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.

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 Ambystoma mexicanum (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, Løvtrup Rein & Løvtrup, 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 al. 1975).

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.

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 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.

Fig. 1

Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs

Fig. 1

Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs

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 10h 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. cal., 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 mM-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 −70°C.

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 μm. A Leitz micromanipulation apparatus was used.

Cytological study

Germs were fixed with Zenker solution, embedded in paraffin and serially sectioned (10 μm) for light microscopy. Nuclei were stained by the Feulgen-Rossenbeck method and cytoplasmic structures with light green dye.

(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.

Table 1

Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs

Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs
Reciprocal injections of cytoplasm between P. waltlii and X. laevis virgin eggs

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).

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.l→X.l 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 P. 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).

Table 2

Injection of the cytoplasm from various amphibian species into P. watllii virgin eggs

Injection of the cytoplasm from various amphibian species into P. watllii virgin eggs
Injection of the cytoplasm from various amphibian species into P. watllii virgin eggs

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).

Table 3

Effects ofX. laevis cytoplasm on cleavage of P. waltlii eggs

Effects ofX. laevis cytoplasm on cleavage of P. waltlii eggs
Effects ofX. laevis cytoplasm on cleavage of P. waltlii eggs

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.

In a second group of eggs, the recipient blastomeres cleaved rapidly and the cells subsequently divided early either once or twice (Fig. 1 D, 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 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.

Table 4

Chronology of the cell cycles of a P. waltlii egg injected with X. laevis cytoplasm

Chronology of the cell cycles of a P. waltlii egg injected with X. laevis cytoplasm
Chronology of the cell cycles of a P. waltlii egg injected with X. laevis cytoplasm
Fig. 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.

Fig. 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.

(4) Comparison of the effects of X. 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.

Table 5

Injection ofX. laevis karyoplasm and cytoplasm into P. waltlii virgin eggs

Injection ofX. laevis karyoplasm and cytoplasm into P. waltlii virgin eggs
Injection ofX. laevis karyoplasm and cytoplasm into P. waltlii virgin eggs

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.

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. It 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.

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 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 al. 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.

Ca2+ 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 Ca2+ in an egg seem to be different from those observed in our experiments. Ca2+ induced constrictions lacked any defined 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 Ca2+ 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 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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