Analysis of the formation of the animal pole plasm in the eggs of Limnaea stagnalis

The animal pole plasm forms a layer of protoplasm immediately beneath the egg cortex in the animal hemisphere, staining dark violet blue with iron hematoxylin. Contrary to the rest of the cytoplasm, it contains no vacuoles. It is, however, very rich in mitochondria (Raven, 1945) (Plate 1, A).

In eggs centrifuged before the first maturation division, an animal pole plasm may be formed at the normal time and in its normal location, irrespective of the stratification of substances brought about by centrifuging. This was explained by assuming that its formation is due to an attraction exerted by a particular region of the egg cortex upon certain cytoplasmic components (Raven, 1945).

These experiments were repeated and extended by Raven & Brunnekreeft (1951), who studied in what manner the formation of the animal pole plasm in centrifuged eggs depends on the moment of centrifuging and on the resulting differences of stratification. These differences are mainly due to the fact that the γ-granules of the proteid yolk during the uncleaved stage become surrounded by vacuoles which gradually increase in size. At centrifugation a granule and its vacuole are not separated but move as a whole (Andrew, 1959), either centrifugally or centripetally, depending on the size of the vacuole which influences the specific gravity of the whole structure. Therefore, in eggs centrifuged immediately after laying, the cytoplasm shows a stratification into four zones : (1) centripetal fat zone, (2) hyaloplasm, (3) α-granules (mitochondria), and (4) centrifugal proteid yolk zone, consisting of β- and γ-granules. The boundaries between the zones are rather sharp (Raven & Bretschneider, 1942). In eggs centrifuged a short time before cleavage, on the contrary, nearly all y-granules have become surrounded by vacuoles, and consequently are accumulated in the centripetal half. The separation between zones 1 and 2 has become indistinct so that the egg contents now show a stratification into three layers: (1) a centripetal zone of fat and frothy alveolar protoplasm, with y-granules in the vacuolar spaces, (2) a layer of mitochondria, (3) a centrifugal dense yolk mass, mainly consisting of β-granules (Raven, 1946b). Eggs centrifuged in the middle part of the uncleaved stage show a condition intermediate between these two.

The egg substances displaced by centrifuging do not remain in their positions but are redistributed about the egg after centrifugation, tending to a restoration of their normal arrangement (Raven & Bretschneider, 1942). Therefore the structure of the eggs at a certain moment depends both on the kind of stratification produced by centrifuging, and on the amount of rearrangement which has taken place afterwards.

Raven & Brunnekreeft (1951) found that a distinct animal pole plasm had been formed in eggs centrifuged shortly after oviposition or immediately after the extrusion of the first polar body, and fixed hr. after the formation of the second polar body. But in eggs centrifuged some time after the extrusion of the second polar body and fixed at the same time as the former ones, no animal pole plasm was ever found. This was explained by assuming that in the latter eggs the time interval between centrifuging and fixation was too short for the necessary rearrangement of egg substances and segregation of pole plasm substance to take place. Moreover, the possibility was taken into account that the frothy structure of the centripetal part of eggs centrifuged during the later part of the uncleaved stage might have inhibited or retarded the processes of ooplasmic segregation.

The conclusion drawn from previous experiments (Raven, 1945), that the formation of the animal pole plasm is due to attractive actions exerted by the egg cortex in the neighbourhood of the animal pole upon certain components of the cytoplasm, was corroborated by these results.

In eggs centrifuged during the first part of the uncleaved stage, Raven & Brunnekreeft (1951) observed that, in addition to the animal pole plasm, a subcortical accumulation of deeply stained protoplasm, showing some resemblance to the animal pole plasm, might be found in other regions of the centripetal half of the egg (for the sake of convenience we will call this a ‘spurious pole plasm’). Two possible explanations of this fact were considered. The attractive forces exerted by the egg cortex, even in normal eggs, might not be entirely restricted to the region of the animal pole, but in a weaker degree might also be present in other parts of the cortex and exert their influence under favourable conditions, so giving rise to a spurious pole plasm. Alternatively, it might be supposed that the special properties of the egg cortex near the animal pole, by virtue of which it attracts the animal pole plasm, arise only secondarily by an interaction of the cortex with the underlying plastin-rich cytoplasm of the polar area in normal eggs. In that case, the cortex overlying the hyaloplasm zone of centrifuged eggs might, by a similar interaction, get similar properties, and therefore attract comparable components of the cytoplasm. No decision between the two alternative explanations could be made at that time.

The present experiments were intended to test the last-mentioned hypothesis. If the parts of the cortex overlying the hyaloplasm zone of centrifuged eggs, by an interaction with the plastin-rich hyaloplasm, acquire properties resembling those of the animal pole region, it is to be expected that the effect will be the more pronounced the longer the contact between the two has lasted. To this end, one can try to prevent the rearrangement of the egg substances after they have been separated by centrifugation. This can be done by prolonging the time of centrifugation, but at the same time this may lead to a sharper separation of the components of the cytoplasm. In order to prevent this the eggs were treated in the following way : first the cytoplasm was stratified by a short treatment with strong centrifugal force; then they were kept for a long time in a stratified condition by weak centrifugal force, insufficient to stratify the eggs but strong enough to prevent the rearrangement of substances once stratified. These eggs were then compared with three groups of controls in order to be able to tell apart the effects of the total centrifugation time, the sequence of weak and strong centrifugation, the moment of stratification and the duration of the stratified condition.

Though the experiments did not produce quite the expected results, they have given a valuable contribution to our understanding of the formation of the animal pole plasm.

In a preliminary series of experiments the right rates and times of centrifugation were tried out. It appeared that 5 min. centrifugation at 3500 rev./min. (1360 g.) produced a sharp stratification of the egg contents. On the other hand, 700 rev./min. (55 g.) gave only a slight displacement of the egg substances, but proved sufficient to maintain a sharp stratification for 2 hr. in eggs previously stratified by stronger centrifugation.

Eight batches belonging to this group have been studied. The stage of development reached at the time of fixation is very different; it varies from early prophase of first cleavage to a late 2-cell stage (st. 8–9, Raven, 1946a). This is probably due to two causes. In the first place, the egg masses are not always laid at the same age. The time between oviposition and extrusion of the first polar body has been observed to vary in different batches from 120 to 25 min. at room temperature (Raven, 1945). Secondly, the rate of development may have varied among different batches, either by slight fluctuations in room temperature or by inherent differences between the egg masses. In the batch which had developed farthest, several of the eggs had giant first-polar bodies. This suggests that it had been centrifuged at or just before the time of extrusion of the first-polar body, therefore centrifugation of the batches probably took place from about 100 to 0 min. before the first maturation division.

The degree of stratification of the eggs also shows a great deal of variation. In some batches the boundaries between the layers of substances separated by centrifuging are still clearly visible. In others, only traces of the original stratification have been left. The latter especially applies to those batches where cleavage had taken place at the time of fixation, whereas in uncleaved eggs the stratification generally had been better preserved. Apparently, the changes in shape of the egg during cell division greatly further the redispersion of the egg substances accumulated during centrifugation.

In the eggs still showing a good stratification (Plate 1, B, C), one often finds a group of big empty vacuoles at the centripetal end, where the fat has been dissolved away during preparation of the sections. Then follows a zone of hyaloplasm, having a rather dense structure and often being somewhat fibrillar in appearance. The mitochondria of the following zone are sometimes widely dispersed, but in many eggs this zone can still be clearly recognized. The proteid yolk occupies the centrifugal half of the egg. It consists of smaller β- and bigger γ-granules. The latter, at the time of centrifugation, were not yet surrounded by their vacuoles and so were accumulated in the heavy yolk region. Subsequently the vacuoles have been formed, so that they are now each surrounded by a clear vacuolar space (Raven & Bretschneider, 1942; Raven, 1945). The yolk region as a whole, therefore, has a spongy, vacuolar appearance, which becomes more pronounced with the age of the eggs (compare Plate 1, B & C with D).

The axis of stratification may make various angles with the egg axis, ranging from 0° to about 135°. Some orientation of the eggs in the centrifuge has taken place, however, as in most cases the centripetal end of the axis of stratification is situated in the animal half of the egg.

In all eggs a distinct animal pole plasm is visible. Both in its structure and in its location it agrees with the animal pole plasm found in normal eggs. It forms a rather narrow layer of deeply-staining protoplasm immediately beneath the egg cortex at the animal side of the egg. In a normal egg this pole plasm is very rich in mitochondria. In some of these centrifuged eggs, however, the animal pole plasm, or some part of it, contains only few mitochondria, although in other respects it looks quite normal. This depends on the position of the layers of the stratified egg with respect to the egg axis. If the zone of mitochondria passes through the animal pole, the animal pole plasm contains many mitochondria, though often somewhat less than in normal eggs (Plate 1, B). If it passes at some distance to one side of the animal pole, the pole plasm on that side is much richer in mitochondria than on the other side (Plate 1, C & D). Finally, if the zone of mitochondria is far removed from the animal pole (as is the case, for instance, when the egg axis and the axis of centrifugation coincide), then the animal pole plasm contains only very little mitochondria. It is clear, therefore, that we must distinguish the ground substance (‘matrix’) of the animal pole plasm from the dense accumulation of mitochondria normally coinciding with it.

As in Raven & Brunnekreeft’s (1951) eggs treated in the same way, a ‘spurious pole plasm’ is clearly visible in all eggs with distinct stratification (but not in those in which the latter has for the most part disappeared). A comparison of its location and structure in different eggs permits the following statement : a ‘spurious pole plasm’ develops at those places where the mitochondria zone of the centrifuged egg reaches the egg surface. Here the mitochondria become heaped up in dense layers beneath the cortex, at the same time fanning out in streams towards both sides (Plate 1, B, C, D). This dense accumulation of mitochondria produces the spurious resemblance to the animal pole plasm of normal eggs. It is evident, however, that the ‘matrix’ of the animal pole plasm has no counterpart in these places. The resemblance between the two is therefore of a superficial nature only.

In the original design of the experiments this was the main treatment group, the other three groups serving as controls. Its purpose was to maintain the stratified condition of the eggs for a longer time, by preventing the readjustment of the layers, in order to study possible interactions of the cortex with the underlying protoplasm.

As in group A, the stage of development reached at the time of fixation varies a great deal. It is evident, however, that the eggs of this treatment group are not so advanced in their development as those of group A belonging to the same egg masses and fixed at the same moment. It can be estimated that, on average, they have lagged behind by about hr.

The stratification of the eggs is generally somewhat more distinct than in group A. This is understandable due to the fact that the redistribution of egg substances has presumably started 2 hr. later in these eggs. The fact that the eggs are not so advanced in their development, and therefore fewer of them have cleaved, may have acted in the same direction. Taking this into account, the difference in degree of stratification between the eggs of groups A and B is even less pronounced than one might have expected. There is therefore no reason to think that the period of slow centrifugation has added to the stratification of substances in any significant degree.

The sequence and composition of the layers is the same as in group A. Specifically, the γ-granules with their vacuoles are also found in the proteid yolk zone in these eggs.

With regard to the animal pole plasm and the ‘spurious pole plasm’, the same observations can be made as in the eggs of group A. The relationships are often even somewhat clearer in this group. For instance, in the egg of Plate 1, E, which is in prometaphase of first cleavage, the egg axis and stratification axis make an angle of about 50°. The original zone of mitochondria passes as a dark band obliquely through the egg. Part of the mitochondria have accumulated around the cleavage spindle. At the animal side there is a well-developed animal pole plasm. Its left half, which coincides with the zone of mitochondria and partly extends into the yolk zone, is very rich in mitochondria. Its right half, on the contrary, which lies in the hyaloplasm and fat zones, contains only few mitochondria. Where the band of mitochondria reaches the egg surface in the vegetative half of the egg, there is a dense accumulation of mitochondria immediately beneath the cortex, but it is only thin in this case and shows little resemblance to a pole plasm.

The egg of Plate 1, F, is likewise in pro-metaphase. One of the asters is visible, and in this case the axis of centrifugation has coincided with the egg axis. The stratification has become very unclear, however, though traces of it are still visible. The animal side of the egg is covered by the animal pole plasm, which exhibits its normal shape and structure but is rather poor in mitochondria. At the two extremities of the crescent-shaped animal pole plasm, however, a little above the egg equator, there are two extremely dense subcortical accumulations of mitochondria. They cause slight bulges of the egg surface. With regard to mitochondrial density they resemble or even exceed a normal animal pole plasm, but they lack ‘matrix’ and therefore represent a ‘spurious pole plasm’. From their position it is evident that they correspond to the original mitochondria zone of the stratified egg.

Plate 2, A, represents a similar egg at a slightly later stage (stage 1, beginning of first cleavage). One of the groups of karyomeres is visible. The stratification has nearly disappeared, but it is evident that in this case the axis of centrifugation and the egg axis have also coincided. The middle part of the animal pole plasm, on either side of the cleavage furrow, is markedly poor in mitochondria. Most mitochondria are heaped up in the supra-equatorial region in dense layers beneath the cortex, forming a ‘spurious pole plasm’ on either side.

This treatment group forms a control to group B. The total rate of centrifugation is the same, but, while the eggs of group B were kept for 2 hr. in a strongly stratified condition, those of group C were at most weakly stratified during this time.

Like the eggs of group B, those of this treatment group are behind in their development to comparable eggs of group A, but they appear to be slightly farther advanced than those of group B, the average difference between the two groups amounting to about 10 min. It is not certain that this difference is significant.

With regard to stratification, these eggs show a marked difference to those of the previous groups. The γ -granules with their vacuoles, which in the latter groups were found in the centrifugal yolk zone only, lie both in centrifugal and centripetal egg regions in most of the eggs of group C. Those in centripetal regions, especially, have very big vacuoles, while the γ -granules in the yolk zone are mostly surrounded by smaller vacuoles. Depending on the number of γ -vacuoles accumulated in the centripetal half of the egg, the latter has got a more-or-less frothy structure. Although in some eggs a few big fat-vacuoles are found at the centripetal end, generally the boundary between layers 1 and 2 has become rather unclear. Furthermore, in most eggs the zone of mitochondria is very diffuse.

The frothy centripetal protoplasm as a rule extends over at least part of the animal pole region. Where this is the case, the animal pole plasm, though certainly present, exhibits an abnormal structure. It is very thin, often it is no more than a third or a quarter of its normal thickness; its inner boundary appears more or less notched, as it is connected with the meshes between the vacuoles beneath it; and, as a rule, it is very poor in mitochondria in these regions, consisting of ‘matrix’ only (Plate 2, B-D).

In view of the unclear delimitation of the zone of mitochondria, it is hardly surprising that a clear ‘spurious pole plasm’ is lacking in most eggs. In some uncleaved eggs a somewhat denser accumulation of mitochondria near the vegetative egg surface in the former mitochondria zone is still clearly visible (Plate 2, B); in more advanced eggs only traces of it remain (Plate 2, C, D).

This group agrees with group A in being centrifuged for 5 min. with high speed only, but differs from it with respect to the stage of the eggs at the time of centrifugation. It differs from group C in that high-speed centrifugation was not preceded by a period of slow centrifugation.

As in the other groups, the stage of development reached at the time of fixation varies a great deal; it ranges from prophase of first cleavage to a medium 4-cell stage. On an average, the eggs in this group are the most advanced in their development. They are even an estimated 20 – 25 min. ahead of those of group A.

In one batch some giant second polar bodies had been formed, suggesting that it had been centrifuged at or just before the second maturation division. Starting from this datum, it could be calculated that the batches of this treatment group were centrifuged from about 40 min. before to 60 min. after the second maturation division, which is in agreement with the conclusion reached above with respect to treatment group A.

The γ-granules, which in the eggs of groups A and B were found exclusively in the centrifugal yolk zone and in those of group C both centripetally and centrifugally, in the eggs of the present group have been accumulated mainly or exclusively in the centripetal egg region. The fat and hyaloplasm zones can no longer be distinguished. In most eggs the zone of mitochondria is hardly visible. As a rule, therefore, only two regions can be distinguished in these eggs: a centripetal mass of frothy, highly vacuolar protoplasm, with γ-granules in the vacuoles; and a centrifugal zone, consisting either of a tightly packed mass of β -granules only (Plate 2, E) or containing in addition some of the γ-granules with small vacuoles around them (Plate 2, F).

Apparently the orientation of the eggs in the centrifuge has been somewhat better than in group A. Therefore in most cases the frothy centripetal protoplasm extends over the animal pole region. Where this is the case, an animal pole plasm is lacking altogether (Plate 2, E, F).

In a few eggs there are traces of a subcortical accumulation of mitochondria in the former mitochondria zone (Plate 2, F), but in most eggs no such thing could be observed.

In some eggs at the 2-cell stage, in which the egg substances were distributed quite unequally, one cell containing most of the β-yolk while the other consisted mostly of frothy centripetal cytoplasm, it was observed that the nuclei were at different stages of their cycle, the nucleus surrounded by β-yolk always being ahead of the other one (e.g., anaphase over against metaphase, or prometaphase over against interphase). Such large differences between the sister nuclei have never been observed in normal eggs of this stage nor in centrifuged eggs with more equal distribution of the substances among the cells. This suggests that the developmental rate of the nuclei is dependent on the surrounding cytoplasm, being more rapid in an environment of β-yolk than in the alveolar centripetal cytoplasm.

1. These experiments were designed to test an hypothesis put forward by Raven & Brunnekreeft (1951) in order to explain the formation of a ‘spurious pole plasm’ in Limnaea eggs centrifuged in the first part of the uncleaved stage. The possibility was considered that the special properties of the egg cortex near the animal pole, by virtue of which it attracts the components of the animal pole plasm, arise by an interaction of the cortex with the underlying cytoplasm surrounding the maturation amphiasters. In centrifuged eggs the cortex overlying the hyaloplasm zone might get similar properties, and so give rise to the formation of a ‘spurious pole plasm’. By prolonging the period of contact between these parts of the cortex and the hyaloplasm, it might be possible to get a stronger effect.

   The results of this investigation have not confirmed the hypothesis. To be sure, on an average a ‘spurious pole plasm’ can somewhat more often and somewhat more distinctly be observed in the eggs of group B than in the corresponding lots of treatment groups A and C, which is in accordance with expectation. But our further observations on these eggs have convinced us that a ‘spurious pole plasm’ has only a superficial resemblance to a true animal pole plasm, and that its formation must be explained in another way.

2. The centrifuged eggs of the four treatment groups show great differences in the nature of the stratification. This is mainly due to the different behaviour of the γ-granules at centrifugation. When the eggs are centrifuged shortly after oviposition (group A), the formation of vacuoles around the γ-granules has hardly begun, therefore these heavy granules are thrown centrifugally and accumulated in the yolk zone. In the 4 hr. following centrifugation vacuoles form around them, so that the yolk zone as a whole becomes more or less spongy, in contrast to the zone of hyaloplasm, which looks rather dense in the sections (Plate 1, B, D).

   When centrifugation takes place 2 hr. later (group D), vacuoles have formed around most of the y-granules. At centrifugation these granules with their vacuoles are thrown mainly or completely to the centripetal side. By their accumulation in the centripetal part of the egg, a frothy mass is formed consisting of γ-vacuoles and fat vacuoles, separated by meshes of protoplasm, which occupies nearly half of the egg volume, and in which the distinction between a fat zone and hyaloplasm zone is no longer possible. The other half of the egg is occupied by the yolk zone, mainly consisting of tightly-packed β-granules. In some eggs it still contains some y-granules that have also become surrounded by vacuoles in the 2 hr. following centrifugation (Plate 2, F); in other eggs all y-granules had been thrown centripetally, and the yolk zone consists nearly entirely of β-granules (Plate 2, E).

   As a result of these displacements of the γ-granules, the appearance of the centrifuged eggs of groups A and D is very different. The apparent density in the sections of the centripetal and centrifugal egg regions is almost entirely reversed (compare Plate 2, E, with Plate 1, B; Plate 2, F, with Plate 1, D).

   It is interesting to compare the behaviour of the y-granules in the other two treatment groups. The structure of the eggs in group B generally corresponds to that of group A: the γ-granules lie mainly in the yolk zone (Plate 1, E; Plate 2, A). This means that once the γ-granules have been accumulated in the centrifugal egg region by short high-speed centrifugation, 2 hr. of low-speed centrifugation do not suffice to displace them toward the centripetal half, even though we may expect that in the meantime vacuoles have formed around them.

   In the eggs of group C the γ-granules are more or less evenly distributed among the centripetal and centrifugal egg regions, in contrast to group D where most of them lie in the centripetal part. We have assumed that the definitive stratification of the egg contents in both groups took place simultaneously during the 5 min. high-speed centrifugation which they underwent together (at time 125–130 min.). The difference between groups C and D in the distribution of γ-granules might be due to a different rate of swelling of the granules in the two groups. The preceding 2 hr. of low-speed centrifugation in group C, besides retarding development (cf. below p. 102), may also slow down the swelling of the y-granules. An alternative explanation would be that this low-speed centrifugation actually causes a weak stratification of the egg substances, and the y-granules, once displaced toward the centrifugal half, are somewhat hindered in their centripetal movement during the subsequent period of high-speed centrifugation.

3. There are further differences in the appearance of the eggs with respect to the zone of mitochondria. In the eggs of group A, this zone, as a rule, is still clearly visible (Plate 1, B-D), even though 4 hr. have elapsed since the end of centrifugation. In group B it is, on average, even clearer and more sharply delimited (Plate 1, E), but in group C, on the contrary, the zone of mitochondria is very diffuse (Plate 2, B) and has often become invisible altogether, especially in older eggs (Plate 2, D). Finally, in most eggs of group D a distinct mitochondria zone is lacking altogether.

   This difference between groups A and B on the one hand, and C and D on the other, might be due to two different causes. First, one might think that the density relationships of the egg components are different at different times. Before the first maturation division, when the eggs of groups A and B become stratified, they may be such that a clear density-dependent separation of the layer of mitochondria occurs. Two hours later, when the final stratification is brought about in groups C and D, this may be otherwise. For instance, the specific weight of the mitochondria themselves may have changed, or may have become so variable that they are no longer accumulated in a special zone. Another possibility is that the extensive displacements of γ-granules with their big vacuoles during centrifugation causes such turbulence that the sedimentation of the mitochondria is disturbed.

   Secondly, the more diffuse distribution of mitochondria in groups C and D might be due to their more rapid dispersal in the 2 hr. following centrifugation. The stratification of egg substances brought about in these eggs, in contrast to that of groups A and B, might facilitate this dispersal of the mitochondria. Though at first sight this possibility appears more remote than the first-mentioned one, there are some empirical results pleading in its favour. In previous experiments in which the eggs were centrifuged some time after the extrusion of the second polar body, or shortly before cleavage, and fixed immediately afterwards, it has been shown that a distinct accumulation of the mitochondria in a narrow zone had taken place (cf. Raven & Bretschneider, 1942, pl. IV; Raven, 1946b, pl. X). Furthermore, in the last-mentioned eggs it was found that the mitochondria (α-granules) had already begun to disperse 35 min. after centrifugation, while the mitochondria zone had disappeared altogether at 100 min. after centrifugation.

4. This investigation has thrown further light on the nature of the ‘subcortical accumulation of dense protoplasm, resembling the animal pole plasm ‘, which was observed by Raven & Brunnekreeft (1951). The following observations were made: (1) This ‘spurious pole plasm’, as it is called in this paper, does not correspond in its localization with zones 2 and 3 (hyaloplasm and mitochondria) of the recently-centrifuged egg, as Raven & Brunnekreeft supposed, but more specifically with the mitochondria zone. As a matter of fact, it tends to extend fan-wise beneath neighbouring parts of the cortex, but rather toward the yolk than toward the hyaloplasm zone (Plate 1, B, C, D). (2) It is only well-developed in those eggs in which the mitochondria zone is still distinctly visible. (3) It consists of a dense accumulation of mitochondria only; in this respect it differs from the animal pole plasm, in which the mitochondria are embedded in a special ground substance (matrix). (4) In this marginal region of the former mitochondria zone the mitochondria are more tightly packed than in its central region (Plate 1, C, D), and especially so immediately beneath the cortex, where several layers of mitochondria are closely applied to the cell membrane.

   We may conclude from these observations that the ‘spurious pole plasm’ has only a superficial resemblance to the animal pole plasm of normal eggs. Presumably its formation is due to an attraction exerted by the cortex upon the mitochondria. As a ‘spurious pole plasm’ may be found in animal, equatorial and vegetative regions of the egg, apparently all parts of the cortex are alike in this respect. The mitochondria tend to accumulate closely beneath the cortex at all places where their density is sufficiently high. In centrifuged eggs, this is especially so in the mitochondria zone.

5. With respect to the animal pole plasm these experiments have enabled us to distinguish between two of its components which are inseparable in normal development : the matrix, on the one hand, and the mitochondria, on the other. In eggs centrifuged during the first part of the uncleaved stage (groups A and B), a distinct animal pole plasm is always formed in its normal location around the animal pole (Plate 1, B-F, Plate 2, A). But, while this pole plasm is characterized by a dense accumulation of mitochondria in the normal egg (Plate 1, A), in centrifuged eggs some parts of it may be nearly devoid of mitochondria and consist of matrix only (Plate 1, C, D, E; Plate 2, A). Apparently it is the matrix which forms the substance proper of the animal pole plasm. It is only for this component that the conclusion reached in previous papers (Raven, 1945; Raven & Brunnekreeft, 1951) can be upheld, viz. that its formation is due to specific attractive actions, exerted only by the egg cortex in the neighbourhood of the animal pole, upon certain substances of the inner cytoplasm. The mitochondria, on the other hand, appear to be attracted in an unspecific way by all parts of the cortex, as we have seen above. Their accumulation in the animal pole plasm during normal development is therefore, in a way, incidental (which does not mean, of course, that it is without importance for further development).

6. Observations on the course of the maturation divisions have shown in what manner the accumulation of mitochondria in the animal pole plasm of normal eggs of Limnaea stagnalis (and, even more clearly, in those of the related species Limnaea ovata and Myxas glutinosa) takes place. The first maturation spindle and its asters are already surrounded by a dense cloud of mitochondria, which partly penetrate in rows between the astral rays (Raven, 1945). Apparently, mitochondria are attracted towards the spindle from a great part of the egg, perhaps by centripetally directed currents between the astral rays, such as described by Chambers (1917) and others. After the extrusion of the first polar body these mitochondria lie near the animal pole, surrounding the centrosphere remaining in the egg, and the second maturation spindle developing from it. The sperm aster emerging from the central part of the egg, and approaching the second maturation spindle, eventually fusing with its inner end (cf. Raven, Escher, Herrebout & Leussink, 1958), is responsible for a new supply of mitochondria. Even when it is still rather small it has gathered a dense cloud of mitochondria around it, which it takes along toward the animal side. In this way the vegetative part of the egg becomes virtually deprived of its mitochondria, which are all transported toward the animal pole region. When the second polar body has been extruded and the remnant of the maturation spindle gradually disappears, there remains a dense cloud of mitochondria in the animal pole region which now concentrates in layers beneath the cortex in the animal pole plasm (Plate 1, A).

   It follows that the transport of mitochondria toward the animal pole region in the normal development takes place mainly indirectly by way of the maturation spindles and their asters. Once accumulated in this region, and set free by the disappearance of the spindle apparatus, they are at liberty to collect beneath the cortex. A specific attraction of the animal pole cortex upon the mitochondria, in excess of the general attraction exerted by all parts of the cortex, need not be assumed.

7. We must now consider the question of why no animal pole plasm is found in eggs centrifuged at or after the time of the second maturation division and fixed 2 hr. later. Raven & Brunnekreeft (1951), who made the same observation, consider two possible explanations. Either the structure of the eggs centrifuged at later phases offers some resistance to the displacements of cytoplasmic substances playing a part in the formation of the animal pole plasm, or the time interval between centrifuging and fixation has been too short in these experiments for the accumulation of pole plasm substance to take place. Raven & Brunnekreeft, after considering the evidence, tend to adopt the latter view. It must be noted that they do not distinguish between the matrix of the animal pole plasm and the mitochondria.

   The present experiments have yielded some further data relating to this question. If it is true, as it has been presumed, that the slow-speed centrifugation applied in these experiments, though insufficient to produce a sharp stratification of the egg contents, prevents the redistribution of substances once stratified, then the processes of oöplasmic segregation leading to the formation of an animal pole plasm could only begin in the eggs of group B at 2 hr. before the moment of fixation, i.e. practically simultaneously with those of groups C and D. Nevertheless, the eggs of group B have formed a clear-cut animal pole plasm (Plate 1, E, F; Plate 2, A), while those of group D have none at all (Plate 2, E, F). Furthermore, if we compare groups C and D, the eggs of C have pole plasms which, though more or less rudimentary, can still be clearly recognized (Plate 2, B, C, D), whereas in the eggs of group D practically all traces of it are lacking (Plate 2, E, F). Still, the time between centrifugation and fixation has been equal in both groups. They only differ more or less in the distribution of the y-granules, so that the frothy structure of the centripetal egg region is more pronounced in the eggs of group D than in those of group C.

   We must come to the conclusion, therefore, that, contrary to the view expressed by Raven & Brunnekreeft (1951), it is not the time interval between centrifugation and fixation which decides whether an animal pole plasm will be formed or not, but rather the structure of the centrifuged eggs. Apparently the frothy structure of the centripetal cytoplasm in eggs centrifuged late, when it extends over the animal pole, inhibits or retards the processes of oöplasmic segregation to such an extent that the formation of an animal pole plasm within 2 hr. becomes impossible. This is the more remarkable since we have seen above that the redistribution of the mitochondria seems to be facilitated in such eggs. The two components of the animal pole plasm (matrix and mitochondria), in this respect, seem to behave differently.

8. Finally, the experiments have produced some results which need further confirmation. The first of these concerns the differences in stage of development reached in the four treatment groups. Though the eggs from each egg mass were distributed in about equal numbers among the groups, and all were fixed at about the same time, there was a distinct stage difference between the eggs of comparable lots, as a rule those of group D being the most advanced while the eggs of groups A, C and B, in that sequence, were progressively more retarded. This seems to show (1) That 5 min. high-speed centrifugation prior to the first maturation division retards development compared with the same treatment applied 2 hr. later. (2) Two hours’ low-speed centrifugation causes a considerable additional retardation of development. (3) This is more pronounced when the eggs are strongly stratified during this time; apparently the delay is not only caused by centrifugation as such, but the degree of stratification of the eggs is important, too.

   Since these conclusions are drawn from a comparison between restricted numbers of sectioned eggs they have a provisional character only. They should be verified by a quantitative study of a sufficient number of centrifuged eggs.

   The second observation that may be mentioned in this connexion concerns the difference in stage of development of sister nuclei in cleaved centrifuged eggs, according to whether they are surrounded by β-yolk or frothy centripetal cytoplasm rich in fat and y-granules. This has been observed in a few eggs only, and cannot be considered as an established fact. It might be worth while to pursue this matter further in future experiments.

1. Eggs of Limnaea stagnalis were centrifuged with various combinations of high speed (3500 rev./min.) and low speed (700 rev./min.) during the first 2 hr. of the uncleaved stage, and fixed 4 hr. after the beginning of the experiment.

2. The eggs of different treatment groups show great differences in the nature of the stratification. This is mainly due to the behaviour of the y-granules, which move centrifugally in eggs centrifuged shortly after oviposition, and centripetally in eggs centrifuged 2 hr. later.

3. Moreover, the mitochondria zone is still clearly visible at the time of fixation in earlier centrifuged eggs, while it is very diffuse or lacking altogether in later centrifuged seggs. This seems to be due to the fact that the dispersal of the mitochondria after centrifuging occurs more rapidly in the latter eggs.

4. In eggs in which the mitochondria zone is still present, the mitochondria tend to accumulate in dense layers beneath the cortex. Apparently, all parts of the cortex exert an attraction upon the mitochondria.

5. In all early centrifuged eggs a well-developed animal pole plasm has been formed. Contrary to normal eggs, it is not always rich in mitochondria but consists mainly of ‘matrix’. Its formation points to the existence of specific attractive actions, exerted by the cortex in the neighbourhood of the animal pole upon certain components of the inner cytoplasm.

6. In normal development most mitochondria of the egg are conveyed toward the animal pole by way of the maturation spindles and asters. After the extrusion of the second polar body and the breakdown of the spindle and aster remnants, they then accumulate beneath the cortex in the area of the animal pole plasm.

7. In the eggs centrifuged 2 hr. after oviposition, no animal pole plasm has been formed at the time of fixation. This is apparently due to the fact that, as a rule, the cytoplasm in the animal pole region has a frothy structure in such eggs; this inhibits or retards the processes of ooplasmic segregation.

8. Differences in developmental rate between eggs centrifuged in a different way, and between nuclei surrounded by different kinds of cytoplasm, are discussed.

1. On centrifuge des oeufs de Limnaea stagnalis en les soumettant à différentes combinaisons de grandes vitesses (3500 tours par minute) et de vitesses réduites (700 tours par minute) pendant les 2 premières heures du stade indivis. On les fixe 4 heures après le début de l’expériencé.

2. Les oeufs des groupes soumis à des traitements différents montrent de grandes différences dans les modalités de stratification. Cela est dû principalement au comportement des granules γ, qui se déplacent en direction centrifuge dans des oeufs centrifugés peu après l’oviposition, et en direction centripète dans des oeufs centrifugés 2 heures plus tard.

3. De plus, la zone des mitochondries est encore bien visible au moment de la fixation dans les oeufs centrifugés aux plus jeunes stades, tandis qu’elle est très diffuse ou manque complètement si la centrifugation est plus tardive. Ce résultat semble dû au fait que la dispersion des mitochondries après centrifugation se produit plus rapidement dans ces derniers oeufs.

4. Dans les oeufs où la zone des mitochondries est encore présente, les mitochondries tendent à s’accumuler en couches denses au-dessous du cortex. Il semble que toutes les régions du cortex exercent une attraction sur les mitochondries.

5. Dans tous les oeufs centrifugés précocement, un plasme polaire bien développé s’est formé au pôle animal. Contrairement aux oeufs normaux, il n’est pas toujours riche en mitochondries, mais il consiste principalement en une trame (‘matrix’). Cette formation suggère l’existence d’attractions spécifiques qu’exercerait le cortex au voisinage du pôle animal sur certains composants du cytoplasme interne.

6. Dans le développement normal, la plupart des mitochondries de l’oeuf sont déplacées vers le pôle animal par les fuseaux de maturation et les asters. Après l’expulsion du deuxième globule polaire et la disparition du fuseau et des résidus astériens, elles s’accumulent sous le cortex dans la région du plasme polaire animal.

7. Dans les oeufs centrifugés 2 heures après la ponte, aucun plasme polaire animal ne s’est formé au moment de la fixation. Ceci semble dû au fait que le cytoplasme du pôle animal a généralement une structure écumeuse dans de tels oeufs, ce qui inhibe ou retarde les processus de la ségrégation oôplasmique.

8. Les différences de vitesse de développement entre les oeufs centrifugés de différentes manières et entre les noyaux entourés par différents types de cytoplasme, sont l’object de la discussion.

All photomicrographs are × 540.

Abbreviations : A.P.P., animal pole plasm; C.P., frothy centripetal protoplasm; CL.SP., cleavage spindle;H., hyaloplasm zone;K.,karyomeres;M., mitochondria zone; 1 P.B., 2P.B., firstand second polar body; PR., pronuclei; SP.N., sperm nucleus; S.P.P., ‘spurious pole plasm’; Y., yolk zone.