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.

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.

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 NaN3 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 NaN3 solution (5 ×10–2 M, pH 6·8) in tapwater at 25 °C. After 1 h in the NaN3 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 microphthalmic, monophthalmic, synophthalmic, 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.

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. 1A–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.

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; PM = polymorphic nucleus; P = prophase; PM — nrnmpfanhacp* M = mptanhase.’ A = ananhaseS = DNA svnthesis ÍGeilen-

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; PM = polymorphic nucleus; P = prophase; PM — nrnmpfanhacp* M = mptanhase.’ A = ananhaseS = DNA svnthesis ÍGeilen-

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).

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).

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 NaN3 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 NaN3 solution (5 × 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 is observed. Treatments during the second cleavage cycle have no influence on the duration of the third cleavage cycle (Fig. 1C).

(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. 1C) 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 and fourth 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 × 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.

Fig. 2.

Percentages of trefoils arising at first cleavage (ordinate) after treatment with Na-azide of developmental stages between oviposition and second maturation division.

Fig. 2.

Percentages of trefoils arising at first cleavage (ordinate) after treatment with Na-azide of developmental stages between oviposition and second maturation division.

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 DMA 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 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.

4. Morphogenetic effects

After measuring the reversible effect of NaN3 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 between first and fourth cleavage

The curve for normal 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 NaN3 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 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.

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).

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 NaN3 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 NaN3 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).

  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.

Retard de clivage et anomalies de la morphogenèse dans des œufs de Lymnaea après des traitements, limités dans le temps, par de P azide à des stades successifs des cycles de segmentation

  1. Des œufs de Lymnaea stagnalis ont été traités par de l’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.

  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.

Geilenkirchen
,
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
.
J. Embryol. 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
.
J. 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
.