Cytokinesis by contraction of the base of the cleavage furrow and the dependence of the position of the future furrow on the arrangement of cytoplasm under it have been demonstrated at relatively earlier stages of cleavage in amphibian eggs. The following experiments and observations on dividing eggs of the newt Cynops pyrrhogaster indicated the presence of the contracting force at the base of the furrow and revealed that the above cleavage mechanisms occurred throughout cleavage. (1) Deformation of the animal surface induced conversion of an early furrow to a ridge, the base of the furrow forming the top of the ridge. (2) A needle placed in the path of the furrow blocked its deepening, not only at the point of contact but also in neighbouring regions. Any part of the plane of cleavage could be blocked in this way. (3) A microfilamentous band was found under the plasma membrane at the base of the furrow at a late stage of cleavage. (4) Alteration in the position of the furrow base could be induced in the middle of cleavage by outflow of egg cytoplasm.
It is now accepted that cytokinesis results from contraction of the equatorial surface in amphibian eggs as well as in marine invertebrate eggs. This view is based on the behaviour of cleavage furrows in eggs after surgical operation (Waddington, 1952; Dan & Kojima, 1963), the presence of microfilaments at the base of advancing furrows (Bluemink, 1970; Selman & Perry, 1970; Kalt, 1971; Singal & Sanders, 1974), and the binding of these to heavy meromyosin (Perry, John & Thomas,1971). The position of the future furrow in amphibian eggs, unlike sea-urchin eggs, depends on the underlying cytoplasm until shortly before the furrow appears (Sawai, Kubota & Kojima, 1969). Nearly all this information has been obtained from studies on relatively earlier furrows formed in the animal half. However, since amphibian eggs are large and contain much yolk in the vegetal half, and their mitotic apparatus is excentrically located on the animal pole side, there may be no direct interaction between the mitotic apparatus and the vegetal half. Moreover cleavage takes a long time (the first cleavage of newt eggs takes more than 1 h at 20 °C). Thus further studies are needed to determine whether this mechanism works at all stages of furrow formation of amphibian eggs.
The present experiments on living, dividing cells offered additional, direct evidence that there is a contractile tension at the base of the furrow during cleavage of newt eggs. Mechanical blocking of the progress of the furrow base, as has been done in experiments on sea-urchin eggs (Mitchison, 1953; Rappaport, 1966), showed that this tensile force caused cytokinesis at all stages. Furthermore, studies showed that there was a filamentous structure under the furrow base in late stage of cleavage, and that the position of the furrow base could be altered by displacing the cytoplasm under it. These findings show that the furrowing mechanism of newt eggs remains fundamentally the same throughout cleavage.
MATERIALS AND METHODS
Fertilized, uncleaved eggs of Cynops (Triturus) pyrrhogaster were obtained by injecting females with chorionic gonadotropin. Eggs were decapsulated and kept in Steinberg’s solution at room temperature (20–22 °C). Operations and observations were made under a binocular dissecting microscope in a dish containing this solution over a layer of agar.
For electron microscopy, intercellular junctions of dividing eggs were cut after the beginning of the second cleavage. Then the eggs were fixed for 2 h at room temperature in 3% glutaraldehyde in 0·13 M phosphate buffer (pH 7-4), rinsed overnight, or for a few days, in cold buffer containing 10% sucrose, and postfixed for 2 h in buffered 2% OsO4 in the cold. Then they were dehydrated in acetone, and embedded in Epon. Sections were cut with a Porter-Blum II ultramicrotome, stained with uranyl acetate and lead citrate, and examined in a JEM 7 electron microscope.
Conversion of the furrow to a ridge
At onset of the first cleavage, a glass rod was pressed down on the egg surface at right angles to the furrow so that the furrow base was pushed in. Soon the depression of the furrow completely disappeared, but a pigmented line remained in its original position. Under the rod the tips of the pigment line gradually extended out in both directions and reached the convex, free surfaces on either side of the rod (Fig. 1 A). There the tips connected with the bases of newly developed, normally shaped furrows. On removal of the rod at this time, the depressed egg surface marked by the pigment line rapidly bulged up, forming a ridge, ‘exofurrow’, between the pre-existing normal furrows (Fig. 1B, C). This ridge persisted until the animal surface returned to its normal shape, but it did not change back to a groove. The formation of this ridge with a pigment line along its top is direct, visible evidence of a tensile force acting at the base of the furrow.
Mechanical blocking of the deepening of the furrow base
Experiments were performed on dividing newt eggs with and without vitelline membranes.
The membrane was removed with forceps from eggs soon after the first cleavage, and a glass needle was placed in the path of the furrow at right angles to the cleavage plane with the aid of a simple micromanipulator. When the base of the furrow was difficult to observe in the late or final stage, the blastomeres were gently pulled apart with a needle or hair loop.
Observations showed that in these eggs the furrow deepened until it came into contact with the needle, but that it never passed the needle regardless of the position of the needle on the cleavage plane (Fig. 2); usually at some time after the furrow came in contact with the needle, it started to regress, beginning from its point of contact. On contact of the furrow with the needle at a very late stage, however, furrowing proceeded on the opposite side and was completed when the second cleavage in the 2 blastomeres was fairly advanced; in these eggs the blastomeres were connected by a narrow cytoplasmic tube covering the needle. The tube was not always formed at the centre of the egg, as judged by viewing the egg from above (Fig. 3).
Eggs with a membrane
The experiment described in the preceding paragraph was repeated on eggs within a vitelline membrane. The membrane prevented the eggs from being distorted and held the needle in place during the experiment. The needle was placed under an early furrow near the animal pole, where the egg surface was relatively flat, without the aid of a micromanipulator. An optical section through the plane of cleavage showed that where the furrow touched the needle, the furrow base adjacent to the point of contact gradually became level, but in no case did it bend inward, and later the furrow regressed.
When needles were inserted into much deeper regions of eggs, the cleavage, viewed from outside, appeared to proceed quite normally. After initiation of the second division, such eggs were dipped in hot water. Then the needle was removed and the 2 blastomeres of the fixed egg were separated manually. In these blastomeres the location of the furrow base was identified by the position of the brown pigment on the border of the smooth surface. (Pigmentation of the furrow base was confirmed by examination of living eggs.) The smooth surface was the area of the furrow wall where the plasma membrane remained unbroken when the cell was bisected; the exact position of the needle was apparent as a circular hole through the blastomere.
In this way, the location of the furrow base relative to the needle was determined in 31 of 41 eggs examined. Findings again showed that at a late or final stage the furrow base never passed the needle; in some fixed eggs the bottom of the furrow was in contact with the needle, and in others it was separated from the needle in an unusual fashion, suggesting that furrowing had regressed. Moreover, in the eggs in which the furrow was in contact with the needle it was confirmed that the furrow base did not bend inwards, as noted above.
Filamentous structure in the furrow base
Immediately after onset of the second cleavage, the intercellular junction on the animal side was cut with a needle. This procedure caused partial, spontaneous separation of the blastomeres, and as a result their cytoplasmic connexion became visible from above. When eggs were left at this stage they completed cleavage. Five minutes after this operation some eggs were fixed for electron microscopy. Sections cut through the base of the furrow, parallel to the plane of cleavage, showed the presence of thin filaments below the plasma membrane (Fig. 4). In median crosssections, the filaments were arranged in a band 0·07–0·1 μm thick in the furrows on both the animal and vegetal sides. The diameters of filaments could not be measured accurately, but their approximate order and the localization and appearance of the bundle of filaments indicated that they were microfilaments.
Change of the furrowing position induced by displacing cytoplasm
In the middle of first cleavage, most of one of the two blastomeres was removed by cutting it parallel to the furrow. As the cytoplasm then gradually exuded, carbon particles deposited on the furrow wall on the cut side migrated across the bottom of the furrow to the other side (Fig. 5). No migration across the furrow base occurred in the reverse direction, and no such migration at all occurred in demembranated but unoperated eggs. Similar observations have been made on the initial furrow around the animal pole (Sawai, Kubota & Kojima, 1969) and were interpreted as being due to a change in the position of furrowing owing to displacement of the underlying cytoplasm.
The existence of contractile activity on the furrow has been demonstrated directly in dividing sea-urchin eggs by deformation of an oil drop introduced into the equator (Hiramoto, 1965) and by the bending of a glass needle placed in the path of the furrow (Rappaport, 1966). The experimental conversion of a cleavage furrow to a ridge in the present work affords additional, visible evidence for this contractile activity in amphibian eggs.
Our finding that the furrow base adjacent to a blocking point progressed until it became straight but never bent inward supports the idea of cytokinesis by a contractile force and, therefore, accords with the recent view that cleavage is caused by contraction of microfilaments located in the furrow base. On the other hand, these findings appear to indicate little or no participation in furrowing of a possible force directed directly inward (reviewed in Rappaport, 1971), although in amphibian eggs there is a sheet of modified cytoplasm, the diastema, under the animal furrow (Selman & Waddington, 1955), and the egg cytoplasm possesses latent contractility (Kubota, 1979). Thus these findings give experimental support to the suggestion from ultrastructural studies that the diastema is not contractile (Selman & Perry, 1970).
It is known that, with the circumferential spread of the cleavage furrow toward the vegetal pole, the capacity of the egg surface to contract in response to ‘furrow-inducing cytoplasm’ spreads over the outer surface as a latitudinal belt (Sawai, 1972). A similar change may spread centripetally with deepening of the furrow base. If it does, then in the present work the period between the time when the furrow base came in contact with the needle and the time when furrowing started to regress, namely, the period when the furrow base was in contact with the needle, may represent the time required for passage of this contractile wave through the point of contact; furrowing was mechanically blocked, but the capacity for normal contraction may have spread.
In the present work a microfilamentous band was observed under the furrow base at a late stage of cleavage. The rapid penetration of fixative on its direct contact with the furrow base may have preserved microfilaments normally situated in deeper regions of the eggs, since when whole eggs were fixed these filaments could be observed at an early stage of cleavage but not at a late state (Selman & Perry, 1970; Kalt, 1971; Singal & Sanders, 1974).
It is concluded from this study that the mechanism of cleavage of nevi eggs is the same in both the animal and vegetal halves and in both outer and inner parts of the eggs: namely, a contractile force generated at the furrow base causes cytokinesis and the position of the future furrow base depends on the underlying arrangement of cytoplasm.
It is clear that there are similarities and dissimilarities in the cleavage mechanisms of amphibian eggs and of smaller cells, such as sea-urchin eggs. In all these eggs cleavage is due to contraction, but the time when the egg surface becomes independent of the subsurface organization differs. This occurs much earlier in sea-urchin eggs (Rappaport, 1971) than in amphibian eggs. This difference is probably due chiefly to the difference in absolute size of the eggs and in the relative sizes and positions of the mitotic apparatus. Further experiments are necessary on the interactions of the mitotic apparatus, the cytoplasmic arrangement, and the egg equatorial surface.
I am grateful to Dr M. Samezima for his expert assistance in the preparation for electron microscopy.