Distribution of visible materials in the egg and in the embryo during cleavage is, however, not always of crucial importance for the localization of parts in the developing embryo. Some substances and cellular inclusions may be displaced without disturbing the normal segregation of the embryo into its subordinate parts.

This displacement can most conveniently be accomplished by centrifugation of the un-cleaved eggs. Centrifuging the eggs of most animals for a few minutes with moderate speeds, at an acceleration of several thousand times gravity, is sufficient to rearrange various cellular inclusions in the interior of the egg according to their specific gravities.

After sufficiently strong centrifugation, the eggs become stratified and show at least three typical layers. At the centripetal pole there is usually an accumulation of fat or lipid droplets, which are the lightest constituents of the egg cytoplasm.

A layer of hyaline cytoplasm, which is the ground substance of the egg, follows. The nucleus or asters with chromosomes (if the centrifuged egg was in meiosis or mitosis) are also found in the hyaline layer. The yolk, as the most dense and heavy constituent of the egg, accumulates at the centrifugal pole.


In the eggs of some animals the vegetal pole is so much heavier, owing to the presence of yolk, that it becomes oriented centrifugally during centrifugation. In this case the yolk is not displaced from its normal site at the vegetal pole but is only more concentrated. To displace the yolk in these cases the eggs have to be fixed in a desired position, so that they cannot freely rotate. This can sometimes be done by sucking them into narrow capillaries or by embedding the eggs in gelatin prior to centrifugation.

If the main axis of the embryo lies at random to the centrifugal force, as often happens, cellular inclusions will be dislocated to different parts in individual eggs. The results of centrifuging eggs of the sea urchin Arbacia. The red pigment granules present in these eggs are concentrated at the centrifugal end of the egg. Some scattering of the granules occurs after the centrifuging is stopped and before cleavage progresses sufficiently to prevent further redistribution of the granules by subdividing the egg into blastomeres.

It now becomes evident that the granules are concentrated in different positions in respect to the egg axis – near the vegetal pole, near the animal pole, or toward one side. Independently of the position of the granules, the invagination of the blastopore occurs at the vegetal pole, so that the region of the blastopore will contain the pigment granules in some embryos but not in others.

It has often been found that the pattern of cleavage may be highly independent of the distribution of cytoplasmic substances inside the egg. The cleavage of the mollusc Dentalium, in which a polar lobe appears at the vegetal pole during the first and second divisions of the egg and contains the cytoplasm necessary for the development of the mesoderm in the larva.


A similar polar lobe is observed during cleavage of another mollusc, Ilyanassa. The polar lobe in this species is normally filled with yolky cytoplasm, while at the animal pole the egg cytoplasm is fairly free from yolk. Eggs of Ilyanassa have been centrifuged “in reverse,” that is, with the vegetal pole fixed in position, facing the axis of the centrifuge. As a result, the heavy yolk is thrown into the animal hemisphere (still marked by the position of the polar bodies), and the hyaline cytoplasm and lipid droplets are concentrated at the vegetal pole.

Nevertheless, the polar lobe appears at the vegetal pole when the egg starts cleaving, although the lobe now contains mainly hyaline cytoplasm and lipid instead of the yolk granules. Obviously, the formation of the polar lobe is not dependent on the yolk normally located at the vegetal pole, but on something that is not displaced by the centrifugal force.

What can this something be? There are two possibilities. The first is that in the cytoplasm some fixed network exists with sufficiently broad meshes to allow for the free movement of yolk granules and other inclusions without itself being torn or distorted. In fact a system of “skeletal” fibers has been found to exist in many kinds of cells.

There is so far no evidence, however, that the polarity of the egg is dependent on the existence of such an internal skeleton, neither is it certain that such an internal skeleton, if it existed in the eggs, would not be dislodged by centrifugation. The other alternative is that the fixed system which is not displaced by centrifugation is the cortical layer of cytoplasm, or the cortex of the egg.


This is in conformity with direct ob­servation, for the cortical granules are not displaced by centrifugation. The layer of cytoplasm in which they are embedded is therefore suf­ficiently viscous to resist the forces usually generated in centrifugation experiments.

Since the immovable cortex of the egg appears to determine the point at which invagination begins in centrifuged Arbacia eggs, as well as the position of the vegetal polar lobe in Ilyanassa, the further suggestion may be made that the cortex is the actual carrier of the polarity of the egg, or that the polarity of the egg is ingrained in its cortex.

If this were the case, the distribution of substances in the interior of the egg might be expected to be controlled or determined by the egg cortex. This suggestion finds support in some further results of centrifugation experiments, namely, the fact that cell con­stituents tend to return to their normal positions after the cessation of centrifuging.

A scattering or mixing up of the strata into which the egg contents had been arranged by centrifugation could be the result of random movement of particles. This explanation, however, does not apply to cases in which after centrifugation certain particles not only move from the position to which they were brought by the centrifugal force but take up a very definite location in the egg.

In the egg of the ascidian Styela different kinds of egg cytoplasm may be displaced by centrifugation. Immediately after centrifugation the eggs show a clear stratification. The yellow granules, which go into the formation of mesoderm, are displaced to the centripetal pole; the yolk is displaced to the centrifugal pole; and the hyaline cytoplasm remains as a layer in between.

When the eggs are left to themselves after centrifugation, the egg substances start flowing and rearranging them in the interior of the egg. If the cell divisions set in sufficiently soon, this rearrangement is stopped by partitions appearing between the cleavage cells, and the result is the formation of the abnormal embryos dealt with earlier.

If, however, the eggs are centrifuged well in advance of the beginning of cleavage, or if the first divisions of the egg are delayed, the redistribution of the egg contents may proceed so far that normal conditions are attained. The end result of such redistribution- the yolk is at the vegetal pole; the animal hemisphere is filled with hyaline cytoplasm; and the yellow granules take up a subequatorial area on one side of the egg, corresponding to the mesodermal “yellow crescent” of normal development.

Another and perhaps even more impressive example is that of the egg of the mollusc Aplasia limacina. In the eggs of this animal there are granules containing ascorbic acid (vitamin C), probably connected with the Golgi bodies. In immature oocytes the ascorbic acid granules are uniformly distributed throughout the egg cyto­plasm.

During maturation the granules accumulate in a ring lying inside the cortical cytoplasm and somewhat above the equator. By centrifugation the granules are concen­trated at the centrifugal pole, but after cessation of centrifugation the ascorbic acid granules soon start moving and again take up their normal position as a supraequatorial ring.


The most plausible explanation of the two preceding experiments is that the displaced cytoplasmic particles tend to return to the proximity of certain regions of the egg cortex which had remained in their respective positions all the time that the egg was being centrifuged.

It follows that in normal development the cytoplasmic substances in the egg are distributed in relation to local differences in the egg cortex, and that it is the egg cortex, therefore, that foreshadows, in some way, the pattern of future development of the embryo.

Local differences in the egg surface have been recorded by various authors, and in some cases the differences are very obvious if the eggs or early embryos are examined with the scanning electron microscope- in molluscs forming a polar lobe during early cleavage the cortex of the lobe has a different sculpture from the rest of the egg (embryo).