As the oocyte grows and as various inclusions are produced in its cytoplasm, a pattern of organization gradually emerges, which will be of importance when the eggs start developing.
The arrangement of various substances and cellular constituents in the advanced oocyte and, later, in the egg shows a polarity, that is, an unequal distribution with respect to what may be called the two opposite poles of the egg and with respect to the main axis of the egg—the line connecting the two poles. The nucleus of the egg is approximated to one pole of the egg, which is termed the animal pole.
The opposite pole is termed the vegetal pole, because the accumulation of yolk at that pole serves for the nutrition of the developing embryo. When the oocyte undergoes meiosis, the nucleus of the oocyte approaches the animal pole, and the polar bodies are always discharged at the animal pole. This process serves to distinguish the animal pole in oligolecithal eggs, if the concentration of the yolk at the vegetal pole is not very distinct, as is often the case.
In amphibian oocytes the first yolk platelets appear around the periphery, in the subcortical cytoplasm. As the yolk platelets increase in size and more yolk platelets are produced, they fill the cytoplasm from the outside inward. In the last stages of the growth of the oocyte, the yolk platelets are formed around the nucleus, so that the cytoplasm becomes filled up with yolk. The yolk platelets are not all the same size.
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At the animal pole of the oocyte, where the polar bodies are to be given off later, the yolk platelets remain relatively small and are not packed quite so densely. At the vegetal pole they reach a larger size (about 1.5 µm in length) and come to lie very close to one another, leaving little cytoplasm in between. The interior of the oocytes, the area surrounding the nucleus, is fairly closely packed with yolk platelets which are not quite as large as those at the vegetal pole.
In bony fishes, reptiles, and birds, the thickened cytoplasmic cap containing the nucleus denotes the animal pole of the egg.
Yolk and the other substances obviously destined to serve as food for the developing embryo are not the only substances that are laid down in the oocyte during its growth. A characteristic feature of the mature eggs in many animals is the presence of granules of pigment, which are elaborated during the growth of the oocyte.
In the oocytes of the ascidian, Styela partita, in addition to the yolk, which accumulates at the vegetal pole, there appears in the later stages a yellow pigment in the form of granules distributed all over the surface of the oocyte in a thin cortical layer of cytoplasm.
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In the sea urchin Paracentrotus lividus a similar distribution of pigment granules is observed, but the pigment is red. The yellow pigment of Styela partita and the red pigment of Paracentrotus lividus, although uniformly distributed over the surface of the maturing oocyte, will later be concentrated and come to lie in specific parts of the embryo—in the muscles and mesenchyme of the former, and in the walls of the gut of the latter.
Although it does not necessarily follow that the pigment is in any way a precursor of muscular or intestinal differentiation, it may well be that the pigment is an indicator of some specialization in the cytoplasm of the oocyte which eventually leads to specific types of differentiation of parts of the embryo.
In the eggs of most amphibians there is present a greater or lesser amount of dark brown or black pigment. Depending on the amount of pigment, the egg may appear to be light fawn, through various shades of brown, to pitch black. However, young oocytes have no pigment.
Pigment granules start to be formed somewhat later than the yolk platelets, in oocytes which have grown to about one half of the final diameter. The greatest number of pigment granules becomes located in the cortical layer of cytoplasm of the mature oocyte, but quite considerable amounts of pigment are distributed in the interior.
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A remarkable feature in the distribution of pigment in the amphibian egg is that it is not uniform. There is much more pigment in the animal hemisphere of the oocyte than in the vegetal hemisphere.
The difference may be very marked, so that while the animal hemisphere may be dark brown or black, the vegetal hemisphere appears clear white, although in reality a small number of pigment granules are practically always present in the vegetal hemisphere as well. The transition from the dark to the light areas is fairly sharp, but there is always a zone of intermediate, gradually fading pigmentation, which can be conveniently referred to as the marginal zone.
In a cross section of a ripe amphibian egg it may be seen that the vegetal half of the egg is filled by a densely packed mass of yolk containing very little pigment. This mass of yolk is slightly concave on the top. The center of the egg is occupied by a roughly lens-shaped mass of cytoplasm with middle-sized yolk platelets and a moderate amount of pigment.
This zone also contains the nucleus in the immature oocyte. On the outer edges of this interior mass of cytoplasm lies a ring-shaped area containing large amounts of pigment. The ring is thicker on one side of the egg, where it also reaches nearer to the surface.
This side of the egg corresponds, to the future dorsal side of the embryo and thus, in conjunction with the differences along the main axis of the egg, indicates a plane of bilateral symmetry. Lastly, on top of the interior mass of cytoplasm lies, like an inverted saucer, a layer of cytoplasm of the animal hemisphere (the “animal cap”), which is rich in pigment, especially in the cortical layer, and relatively poor in yolk.
The pigment granules in themselves may perhaps not be very important for the development of the embryo; in fact, there are some species of amphibians which do not have any pigment in their eggs (the large European crested newt, Triturus cristatus, and some frogs making foam nests, like the African Chiromantis xerampelina) and yet develop in the same way as related species which have pigmented eggs.
However, the uneven distribution of the pigment may be considered as an indicator of qualitatively different areas in the cytoplasm of the egg. This can be corroborated by some further observations. We have seen that the cytoplasm of young frog oocytes contains large amounts of ribonucleic acid. It has been observed that in oocytes of 400 to 500 µ in diameter the ribonucleic acid is especially concentrated in the surface layer of the animal hemisphere of the oocyte.
With the development of yolk platelets in the subcortical layer of cytoplasm, the ribonucleic acid concentration disappears, but it seems very probable that the localization of pigment in the animal hemisphere reflects some peculiarities in cytoplasm—peculiarities which are related to the distribution of the ribonucleic acid in the preceding stage.
In the elephant tusk mollusc, Dentalium, very distinct cytoplasmic areas can be seen in the egg even before it leaves the ovary. The fully grown oocyte contains a pigment varying in different individuals from olive green to brick red. However, the pigment does not encroach on two areas at the opposite sides of the oocyte, which are therefore colorless.
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One of the two colorless areas lies on the side of the oocyte, which is attached to the wall of the ovary. While the oocyte is in the ovary, this part is somewhat drawn out, so that the oocyte is more or less pear-shaped. The second pigment-free area is on the free surface of the oocyte, and in the center of this area the polar bodies are given off, thus marking this side as the animal pole.
The egg is released from the ovary with the germinal vesicle still intact, and it proceeds to the meiotic divisions only afterwards. After release from the ovary, the egg partially rounds up but remains slightly flattened from animal to vegetal pole. In a vertical section through the egg, the light area at the vegetal pole (the pole that was attached to the wall of the ovary) is seen to be made up of clear cytoplasm which contains no yolk granules.
This cytoplasm reaches inward to the nucleus and surrounds it, and at the outer edges it is continuous with a very thin cortical layer covering the entire egg surface. There is also a small patch of clear cytoplasm at the opposite pole which marks the place where the polar bodies will be given off. The rest of the cytoplasm is filled by rather densely packed yolk granules.
The yolk-free and unpigmented cytoplasm at the vegetal pole may be termed the vegetal polar plasm. The pigment-free animal polar plasm is only partly free from yolk. Of all the developments in the cytoplasm of the oocytes, perhaps the most important are those which concern the surface layer of the oocytes. The superficial layer of cytoplasm in oocytes differs in its physical properties from the rest of the cytoplasm.
Most of the oocyte cytoplasm is in the physical state of a suspension, with a continuous liquid phase (containing in solution various large and small particles, ranging in size from simple inorganic ions to protein molecules) and with larger inclusions, such as mitochondria and ribosomes, dispersed more or less at random in the continuous phase.
These inclusions are freely movable, under natural conditions, owing to cytoplasmic streaming, or in experiments in which the cells are exposed to a centrifugal force. The superficial layer is, on the other hand, in a gelated state or at least in a very much more viscous state, so that its components are not readily displaced during cytoplasmic streaming or in moderately strong centrifugation.
This superficial layer of the cytoplasm is known as the cortex, and it plays the role of a fixture in the cell, remaining in the same position for continuous periods of growth and development. The thickness of the cortex has been measured by various means, and the results have been somewhat contradictory.
It has been claimed on the grounds of centrifugation experiments that the only immovable parts of the egg are the cell membrane and the particles immediately attached to the cell membrane, which would then represent the cortex of the egg. It is more generally recognized, however, that in addition to the cell membrane a layer of cytoplasm, about 2 to 3 µm in thickness, is in a gelated state and thus constitutes the cortex of the egg.
The larger cytoplasmic inclusions are either embedded in or attached to the cortex and so remain in a fixed position. In addition to fixing the position of parts of the oocyte cytoplasm, the cortex is the bearer of some differentiations which are highly characteristic of the late oocyte and mature egg.
Both seem to be concerned with the transport of substances from the follicle cells into the oocyte. An entirely different aspect of cortical differentiation is seen in the formation of special structures known as the cortical granules.
These are spherical bodies, varying in diameter from 0.8 µm (in sea urchins) to 2 µm (in frogs), surrounded by a simple membrane and containing acid mucopolysaccharides. In mature oocytes the cortical granules are arranged in a layer close to the plasmalemma. Cortical granules have been found in oocytes of many animals, but not in all.
They are present in sea urchins, frogs, fishes, bivalve molluscs, some annelids, and some mammals, such as the hamster, the rabbit, and man. They are absent, however, in other mammals, such as the rat and the guinea pig. They are also absent in gastropod molluscs, urodele amphibians, insects, and birds.
In most cases the mucopolysaccharide content of the cortical granules is fairly homogeneous, finely granular or floccular, but in sea urchins the cortical granules show a complicated internal structure consisting of a system of concentric or possibly spirally arranged dense lamallae, separated by less dense material.
The cortical granules are known to be formed in the interior of the oocyte, and in sea urchins, frogs, and humans, their origin has been traced to the Golgi bodies. The cortical granules when first formed lie inside the cup-shaped space formed by the Golgi membranes.
Later, they move to the periphery, to the cortical layer of cytoplasm. The cortical granules appear to play an important role at the time of fertilization, when the granules burst and their contents contribute to the accumulation of fluid around the fertilized egg.
In the last stage of maturation, the nuclear membrane of the oocyte breaks down, and the chromosomes move to the surface at the animal pole to take part in the maturation divisions. At the same time the nuclear sap merges with the cytoplasm of the oocyte. It has been observed in some animals that the nuclear sap does not mix completely with the cytoplasm, but that it forms a more or less separate mass, thus increasing the diversity of the cytoplasmic areas of the eggs.
As a general rule mature eggs are spherical in shape, though elongated eggs are not infrequently found, especially among insects. Among vertebrates, oval-shaped eggs are found in the hagfish Myxine and in the ganoid fishes. The elongated shape of the bird’s egg, on the other hand, is not due to an elongated shape of the egg cell itself. The egg cell, which is the yellow of the egg, is in this case spherical.
Before concluding this article on the organization of the egg and the processes which provide for the arrangement of the various cytoplasmic substances in the egg, we must return once more to the polarity of the egg and consider its origin. The polarity of the egg is discernible from the position of the parts in the egg cell – the nucleus, yolk, and other cytoplasmic substances.
Many efforts have been made to elucidate what factors are responsible for the unequal distribution of these parts. It has been claimed that the polarity of the egg may be imposed on it by the direction of flow of the nutrient substances during the growth of the oocyte. In the molluscs and echinoderms the vegetal pole of the egg develops from that end of the oocyte which is attached to the wall of the ovary.
The nutrient substances, at the expense of which the oocyte grows, presumably enter the ovary from outside, from the body cavity. Therefore, it stands to reason that greater amounts of yolk might be deposited in the part of the cell nearest to the proximal surface of the ovary, thus causing this part to become the vegetal pole.
It was shown, however, that in sea urchins the oocytes are nourished not by diffusion from outside the ovary, but by “wandering cells” which surround the oocytes inside the ovary. It is the initial position of the early oocytes, as cells of the epithelial lining of the ovary that determines the axis of the animal-vegetal polarity of the mature egg.
In a hydroid Amphisbetia operculata the polarity of the egg can be traced visually to the position of the oocyte in the epithelium from which the oocyte is derived (the epithelium of the gastric canal in this case). These epithelial cells have an inclusion of yellow pigment granules in the distal (free) part of the cytoplasm.
The pigment is retained after the oocyte has matured, and even after the eggs have started to cleave. The part of the developing embryo which contains the yellow pigment becomes the part in which the endoderm is formed by ingression of cells into the blastocoele.
These older data on polarity in hydroid eggs have been confirmed and extended with the use of vital staining in species that do not have pigmentation to help in tracing the fate of parts of the egg. In Hydractinia echinata and Phialidium gregarium the polar bodies are produced at the free, distal end of the oocyte (the end furthest from the attachment at the base of the gamete-producing epithelium).
After the end of the egg, where the polar bodies have been formed, is marked with vital dye, and the eggs are fertilized and allowed to develop, it is found that the first cleavage furrow starts within the stained part of the egg, and subsequently this part of the egg gives rise to the posterior end of the larva (planula), which after attachment gives rise to the oral end of the polyp.
Thus in normal development, provided that it is not interfered with by the experimenter, the polarity of the fully developed animal (the polyp) can be traced, through the larvae, through the stage of cleavage of the embryo, through the stage of polar body formation, to the position of the oocyte in the epithelium from which it is derived.
The position of the oocyte in the epithelial layer of the gonad cannot be the cause of the development of the polarity in oocytes which are surrounded by follicle cells from all sides, as are the oocytes of amphibians or mammals. It has been suggested that the course of the nearest blood vessel supplying parts of the ovary with nourishment might cause the parts of the oocyte nearest to the vessel to develop into the vegetal pole.
But according to the views of Child (1941), the animal pole, as the more active one, should develop from that part of the oocyte which has a better oxygen supply, and on this principle the part of the oocyte nearest to a blood vessel should become the animal pole. Actually, there does not seem to be a clear connection between the position of the animal and vegetal poles of the egg and the course of the blood vessels.
In view of the differences in the structure of ovaries in different animals, it would seem rather hopeless to try to find a common factor in the environments of growing oocytes which could be held responsible for the origin of polarity of the egg.
A method has been worked out to allow oocytes of a frog, Xenopus laevis, to grow and develop in complete isolation from the female body, in a culture medium. An essential ingredient of the medium has to be vitellogenin—the precursor of the yolk accumulated in the oocyte (vitellogenin = lipovitellin). This is necessary because the oocyte does not synthesize yolk in its cytoplasm, but draws on a supply provided from the blood.
The smallest oocytes used in the experiments had a diameter of 0.66 mm. and were barely pigmented. In the course of cultivation they became fully pigmented over their entire surface, and subsequently the pigment migrated to the animal hemisphere of the oocyte. The oocyte in this way acquired the typical heteropolar structure, with a pigmented animal hemisphere and a vegetal hemisphere largely devoid of pigment.
Oocytes, which reached a size of 1.1 mm. in diameter in vitro, when treated with progesterone, matured; the germinal vesicle was ruptured; and the first polar body was formed—the oocytes were thus converted into mature eggs. The important fact in this experiment is that the polar structure of the egg (animal-vegetal polarity) could be achieved as a result of intrinsic factors contained in the oocyte itself, quite independently of the maternal body.
Polarity is, however, a phenomenon which is found not only in egg cells but in other cells as well. In epithelial cells there is a distinct difference between the proximal end of the cell (the end resting on the underlying basement membrane) and the distal end (which forms the free surface of the epithelium). In the nerve cells the polarity of the cell takes the form of the opposite differentiations of axon and dendrites.
In the interior of any cell, the position of the centrosome with respect to the nucleus establishes a general form of polarity, the main axis of which is the line drawn through the centrosome and the nucleus. This polarity not only affects the distribution of cytoplasmic inclusions (the Golgi bodies are often found grouped around the centrosome) but may involve the intimate structure of the nucleus itself.
When the oocytes are in the early leptonema stage of the meiotic prophase, the chromosomes become arranged in a definite way, converging to that side of the nucleus which is nearest to the centrosome. This stage is known as the “bouquet stage”. Early oocytes thus already possess a polarity, and this intrinsic polarity may well serve as a basis for the distribution of cellular constituents during the growth and maturation of the oocyte.
The structural aspects of polarity may be only a final result of physiological processes that cannot be detected by the examination of the morphology of cells, oocytes in particular. By applying specially adapted electrodes to oocytes of a frog (Xenopus) it was found that there is a continuous current, up to 1 µA/cm2 in strength passing through the oocyte in the direction from the animal to the vegetal pole, taken as the movement of positive charges.
The positive current is maximal at the animal pole, is reduced to zero just below the equator, and becomes negative in the vegetal hemisphere with a negative maximum at the vegetal pole.
The actual carrier of the charges was found to be the Cl– ion moving through the oocyte from the vegetal to the animal pole, with the permeability of the cell membrane to Ca++ ions being an indispensable factor.
The current inevitably produces a potential difference along the animal-vegetal axis, and this in turn could cause a directed movement of charged particles inside the oocyte, establishing, as it were, an electrophoresis system in the cell. Such a system could well contribute to the morphological animal-vegetal polarization in the oocyte.
The electric current through the oocyte has been recorded in oocytes of a diameter of 0.5 mm. in which pigment is just starting to be formed, as well as in fully grown oocytes. With maturation of the oocyte, either natural, or caused experimentally, the current stops within a short time.
This refer to the polarity along the main axis of the egg (along the axis extending from the animal to the vegetal pole). A similar sort of polarity may be responsible for the difference between the side which is to develop into the dorsal side of the embryo and that which is to develop into the ventral side of the embryo. However, even less is known about the origin of this polarity than about the origin of polarity along the main axis.