with LiCl (Geilenkirchen, 1967). It appeared that treatments of short duration between prophase
of the second division and prophase of the third division cause exogastrulation
whereas before and after this period of development LiCl rarely causes exogastrulation. It was also found that the sensitivity of the eggs to LiCl, with
respect to exogastrulation, can be correlated with a change in the oxygen
consumption.
In relation to these results it is of interest to know whether the processes by
which division and morphogenesis are carried out in the egg cell are equally
dependent on energy expenditure. In this paper the results are given of a study
of the effects on cleavage and morphogenesis of treatments of short duration
with sodium azide, an inhibitor of phosphorylating respiration. The developmental stages between oviposition and the 12-cell stage have been studied.
1
Author's address: Instituto Nacional de Endemias Rurais, Centro de Pesquisas 'René
Rachou', Av. Augusto de Lima, 1715-Caixa Postal 1743, Belo Horizonte, MG. Brazil.
2
Author's address: Zoologisch Laboratorium, Janskerkhof 3, Utrecht, The Netherlands.
25
EMB 23
386
T. CAMEY AND W. L. M. G E I L E N K I R C H E N
The applied criteria for disturbance are (a) retardation of the cleavages which
follow treatment and (b) the ultimate stage of development reached by the
embryo after a pulse treatment has been given.
MATERIAL AND METHODS
Eggs of Lymnaea stagnalis were obtained by exposing the snails to fresh
water and a temperature rise of 5 °C (Geilenkirchen, 1961). In each experiment
one egg mass only was used. Even in one egg mass, however, the egg cells do not
divide synchronously. Groups of 10 almost synchronously dividing eggs can be
obtained by selecting eggs in which the first division starts within 1-2 min. The
first group selected was used as a control. Treatment groups A-D, etc. were given
a treatment with NaN 3 at 0, 10, 20, 30, etc. or 0, 15, 30, 45, etc. min after the
eggs started first cleavage. The last group selected was used as a second control.
The treatment was started by transferring the eggs to a NaN 3 solution (5 x
10~2 M, pH 6-8) in tapwater at 25 °C. After 1 h in the NaN 3 solution the eggs
were carefully rinsed in tapwater at 25 °C and kept in tapwater at the same
temperature. After treatment the time for the next cleavage or the next two
cleavages to occur was determined in each sample, and hence the cleavage delay
of the next cleavage and possible extensions of following cleavage cycles, with
respect to the control groups, were calculated. After 24 h in tapwater, the eggs
were laid out on moist agar in Petri dishes and cultured at 25 °C. The development of each embryo was followed from day to day and recorded until a stage
at which the normal embryos were well-developed young snails. In all, three
grades of developmental disturbances could be distinguished.
First-period death. Under this heading were included all those embryos which
died before or during the gastrula stage. In these experiments this category plays
an important role. It is noteworthy that more than 95% of all embryos scored
under this heading develop into blastulae. They are alive for 3 or 4 days and die
without showing any further development.
Exogastrulation. Exogastrulae are vesicular embryos in which the invagination
of the archenteron is suppressed. These embryos die within a few days.
Head malformations: All microphthalmia monophthalmic, synophthalmia
triophthalmic, cyclopic and anophthalmic embryos were classed in this group.
Similar experiments have been carried out starting at the second and the third
division and at oviposition. The cleavage cycle ending with first cleavage is called
the first cleavage cycle, the cycle between first and second cleavage the second
cleavage cycle, etc. In experiments covering the developmental time between
oviposition and first cleavage, a difficulty arises with respect to the selection of
groups of synchronized eggs. In one egg mass the developmental age of the egg
cells increases gradually from the front end to the rear end. Therefore the egg
mass was divided in pieces containing eggs of increasing age. Every fifth group
was taken as a control. In all groups the time of extrusion of the first and second
polar bodies and the time of first cleavage were observed.
Azide and cleavage
abnormalities
387
Representation of results
Abscissae. The method used by Geilenkirchen (1966) was followed. Since eggs
of different egg masses vary with respect to the duration of the cleavage cycles,
the average duration of a cycle in the control groups of each egg mass is set at
100 %. The preset times of the start of treatment 10, 20, 30, etc. min after first
(2nd or 3rd) cleavage are then expressed in percentages of the duration of the
cleavage cycle. The results of the separate experiments falling within successive
10 % time periods are taken together. The arithmetic means of the data in such
a period are given in the Figs. In case of experiments starting at oviposition the
time between oviposition and first cleavage is set at 100%. In this procedure
the assumption is made that if a cleavage cycle takes a shorter or longer time,
all stages of the cycle are proportionately shorter or longer. Control experiments
have confirmed this assumption.
Ordinates. On the ordinates of Fig. IA-D the extension of cleavage cycles is
given in minutes. On the ordinate of Fig. 3 the percentages of normal and
abnormal development are indicated.
RESULTS
1. The extension of cleavage cycles after treatment of
successive stages in a cycle
The period of development from oviposition to fourth cleavage (formation of
the second quartet of micromeres) was studied for sensitivity to NaN 3 treatment
in terms of extension of cleavage cycles (Fig. 1A-D).
(a) Treatment of stages between oviposition and first cleavage. At regular
time intervals, read on the abscissae, groups of eggs were transferred to a NaN 3
solution (5 x 10~2 M) in tapwater. After 1 h the treatment was terminated by
transferring the eggs to tapwater. The time of first, second and third cleavage
was determined in each group. The delay of first cleavage (Fig. 1A) and the
possible prolongation of the second (Fig. IB) and the third cleavage (Fig. 1C)
cycle with respect to control groups were calculated.
The treatment causes development to stop immediately. First cleavage is
delayed at all stages for about 100 min. This is about 40 min in excess of the
duration of treatment. A prolongation of the second and the third cleavage cycle
(Fig. IB, C) is not observed. It is concluded that treatment between oviposition
and first cleavage causes a delay of the first cleavage only. It was observed, however, that treatments up to the extrusion of the first polar body quite often
caused an abnormal first cleavage. Instead of two blastomeres, three equally
large blastomeres in a trefoil arrangement were formed.
(b) Treatment of stages between first and second cleavage. Development ceases
soon after the start of treatment. The delay of the second cleavage is most pronounced after treatment at the time of first cleavage (Fig. IB). During interphase the extension of the cycle decreases. From prophase onwards an increase
25-2
388
T. CAMEY AND W. L. M. G E I L E N K I R C H E N
140
140
120 _ Delay of first cleavage
100
A
:
80
60
40
r-sH
20 :
'
I I I
0 "l ' I I ' ' ' I I I I '
10 20 30 40 SO 60^70 80 90 100%20 40 60
140
120 Z Extension of the second cleavage cycle
100
;
I I I I I I I I'
I I I I I I
80 100%20 40 60 80 100%20 40 60
60
'I
I I I I I I I I I I I I
90 100%20
40
60
100
80
60
40
20
0
140
B I 120
80
40
20
0
120
80 100%20
40
60
80 100%20
40
60
80
100
80
60
40
20
0
140
140
120 _ Extension of the third cleavage cycle
120
100
100
80
80
60
60
40
40
20
0
20
100%20
40
60
80
140
120 Z Extension of the fourth cleavage cycle
100
80
60
40
20
'I
10 20
Oviposition
30
140
120
100
80
60
40
20
I I I I I I l I l l I
40 50 60 70 80
A
A
A
1st mat. div. 2nd mat. div.
90 100%20
1 se cl.
0
40
'
60
80 100%20
A
2nd cl.
40
60
I I I I I I I I II
80 100%20
A
3rd cl.
40
60
80 100%
A
4th cl.
Time of treatment
FIGURE 1
(A) Retardation of first cleavage (ordinate) after a Na-azide treatment (60 min) in
relation to time after oviposition at which treatment is started (abscissa).
(B) Extension in excess of the second cleavage cycle (ordinate) after Na-azide
treatment in relation to time after oviposition and first cleavage at which treatment
is started (abscissa).
(C) The same as in (B) for the third cleavage cycle.
(D) Extension in excess of the fourth cleavage cycle (ordinate) after Na-azide
treatment in relation to time after second and third cleavage at which treatment is
started (abscissa). Ordinate = time in minutes. Abscissa = time scale: oviposition to
first cleavage, time 100% = 220 min; first cleavage to second cleavage, time .100% =
90 min on average; second cleavage to third cleavage, time 100% = 85 min on average; third cleavage to fourth cleavage, time 100%= 85 min on average. T =
telophase; K = karyomere nucleus; P jr = polymorphic nucleus; P = prophase;
Azide and cleavage abnormalities
389
is observed. Treatments during the second cleavage cycle have no influence on
the duration of the third cleavage cycle (Fig. IC).
(c) Treatment of stages between second and third cleavage. Again it was
observed that development stops soon after the start of treatment. In the third
cleavage cycle (Fig. IC) the same pattern of delays is found as in the foregoing
cycle. At the end of mitosis, however, during meta-anaphase, the delay tends to
diminish. Treatments during the third cleavage cycle have no influence on the
duration of the fourth cycle (Fig. 1D).
(d) Treatment of stages between third andfourth cleavage. The pattern of delay
found after treatment of successive stages in this cycle is similar to that found
during the third cleavage cycle, and needs no further comments (Fig. 1D).
Conclusion
Treatment with 5 x 10 -2 M-NaN3 for 1 h extends the duration of the cleavage
cycle in which the treatment is given. The treatments do not extend the duration
of subsequent cleavages.
2. Abnormal first cleavage
Eight experiments were carried out to study the abnormal first cleavages
observed after treatment of stages around the time of the first maturation
division.
Groups of eggs at stages between oviposition and second maturation division
were treated as described. The number of abnormal first cleavages was determined in each group. Fig. 2 shows the results. The number of trefoils is maximal
after treatment shortly before the first maturation division. The first polar body
always forms, whereas the second polar body is suppressed. At first cleavage a
triaster develops and three blastomeres of equal size are formed. The 3-cell
stage is followed by 6- and 12-cell stages at succeeding divisions.
In one case the trefoil developed into a normal embryo and in that case cells
were extruded during the blastula stage. In other cases development was interrupted at the blastula stage and the embryos died. All other trefoils reached a
late blastula stage. About one-third developed into exogastrulae and two-thirds
died around the time of gastrulation.
3. The immediacy of azide treatment
Morphological observations of the living eggs during treatment showed that
development was stopped very soon after the start of treatment. This is confirmed by the observation that DNA synthesis stops immediately after the start
of treatment. As indicated in Fig. 1, reduplication of DNA starts in normal development at about 10 % in the time scale on the abscissae. (J. A. M. v.d.
Biggelaar, in preparation.) This is about 8 min after cleavage. Eggs incubated right
after second cleavage in [3H]thymidine, and exposed to azide for 1 h, showed no
[3H]thymidine incorporation in autoradiographs. If the eggs after incubation and
exposure to azide for 1 h are left in tapwater for 15 or 45 min low incorporation is
390
T. CAMEY AND W. L. M. G E I L E N K I R C H E N
observed over the nucleus. Since azide treatment may affect the uptake of
[3H]thymidine, experiments were also carried out with incubation in [3H]thymidine starting 15 min before second cleavage. At second cleavage azide was
added to the incubation medium. After exposure to azide for 1 h, the eggs were
fixed immediately. No incorporation was found. If the eggs after treatment were
left for 45 min in tapwater, fairly strong incorporation was observed over the
nucleus. These results indicate that azide treatment indeed impairs [3H]thymidine uptake. But they also prove that DNA synthesis is not possible during
treatment with azide, and that it is resumed after treatment. Furthermore,
they support the morphological observations that the azide effect is shown
immediately.
100
^ 6 0 -
T
40
50
1st polar body
r
60
70
80
2nd polar body
100%
1st cleavage
Stage of development
Fig. 2. Percentages of trefoils arising at first cleavage (ordinate) after treatment
with Na-azide of developmental stages between oviposition and second maturation division.
4. Morphogenese effects
After measuring the reversible effect of NaN 3 on the duration of cleavage
cycles, the eggs were reared in the normal way as described above. In Fig. 3 the
percentages of normal and abnormal development are plotted against the time
of start of treatment.
(a) Treatment of stages between oviposition and first cleavage. The eggs are
rather sensitive to a treatment with azide at stages before the first maturation
division. Normal development lies between 50 and 80 %. Between the first and
second maturation divisions sensitivity decreases and up to 90% normal development is observed. After the second maturation division sensitivity increases
again and at first cleavage only 60% of the eggs develop in a normal way.
Abnormal development consists mainly of first-period death. Some embryos
develop atypically and a few embryos develop shell malformations.
(b) Treatment of stages betweenfirstand fourth cleavage. The curve for normal
Azide and cleavage abnormalities
391
development in Fig. 3 shows that successive cleavage cycles have a similar
sensitivity pattern. A treatment at the moment of first, second, third or fourth
division has a severe effect on development. Normal development occurs in
only 40-60 % of the eggs. Immediately after division the sensitivity to N a N 3
treatment decreases rapidly. Normal development is found in 80-90 % of eggs
until prophase sets in. Between prophase and telophase the egg cells become
increasingly more sensitive. The same sensitivity pattern is found in all the
100
0s-
90
80
70
60
50
40
30
20
'X
10
0
1
10
Oviposition
P
1
-
20
PM M A T T
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30 | 40
50
1st polar body
K
PM
-l—Ï—l
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80
2nd polar body
P PM M A T T
K
90
>sN^
100%10 20 30 40 50
1st cleavage
PM
P PM M A T
100
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10-
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60 70 80 901Ö0%10 20 30 40 50 60 70 80 90100%10 20 30 40 50 60 70 80 90100%
2nd cleavage
3rd cleavage
4th cleavage
Stage of development
Fig. 3. The percentages of normal and abnormal development after Na-azide
treatment, in relation to time after first, second and third cleavage at which treatment is started. Ordinate = percentages of normal or abnormal development.
Abscissa = time scale: oviposition to first cleavage, time 100% = 220min; first
cleavage to second cleavage, time 100% = 90 min on average; second cleavage to
third cleavage time 100 % = 85 min on average; third cleavage to fourth cleavage,
time 100% = 85 min on average; T = telophase; K = karyomere nucleus; PM =
poylmorphic nucleus; P = prophase; PM = prometaphase; M = metaphase; A =
anaphase; S = DNA synthesis (Gielenkirchen, 1961).
O, Normal; X, first-period death; # , exogastrulae; A, head malformations.
392
T. CAMEY AND W. L. M. G E I L E N K I R C H E N
cleavage cycles studied. Concomitantly this pattern is reflected in first-period
death. With respect to exogastrulation increasing percentages are found after
treatment around the time of second, third and fourth cleavage. Similarly, head
malformations are found in small numbers after treatment around the time of
second, third and fourth cleavage. Shell malformations are found incidentally
at all stages treated.
Conclusion
Treatment with azide during the mitotic stages from prophase to telophase
causes abnormalities and/or death during subsequent development. Telophase is
the most sensitive stage.
DISCUSSION
Delay of cleavage
When eggs of Lymnaea are treated with azide, an inhibitor of oxidative
phosphorylation, development is stopped immediately.
The variance of the excess delay per cycle after treatment presumably reflects
phases of different energy expenditure. A period in the cycle with high energy
expenditure as far as ATP is involved, will take more time to recover after
treatment. This is corroborated by the fact that right after first, second and
third cleavage a sharp increase in the rate of oxygen consumption is observed
in Lymnaea eggs (Geilenkirchen, 1961, 1967). The high excess delay immediately
following cleavage may be correlated with the energy used in the preparation
for DNA reduplication. DNA reduplication is postponed following azide treatment. The smaller increase in excess delay during prophase and prometaphase
reflects energy expenditure in relation to mitotic events.
Comparing the effect of azide on the duration of a cleavage cycle of Lymnaea
and Tetrahymena (Hamburger, 1962) quite different results are obtained. In
Tetrahymena the excess delay increases when the treatment starts at successively
later stages in the cell cycle. In Lymnaea the delay decreases in that period. In
Tetrahymena a treatment during the last one-third of the cycle has no delaying
effects, whereas in Lymnaea a delaying effect is definitely observed.
Morphogenetic effects
Morphogenetic effects have been mainly observed after treatment of stages
between prophase and telophase in successive cleavage cycles. These are irreversible effects of the treatment, in contrast with the effects on division which
were shown to be reversible. This leads to the conclusion that in the blastomeres
one set of processes is involved in cell division and a different set in morphogenesis and differentiation at a later stage.
The same conclusion was obtained from the results of heat-shock experiments
with Lymnaea eggs and Arbacia eggs (Geilenkirchen, 1964, 1966) and from coldshock experiments with Lymnaea eggs (unpublished results).
Azide and cleavage abnormalities
393
The heat-shock experiments lead moreover to the hypothesis that successive
divisions represent well-defined steps of differing significance to later development and differentiation. This hypothesis was derived from the observation
that treatment of successive cleavage cycles caused head malformations with a
different pattern of malformations according to the cleavage cycle treated. The
head malformations obtained after NaN 3 treatment are similar to those found
in this period of development after heat shocks. The processes which are involved in morphogenesis in each cycle depend apparently on oxidative phosphorylations and can be nullified by heat, cold or NaN 3 treatment. Once disturbed in one cell cycle they cannot be established in later cell cycles.
The heat-shock experiments led us to surmise that centriole splitting and
reduplication are involved (Geilenkirchen, 1966). Another possibility is a disturbance of specific protein synthesis or enhanced decay of template RNA as
found after heat shocks in Tetrahymena (Zeuthen, 1964; Moner, 1967).
SUMMARY
1. Eggs of Lymnaea stagnalis were treated with sodium azide for 60 min at
successive stages between oviposition and the 12-cell stage. The effects of the
treatment on cleavage and morphogenesis were studied.
2. The division following treatment is delayed. The delay, in excess of the
time of exposure, of the cleavage following treatment varies with the stage
in the cell cycle treated. It is maximal for a treatment during the preceding
cleavage. The effect on division is reversible.
3. Treatment of stages around the first maturation division may suppress the
formation of the second polar body. In that case a triaster forms at first cleavage
and three equally large blastomeres in a trefoil arrangement are formed.
4. DNA synthesis, which normally starts right after cleavage, is immediately
stopped by the treatment. Synthesis is resumed if the eggs are washed free of
azide after treatment.
5. Morphogenesis is disturbed irreversibly after treatment during mitosis and
the ensuing division.
6. The effects of azide on morphogenesis are comparable with the effects of
heat shocks on morphogenesis.
RÉSUMÉ
Retard de clivage et anomalies de la morphogenèse dans des œufs de Lymnaea
après des traitements, limités dans le temps, par de Vazide à des stades successifs
des cycles de segmentation
1. Des œufs de Lymnaea stagnalis ont été traités par de F azide de sodium
pendant 60 min aux stades successifs qui s'étendent entre la ponte et celui de
douze cellules. Les effets du traitement sur le clivage et la morphogenèse sont
étudiés.
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T. CAMEY AND W. L. M. G E I L E N K I R C H E N
2. La division qui suit le traitement est retardée. Le taux de ce retard qui
dépasse le temps du traitement dépend de la période, entre deux divisions
successives, pendent laquelle les œufs sont traités. L'effet sur la division est
réversible.
3. Un traitement aux environs du moment de la première division de
maturation peut supprimer la formation du deuxième globule polaire. Dans ce
cas-là, un triastre se forme au premier clivage qui entraîne la production de
trois blastomères en forme de trèfle.
4. La synthèse de l'ADN qui commence normalement juste après la division,
est retardée immédiatement par le traitement. Cette synthèse recommence après
un lavage des œufs dans l'eau de ville.
5. La morphogenèse est altérée irréversiblement après un traitement qui
s'étend au cours d'une mitose et de la division suivante.
6. L'azide a, sur la morphogenèse, un effet comparable à celui d'un choc
thermique.
REFERENCES
W. L. M. (1961). Effects of Mono- and Divalent Cations on Viability and
Oxygen Uptake of Eggs of Limnaea stagnalis. Thesis, Utrecht.
GEILENKIRCHEN, W. L. M. (1964). The cleavage schedule and the development of Arbacia eggs
as separately influenced by heat shocks. Biol. Bull. mar. biol. Lab., Woods Hole 127, 370.
GEILENKIRCHEN, W. L. M. (1966). Cell division and morphogenesis of Limnaea eggs after
treatment with heat pulses at successive stages in early division cycles. /. Embryo/, exp.
Morph. 16, 321-37.
GEILENKIRCHEN, W. L. M. (1967). Programming of gastrulation during the second cleavage
cycle in Limnaea stagnalis: a study with lithium chloride and actinomycin D. /. Embryol.
exp. Morph. 17, 367-74.
HAMBURGER, K. (1962). Division delays induced by metabolic inhibitors in synchronized
cells of Tetrahymena pyriformis. C. r. Trav. Lab. Carlsberg 32, 359-70.
MONER, J. G. (1967). Temperature, RNA synthesis and cell division in heat synchronised
cells of Tetrahymena. Expl Cell Res. 45, 618—30.
ZEUTHEN, E. (1964). The temperature-induced synchrony in Tetrahymena. In Synchrony in
Cell Division and Growth. Ed. E. Zeuthen. London : Interscience.
GEILENKIRCHEN,
(Manuscript received 30 May 1969, revised 25 September 1969)